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Transcript
Identification of Novel Lysophospholipid Acyltransferases in Yeast (LPT1) and Humans (LPCAT3) and Regulation of Triglyceride Synthesis in Yeast A Thesis Submitted to the Faculty of Drexel University By Shilpa Jain in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 2008 © Copyright 2008 Shilpa Jain. All Rights Reserved. ii Dedications To Mummy, Papa, Didis and Prosenjit iii Acknowledgements I would like to express my gratitude for my advisor, Dr. Peter Oelkers, Dept. of Bioscience and Biotechnology, Drexel University. Under his guidance I learned lot of lab techniques and thus acquired tools to answer research questions and think like a scientist. He taught me all. I don’t remember one time when walk-ins were not welcome in his office. No matter what experiment I plan I will always hear his voice in my head “Think about the scientific question you are asking”. No idea was a stupid idea. It was so much fun to discuss research ideas with him. Peter can explain an experiment on a small piece of paper with the same ease as he can tell you about places to see in a hand drawn paramecium shaped map of New York. Special thanks to my committee members for their time and suggestions. Dr. Bentz, the Chair of the committee brought interesting perspective on membranes and acyl-CoA concentrations, Dr. Nickels had very good suggestions to offer about tools to use in my project. His suggestion about using automated synthetic lethality screen in collaboration saved us a lot of time. Dr. Saunders always had good comments not only related to research but also how to make effective presentations. Dr. Elefant in spite of being a geneticist by training always had relevant questions and made me think of the bigger picture in terms of the physiological role of enzymes studied in the project. I am lucky to have all of these great scientists in my committee. I am grateful to Dr. Howett, Head, Dept. of Bioscience and Biotechnology for sponsoring registration fees to local and regional conferences. She is a role model for all the Graduate students in the department. Nanette, Brenda and Kerin in bioscience office your help is appreciated. iv It is important to have fellow mates who keep you sane in graduate school. I am lucky I had a great support system. Annie (Annette) thanks for the movie-ice cream dates and all fun things we did together. It was always nice to know (with your laughter streaming in our lab and our music vice versa) that you were sitting next door and I could simply walk over and talk to you. I cannot forget lazy eat-sleep-play ludo-eat-ludo-sleep weekends with Poulomi and Vilas. Thanks, to both of you for always being there in the ups and downs of the “to be Ph.D.” life. Kavi, Adetoun, Ang, Mei, Neha and Soumya you all were great lab mates. Sara, you were a cool buddy. Maggie, thanks for teaching me how to swim and overcome my fears of deep water. Shaya, Eugenia and Aiman thanks for your help. The set of instructions in thesis manual makes thesis formatting look like a daunting task. I am glad I had Ellen to help me out. I am indebted to my family: my parents, Alka didi, Vandi and Ritu for their unconditional support and encouragement. Mummy thanks for your prayers (especially before submitting the papers or appearing before job interviews). Ritu and Ken thanks for always checking on my progress in school and supporting me during my job search. Dids by now it has been established that you are my 24 hr. emergency hotline. Prashant thanks for being my personal physician and I am sure we will keep in touch. Roni you are a great housemate and having Pari at home was a great stress buster. Last but not least my special thanks to my husband, Prosenjit for all the support and love. Thanks for talking to me on phone at odd hours in night, (although perfectly normal hours for me...). I always looked forward to week long vacations with you in the entire year. Whenever things were tough for me I always thought of how someone else across Atlantic was working hard to realize his dreams. Your love just keeps me going. v Table of Contents LIST OF TABLES............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii ABSTRACT....................................................................................................................... xi 1. INTRODUCTION .........................................................................................................1 1.1 Triglyceride synthetic pathway..............................................................................1 1.1.1 Steps and enzymes of triglyceride synthetic pathway ..................................6 1.1.2 Regulation of triglyceride synthesis............................................................21 1.2 Phospholipid Biosynthesis .....................................................................................23 1.2.1 Acyl chain asymmetry in phospholipids ....................................................27 1.2.1.1 Mechanisms to incorporate acyl chains in phospholipids..................28 1.2.1.2 Acyl chain remodeling of phospholipids by acyl-CoA dependent lysophospholipid acyltransferases .................................................................32 1.2.1.3 Phospholipid remodeling in Saccharomyces cerevisiae ....................34 1.2.1.4 Physiological role of lysophospholipid acyltransferases ...................35 1.3. Specific Aims .......................................................................................................37 2. REGULATION OF TRIGLYCERIDE SYNTHESIS IN SACCHAROMYCES CEREVISIAE................................................................................................................38 2.1 Abstract ................................................................................................................38 2.2 Introduction..........................................................................................................39 2.3 Experimental Procedures .....................................................................................42 2.4 Results..................................................................................................................46 2.5 Discussion ............................................................................................................67 vi 3. IDENTIFICATION OF A NOVEL LYSOPHOSPHOLIPID ACYLTRANSFERASE (LPT1) IN SACCHAROMYCES CEREVISIAE............................................................76 3.1 Abstract ................................................................................................................76 3.2 Introduction..........................................................................................................78 3.3 Experimental Procedures .....................................................................................84 3.4 Results..................................................................................................................89 3.5 Discussion ..........................................................................................................109 4. IDENTIFICATION AND BIOCHEMICAL CHARACTERIZATION OF HUMAN LYSOPHOSPHATIDYLCHOLINE ACYLTRANSFERASE-3 (LPCAT3)............116 4.1 Abstract ..............................................................................................................116 4.2 Introduction........................................................................................................118 4.3 Experimental Procedures ...................................................................................120 4.4 Results................................................................................................................124 4.5 Discussion ..........................................................................................................139 5. SUMMARY................................................................................................................146 LIST OF REFERENCES.................................................................................................155 APPENDIX 1: GENERAL STRUCTURE OF GLYCEROLIPIDS ..............................173 APPENDIX 2: MISCELLANEOUS EXPERIMENTS...................................................176 vii List of Tables 1.1 Amino acid alignment score of human AGPAT family members...............................9 3.1 Comparison of different methods used to calculate kinetic parameters of Lpt1 .....104 3.2 Kinetic parameters of Lpt1 ......................................................................................104 3.3 Subcellular localization of acyl-CoA dependent LPCAT activity...........................106 4.1 Kinetic parameters of human LPCAT3 ...................................................................135 viii List of Figures 1. Figure 1.1: Triglyceride synthetic pathway ...................................................................2 2. Figure 1.2: Triglyceride synthetic pathway in mammals...............................................4 3. Figure 1.3 Triglyceride synthetic pathway in Saccharomyces cerevisiae .....................5 4. Figure 1.4: Phylogenetic tree based on amino acid sequence similarity of AGPAT family members .............................................................................................................9 5. Figure 1.5 Phospholipid synthetic pathway in mammals ............................................24 6. Figure 1.6 Phospholipid synthetic pathway in Saccharomyces cerevisae...................25 7. Figure 1.7 Phospholipid remodeling (Lands Cycle) ....................................................31 8. Figure 2.1 Inability to oxidize fatty acids significantly increases triglyceride levels in pox1∆ strain .................................................................................................................47 9. Figure 2.2 Triglyceride levels in glycogen deficient strains........................................48 10. Figure 2.3 Effect of addition of water-soluble phospholipid precursors in media on triglyceride levels.........................................................................................................50 11. Figure 2.4 Glycogen levels in triglyceride deficient yeast strain.................................52 12. Figure 2.5 In vitro Diacylglycerol acyltransferase (DGAT) activity with increasing concentrations of [14C] oleoyl-CoA. ............................................................................55 13. Figure 2.6 Effect of 5mM ATP on in vitro Diacylglycerol acyltransferase (DGAT) activity..........................................................................................................................59 14. Figure 2.7 Effect of 0.8mM NADH on in vitro Diacylglycerol acyltransferase (DGAT) activity...........................................................................................................60 15. Figure 2.8 Effect of 0.2mM acetyl-CoA on in vitro Diacylglycerol acyltransferase (DGAT) activity...........................................................................................................61 16. Figure 2.9 Effect of 0.1 mM malonyl-CoA on in vitro Diacylglycerol acyltransferase (DGAT) activity...........................................................................................................62 17. Figure 2.10 Effect of 0.1 mM and 1mM AMP on in vitro Diacylglycerol acyltransferase (DGAT) activity..................................................................................65 ix 18. Figure 2.11 Effect of 0.1mM ADP and 0.8 mM NAD on in vitro Diacylglycerol acyltransferase (DGAT) activity..................................................................................66 19. Figure 3.1 Pathways for phospholipid synthesis in Saccharomyces cerevisiae. ......... 81 20. Figure 3.2 Incorporation of [3H] oleate into phospholipids and neutral lipids in slc1Δ, ydr018Δ, ybr042Δ ................................................................................................................. 91 21. Figure 3.3 Incorporation of [3H] oleate into diglyceride, triglyceride and phospholipids in ybr042∆, ydr018∆ and ybr042∆ydr018∆ in slc1∆ background .......92 22. Figure 3.4 Alignment of LPT1 and 3 human members of the MBOAT gene family.94 23. Figure 3.5 In vitro 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT) assays ......... 96 24. Figure 3.6 Lysophosphatidylcholine acyltransferase (LPCAT) activity is absent in lpt1Δ.............................................................................................................................97 25. Figure 3.7 Lysophospholipid substrate specificity of Lpt1p ................................................ 99 26. Figure 3.8.1 Acyl-CoA substrate specificity of Lpt1p..............................................101 27. Figure 3.8.2-3 Acyl-CoA substrate specificity of Lpt1p .................................. 102-103 28. Figure 3.9 Incorporation of [3H] oleate into phospholipids and neutral lipids .........107 29. Figure 4.1 Alignment of Lpt1p and MBOAT5 / C3F...............................................125 30. Figure 4.2 Predicted transmembrane domains of LPCAT3......................................125 31. Figure 4.3 Lysophosphatidylcholine acyltransferase (LPCAT) activity in infected Sf9 insect cells..................................................................................................................127 32. Figure 4.4 Lysophospholipid substrate specificity of LPCAT3 ...............................128 33. Figure 4.5 Kinetics of lysoPC utilization by LPCAT3 .............................................129 34. Figure 4.6 Kinetics of Stearoyl-CoA (18:0-CoA) utilization by LPCAT3........................131 35. Figure 4.7 Kinetics of Oleoyl-CoA (18:1-CoA) utilization by LPCAT3 ........................... 132 36. Figure 4.8 Kinetics of Arachidonoyl-CoA (20:4-CoA) utilization by LPCAT3............... 133 37. Figure 4.9 Kinetics of Linolenoyl-CoA (18:3-CoA) utilization by LPCAT3..................... 134 38. Figure 4.10 Effect of LPCAT3 expression on Sf9 phospholipid species. ........................... 137 x 39. Figure 4.11 Dendogram showing members of human MBOAT family...................140 40. Figure 5.1 Evolutionary dendogram showing homologs of Lpt1 in other eukaryotes151 41. Figure 5.2 Dendogram showing Lpt1 homologs in human MBOAT family ...........152 42. Figure A1. Glycogen levels in pox1, tgl3∆tgl4∆tgl5∆ yeast strains.........................176 43. Figure A2. Effect of cerulenin on glycogen levels in wild type yeast strain ............177 44. Figure A3. Effect of inositol and choline on triglyceride synthesis in wild type yeast strain at different growth phases ..............................................................................178 xi Abstract Identification of Novel Lysophospholipid Acyltransferases in Yeast (LPT1) and Humans (LPCAT3) and Regulation of Triglyceride Synthesis in Yeast Shilpa Jain, MS Peter Oelkers, Ph.D. Cells require membranes for survival and need energy to meet the costs of living. Phospholipids and triglyceride together fulfill both the requirements. Phospholipids are predominant membrane lipids and triglycerides allow for storage of surplus energy therby enhancing survival in energy poor conditions. Therefore, an understanding of how cell regulates triglyceride synthesis and maintains membrane composition by acyl chain remodeling of phospholipids is important. Saccharomyces cerevisiae was used as a model system to study the intracellular regulation of triglyceride synthesis at the step of diglyceride esterification. Using metabolic labeling with [3H] oleate, we found that the inability to hydrolyze fatty acids, significantly (p<0.05) increased triglyceride synthesis by 75%. Disabling glycogen synthesis (alternative energy reserve) did not alter triglyceride synthesis. Additionally, inability to store energy as triglyceride did not result in a compensatory increase in glycogen synthesis. To address the possibility of regulation at the level of enzyme activity, microsomal diacylglycerol acyltransferase assays were performed in the presence of a variety of metabolites (AMP, ADP, ATP, NAD+, NADH, acetyl-CoA and malonyl-CoA). DGAT activity was not regulated at physiological concentrations of these metabolites. Acyl chains in the sn-2 position of phospholipids are introduced by de novo synthetic pathway involving 1-acylglycerol-3-phosphate acyltransferase (AGPAT) or by acyl chain remodeling that requires lysophospholipid acyltransferases (LPLAT). LPT1 xii (YOR175c) an acyltransferase with unknown function was identified by its synthetic lethal interaction with Slc1, the known AGPAT in yeast. LPT1 was shown to encode for a novel AGPAT/ LPLAT in yeast with high apparent affinity for saturated acyl-CoAs but higher apparent Vmax for monounsaturated acyl-CoAs. Lpt1 does not share sequence similarity with known LPLAT/AGPAT family members and belongs to membranebound-O-acyltransferase (MBOAT) super family. Homology search with Lpt1 identified the human MBOAT5, a protein with unknown function. Cell lysates from insect cells expressing human MBOAT5 (renamed as LPCAT3) showed a 10 fold increase in LPCAT activity over control. In summary, the identification of a novel LPLAT in yeast has proved to be the founding member of LPLAT family in humans. Future characterization of this family is likely to shed light on how membrane composition is adjusted and controlled in humans. xiii 1 CHAPTER 1: INTRODUCTION 1.1 Triglyceride synthetic pathway Triglyceride, also known as triacylglycerol, is composed of a glycerol molecule joined to three acyl chains by ester bonds. This is an energy-rich molecule that allows for compact energy storage and yields twice the amount of energy in comparison to a polysaccharide such as glycogen or starch. All eukaryotes and a subclass of prokaryotes (Actinomycetes), have intracellular triglyceride stores in structures known as lipid droplets, lipid particles and oil bodies. Probably, the ability to store surplus energy in form of triglyceride offers a metabolic advantage during the periods of nutritional surplus and nutritional stress (Alvarez 2002). Triglyceride also sequesters fatty acids and diacylglycerol, precursors for phospholipid biosynthesis. The Kennedy pathway or glycerol -3- phosphate pathway is the de novo route for intracellular triglyceride synthesis (Kennedy 1961). Initial steps of this pathway are common for triglyceride and glycerophospholipid biosynthesis. The Kennedy pathway requires glycerol 3-phosphate and fatty acyl-CoA as substrates (Appendix 1). Glycerol 3phosphate can be generated either by reduction of dihydroxyacetone phosphate, an intermediate of glycolysis, or from the phosphorylation of glycerol. Acyl-CoAs, the other substrates, come from activation of fatty acids by coenzyme A. Glycerol 3-phosphate undergoes sequential acylation by two fatty acyl CoA molecules to form phosphatidic acid (PA) (Figure 1.1). 2 Figure 1.1: Triglyceride synthetic pathway 3 Acyl-CoA: glycerol-sn-3-phosphate acyltransferase (GPAT) catalyzes the first step in the Kennedy Pathway to form 1-acylglycerol-sn-3-phosphate (lysophosphatidic acid) (Figure 1.1). The subsequent acylation of 1-acylglycerol-3-phosphate to phosphatidic acid is catalyzed by acylglycerol-3-phosphate acyltransferase (AGPAT) also called lysophosphatidate acyltransferase (LPAAT). Phosphatidic acid is then converted to diacylglycerol in a reaction catalyzed by phosphatidic acid phosphatase (PAP). Phosphatidic acid and diacylglycerol generated in this pathway can be converted into triglyceride or phospholipid. In the pathway to generate triglyceride, diacylglycerol acyltransferase (DGAT) catalyzes diacylglycerol esterification with a third acyl-CoA to generate triglyceride. This is the terminal and unique step in the de novo triglyceride synthesis. Triglyceride Synthesis in Yeast The triglyceride synthesis occurs in yeast by Kennedy pathway reactions, as discussed above, with a few differences. The first step of acylation reaction starts with either glycerol 3-phosphate or dihydroxyacetone phosphate (DHAP) and acyl-CoA (Figure 1.3). If dihydroxyacetone phosphate is the substrate, it is acylated, and the product 1-acyl DHAP is reduced to form lysophosphatidic acid. Unlike mammals, DHAP in yeast is not utilized to synthesize ether lipids but contributes to the formation of glycerolipids (Coleman 2004). However, the relative contributions of the glycerol 3-phosphate and DHAP to triglyceride and phospholipid synthesis are not known (Racenis 1992). Lysophosphatidic acid is further acylated to form phosphatidic acid. 4 Figure 1.2: Triglyceride synthetic pathway in mammals 5 Figure 1.3 Triglyceride synthetic pathway in Saccharomyces cerevisiae 6 Diacylglycerol formed from phosphatidic acid is acylated to triglyceride by two reactions, acyl-CoA dependent and acyl-CoA independent. The acyl-CoA dependent reaction is similar in mammals and yeast. In the acyl CoA independent reaction the diacylglycerol esterification occurs by using the sn-2 acyl group of glycerophospholipids preferentially phosphatidylcholine and phosphatidylethanolamine (Oelkers 2000). This reaction has not been characterized in humans. 1.1.1 Steps and enzymes of triglyceride synthetic pathway Step 1: Synthesis of 1-acylglycerol 3-phosphate (Lysophosphatidic acid) This is the first step of glycerolipid synthesis. Glycerol-3-phosphate is acylated to 1-acylglycerol-3-phosphate (also known as lysophosphatidic acid) and is catalyzed by acylCoA: glycerol -3-phosphate acyltransferase (GPAT) (Figure 1.1, Appendix 1). In mammals In mammals, four GPAT isoforms, GPAT1-4 have been cloned. GPAT1 and GPAT2 encoded activity is associated with mitochondrial fraction while GPAT3 and GPAT4 encoded enzymes are microsomal (Coleman 2004; Cao 2006; Wang 2007; Chen 2008; Nagle 2008) (Figure 1.2). The microsomal GPAT contributes to about 90% of total cellular activity in most tissues. This activity has only been recently characterized hence not much is known about its regulation. Regulation of the mitochondrial GPAT has been intensively studied and found to occur by the transcriptional activation during adipocyte differentiation and by sterol regulatory element binding protein-1c (SREBP-1c) (Ericsson 1997). 7 The mitochondrial activity is also subject to nutritional and hormonal regulation. In rats, 48 hours of fasting has been shown to reduce hepatic mitochondrial GPAT1 protein expression and activity by 30% while refeeding for 24 hours, a high carbohydrate or high sucrose diet increases mRNA abundance and activity by 60% (Lewin 2001). Fasting and refeeding result in an increase in insulin secretion hence the mitochondrial GPAT mRNA level and activity was examined in streptozotocin induced diabetic mice. In the diabetic mice the mRNA levels of the mitochondrial GPAT could not be detected and upon insulin injection the mRNA levels increased about 20 fold after 6 hours (Shin 1991). In energy rich conditions, there is up regulation of triglyceride synthesis while fatty acid utilization by beta-oxidation is reduced. Over expression of mitochondrial GPAT in primary cultures of rat hepatocytes resulted in 81% reduction in fatty acid oxidation and a significant increase in hepatic diacylglycerol and phospholipid biosynthesis. Interestingly, the increase in triglyceride synthesis was seen only in cells provided with additional fatty acids in media, akin to energy replete condition favoring triglyceride synthesis (Linden 2004). The mitochondrial GPAT activity is affected by energy rich and poor conditions in a cell. Low ATP levels result in activation of AMP kinase that inhibits mouse mitochondrial GPAT activity in liver and adipose tissue (Coleman 2004). Besides these studies, on regulation of mitochondrial GPAT isoform the regulation of microsomal isoforms and their contribution to triglyceride synthesis still requires future studies. 8 In Yeast In yeast, Gat1 and Gat2 are responsible for the GPAT activity in endoplasmic reticulum (ER) and lipid particles (Athenstaedt 1997; Zheng 2001) (Figure 1.3). Both Gat1 and Gat2 are able to utilize glycerol-3-phosphate and dihydroxyacetone phosphate as substrates (Zheng 2001). Both yeast enzymes share sequence similarity (12%) with the mammalian mitochondrial GPATs. A gat1Δgat2Δ double mutation is lethal for yeast cells indicating that these two genes encode for the principal GPATs in yeast. GAT1 deletion results in about 80% decrease in GPAT activity while GAT2 deletion results in 30% decrease in GPAT activity. The gat1Δ and gat2Δ single deletion yeast cells when grown to mid log phase and pulse labeled with [14C] acetate show 50% decrease in labeling of triglyceride in gat2Δ and 50% increase in gat1Δ yeast cells. This results suggests that acylation through Gat2 plays a larger role in triglyceride synthesis (Zaremberg 2002). The GPAT activity increases by 3-fold in yeast grown in media containing non-fermentable carbon sources glycerol and ethanol versus fermentable carbon source glucose (Minskoff 1994). Apart from this observation, nothing is known about the regulation of GPAT activity in yeast. Step 2: Synthesis of phosphatidic acid Lysophosphatidic acid is acylated by acyl-CoA:1-acylglycerol-3-phosphate acyltransferase (AGPAT) also known as lysophosphatidic acid acyltransferase (LPAAT). Phosphatidic acid formed by AGPAT is an important intermediate in the Kennedy pathway because it can serve as precursor for phospholipids as well as triglyceride (Figure 1.1; Figure 1.4; Appendix 1). 9 Gene Identity (%) AGPAT1 AGPAT2 AGPAT3 AGPAT4 AGPAT5 AGPAT1 47 7 13 11 AGPAT2 47 4 10 9 AGPAT3 7 4 61 18 AGPAT4 13 10 61 18 AGPAT5 11 9 18 18 AGPAT6 16 8 2 2 3 AGPAT7 16 16 6 4 8 AGPAT8 16 6 25 22 18 AGPAT6 16 8 2 2 3 12 8 AGPAT7 16 16 6 4 8 12 8 AGPAT8 16 6 25 22 18 8 8 - Table 1.1: Amino acid alignment score of human AGPAT family members Figure 1.4: Phylogenetic tree based on amino acid sequence similarity of AGPAT family members 10 In mammals The human AGPAT1 and AGPAT2 were identified based on sequence homology to the bacterial (plsC) and yeast AGPAT (SLC1) genes (Eberhardt 1997; Stamps 1997; West 1997; Aguado 1998) (Figure 1.2). Seven other putative members of AGPAT family AGPAT3-9 have been identified. By analyzing the conserved sequences of all the AGPAT family members AGPAT1 and AGPAT2 belong to the same sub group and have been confirmed as AGPATs. AGPAT3, AGPAT4 and AGPAT5 belong to the same subgroup and have very weak AGPAT activities (Figure 1.4, Table1.1) (Agarwal 2007). AGPAT1 and AGPAT2 have been shown to convert lysophosphatidic acid to phosphatidic acid; the same cannot be said about the biochemical properties of other member AGPATs (Leung 2001; Lu 2005) Cell lysates from insect cells expressing AGPAT1 showed 3-fold incorporation of [14C] oleoyl CoA in lysophosphatidic acid over background (Aguado 1998). The transient expression of AGPAT2 in COS 7 cells showed 6 to 17-fold increase in AGPAT activity compared to mock transfected cells. The AGPAT1 and AGPAT2 encoded activity was specific for lysophosphatidic acid and did not utilize other acyl acceptors, glycerol 3phosphate, lysophosphatidylcholine, lysophosphatidylethanolamine (Eberhardt 1997). Murine AGPAT3, AGPAT4 and AGPAT5 expressed in COS-1 cells showed only 1.4 fold AGPAT activity over the background. The acyltransferase activity in cell lysate was measured by incorporation of radioactive [14C]oleoyl-CoA in oleoyl-lysophosphatidic acid (Lu 2005). The substrate specificity of AGPAT3, AGPAT4 and AGPAT5 for other acyl acceptors has not been determined. 11 Recently AGPAT8 and AGPAT6 were characterized as microsomal GPAT3 and GPAT4 (Cao 2006; Chen 2008; Nagle 2008). Human AGPAT7 upon ectopic expression in E.coli shows weak acyltransferase activity with lysophosphatidylethanolamine (lysoPE) and 1alkeny lysoPE (Cao 2008) while mouse AGPAT9 displays lysophosphatidylcholine acyltransferase activity (LPCAT1) (Chen 2006; Nakanishi 2006). AGPAT1 mRNA is detectable in all human tissues with highest expression in skeletal muscle (Agarwal 2002). Overexpression of AGPAT-1 in C2C12 myotubes and 3T3-L1 adipocytes resulted in 25% increase in AGPAT activity. In AGPAT1 over expressing 3T3-L1 adipocytes, the uptake of [3H] oleate and its incorporation in triglyceride increased by two fold compared to the mock transfected cells. AGPAT1 over expressing C2C12 myotubes upon stimulation by insulin showed 30% decrease in incorporation of [14C] glucose in glycogen concomitant with 33% increased incorporation in total lipids. In the same study, the immunofluorescence microscopy of 3T3L1 adipocytes and myotubes showed that the overexpressed AGPAT1 protein was localized to cytosol (Ruan 2001). However, AGPAT1 when expressed in mammalian Chinese hamster ovary cell line, shows endoplasmic reticulum staining and lack of plasma membrane localization (Aguado 1998). Thus, the physiological role of AGPAT1 cannot be predicted based on the results of this study. AGPAT2 is expressed in most of the human tissues with high expression levels in heart and liver. AGPAT2 mRNA levels in adipose are two fold higher than AGPAT1 while other AGPATs 3, 4, 5 are not expressed at detectable levels in adipose tissue (Agarwal 2002). 12 AGPAT2 gene expression is induced during adipocytes differentiation and is required for the early induction of transcription factors (c/EBP and PPARγ) required for adipocyte differentiation (Gale 2006). Murine AGPAT3 mRNA shows equal expression in all the tissues while AGPAT4 and AGPAT5 are highly expressed in brain and epidermis respectively (Lu 2005). The biological significance of having multiple (at least 2) isoforms of AGPATs has not been determined. One obvious explanation might be the redundancy in AGPAT family. This possibility is ruled out because AGPAT2 gene mutation in individuals results in congenital generalized lipodystrophy type 1, a disorder characterized by lack of body fat, insulin resistance and hypertriglyceridemia (Agarwal 2002). In this condition, other members of the AGPAT family cannot compensate for the critical role of AGPAT2 in triglyceride synthesis and its role in adipocyte differentiation and maturation. Another possibility is that AGPAT isoforms perform different biological functions in different tissues. Due to the lack of data about the physiological role of AGPAT isoforms, nothing is known about the regulation of multiple AGPAT isoforms. Further studies are required to know whether there is coordinated or independent regulation of isoforms and what is their contribution in triglyceride synthesis and regulation. In Yeast In yeast, Slc1 mediates all of the AGPAT activity in the lipid particles and 54% of the total enzyme activity in microsomes (Athenstaedt 1997). SLC1 (sphingolipid compensation-1) was identified by a screen to search for suppressors of a genetic defect in sphingolipid synthesis and was found to be homologous to bacterial AGPAT (plsC)(Coleman 1990). 13 A mutation in the plsC gene causes the strain to grow slowly at 37ºC and not at all at 42ºC. The ectopic expression of SLC1 in a bacterial strain mutated for plsC gene enabled the strains to grow normally at 37ºC and promoted growth at 42ºC. This suggested that SLC1 encodes for AGPAT activity in yeast (Coleman 1990; Nagiec 1993). Further studies showed that slc1Δ mutants showed no AGPAT activity in lipid particles but microsomes prepared from slc1∆ strain retained 46% of the total AGPAT activity. This indicates that alternative pathway or another enzyme involved in glycerolipid biosynthesis must exist in yeast, which contributes to the formation of phosphatidic acid in the absence of Slc1(Athenstaedt 1997). The enzyme, which mediated phosphatidic acid synthesis in yeast in the absence of Slc1 was not known before the current study was undertaken (Jain 2007). Triglyceride levels in slc1∆ mutants grown to log phase are 3-4 times higher than normal (Athenstaedt 1997). This unidentified enzyme with AGPAT activity might be primarily involved in triglyceride synthesis. This uncharacterized AGPAT activity might have higher expression and activity in specific growth phase, such as log phase or stationary phase, a characteristic shared with diacylglycerol esterifying enzymes in yeast (Oelkers 2002). Identification of the enzyme or enzymes responsible for the Slc1 independent AGPAT activity will complete the characterization of almost all of the enzymes involved in triglyceride synthesis in yeast. The only enzyme which has not been characterized yet is the residual magnesium dependent phosphatidic acid phosphatase (PAP) activity in cells lacking the known PAPs DPP1, LPP1 and PAH1. Acetate labeling of cells lacking PAH1, DPP1, and LPP1 show a minor contribution (~ 10%) of this unknown enzyme to triglyceride synthesis. 14 Thus the characterization of novel AGPAT in yeast will help to understand intracellular regulation of triglyceride synthesis in total. Step 3: Synthesis of diacylglycerol Phosphatidic acid phosphatase (PAP) dephosphorylates phosphatidic acid to form diacylglycerol (Figure 1.1). Two kind of PAP activity exist in yeast and mammalian tissues. PAP1 is magnesium dependent and is sensitive to N-ethylmaleimide (NEM), a cysteine modifying reagent, while PAP2 is neither dependent on magnesium nor is NEM sensitive. (Carman 2006). In mammals The PAP1 activity is thought to be involved in the synthesis of triglyceride, phosphatidylethanolamine and phosphatidylcholine (Cascales 1984). Studies with rat lung microsomes have shown that the magnesium dependent PAP1, was removed from microsomes when washed with (0.5M) of sodium chloride while magnesium independent PAP-2 remained associated with microsomes. The washed microsomes lost their ability to incorporate [14C] glycerol-3-phosphate through phosphatidic acid into triglyceride and phosphatidylcholine. There was also a concomitant increase in labeling of phosphatidic acid in washed microsomes. Addition of magnesium dependent PAP1 isolated by gel filtration from wash supernatant restored the labeling of triglyceride and phosphatidylcholine. Magnesium independent PAP2 isolated from cytosol when added to the washed microsomes was incapable of restoring glycerolipid synthesis (Walton 1984). 15 PAP1 activity is cytosolic and upon stimulation by fatty acids or acyl-CoAs the enzyme has been shown to translocate from cytosol to the endoplasmic reticulum, where it is active (Reue 2008). The molecular identity of PAP1 was unknown until recently. In yeast PAP1, activity was purified and the amino acid sequencing identified it as Pah1, the yeast ortholog of mammalian lipin protein (Han 2006). LPIN-1 a member of the LPIN gene family was initially identified by positional cloning as the gene responsible for fatty liver dystrophy (fld), diabetes and diminished adipose tissue in mice (Péterfy 2001). The mechanism by which this gene would cause lipodystrophy was not clear. Subsequent biochemical characterization of human LPIN 1-3 encoded lipin proteins (lipin-1A, lipin-1B, lipin-2 and lipin-3) confirmed their role as PAP activity specific for phosphatidic acid. It was an exquisite example of the power of using model organisms as tools to establish identity of mammalian lipid metabolizing enzymes. Recombinant lipin proteins, when expressed in 293T cells, show that lipin-2 and lipin-3 exhibit 4- fold less PAP activity than lipin-1. In the fld mice, PAP1 activity encoded by LPIN-1 was completely absent in white and brown adipose tissue and skeletal muscle. Thus Lipin-1 accounts for all PAP activity in these metabolic tissues (Han 2006; Donkor 2007). Human LPIN-1 mRNA levels are abundant in liver and brain while LPIN2-3 are significantly expressed in stomach, pancreas and intestine (Reue 2008). The lipin-1 activity is regulated transcriptionally. In primary culture of mouse hepatocytes the mRNA level of LPIN-1 increased upon 4 hour incubation in media containing glucocorticoids, cAMP and glucagon. There were no changes observed in the mRNA levels of LPIN-2 and LPIN-3 isoforms (Manmontri 2008). 16 Lipin-1 undergoes phosphorylation upon insulin stimulation and in sharp contrast, epinephrine leads to its dephosphorylation. The phosphorylation did not affect PAP-1 activity but mediated cytoplasmic to microsomal localization of lipin-1 (Reue 2008). The PAP-1 activity levels increased in liver in fasting conditions and this correlated with increase in mRNA levels of Lpin-1. Most of these studies have shown lipin-1 to be the regulated isoform and the contribution of lipin-2 and lipin-3 to triglyceride synthesis is not known. PAP2 activity is less selective for its substrate and dephosphorylates lysophosphatidic acid, diacylglycerol pyrophosphate and sphingosine1-phosphate in addition to phosphatidic acid. Therefore, this activity is referred to as lipid phosphate phosphatase (LPP). LPP1-3, DPPL1 and DPPL2 encode for PAP2 activity in mammals (Brindley 2004; Takeuchi 2007). The members of PAP2 (LPP) family are predicted to regulate cell signaling by lipid phosphates and their dephosphorylated forms such as diacylgycerol and sphingosine (Carman 2006). In Yeast In yeast, PAH1 encodes for PAP1 activity while DPP1, LPP1 contribute to PAP2 activity (Carman 2006) (Figure 1.3). Pah1 shares sequence similarity with mammalian PAP1 enzyme, Lipin1. Pah1 is localized to the cytosolic and membrane fractions of cell. The pah1∆ deletion mutants when labeled with 32 Pi to steady state show an increase in cellular PA levels by 120-160% consistent with its role as PAP. The pah1Δ mutants showed a decrease in phosphatidylcholine (43%) phosphatidylethanolamine (50%) and phosphatidylinositol (80%). and increase in 17 The incorporation of [2-14C] acetate in triglyceride was reduced by 92% in pah1Δ mutant stationary phase and by 62% in exponential phase. All these results show that PAH1 is responsible for the generation of diglyceride required for the synthesis of phospholipids and triglyceride in S. cerevisiae (Han 2006). PAP activity encoded by PAH1 is inhibited by phosphorylation and ATP (O'Hara 2006). DPP1 and LPP1 encoded enzymes have broad substrate specificity and they are able to utilize lysophosphatidic acid and diacyglycerol pyrophosphate besides phosphatidic acid. The dpp1Δlpp1Δ mutants when labeled with [2-14C] acetate did not show any major effect on neutral lipid composition in either the exponential or stationary growth phase. DPP1 and LPP1 are responsible for regulation of phosphatidic acid levels in vacuoles and Golgi but they might not contribute to the de novo synthesis of triglyceride that occurs in endoplasmic reticulum (Carman 1997). A dpp1∆lpp1∆pah1∆ triple deletion yeast mutant still shows some PAP1 activity, suggesting the presence of an additional gene or genes that are yet to be identified (Han 2006). Step 4: Synthesis of triglyceride In this terminal and unique step of triglyceride synthesis the diglyceride is esterified to triglyceride by an acyl CoA: diacylglycerol acyltransferase (DGAT) reaction (Figure 1.1, Appendix 1) In mammals DGAT1 and DGAT2 catalyze diglyceride esterification reaction in mammals (Figure 1.2). DGAT1 was identified by sequence similarity to acyl-CoA: cholesterol acyltransferase (ACAT1). Mouse DGAT-1 protein is 20% identical to the mouse ACAT1. 18 DGAT1 cDNA when expressed in H5 insect cells, showed acyl CoA dependent activity capable of utilizing only diglyceride (Cases 1998). Normal triglyceride levels in plasma and adipose tissue of DGAT1-/- mice indicated the presence of another triglyceride synthetic mechanism. Subsequently DGAT2 was identified by sequence homology to a DGAT purified from the oleaginous fungus M. rammaniana using column chromatography. DGAT2 does not share sequence similarity to DGAT1 that is a member of ACAT family. DGAT2 when expressed in insect cells showed DGAT activity which was specific for diglyceride and sensitive to magnesium chloride concentrations higher than 20mM (Cases 2001). DGAT1 and DGAT2 have similar substrate specificities for acyl-CoAs and prefer oleoyl-CoA to palmitoyl-CoA, arachidonoyl CoA and linolenoyl CoA at a fixed concentration of 25μM (Cases 2001). Both DGATs are expressed in tissues that are active in triglyceride synthesis, such as white adipose tissue (WAT), small intestine, liver, and mammary gland. DGAT1 mRNA levels are highest in small intestine, while DGAT2 is expressed at high levels in liver and white adipose tissue (Cases 1998; Cases 2001; Stone 2004). DGAT1-/- mice are viable, have 50% less body fat and are resistant to diet induced obesity. Disruption of DGAT2 gene in mice leads to perinatal lethality (~ 6 hours after birth), decrease in plasma triglyceride by 64% and undetectable triglyceride in liver (Stone 2004). The inability of DGAT1 to compensate for DGAT2 suggests that these two enzymes have different functions. DGAT1 and DGAT2 overexpression in liver increased their mRNA levels by ninety fold and two fold and increased the hepatic triglyceride content by three fold and five fold respectively. In comparison to DGAT1, small increase in DGAT2 mRNA resulted in greater increase in triglyceride content (Monetti 2007). 19 This study reinforced that DGAT2 might be more specific and effective than DGAT1 in promoting triglyceride synthesis. There are very few studies, which have examined the regulation of DGATs. In differentiating 3T3-L1 adipocytes, the DGAT1 mRNA increases by seven fold whereas the protein level increases by 90 fold, suggesting posttranscriptional regulation of DGAT1 gene. Increase in DGAT1 protein level by 90 fold correlated with 100 fold increase in DGAT activity in differentiated adipocytes. Overexpression of DGAT1 in 3T3-L1 adipocytes by an adenovirus construct increased mRNA levels by 20 fold but the protein levels by only two fold. The discordant DGAT1 protein and mRNA expression were not due to changes in protein stability. The steady state DGAT1 protein levels in 3T3-L1 adipocytes were not altered in the presence of proteolytic inhibitor N- acetyl-Leu-Leu-norleucinal (ALLN). In addition, treatment of 3T3-L1 adipocytes with cycloheximide did not show change in the protein turnover. DGAT1 when mutated at conserved tyrosine phosphorylation site to phenylalanine (T316F) did not affect DGAT activity in 3T3-L1 adipocytes. DGAT1 encoded enzyme activity does not seem to be regulated by phosphorylation. Thus it was speculated that regulation of DGAT1 expression occurs at the translational level (Yu 2002). Total DGAT specific activity was found to decrease by 86% upon 24 hours incubation of 3T3-L1 adipocytes in media not containing glucose. Addition of glucose to the media of glucose-starved adipocytes for additional 24 hours increased the DGAT activity by 2- fold. DGAT1 mRNA were abolished in the adipocytes incubated in media not containing glucose and were replenished upon addition of glucose to the media. The DGAT2 mRNA levels were unaffected by similar treatment (Meegalla 2002). To further, study the effect of insulin on DGAT1 and DGAT2 mRNA levels the mature adipocytes 20 were serum starved for 16 hrs followed by addition of insulin for 24 hours. Insulin was shown to preferentially enhance DGAT2 mRNA levels (Meegalla 2002). CAAT/enhancer binding proteins (alpha and beta) play an important role in adipogenesis and these transcription factors have been shown to increase DGAT2 mRNA levels during adipocyte differentiation (Payne 2007) Studying DGAT2 regulation is complicated because DGAT2 gene family in humans has six members, identified by sequence similarity to DGAT2 the original member of the family. Three members are monoacylglycerol acyltransferases (MGAT13), which synthesize diacylglycerol by esterification of monoacylglycerol. MGAT1-3 when expressed in insect cells also show weak DGAT activity(Cao 2007). Two other members are wax-alcohol acyltransferases, that synthesize wax esters and the last member hDC3 remains to be biochemically characterized (Sturley 2007). In Yeast All the enzymes responsible for mediating diglyceride esterification in Saccharomyces cerevisiae have been identified. DGA1, LRO1 and ARE2 encoded enzymes mediate diglyceride esterification in yeast (Oelkers 2002) (Figure 1.3). These three genes belong to different gene families. DGA1 is the yeast homolog of human DGAT2, and is responsible for acyl CoA dependent DG esterification. LRO1 is a member of lecithin cholesterol acyltransferase (LCAT) family and mediates triglyceride synthesis by an acyl CoA independent mechanism that utilizes phospholipids to esterify diacylglycerol (phospholipid diacylglycerol acyltransferase or PDAT) (Oelkers 2002). Dga1 and Lro1 mediate most of triglyceride synthesis (98%) while ARE2, a homolog of 21 DGAT1, has a minor role. Depending on the growth conditions, Dga1 and Lro1 contribute differently to the triglyceride synthesis (Oelkers 2000; Oelkers 2002). The dga1Δ yeast strain when grown to stationary phase and pulse labeled with [3H] oleate for 30 minutes showed 50% reduction in incorporation of radioactive oleate into triglyceride. The lro1∆ deletion mutants grown to stationary phase in the similar experiment did not show significant change in triglyceride synthesis. However, during exponential growth phase lro1∆ deletion led to 75% loss of triglyceride synthesis while dga1∆ deletion caused 25% reduction. This suggests that Dga1 can mediate all of the triglyceride synthesis in stationary phase in the absence of Lro1, but not in exponential phase where Lro1 plays a major role in diglyceride esterification (Oelkers 2000; Oelkers 2002). A dga1∆lro1∆are2∆ strain lacks the ability to synthesize triglyceride (Oelkers 2002), and has been used a model system to study the role of DGAT2 family members in triglyceride synthesis (Turkish 2005). 1.1.2 Regulation of triglyceride synthesis Since several enzymes in the triglyceride synthetic pathway have only been recently identified, their regulation has just begun to be studied. The role of transcription factors, liver X receptors (LXR), sterol regulatory element binding protein (SREBP-1c), peroxisome proliferator-activated receptor-γ (PPARγ) and hormonal and nutritional regulators has been acknowledged (Coleman 2004). However, few studies have focused on the enzymes involved in triglyceride synthesis and the intracellular regulation of the pathway. There can be multiple points of regulation of a biochemical pathway. 22 The first enzyme of the metabolic pathways is often the regulated step, and several studies have shown that the mitochondrial GPAT regulation occurs at the transcriptional level. Interestingly, animal models lacking each of the four reactions of the triglyceride synthesis show profound effect on triglyceride levels. The intermediates of the pathway lysophosphatidic acid, phosphatidic acid and diglyceride, are signaling molecules and is possible that these alter the gene expression of triglyceride synthetic enzymes. Regulation is also likely to occur at branch points of the pathway. Diacylglycerol esterification, mediated by DGAT is one such branch point. DGAT is the only reaction that exclusively mediates the terminal step of triglyceride synthesis. In cultured permeabilized rat hepatocytes, under the experimental conditions when the substrates for the triglyceride synthetic pathway are available, DGAT has been regarded as a rate-limiting step (Mayorek 1989). In comparison to mammals, yeast enzymes that mediate DG esterification have been completely identified. Yeast cells have fewer and conserved isoforms of triglyceride synthetic enzymes and the gene enzyme relationship has been established for almost all of them. This, coupled with the availability of powerful genetic tools, can allow studies on regulation of intracellular triglyceride synthesis. 23 1.2 Phospholipid Biosynthesis Phospholipids are structural and functional components of biological membranes. They typically have a glycerol backbone with acyl chains linked by ester linkage to the first and second carbon of glycerol, and a polar group attached by a phosphodiester linkage to the third carbon. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) are the major phospholipids. Phosphatidylglycerol (PG) and cardiolipin (CL) are also present in mitochondrial membranes (Appendix 1). Phospholipid biosynthesis initially described by Kennedy occurs mainly by CDP-diacylglycerol (CDP-DAG) and CDP-choline pathway (Figure 1.5) (Kennedy 1961). The synthesis of phosphatidic acid, a major building block for phospholipids occurs by reactions common to triglyceride synthesis described earlier. Phosphatidic acid is an important branch point intermediate as it can form either diacylglycerol or CDP-DAG (Figure 1.5). Phosphatidic acid is converted to diacylglycerol, a reaction catalyzed by phosphatidic acid phosphatase (PAP). This diacylglycerol can be utilized either to synthesize triglycerides or PC, PE and PS. The conversion of phosphatidic acid to CDP-DAG is mediated by CDP-DAG synthase (CDS). CDP-DAG is converted to PI, a reaction catalyzed by phosphatidylinsotiol synthase (PIS). Cardiolipin, an exclusive mitochondrial phospholipid is also synthesized from CDP-DAG. 24 Figure 1.5 Phospholipid synthetic pathway in mammals The abbreviations used are: GPAT: Glycerol-3-phosphate acyltransferase, AGPAT: Acylglycerol-3-phosphate acyltransferase, PAP: Phosphatidic acid phosphatase, DGAT: Diacylglycerol acyltransferase, CDP-DAG: CDP-diacylglycerol, CDS: CDP-diacylglycerol synthase, CL: cardiolipin CLS: Cardiolipin synthase, PI: Phosphatidylinositol, PIS: PI synthase, PS: Phosphatidylserine, PC: Phosphatidylcholine, PSS1: PS synthase1, PSS2: Phosphatidylserine synthase 2, PSD: PS decarboxylase, PE:Phosphatidylethanolamine, PEMT: PE methyl transferase, EK: Ethanolamine kinase, ET: CTP:phosphoethanolamine cytidylyltransferase, EPT: Ethanolamine phosphotransferase, CK: Choline Kinase, CT: CTP:phosphocholine cytidylyltransferase, CPT: Choline phosphotransferase 25 Figure 1.6 Phospholipid synthetic pathway in Saccharomyces cerevisiae The abbreviations used are: GPAT: Glycerol-3-phosphate acyltransferase, AGPAT: Acylglycerol-3-phosphate acyltransferase, PAP: Phosphatidic acid phosphatase, DGAT: Diacylglycerol acyltransferase, CDP-DAG: CDP-diacylglycerol, CDS: CDPdiacylglycerol synthase, CL: cardiolipin CLS: Cardiolipin synthase, PI: Phosphatidylinositol, PIS: PI synthase, PS: Phosphatidylserine, PC: Phosphatidylcholine, PSS1: PS synthase1, PSD: PS decarboxylase, PE:Phosphatidylethanolamine, PEMT: PE methyl transferase, EK: Ethanolamine kinase, ET: CTP:phosphoethanolamine cytidylyltransferase, EPT: Ethanolamine phosphotransferase, CK: Choline Kinase, CT: CTP:phosphocholine cytidylyltransferase, CPT: Choline phosphotransferase 26 PC and PE can be synthesized via the CDP-choline and CDP-ethanolamine pathway. Choline kinase (CK) mediates the formation of phosphocholine, the first reaction of the CDP-choline pathway. The phosphocholine is then converted into CDPcholine by CTP: phosphocholine cytidylyltransferase (CT). This reaction requires activation by cytosine triphosphate (CTP). In the subsequent and last reaction, catalyzed by cholinephosphotransferase (CPT), a diacylglycerol molecule is utilized to form PC. A similar pathway converts ethanolamine to PE. This PE can get methylated to PC by phosphatidylethanolamine methytransferase (PEMT). PC synthesis by methylation in addition to CDP-Choline pathway occurs only in liver. In all other tissues CDP-choline pathway is the only pathway to synthesize PC. PS is synthesized from PC and PE by a base dependent reaction. PS synthase-1 (PSS-1) mediates exchange of serine for choline while PS synthase-2 (PSS-2) uses PE. PS is also converted to PE by PS decarboxylase (PSD). In Yeast The basic pathways of phospholipid biosynthesis in S.cerevisiae are similar to those in higher eukaryotes with the exception of PS synthesis (Figure 1.6). In yeast, PS is synthesized from CDP-DAG and serine whereas in mammalian cells, the only pathway to synthesize PS is a base-exchange reaction in which the head group of PC and PE is replaced by serine. PC synthesis in yeast occurs primarily by CDP-DAG pathway (Carman 1989), while in mammals with the exception of liver, CDP-choline pathway is the only pathway to synthesize PC. 27 1.2.1 Acyl chain asymmetry in phospholipids Phospholipids exhibit a variety in their molecular species, because of different combinations of acyl chains present at the sn-1 and sn-2 positions. In general, there is an asymmetrical distribution of acyl group on phospholipids (Hanahan 1960). Saturated fatty acids are usually esterified at the sn-1 position whereas unsaturated fatty acids are esterified at the sn-2 position. This asymmetrical distribution of acyl chains in phospholipids might influence the degree of order and motion of phospholipid molecules in lipid bilayer, a property referred to as membrane fluidity. The temperature, at which the membrane changes from gel like to a fluid like physical state, is dependent on the acyl chain composition of phospholipids. This transition temperature (Tt) is higher for distearoyl phosphatidylcholine (58ºC), phospholipid with two saturated acyl chains compared to 1-stearoyl-oleoyl phosphatidylcholine (3ºC), phospholipid with one saturated and one unsaturated acyl chain (Cronan 1975). Membrane properties like carrier mediated transport, phagocytosis and endocytosis are affected with changes in acyl chain composition of phospholipids (Spector 1985). It is acknowledged that membrane fluidity and properties are also influenced by other components of membrane such as cholesterol and proteins. This positional distribution of acyl groups in phospholipids is particularly important when arachidonic acid is present at the sn-2 position. This fatty acid is a precursor for biologically active eicosanoids (Funk 2001). The distinct acyl chain composition of phospholipids has been observed in various cells and tissues. It is possible that acyl chain composition of phospholipids is required for a particular cellular function, an example being dipalmitoylphsopahtidylcholine, a 28 major component of the pulmonary surafactant (Yamashita 1997). Mostly the physiological relevance of individual molecular species of phospholipids is not known. 1.2.1.1 Mechanisms to incorporate acyl chains in phospholipids There can be two mechanisms to incorporate acyl chains in phospholipids: (1) de novo phospholipid synthesis and (2) acyl chain remodeling of existing phospholipids by deacylation/reacylation reaction. Incorporation of acyl chains by de novo phospholipid synthesis De novo synthetic enzymes catalyzing the fatty acid esterification at sn-2 position might determine positional distribution of acyl groups in phospholipids. 1-acylglycerol3phosphate acyltransferases (AGPAT) introduce acyl chains at the sn-2 position of the glycerol backbone of lysophosphatidic acid to form phosphatidic acid (PA), an important intermediate in the de novo synthesis of membrane phospholipids and triglycerides (Figure 1.1) (Kennedy. 1961). Among the AGPATs 1-9, only AGPAT-1 and AGPAT-2 show robust enzyme activity. Cell lysates from insect cells expressing human AGPAT-1 using 10μM of acyl-CoAs showed highest activity for palmitoyl-CoA (16:1) and then the rank order was C16:0=C14:0=C12:0=C18:2>C18:3=C18:0=C20:4>C18:1. In the same study using 45 μM of acyl-CoAs the preference was in the rank order C16:1=C16:0=C14:0>C18:3=C18:2>C20:4=C18:0=C18:1. At these two fixed concentrations AGPAT-1 seems to utilize wide range of acyl-CoAs from C12- C18 with no clear preference for acyl groups by degree of saturation (Aguado 1998). The preference of the AGPAT-2 for different molecular species of acyl-CoA was studied 29 using membrane preparations of Sf9 cells expressing the human AGPAT2. At a single concentration of 8μM acyl-CoAs were preferred in the following order: C18:1>C18:2=C14:0=C16:0>C18:0>C20:4. The AGPAT2 activity displayed a Vmax of 200 nmol/min/mg and an apparent Km of 2μM with varying concentrations of oleoylCoA. In the same study competition experiments were performed with Sf9 membrane preparations containing AGPAT2, [3H] lysophosphatidic acid, 0.6μM [14C]18:1-CoA and 1.2 μM unlabeled acyl-CoAs. AGPAT2 displayed equal preference for C18:0-CoA and C16:0-CoA in comparison to 18:1-CoA (Eberhardt 1997; Aguado 1998; Leung 2001 Hollenback 2006). Due to single concentration of acyl-CoAs used in these studies to determine the acyl-CoA specificity of AGPAT1 and AGPAT2 it is difficult to compare substrate preferences of the enzyme in terms of catalytic efficiency. However, these results suggest that AGPAT1 and AGPAT2 might have a generic role in synthesizing precursors with a variety of acyl chain composition for phospholipids and triglyceride synthesis and might not contribute to asymmetric distribution of acyl chains in phospholipids. Acyl chain remodeling of existing phospholipids Various studies have shown that the distribution of fatty acids in phospholipids present in tissues and cells was different from its precursor molecule phosphatidic acid. This suggested that the phospholipid undergo fatty acid remodeling after de novo synthesis. Experiments using [14 C]acetate to label fatty acid residues and [14C] glycerol to label the glycerol backbone of phospholipids showed for the first time differences in turnover of acyl chains and glycerol in phospholipids (Lands 1958). 30 These results introduced the concept that the phospholipid species synthesized by the de novo synthetic reaction undergo deacylation at the sn-2 position by phospholipase A2 and reacylation by lysophospholipid acyltransferases to form remodeled phospholipid species (Lands cycle) (Figure 1.7). The presence of acyl-CoA dependent lysophosphatidylcholine acyltransferase (LPCAT) activity in rat liver microsomes supported the idea of phospholipid remodeling (Lands 1960). The rat liver microsomal LPCAT activities selectively incorporated palmitoyl-CoA at the sn-1 position of 2- acyl lysophosphatidylcholine and oleoyl-CoA at the sn-2 position of 1-acyl lysophosphatidylcholine. These experiments were done with mixtures of 1-acyl lysoPC and 2-acyl lysoPC with a single concentration of 0.5mM [14C] fatty acid and 0.2mM CoA (Lands 1963). This demonstrated the role of acyl-CoA specificity of these activities in maintaining asymmetrical distribution of acyl chains in phospholipids. There are very few in vivo studies on the deacylation-reacylation mechanism for acyl chain remodeling in phospholipids. Labeled lysophosphatidylcholine (lysoPC) and lysophosphatidylethanolamine (lysoPE) when injected intravenously disappeared from blood and labeled PC and PE were recovered in organs such as liver, small intestine, skeletal muscles, lungs, kidney and heart. To study the mechanism of formation of PC, lysoPC labeled in both phosphate (32P) and acyl chain (14C) was injected into rats. The labeled PC was found to have a similar 14C/32P ratio suggesting that majority of PC was synthesized from lysoPC by acylation reaction and not by transacylation reaction between two lysophospholipid molecules (Stein 1966). 31 Figure 1.7 Phospholipid remodeling (Lands Cycle) 32 Another study showed that remodeling of PC occurred in tissues post synthesis. Hamster hearts were perfused with, [1-3H] glycerol and then the lipids were extracted at various time points (0, 30, 60, 120 and 240 minutes). Immediately after label was introduced, the acyl chain composition of diacylglycerol and newly synthesized PC was similar. The composition of newly synthesized PC was different from that of endogenous PC. During the chase period the medium chain and saturated species showed gradual decrease and dienoic species increased. At the end of chase (240 minutes post labeling) the acyl chain distribution in PC was found to be similar to that of the endogenous PC. Since the labeling in diacylglycerol decreased by 90% after 30 minutes the redistribution of label in molecular species of PC did not occur due to de novo synthesis of phospholipids but was a result of acyl chain remodeling of phospholipids by deacylation and reacylation (Arthur 1984). 1.2.1.2 Acyl chain remodeling of phospholipids by acyl-CoA dependent lysophospholipid acyltransferases Lysophospholipids are acylated by two types of reactions. The acyl-CoA dependent reactions involve transfer of acyl group from acyl-CoA to lysophospholipids to form phospholipids and these reactions are catalyzed by acyl-CoA: lysophospholipid acyltransferases (LPLAT). The acyl-CoA independent reactions also known as transacylation, involve the transfer of acyl group of a phospholipid directly to another lysophospholipid to synthesize phospholipids. The focus of this study is the acylation mediated by acyl-CoA dependent 1-acyl lysophospholipid acyltransferase. 33 Since LPLAT activity is involved in manipulating the acyl chain composition of the phospholipids it is likely that this activity is present in most of the tissues. In rat brain in vitro LPCAT activity was demonstrated (Webster 1964). Acylation activity for 1-acyl lysophosphatidylinositol and 1-acyl lysophosphatidyserine and 2-acyl lysophosphatidylserine was reported in rat liver (Yamashita 1997). In rat liver microsomes 0.5mM linoleate in the presence of 0.1mM Co-A was incorporated preferentially in the sn-2 position of 1-acyl lysophosphatidylethanolamine in comparison to stearate (Lands 1963). Also in rat pancreas, enzyme activity in homogenate responsible for acylation of 2-acyl lysoPC, 2-acyl lysoPE, 2-acyl lysoPI and 2-acyl lysoPS showed preference for arachidonate over stearate at 17μM in the presence of 0.1 mM CoASH and 6mM ATP (Rubin 1983). In general, in these single concentration studies LPLAT activities seem to prefer saturated acy-CoA at the sn-1 position and unsaturated acyl-CoA for the sn-2 position, thus maintaining the acyl chain asymmetry in phospholipids. There are exceptions to this selectivity, in adult rat lung type II epithelial cells 1-acyl lysophosphatidylcholine acyltransferase (1-acyl-LPCAT) shows preference for palmitoyl-CoA at varying concentrations ranging from 0.5-22 μM. The Km and Vmax values were not reported in the study (Tansey 1975). However, in rabbit lung cells 1-acyl LPCAT showed only mild preference for palmitoyl-CoA (Km=15μM, Vmax=137nmol/min/mg) over oleoyl CoA (Km=16μM, Vmax= 100 nmol/min/mg). The reacylation system was suggested to catalyze the synthesis of dipalmitoyl phosphatidylcholine, a major constituent of pulmonary surfactant system (Yamashita 1997). It is possible that physiological concentrations of palmitoyl-CoA in lung cells favor its incorporation in the sn-2 position. 34 Almost 50 years after the lysophospholipid acyltransferase activity was reported, there are very few studies on molecular nature of these enzymes. The genes encoding mouse lysophsophatidylcholineacyltransferase-1(LPCAT1) and lysoplatelet activating factor acetyltransferase (LysoPAFAT)/ LPCAT2 were recently identified (Nakanishi 2006; Shindou 2007). LPCAT1 has been suggested to catalyze synthesis of dipalmitoyl PC in alveolar type II cells of lung. Platelet activating factor has an ether linked alkyl chain at the first carbon of glycerol, acetyl group in the second carbon and choline head group linked to the third carbon similar to phosphatidylcholine (Appendix 1). LysoPAFAT/LPCAT2 exhibited acetyltransferase activity also capable of incorporating arachidonoyl-CoA into sn-2 position of lysoPAF to synthesize PAF (Nakanishi 2006; Shindou 2007). The gene or genes encoding the lysophospholipid acyltransferase activity in other tissues reported in literature, responsible for asymmetrical distribution of fatty acids in phospholipids are yet to be identified. 1.2.1.3 Phospholipid remodeling in Saccharomyces cerevisiae Similar to mammals, yeast phospholipids have saturated acyl groups (mostly palmitate) localized to sn-1 position and unsaturated acyl group (predominantly oleate) to the sn-2 position in phospholipids (Yamada 1977). As reviewed earlier, yeast can synthesize PC by either methylation of PE or via CDP-choline pathway (Figure 1.6). However, in wild type yeast the steady state acyl chain composition of PC is different from that of PC newly synthesized by either of these pathways (Boumann 2003). Yeast cells when incubated with [3H]oleic acid, just after 2 minutes, show rapid and exclusive incorporation of oleate preferentially in sn-2 position of PC, PE , PI and PS. 35 The deacylation mediated by phospholipase and reacylation was suggested to explain these findings (Wagner 1994). Later, an acyl-CoA dependent 2-acyl lysophosphatidylcholine activity localized to endoplasmic reticulum was reported in yeast (Richard 1998). Acyl-CoA independent mitochondrial 1-acyl lysoPC acyltransferase activity encoded by TAZ1 has been described in yeast (Testet 2005). However, for the acyl CoA dependent lysophospholipid acyltransferase activity the gene enzyme relationship was not established until the present study was undertaken (Jain 2007). Identification of yeast gene would enable the identification of the human homologs responsible for the reacylation arm of phospholipid remodeling in higher eukaryotes. 1.2.1.4 Physiological role of lysophospholipid acyltransferases Lysophospholipid acyltransferases (LPLAT) play an important physiological role in selective placement of acyl chains in the sn-1 and sn-2 position of phospholipids. Remodeling of phospholipids helps to maintain acyl chain composition of membrane lipids and allows for the incorporation of arachidonic acid in the sn-2 position of phosphatidylcholine which when released by cytosolic PLA2 is a substrate for eicosanoids (Funk 2001). The acyl chain composition of the peritoneal macrophage membrane phospholipids when enriched for saturated fatty acids, showed a reduction in endocytosis and bilayer fluidity (Mahoney 1980). Although it is agreed that lysophospholipid acyltransferases are involved in remodeling of acyl chains in phospholipids but this might not be the only function of acyltransferases in all tissues. 36 In intestinal mucosa, 1-acyl-lysophosphatidylcholine acyltransferase activity is enriched in the brush border free microsomal fraction. At a fixed concentration of 125μM, when equimolar amounts of labeled oleoyl-CoA and unlabeled palmitoyl-CoA were used, the enzyme incorporated 60% less palmitoyl-CoA in the sn-2 position of lysoPC to synthesize PC. This enzyme synthesizes PC from lysoPC formed after digestion of phospholipids. This PC can then be packaged into chylomicrons and secreted into lymph. Thus this enzyme has an important physiological role in absorption of phospholipids (Subbaiah 1969). Few studies have suggested that lysophospholipid acyltransferases might play an important role in vesicular trafficking. Lysophospholipids produced by the action of cytoplasmic phospholipase A2 on membrane phospholipids, have been shown to induce the formation of Golgi membrane tubules. These tubules most likely play an important role in intracellular trafficking pathways, that include transport between Golgi to endoplasmic reticulum (ER) and maintenance of Golgi structure. It has been proposed that lysophospholipid acyltransferases tightly associated with Golgi membranes reduces the lysophospholipid levels and promotes fission of tubules leading to vesicle formation. This hypothesis is supported by the in vitro studies in which Golgi membrane incubated with [14C] palmitoyl-CoA incorporated majority of labeled acyl chain into PC and PE. However, in the presence of CI-976, an ACAT inhibitor, the lysophospholipid acyltransferase activity associated with Golgi membrane was inhibited. In vitro and in vivo studies on the same inhibitor have shown that it induces tubule formation in Golgi and increase lysophospholipid content in Clone 9 cells (Drecktrah 2003). 37 High levels of lysophosphatidylcholine lead to apoptosis (Takahashi 2002; Han 2008), cell lysis (Weltzien 1979) and inflammation (Kougias 2006). Thus in addition to the above mentioned functions lysophospholipid acyltransferases might play an important role in regulation of lysophospholipid levels, thus mitigating their harmful effects. Characterization of molecular nature of lysophospholipid acyltransferases will provide us with tools (RNAi or knock out animals) to understand the physiological role of these enzymes. 1.3. Specific Aims 1. Examine the intracellular regulation of triglyceride synthesis at the step of diglyceride esterification in Saccharomyces cerevisiae. 2. Identify novel yeast acylglycerol-3-phosphate acyltransferase(s) (AGPAT) in Saccharomyces cerevisiae. 3. Identify uncharacterized human genes with sequence similarity to the novel yeast AGPAT. 38 CHAPTER 2: REGULATION OF TRIGLYCERIDE SYNTHESIS IN SACCHAROMYCES CEREVISIAE 2.1 Abstract Dga1, Lro1 and Are2 are the only enzymes that mediate the terminal step of diglyceride esterification to synthesize triglyceride in Saccharomyces cerevisiae. Identification of all the enzymes responsible for diglyceride esterification allows yeast to be used as a model system to study the intracellular regulation of triglyceride synthesis. Initially, various genetic and growth conditions were studied where triglyceride synthesis varies. Using metabolic labeling with [3H] oleate, we found that the deletion of POX1, the enzyme that mediates the initial step of fatty acid oxidation, significantly increased triglyceride synthesis by 75%. However, deletion of two glycogen synthase enzymes (GSY1 and GSY2) did not alter triglyceride synthesis. To identify additional growth conditions in which triglyceride synthesis is regulated the effect of water soluble phospholipid precursors, inositol (75 μM) and choline (1 mM) on triglyceride synthesis was studied. Addition of inositol and choline or both to growth medium did not alter triglyceride synthesis. Additionally, deletion of the two enzymes that mediate the vast majority of triglyceride synthesis, LRO1 and DGA1, did not result in an increase in glycogen synthesis. Therefore, an inability to hydrolyze fatty acids seems to promote their storage while an inability to store energy as carbohydrate does not result in a compensatory increase in energy storage in lipid form. 39 We further hypothesized that Dga1 mediated diglyceride esterifying activity will be regulated by the level of certain cellular metabolites. Specifically, those metabolites that indicate a relatively energy poor environment (AMP, ADP and NAD+) would likely inhibit the enzyme and the metabolites that indicate a relatively energy rich environment (ATP, NADH, Acetyl CoA) would activate it. A lro1Δare1Δare2Δ triple deletion yeast strain was created in which Dga1 was the only diglyceride esterifying enzyme. In vitro diacylglycerol acyltransferase (DGAT) activity in microsomes from this strain was measured in the presence of physiological concentration of the metabolites of interest. DGAT activity was not regulated in response to the metabolites. 2.2 Introduction Triglycerides are important energy storage molecules in all eukaryotes. The intracellular triglyceride synthetic pathway was largely identified by Kennedy (Kennedy 1961) and shares initial steps with phospholipid synthesis. This de novo synthetic pathway requires glycerol 3-phosphate and acyl-CoA as substrates (Figure 1.1). Glycerol 3- phosphate is acylated at the sn-1 position to form 1-acylglycerol-3-phosphate (lysophosphatidic acid), and then at sn-2 position yielding phosphatidic acid. Phosphatidic acid is then dephosphorylated to form diglyceride. The terminal and unique step in triglyceride synthesis is the esterification of diglyceride catalyzed by diacylglycerol acyltransferase (DGAT). In mammals, two independent enzymes DGAT1 and DGAT2 catalyze this reaction (Cases 1998; Cases 2001). DGAT1 is a member of the acyl CoA: cholesterol acyltransferase (ACAT) gene family. 40 DGAT2, the founding member of the DGAT2 gene family, was identified by its sequence similarity to two proteins DGAT enzymes purified from the fungus, Mortierella ramanniana (Cases 2001). Members of the DGAT2 family share no sequence homology to the ACAT family which includes DGAT1. In humans, the DGAT2 family is complex and has seven members: DGAT2, three monoacylglycerol acyltransferases (MGAT1-3), two wax alcohol acyltransferases (AWAT1-2) and hDC3 which remains to be characterized. MGAT catalyzes the esterification of monoacylglycerol (MG) to synthesize diglyceride, an important reaction for the absorption of dietary fat in small intestine (Lehner 1996). MGAT1-3 when expressed in insect cells also show weak DGAT activity (Cao 2007), hence the physiological role of the members of DGAT2 family is not well defined. In yeast, enzymes mediating diglyceride esterification have been completely identified. Three different enzymes mediate diglyceride esterification. Dga1, the yeast ortholog of human DGAT2 enzyme mediates diglyceride esterification by a DGAT reaction (Oelkers 2002). Lro1, a homolog of mammalian lecithin cholesterol acyltransferase (LCAT) mediates triglyceride synthesis by phospholipid diacylglycerol acyltransferase (PDAT) reaction (Oelkers 2000). This reaction utilizes phospholipids in an acyl-CoA independent esterification of diglyceride. PDAT activity has also been identified in plant species (Stahl 2004). However, in mammals LCAT specifically catalyzes the cholesterol esterification. Dga1 and Lro1 contribute to 98% of triglyceride synthesis in yeast cells grown in media containing low nitrogen or 0.1% oleate and in logarithmic and stationary growth phase. 41 Are2, one of the sterol esterifying enzymes in yeast and homolog of DGAT1 contributes to the residual (2%) triglyceride synthesis (Oelkers 2002). Deletion of DGA1, LRO1, and ARE2 leads to complete loss of triglyceride synthesis in yeast (Oelkers 2002). Thus, Saccharomyces cerevisiae is a good model system to study the intracellular regulation of triglyceride synthesis. In the current study we hypothesized that digyceride esterification, the only committed step in triglyceride synthetic pathway, is responsible for the regulation of triglyceride synthesis and the enzymes which mediate digyceride esterification are regulated. In addition to this central hypothesis we also pursued the idea that there is a coordinated regulation of triglyceride and glycogen (cellular energy reserves) synthesis in a cell. Inability to synthesize triglyceride by digyceride esterification might result in increased glycogen synthesis and vice versa. To address the hypothesis the specific objectives of the study were: 1) identify genetic backgrounds and growth conditions where triglyceride synthesis is altered, 2) determine if the changes in the protein and activity levels of Dga1 and Lro1 are responsible for the alteration in triglyceride synthesis in conditions identified in aim1 and 3) examine whether glycogen synthesis is altered in the absence of triglyceride synthesis and vice versa. 42 2.3 Experimental Procedures Material Yeast extract, peptone, and Bacto-agar were from Fisher Scientific. Dextrose, inositol and choline were from Sigma. Synthetic defined media, yeast nitrogen base were obtained from Qbiogene (MP Biomedicals). Yeast extract peptone, plus 2% dextrose (YPD), synthetic complete (SC), and sporulation media (1% Potassium acetate) were prepared as described (Sherman 2002). [1-14C] oleoyl CoA (20,000 dpm/nmol) and [3H] oleate (2.63 Ci/mmol) were from PerkinElmer Life Sciences. All other chemicals were obtained from either Sigma or Fisher. Yeast Strains Molecular biology and yeast genetic procedures were performed according to conventional protocols. Deletion mutant strains for GSY1 and GSY2 were generated by homologous recombination in a W303-1B haploid (MATa ade2–1, can1–1, trp1–1, ura3– 1, his3–11, 15, leu2–3, 112) (Thomas 1989) by transformation with PCR derived products containing 50 base pairs of GSY1 or GSY2-specific sequence flanking the Kluyveromyces lactis URA3 sequence generated (Ito 1983; Rothstein 1991). GSY1 knockout oligos were as follows: GSY1F, 5’ ATGGCTCGTGACTTACAAAATCATCTACTGTTCGAAGTAGCAACAGAAGTAC ATGGCAATTCCCGGGGATCG and GSY1 R, 5’ TTAATTATCCTCGTAGTATGCAGACGTATCGTTGTCATCATCGTCATTGTTCA ACATGGTGGTCAGCTGGAATT. GSY2 knockout oligos were as follows GSY2F, 5’ ATGTCCCGTGACCTACAAAACCATTTGTTATTCGAGACTGCGACTGAGGTACA TGGCAATTCCCGGGGATCG and GSY2 R, 5’ 43 TTAACTGTCATCAGCATATGGGCCATCGTCGTCATCGTCAGCTGCAGGATTCA ACATGGTGGTCAGCTGGAATT. Single deletion transformants were confirmed by PCR using gene specific primers. Conventional inter strain crosses were used to generate double or triple deletion strains. The generation of yeast strains with single or multiple deletions in the DGA1, LRO1, ARE1 and ARE2 genes have been described previously (Oelkers 2002). The pox1Δ strain was obtained from Dr. Peter Oelkers. Yeast strains were grown in YPD media , SC media (Adams 1997). Pulse labeling A dilute inoculation (1/1000 of a saturated culture) of each strain into YPD was grown overnight at 30°C into logarithmic (log) phase (OD660 0.55–0.80). 4 ml of cells were labeled with 4μl of [3H] oleate (2.63 Ci/mmol) at 30°C for 30 minutes with shaking, washed twice with 0.5% tergitol and once with water, and frozen. Lipids were extracted from cell pellets by lyticase treatment and organic extraction. The extracted lipids were resolved by thin layer chromatography (TLC) in hexane: diethyl ether: acetic acid (70:30:1) as described (Oelkers 2000). For pulse labeling experiments two independent strains of each genotype were assayed on three different days. Isolation of Microsomes Yeast cells were grown in YPD into early stationary phase (O.D.660 = 1.0). The cells were harvested, washed and lysed, and microsomes prepared from a 100,000 x g spin as described (Leber 1994). 44 In vitro Diacylglycerol Acyltransferase Assay All DGAT assays were performed in a final volume of 200 µl for 5 minutes at 23°C. The final concentration of assay components was 175 mM Tris, pH 7.8, 10 mM sodium acetate, 2 mg/ml BSA (essentially fatty acid free), 8 mM MgCl2, 160 μM dioleoylglycerol (in ethanol), 7.5 - 60 µM [1-14C] oleoyl-CoA (20,000 dpm/nmol) and 40 μg of microsomal protein. The reactions were stopped by adding chloroform: methanol (2:1) and lipids were extracted and resolved by TLC as described earlier. The background was determined as the activity observed when the radiolabel was added after the chloroform/methanol. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), oxidized nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), acetyl-CoA and malonyl-CoA were individually added to the reaction mixture to study their effect on in vitro DGAT activity. Parallel DGAT reactions were also conducted in the absence of these metabolites. Kinetic data was analyzed by using EZ -Fit and GraphPad Prism 5 software. The Hill plot was used to estimate the Hill number and apparent Km for oleoyl-CoA. Glycogen estimation Glycogen levels were assayed by alkaline digestion at 95ºC of yeast cells in 0.25 M Na2CO3 as described in literature (Parrou 1997). Briefly, yeast cells grown to different growth phases (O.D.660nm 1.2-3.6) were harvested by centrifugation (3000g for 5 minutes and at 0-4ºC), carefully drained to remove culture media and resuspended in 0.25 ml of 0.25M Na2CO3 in screw top eppendorf tubes. The tubes containing the cell suspension was then incubated in water bath at 95ºC for 4 hours. 45 After 4 hours the pH of the cell suspension was brought down to 5.2 by addition of 0.15 ml acetic acid and 0.6 ml of 0.2M sodium acetate, pH 5.2. The mixture was then incubated overnight with 2.5 μl amyloglucosidase (10 mg of enzyme/ml of acetate buffer) at 57 ºC with constant agitation in either a hybridization oven or in a water bath. After the overnight incubation the suspensions were centrifuged for 3 minutes at 5000g, and glucose was determined in 20μl of supernatant by addition of 200μl of glucose oxidase reagent (Parrou 1997). Statistical tests The statistical analyses involved comparing two means by t tests and greater than two means by employing ANOVA. F test for equality of variance among two samples was conducted before comparing means of two samples by t test. Levene’s test of homogeneity was used to establish equality of variances before analyzing the data by ANOVA. Post hoc tests were conducted after ANOVA analysis to find out which means significantly differed from each other. Tukey’s test was used for post hoc analysis if the variances were equal and Tamhane’s T2 or Dunnett’s T3 were used if the variances were not equal. The statistical analysis was done by using SPSS 16 and Excel software. All the experiments were done at least 3 times on 3 different days with at least two different strains with the same genotype to account for day to day and strain to strain variation. All the data was expressed as mean ± S.D. 46 2.4 Results Identification of genotypes and growth conditions that affect triglyceride synthesis POX1 encodes for acyl-CoA oxidase, the first and rate-limiting enzyme in the βoxidation pathway in peroxisomes. The deletion of POX1 gene would disable the hydrolysis of fatty acids and might induce triglyceride synthesis to store excess fatty acids. The normal and pox1Δ deletion strains were pulse labeled with [3H] oleate for 30 minutes. Indeed pox1Δ showed 75% more [3H] oleate incorporation into triglyceride in comparison to the normal yeast strain (Figure 2.1) (p<0.05). To further probe whether the removal of the ability of cell to store energy as glycogen would result in an increased storage of energy in the form of triglyceride, strains lacking glycogen synthetic enzymes gsy1Δ, gsy2Δ and gsy1Δgsy2Δ deletion mutants were created. The deletion strains when pulse labeled with [3H] oleate did not show significant alteration in triglyceride synthesis in comparison to the normal strain (Figure 2.2). Previous studies have shown that triglyceride synthesis is up regulated when yeast enter stationary growth phase, undergo sporulation and when oleate is added to the growth media (Illingworth 1973; Taylor 1979). To identify additional growth conditions in which triglyceride synthesis is regulated the effect of inositol and choline, water soluble phospholipid precursors on triglyceride synthesis was studied. The reason for using these phospholipid precursors is that addition of 75µM inositol to the growth medium decreased phosphatidic acid (PA), an important intermediate in triglyceride and phospholipid synthesis, by 4.6 fold. 3 40 (% of total lipids) [ H] Oleate incorporation in TG 47 * 30 20 10 0 Normal pox1Δ Figure 2.1 Inability to oxidize fatty acids significantly increases triglyceride levels in pox1∆ strain. Indicated yeast strains were grown in YPD media to (O.D. 0.6-0.8, logarithmic phase), labeled with [3H] oleate for 30 minutes and harvested. Lipids were extracted from cell pellets, resolved by thin layer chromatography and quantified by scintillation counting of the cut TLC plates.( * indicates p< 0.05). The data represent mean ± S.D of 3 independent experiments. 3 25 (% of total lipids) [ H] oleate incorporation in TG 48 20 15 10 5 0 Normal gsy1Δ gsy2Δ gsy1Δgsy2Δ Figure 2.2 Triglyceride levels in glycogen deficient strains. Yeast cells were grown in YPD media to (O.D. 0.6-0.8, log phase), labeled with [3H] oleate for 30 minutes and harvested. Lipids were extracted from cell pellets, resolved by thin layer chromatography and quantified by scintillation counting of the cut TLC plates. The data represent mean ± S.D of 3 independent experiments. 49 Also, phosphatidylinositol (PI) synthesis increased by 6 fold at the expense of decrease in phosphatidylserine (PS) and phosphatidylcholine (PC) synthesis by 1.2 and 3.8 fold respectively (Kelley 1988). Also, phosphatidylinositol (PI) synthesis increased by 6 fold at the expense of decrease in phosphatidylserine (PS) and phosphatidylcholine (PC) synthesis by 1.2 and 3.8 fold respectively (Kelley 1988). Thus inositol, by perturbing the synthesis of phospholipids and intermediates common to phospholipids and triglyceride, might alter the synthesis of triglyceride. The addition of choline (1mM) to the medium in addition to inositol enhances these effects (Homann 1985). Therefore, we decided to study the effect of addition of inositol, choline or both on triglyceride synthesis. In comparison to the YPD growth media all the components of synthetic complete (SC) media are defined and allow easy manipulation of growth media by systematic omission or inclusion of individual components. Normal yeast strains were grown in SC media (lacking inositol and choline) with 2% glucose or the same media supplemented with inositol (75 μM) or choline (1 mM) or both to logarithmic phase and pulse labeled with [3H] oleate (Figure 2.3). The addition of inositol resulted in a slight decrease in incorporation of [3H] oleate in triglyceride but it was not statistically significant. Addition of both inositol and choline or just choline to growth medium did not alter triglyceride synthesis. 3 40 (% of total lipids) [ H] oleate incoporation in TG 50 30 20 10 0 Control Inositol Choline Inositol + Choline Figure 2.3 Effect of addition of water-soluble phospholipid precursors in media on triglyceride levels. Normal strain was grown to log phase (O.D. 0.60.8) in synthetic complete media with 75µM inositol, 1 mM choline or both to log phase and labeled with [3H] oleate for 30 minutes and harvested. Lipids were extracted from cell pellets, resolved by thin layer chromatography and quantified by scintillation counting of the cut TLC plates. The data represent mean ± S.D of 3 independent experiments. 51 The synthesis of energy reserves in yeast cell is not coordinately regulated. DGA1 and LRO1 contribute to 98% of triglyceride synthesis in yeast (Oelkers 2002). Absence of the ability to esterify diglyceride to form triglyceride, which is a major energy store, might result in a compensatory increase in another energy reserve, glycogen. To test this hypothesis, the glycogen content of normal and dga1Δlro1Δ yeast strains grown in YPD to late exponential phase to stationary growth phase (O.D.660 1.23.6) was determined (Figure2.4). The cellular glycogen levels of normal and dga1Δlro1 yeast strains increased by 15 and 17 fold respectively, as yeast cells progressed from late logarithmic to stationary growth phase. This increase in glycogen levels in normal and dga1Δlro1Δ yeast strains with growth phase is consistent with previous studies (Lillie 1980; François 2001). However, there was no significant difference in glycogen levels between yeast strains lacking triglyceride synthesis, dga1Δlro1Δ, and normal even at a high cell density (O.D660 = 3.6). This shows that there is no coordinated regulation between triglyceride and glycogen synthesis in yeast in the culturing conditions used in the present study. It is possible that the loss of triglyceride synthesis has a pronounced effect on glycogen synthesis in a particular culturing condition. A quadruple deletion mutant dga1Δlro1Δgsy1Δgsy2Δ was created. This yeast strain lacks the ability to synthesize glycogen and triglyceride and is viable on YPD growth media at 30°C. This suggested that in laboratory conditions on energy rich media (YPD) energy reserves triglyceride and glycogen are non-essential for viability. Future studies on the ability of this strain to grow in energy poor media would be interesting. 52 7 Glycogen (mg/10 cells) 1.2 0.8 0.4 0 1.2 1.8 2.4 3 3.6 O.D.660nm Figure 2.4 Glycogen levels in triglyceride deficient yeast strain. Normal and dga1Δlro1Δ haploids were grow in YPD to an O.D. of 1.2 to 3.6. The cells were lysed, boiled, treated with amyloglucosidase and glucose oxidase in order to determine the amount of glycogen present. Data are normalized to the number of cells. The data represent mean ± S.D of three independent experiments. Open bars-normal yeast strain, filled bars- dga1Δlro1Δ deletion mutant 53 Determination of protein and activity levels of diacylglycerol esterifying enzymes The regulation of an enzyme can occur at the level of transcription, translation and / or changes in enzyme activity due to covalent modifications or modulator induced conformational changes. The next step involved the determination of protein and activity levels of diglyceride esterifying enzymes in the identified genetic and growth conditions in which triglyceride levels are altered. The results would test the hypothesis whether changes in expression and activity of diglyceride esterifying enzymes are associated with changes in triglyceride synthesis. This study was part of an ongoing larger study on the regulation of yeast triglyceride synthetic enzymes Dga1 and Lro1. Hence, the focus was on the in vitro microsomal DGAT activity mediated by Dga1, one of the diglyceride esterifying enzymes. The experiments on mRNA abundance and protein expression by Western blotting were conducted by other members of the Oelkers lab. Real time PCR was conducted to quantify DGA1 and LRO1 mRNA levels in normal yeast cells grown in YPD to exponential and stationary phase. The mRNA levels of DGA1 and LRO1 (normalized to actin) increased by 3-fold in stationary phase which correlates with increased incorporation of [3H] oleate in triglyceride in a normal strain in stationary phase (Oelkers et al., unpublished data). The measurement of mRNA levels of DGA1 and LRO1 in other genetic and growth conditions yielded results with high variability and hence further studies were not carried. In order to study regulation at the posttranscriptional level, an attempt was made to measure the protein levels of Dga1 and Lro1 in conditions where triglyceride synthesis is altered. 54 Polyclonal antibodies were raised in rabbits against synthetic peptides derived from the protein sequences of DGA1 and LRO1. The experimental conditions were optimized to see if the specific antibodies could detect Dga1 and Lro1 immobilized on membrane. Western blot indicated non specific bands and failed to detect Dga1 and Lro1 with reproducible results. Thus the changes in protein expression levels by Western blotting could not be pursued (Oelkers et al., unpublished data). Next we sought to study whether regulation of triglyceride synthesis occurred by change in enzymatic activity of diglyceride esterifying enzymes. In vitro microsomal diacylglycerol acyltransferase (DGAT) activity To measure in vitro DGAT activity mediated specifically by Dga1 and to rule out any minor contribution from Are2, a lro1Δare1Δare2Δ yeast strain was created. Dga1 was the only enzyme catalyzing acyl-CoA dependent diglyceride esterification in this yeast strain. To enrich Dga1 mediated activity microsomes were prepared from lro1Δare1Δare2Δ triple deletion strain. The assay conditions were optimized with regards to the protein mass and temperature. The enzyme activity was found to be linear for 5 minutes with 40µg of microsomal protein at 23°C. The DGAT activity was recorded with a range (5µM-60μM) of [14C] oleoyl CoA concentrations (Figure 2.5). EZ-Fit and Graphpad Prism 5.0 software were used to analyze the kinetic data. Sigmoidal curve fitted better to the data in comparison to a Michaelis Menten curve (r2 = 0.99 versus 0.87). Therefore, the kinetic data were analyzed on a Hill plot which yielded a Hill number of 3.0. This indicated positive cooperativity of enzyme for oleoyl-CoA (Figure 2.5). The apparent K0.5 and Vmax for oleoyl-CoA were determined to be 16.1µM and 6.7 nmol/min/mg respectively. 55 A DGAT activity (nmol/min/mg) 8 6 4 2 0 0 20 40 60 14 [ C] Oleoyl CoA (Conc.µM) B 3 Log (v/Vmax-v) 2 1 0 -1 0 0.5 1 1.5 2 -2 -3 -4 Log (Oleoyl-CoA (µM) ) Figure 2.5 In vitro diacylglycerol acyltransferase (DGAT) activity with increasing concentrations of [14C] oleoyl-CoA. (A) Microsomes from lro1Δare1Δare2Δ deletion strain were incubated in the presence of diacylglycerol and varying concentration of [14C] oleoyl-CoA for 5 minutes at 23°C as described in “Experimental Procedures”. The data represent mean ± S.D. n=3. (B) Hill plot of the data in panel A. 56 Effect of ATP, NADH, Malonyl-CoA and Acetyl-CoA on in vitro DGAT activity Acute regulation of triglyceride synthetic enzymes has been reported by two studies. In vitro glycerol 3-phosphate acyltransferase (GPAT) and DGAT activity in post mitochondrial supernatant (PMS) from rat adipose tissue, upon incubation with 1mM adenosine 5’- triphosphate (ATP) for 15 minutes showed 25% and 35% reduced activity respectively. DGAT activity has been shown to be predominantly (90%) associated with microsomal fraction. However, in the same study, the microsomal fraction obtained from the PMS did not show reduction in DGAT activity. The addition of cytosolic fraction obtained from PMS when added to the in vitro DGAT assay with microsomal protein as enzyme source resulted in reduction of enzyme activity in the presence of 1mM ATP (Rodriguez 1992). The cytosolic activity responsible for reduction in in vitro DGAT activity was not inhibited by protein kinase C inhibitors. It was suggested that phosphorylation by tyrosine kinase was responsible for the reduction in DGAT activity (Walsh 1989; Rodriguez 1992). Mouse and human DGAT1 contain a single conserved tyrosine residue while DGAT2 contains 6 potential protein kinase C phosphorylation sites (Cases 2001). However, no change in DGAT activity was observed when conserved tyrosine residue in human DGAT1 was mutated to phenylalanine (Oelkers 1998). We hypothesized that the presence of metabolites which indicate energy status of the cell would affect in vitro microsomal DGAT activity. The metabolites that increase in concentration in energy replete cells, namely ATP, NADH, acetyl-CoA and malonylCoA would activate DGAT activity so that energy can be stored in the form of triglyceride and vice versa. 57 It was suggested as early as 1960s that adenosine phosphates regulate reactions involved in energy storage or energy production. Two classic examples include phosphofructokinase-1 and muscle glycogen phosphorylase that have been shown to be allosterically regulated by ATP and adenosine monophosphate (AMP) in opposite directions (Ramaiah 1964). Previous studies have shown that ATP levels are 5 fold higher in yeast cells grown in the presence of 2% glucose in media in comparison to the starved cells (Wu 1994). The cellular concentration of ATP has been estimated to be 2.3 – 5.2 mM in yeast cells grown to logarithmic phase in regular YEP growth media containing 2% dextrose and in starved cells the ATP concentration is 0.57mM (OzierKalogeropoulos 1991; Ingram 2000). 5mM ATP was added to DGAT assay components and DGAT activity was recorded for varying concentrations of oleoyl-CoA. Hill plot analysis of kinetic data was performed (Figure 2.6). The apparent K0.5 for oleoyl-CoA with ATP and without ATP was 14.3 ± 4.3 μM and 17 ± 5.6 μM respectively. The Vmax values with and without ATP were 6 ± 3 nmol/min/mg and 5.8 ± 2 nmol/min/mg respectively. The decrease in apparent K0.5 with the addition of ATP was statistically not significant in comparison to the control reactions. Two way ANOVA and post hoc analysis of kinetic data did not indicate statistically significant difference in enzyme activity with and without ATP. Nicotinamide adenine dinculetoides work in conjunction with adenosine phosphates as energy transfer system in the cell. The ratio between reduced and oxidized forms of nicotinamide adenine dinculetoides is indicative of the cellular metabolic state (Krebs 1967). 58 Yeast cells grown in 0.5% glucose show a 50% decrease in NADH levels in comparison to cells grown in 2% glucose. The cellular concentration of NADH in yeast cells has been reported to be around 0.8mM in cells grown in YPD (2%glucose) (Lin 2004). 0.8 mM NADH was added to the mixture for DGAT assay and Hill plot analysis was done (Figure 2.7). The K0.5 values with NADH and without NADH were 24.7 ± 9.8 μM and 17 ± 4.2 μM respectively. The Vmax values with NADH and without NADH were 5.5 ± 1.4 nmol/min/mg and 4.8 ± 1.6 nmol/min/mg respectively. However due to the high variability in the kinetic data the t-test did not detect significant difference between K0.5 values with and without NADH. No statistically significant changes in DGAT activity at varying concentrations of oleoyl-CoA were seen with the addition of NADH. Acetyl-CoA is a primary substrate for citric acid cycle with a marked function in energy production. Acetyl-CoA has been shown to allosterically activate pyruvate carboxylase, an enzyme involved in gluconeogenesis (Cooper 1966). The reported cellular concentrations of acetyl-CoA range from 25 - 100 μM (McCormick 1983). 0.25 mM acetyl-CoA has been shown to allosterically activate yeast pyruvate carboxylase and increase the enzyme activity by 100 fold (Rhode 1986) and the same concentration of acetyl-CoA was added to the DGAT assay mixture (Figure 2.8). In the presence of acetylCoA the apparent K0.5 was 17.9 ± 3.2 μM which was not significantly different from K0.5 of 17± 4.2 μM of reaction without acetyl-CoA. The apparent Vmax upon addition of acetyl-CoA was 4.5 ± 0.9 nmol/min/mg and was similar to that of control reaction (4.8 ± 1.6 nmol/min/mg). 59 A DGAT activity (nmol/min/mg) 8 6 4 2 0 0 10 20 30 40 50 14 ATP Control [ C]Oleoyl CoA (µM) B Log (v/Vmax-v) 2 1 0 0 0.5 1 1.5 2 -1 -2 ATP -3 Log (Oleoyl-CoA (µM)) Control Figure 2.6 Effect of 5mM ATP on in vitro Diacylglycerol acyltransferase (DGAT) activity. (A) Microsomes from lro1Δare1Δare2Δ deletion strain were incubated in the presence of 5mM ATP, diacylglycerol and varying concentration of [14C] oleoyl-CoA for 5 minutes at 23°C as described in “Experimental Procedures”; The data represent mean ± S.D. n=3. (B) Hill plot of the data in panel A. 60 A DGAT activity (nmol/min/mg) 4 3 2 1 0 0 10 20 30 40 NADH Control 14 [ C]Oleoyl CoA (µM) B Log (v/Vmax-v) 1 0 0 0.5 1 1.5 2 -1 -2 -3 Log (Oleoyl-CoA (µM)) NADH Control Figure 2.7 Effect of 0.8mM NADH on in vitro Diacylglycerol acyltransferase (DGAT) activity. (A) Microsomes from lro1Δare1Δare2Δ deletion strain were incubated in the presence of 0.8mM NADH, diacylglycerol and varying concentration of [14C] oleoyl-CoA for 5 minutes at 23°C as described in “Experimental Procedures”; The data represent mean ± S.D. n=3. (B) Hill plot of the data in panel A. 61 A DGAT activity (nmol/min/mg) 5 4 3 2 1 0 0 10 20 30 14 [ C]Oleoyl CoA (µM) 40 Acetyl CoA Control B Log (v/Vmax-v) 1 0 0 0.5 1 1.5 2 -1 -2 Acetyl-CoA -3 Log(Oleoyl-CoA (µM)) Control Figure 2.8 Effect of 0.2mM acetyl-CoA on in vitro Diacylglycerol acyltransferase (DGAT) activity. (A) Microsomes from lro1Δare1Δare2Δ deletion strain were incubated in the presence of 0.2mM acetyl-CoA, diacylglycerol and varying concentration of [14C] oleoyl-CoA for 5 minutes at 23°C as described in “Experimental Procedures”; the data represent mean ± S.D. n=3. (B) Hill plot of the data in panel A. 62 A DGAT activity (nmol/min/mg) 4 3 2 1 0 0 20 Malony-CoA 40 Control 14 [ C]Oleoyl CoA (µM) B Log (v/Vmax-v) 1 0 0 0.5 1 1.5 2 -1 -2 -3 Log(Oleoyl-CoA (µM)) Malonyl-CoA Control Figure 2.9 Effect of 0.1 mM malonyl-CoA on in vitro Diacylglycerol acyltransferase (DGAT) activity. (A) Microsomes from lro1Δare1Δare2Δ deletion strain were incubated in the presence of 0.1mM malonyl-CoA, diacylglycerol and varying concentration of [14C] oleoyl-CoA for 5 minutes at 23°C as described in “Experimental Procedures”; the data represent mean ± S.D. n=3. (B) Hill plot of the data in panel A. 63 Malonyl-CoA is an intermediate in the de novo synthesis of fatty acids and is an allosteric inhibitor of carnitine palmitoyltransferase I (CPT I), an enzyme that regulates the rate at which long chain fatty acyl-CoAs are transported into mitochondria for βoxidation (Ruderman 1999). The effect of malonyl-CoA on in vitro DGAT activity was studied. 0.1 mM malonyl-CoA has been shown to inhibit 92-96% of rat liver CPT I activity upon ectopic expression in yeast (de Vries 1997). The apparent K0.5 in the presence of malonyl-CoA was 18.7 ± 1.6 μM compared to 17 ± 4.2 μM of control reaction (Figure 2.9). This slight increase in K0.5 in the presence of malonyl-CoA was not found to be statistically significant. Vmax of the enzyme was unaffected upon addition of malonyl-CoA. Overall the addition of metabolites which indicate energy rich status of cell did not affect the Vmax and Km of DGAT for oleoyl-CoA. Statistical analysis of kinetic data in the presence of modulators by two way ANOVA and post hoc tests did not detect any changes in DGAT activity at varying concentrations of oleoyl-CoA. Next, we proceeded to determine kinetic parameters of DGAT in the presence of metabolites that indicate energy poor status of cell: AMP, ADP and NAD+. We hypothesized that these metabolites should lead to decrease in DGAT activity so that energy storage in form of triglyceride is not favored. The cellular AMP levels in yeast range from 0.1mM to 1mM (Polakis 1966; Betz 1967). In the presence of 0.1mM AMP the apparent K0.5 was 15.4 ± 5.5 µM while in the presence of 1mM AMP it was 16.4 ± 5.8 µM (Figure 2.10). The K0.5 values were not statistically significant in comparison to the K0.5 of 17.6 ± 8.7 µM in the absence of AMP. There was no change in Vmax of the enzyme with increase in concentration of AMP. 64 The Vmax in the presence of 0.1m M and 1 mM AMP was 5.5 ± 1 nmol/min/mg and it was not significantly different from Vmax in the absence of AMP. Statistical analysis by two way ANOVA followed by post hoc tests did not detect any effect of AMP on DGAT activity at varying concentrations of oleoyl-CoA. The cellular concentration of ADP in yeast has been reported to range from 0.3 mM to 1 mM (Wu 1994; Ingram 2000). At concentration as high as 6 mM, ADP has been shown to enhance glucose binding by yeast hexokinase (Woolfitt 1988). Microsomes of liver rat when incubated for 15 minutes with 5 mM ADP show 14% reduction in DGAT activity (Haagsman 1982). 5mM ADP was added to the DGAT assay components to investigate whether ADP modulates in vitro DGAT activity. In the presence of 5 mM ADP the DGAT activity was linear for 7.5-20 μM of oleoyl-CoA (Figure 2.11). We did not study the effect of ADP on DGAT activity at concentrations higher than 20 μM of oleoyl-CoA. Therefore, it was not possible to determine a K0.5 or Vmax for oleoyl-CoA using Michaelis–Menten equation and Hill plot analysis. The change in DGAT activity upon addition of ADP to the DGAT assay components was not found to be statistically significant. Next we studied the effect of addition of 0.8 mM NAD on DGAT activity (Figure 2.11). Intracellular concentration of NAD+ of 1-1.2 mM has been reported in yeast grown on YPD (Lin 2004). The kinetic data was analyzed by Hill plot (Figure 2.11). The Km of DGAT for oleoyl-CoA in the presence and absence of NAD+ was 16.7 ± 8 μM and 17.6 ± 8.7 μM respectively. The Vmax of the enzyme upon addition of NAD+ was 5.6 ± 0.3 nmol/min/mg in comparison to 4.8 ± 1.6 nmol/min/mg for the control reaction. Addition of NAD+ did not have statistically significant effect on Vmax or Km of enzyme for oleoyl-CoA. 65 A DGAT activity (nmol/min/mg) 5 4 3 2 1 0 0 10 20 14 [ C]Oleoyl CoA conc. (µM) AMP (0.1mM) AMP (1mM) Control B Log (v/Vmax-v) 1 0 0 1 2 -1 -2 -3 -4 Log (Oleoyl-CoA (µM)) Control AMP 0.1mM AMP 1mM Figure 2.10 Effect of 0.1 mM and 1mM AMP on in vitro Diacylglycerol acyltransferase (DGAT) activity.(A) Microsomes from lro1Δare1Δare2Δ deletion strain were incubated in the presence of 0.1mM AMP and 1mM AMP, diacylglycerol and varying concentration of [14C] oleoyl-CoA for 5 minutes at 23°C as described in “Experimental Procedures”; the data represent mean ± S.D. n=3. (B) Hill plot of the data in panel A. 66 A DGAT activity (nmol/min/mg) 6 4 2 0 0 10 20 14 [ C]Oleoyl CoA conc. (µM) 30 Control ADP NAD B Log (v/Vmax-v) 1 0 0 0.5 1 1.5 -1 -2 -3 Log (Oleoyl-CoA(µM)) NAD Control Figure 2.11 Effect of 0.1mM ADP and 0.8 mM NAD+ on in vitro Diacylglycerol acyltransferase (DGAT) activity. (A) Microsomes from lro1Δare1Δare2Δ deletion strain were incubated in the presence of 0.1mM ADP and 0.8 mM NAD+, diacylglycerol and varying concentration of [14C] oleoyl-CoA for 5 minutes at 23°C as described in “Experimental Procedures”; the data represent mean ± S.D. n=3. (B) Hill plot of the data in panel A. 67 2.5 Discussion Diglyceride esterification is the terminal and unique step in triglyceride synthesis. Diglyceride is common intermediate for triglyceride and phospholipid synthesis. Cells must maintain a balance between storage of energy in triglyceride and membrane phospholipid synthesis, therefore it is likely that this branch point is subject to regulation. The main hypothesis of the study was that the regulation of triglyceride synthesis occurs at the step of diglyceride esterification and the enzymes that mediate this step are regulated. Another hypothesis was that inability of cells to esterify diglyceride to form triglyceride will increase glycogen synthesis as an alternative to store energy. We used Saccharomyces cerevisiae as a model system to study the intracellular regulation of triglyceride synthesis. In yeast Lro1, Dga1 and Are2 are the only diglyceride esterifying enzymes and a yeast strain lacking all of these enzymes is devoid of triglycerides (Oelkers 2002). Identification of all the diglyceride esterifying enzymes allows genetic manipulation to study their role individually. In addition, yeast has conserved glycerolipid synthetic pathways homologous to those in mammalian cells. The amino acid sequence of Dga1 shares 35% identity with mammalian DGAT2 protein and like its mammalian counterpart catalyzes acyl CoA dependent diglyceride esterification (Oelkers 2002). Lro1 is the yeast ortholog of mammalian lecithin-cholesterol acyltransferase (LCAT) with which it shares 27% identity.However, Lro1 catalyzes triglyceride synthesis by a PDAT reaction which is acyl-CoA independent. Together Dga1 and Lro1 conribute to 98% of triglyceride synthesis and the residual diglyceride esterification is mediated by Are2, with a predominant role in sterol esterification (Oelkers 2000; Oelkers 2002). 68 To address our hypothesis that triglyceride synthesis is regulated at the step of diglyceride esterification, it was necessary to first identify genetic backgrounds and growth conditions in which triglyceride synthesis is regulated. Previous studies have described few conditions in which triglyceride synthesis is altered. Yeast cells grown in media containing 0.1% oleate as the only carbon source, show seven fold increase in total lipids, the majority of which is triglyceride (Dyer 2002). Triglyceride synthesis also increases in yeast cells in stationary growth phase and when they undergo sporulation in nitrogen poor media (Henry 1973; Taylor 1979). In lro1∆ yeast strains triglyceride synthesis is reduced by 75% in logarithmic growth phase while dga1∆ shows 50% reduction in stationary phase (Oelkers 2002). To identify additional conditions in which triglyceride synthesis might be altered, we examined yeast strains with gene deletions which disable fatty acid hydrolysis (pox1∆) or glycogen synthesis (gsy1∆, gsy2∆). We hypothesized that in these three genetic backgrounds, triglyceride synthesis will be increased to store the surplus energy. Triglyceride synthesis was measured by pulse labeling pox1Δ and wild type yeast strain with [3H] oleate. Indeed, there was a 75% increase in incorporation of the labeled oleate in triglyceride in pox1Δ strain (p<0.05). Phospholipid and sterol ester synthesis were unaffected suggesting that the effect of pox1Δ was triglyceride specific. However, triglyceride and phospholipid synthetic pathways have common intermediates, substrates and initial steps; it is less likely that the availability of acyl-CoA will favor one process over another. 69 There can be two possibilities: first being that the enzymes in glycerolipid synthetic pathway selectively channel substrates towards either phospholipid or triglyceride synthesis or the second possibility being that the enzymes utilizing phosphatidic acid and diglyceride as substrates for phospholipid and triglyceride synthesis are regulated. Glycerol 3-phosphate acyltransferase (GPAT) mediates the first step of triglyceride and phospholipid synthetic pathway. In yeast this reaction is mediated by Gat1 and Gat2. The gat1∆ yeast cells show 50% increase in triglyceride synthesis while gat2∆ yeast show 50% decrease in triglyceride synthesis (Zaremberg 2002). This result raises the question whether lysophosphatidic acid species synthesized by Gat1 are utilized for phospholipid synthesis while that synthesized by Gat2 are utilized to synthesize triglyceride. So far no studies have been carried out to show that lysophosphatidic acid species synthesized by Gat1 and Gat2 are different. At least two isoforms exist for all the enzymes involved in synthesizing diglyceride, an important intermediate in triglyceride and phospholipid synthesis. It is also possible that these isoforms play an important role in directing intermediates of triglyceride and phospholipid synthesis to a particular pathway. However, there is lack of information about protein-protein interaction among different isoforms of the pathway that would facilitate substrate channeling towards particular pathway. Next we examined triglyceride synthesis in yeast strains with deletions in glycogen synthetic enzymes (gsy1∆, gsy2∆ and gsy1∆gsy2∆). GSY1 and GSY2, encode for yeast glycogen synthases with 50% similarity with mammalian muscle and liver glycogen synthases. Glycogen synthase catalyzes the rate-limiting step of glycogen synthesis. 70 Between the two yeast isoforms, Gsy2 mediates 90% of the enzyme activity in YPD in stationary phase. Deletion of both the genes results in loss of glycogen synthesis (Francois, 2001). Loss of glycogen as an energy reserve was expected to stimulate storage of energy in form of triglyceride as a compensatory mechanism to maintain total cellular energy reserves. Yeast single and double deletion strains gsy1Δ, gsy2Δ, gsy1Δgsy2Δ, and wild type strains were pulse labeled with [3H]oleate to monitor triglyceride synthesis. There was no significant difference in triglyceride synthesis in glycogen synthesis deficient and wild type strains. Next, we addressed the question whether the loss of triglyceride synthesis increases glycogen accumulation in the cells. There is only one study which has examined whether there is coordination between synthesis of cellular energy stores. Glycogen synthesis in obese and lean rats was monitored by 3H2O after feeding them isocaloric meal. Obese rats showed 3-fold reduction in hepatic glycogen synthesis and 5fold increase in lipogenesis when compared to the lean rats. However, the plasma insulin levels were 4 times higher in obese animals in comparison to lean animals and it is possible that the changes in glycogen synthesis were due to insulin resistance rather than due to increase in lipogenesis (Obeid 2000). Since triglyceride and glycogen synthetic enzymes have been characterized in yeast it is possible to directly examine the effect of loss of intracellular triglyceride synthesis on glycogen synthesis The dga1Δlro1Δ double deletion strain is deficient in triglyceride synthesis yet viable and synthesizes only 2% of the normal amount of triglyceride (Oelkers 2002). 71 Glycogen levels in dga1Δlro1Δ yeast cells grown (in YPD) to late exponential phase and stationary phase were measured. Both the yeast strains showed an increase in glycogen levels as they progressed from exponential to stationary phase. This agreed with a previous study that found six-fold increase in glycogen levels (% of dry weight of cells) as yeast cells progress from exponential to stationary phase (Lillie 1980). The glycogen levels in dga1Δlro1Δ strain did not show significant increases in comparison to the wild type cells. This result suggested that cellular glycogen and triglyceride synthesis cannot compensate for the absence of each other and hence, there is no coordinated regulation in them in the present assay conditions. It is possible that culturing of the yeast strains in YPD, energy rich media might have masked any kind of coordinated regulation between triglyceride and glycogen synthesis. Further studies in different growth conditions will be required to establish how a cell adapts to loss in triglyceride synthesis. To identiy additional growth condition in which triglyceride synthesis is altered, we studied the effect of inositol and choline on triglyceride synthesis. Addition of water soluble phospholipid precursors, inositol and choline to the growth media has been shown to regulate a number of yeast phospholipid synthetic enzymes (Carman 1999).The levels of phosphatidic acid (PA), an important intermediate in triglyceride and phospholipid synthesis decreased by 2 fold in the yeast cells grown in presence of inositol (Kelley 1988). Phosphatidic acid can be either dephosphorylated to form diglyceride for triglyceride synthesis or can be converted to CDP-diacylglycerol (CDP-DG) for phospholipid synthesis. Triglyceride content of yeast cells has been shown to increase in stationary phase with simultaneous decrease in phospholipid synthesis (Taylor 1979). 72 This increase in triglyceride content is thought to occur by induction of phosphatidic acid phosphatase (PAP) activity (Hosaka 1984). In stationary phase, the activity of CDP-DG synthase is decreased by 2.5 fold, thus increasing the availability of phosphatidic acid for diglyceride synthesis mediated by PAP (Homann 1987). Loss of triglyceride synthesis in yeast cells lacking diglyceride esterifying enzymes increases the phospholipid synthesis significantly in logarithmic and stationary growth phases and when grown in the presence of oleate or nitrogen poor media (Oelkers 2002). Thus according to these studies phospholipid synthetic pathway and triglyceride seem to be coupled to each other rather than former being regulatory. Interestingly, in the present study addition of 75 µM inositol or 1mM choline and both to the growth media, known to affect the activity of phospholipid synthetic enzymes showed no effect on triglyceride synthesis in wild type yeast grown to exponential phase. Similar result was obtained with wild type yeast grown to different growth phases (Appendix 3). A previous study reported that inositol requiring yeast mutant (ino1-13) incorporated twice the amount of [14C] acetate in triglyceride and phospholipids per cell six hours after being shifted to inositol starved culture and losing viability (McCammon 1982). At earlier time points (2 and 4 hours) there was no difference in incorporation of acetate label in total phospholipids and triglyceride in cells starved for inositol and supplemented control containing 27.5µM of inositol (McCammon 1982). This results show that changes in phospholipid synthesis might not be tightly coupled to triglyceride synthesis required for storing excess fatty acids in a cell. 73 After identifying conditions which alter triglyceride synthesis, we studied whether in these conditions expression and activity of diglyceride esterifying enzymes are regulated. Regulation of diglyceride esterifying enzymes can occur at the level of mRNA abundance, protein abundance and enzymatic activity. Triglyceride synthesis is increased in yeast cells when they enter stationary phase from exponential growth phase (Taylor 1979). Real time PCR was conducted to quantify DGA1 and LRO1 mRNA levels in wild type yeast cells grown in YPD to exponential and stationary phase (Oelkers et al., unpublished data). The mRNA levels (normalized to actin) of DGA1 and LRO1 increase by 3-fold in stationary phase which correlates with increased incorporation of [3H]oleate in triglyceride in a wild type strain in stationary phase. The measurement of mRNA levels of DGA1 and LRO1 in other genetic and growth conditions yielded results with very high variability and hence further studies were not carried. In order to study regulation at the posttranscriptional level, we sought to measure the protein levels of Dga1 and Lro1 in conditions where triglyceride synthesis is altered. To do so polyclonal antibodies were raised in rats using synthetic peptides derived from the protein sequences of Dga1 and Lro1. The changes in protein expression levels by Western blotting could not be pursued due to inability to detect proteins by the antibodies (Oelkers et al., unpublished data). To address the possibility that Dga1 and Lro1 enzyme activity are regulated by post translational modification or by allosteric mechanism, the in vitro DGAT enzyme activity was measured in microsomes, an enriched source of the diglyceride esterifying enzymes. A yeast triple deletion strain lro1Δare1Δare2Δ was created, in which diglyceride esterification was only carried out by Dga1. 74 The microsomal DGAT activity with varying concentrations of oleoyl-CoA when plotted resulted in a sigmoidal shape plot. The enzyme activity when analyzed by Hill equation exhibited positive cooperativity (Hill number: 3). Cooperativity with respect to oleoyl-CoA might be important in regulating the rate of triglyceride synthesis. Studies done on permeabilized hepatocytes have suggested that at physiological levels of glycerol-3-phosphate (1mM) when DGAT is saturated with diglyceride the rate of triglyceride synthesis is determined by the affinity of DGAT for acyl-CoA (Stals 1994). The apparent Vmax and K0.5 of DGAT for oleoyl-CoA were 6.7 nmol/min/mg and 15.5 µM respectively. Previous study on microsomal DGAT activity in HepG2 cells has reported apparent Km of 8.3 µM for oleoyl-CoA (2.5-17 µM). The total DGAT activity was representative of 2 DGAT isoforms, DGAT1 and DGAT2 expressed in HepG2 cells (Ganji 2004). It is difficult to compare our data with that of this study because DGAT1 and DGAT2 might have characteristic enzyme kinetic parameters and contribution to total DGAT activity in HepG2 cells. Nothing is known about the kinetic parameters of DGAT1. However, a study reported that human DGAT1 was a multimeric protein but the biochemical significance of tetramer formation was not examined (Cheng 2001). In our assays, the microsomal DGAT activity was only mediated by Dga1p, a member of DGAT2 family. Monoacylglycerol acyltransferase1 and 2 (MGAT1 and MGAT2) also belong to the DGAT2 family and it was suggested that they exhibit cooperative kinetics with varying concentrations of oleoyl-CoA ranging from 0-400 µM. No analysis was done in these studies to estimate kinetic parameters (Yen 2003). Thus this is the first study in our knowledge to describe the kinetic parameters of a member of DGAT2 family. 75 To further characterize microsomal DGAT activity we studied the effect of metabolites which indicate cellular energy status: ATP,ADP,AMP,NAD,NADH, acetylCoA, malonyl-CoA on Dga1. We hypothesized that during energy rich conditions in a cell, when the levels of ATP, NADH, acetyl-CoA and malonyl-CoA are high these metabolites may bind to Dga1 and allosterically activate the enzyme activity. This will result in increased triglyceride synthesis. On the other hand when ADP, AMP and NAD+ levels are high when cells are depleted of energy, the triglyceride synthesis will be inhibited. Enzymes involved in glycolysis and citric acid cycle such as pyruvate dehydrogenase, isocitrate dehydrogenase and phosphofructokinase-1 (PFK1) have been shown to be regulated in similar manner (Ramaiah 1964). Addition of ATP, ADP, AMP, NADH, NAD+, acetyl-CoA and malonyl-CoA individually to the assay components at cellular concentration did not alter the microsomal DGAT activity. In Brassica napus (oil seed rape) in vitro microsomal DGAT activity was shown to increase by 2 fold when 1- 2 mM ATP was added to the assay mixture containing 15µM of oleoyl-CoA and 330µM of diglyceride (Byers 1999). No changes in DGAT activity were observed upon addition of ATP to the DGAT assay components used in the present study. It is acknowledged that our results are limited because of the single concentrations of most of the metabolites. However, the results are relevant because we tried to mimic the physiological conditions by adding metabolites at cellular concentration or at concentrations known to allosterically regulate activity of other enzymes. . 76 CHAPTER 3: IDENTIFICATION OF A NOVEL LYSOPHOSPHOLIPID ACYLTRANSFERASE (LPT1) IN SACCHAROMYCES CEREVISIAE (Published as Jain et al. 2007) 3.1 Abstract The incorporation of acyl chains in the sn-2 position of phospholipids during de novo synthesis is primarily mediated by the 1-acyl-glycerol-3-phosphate acyltransferase (AGPAT) reaction. In S. cerevisiae, Slc1 has been shown to mediate this reaction but AGPAT activity remains after SLC1 deletion. To identify the enzyme that mediates the remaining activity, synthetic genetic array (SGA) analysis was performed using a slc1∆ strain. SGA analysis was performed in collaboration with Dr. Charles Boone and Michael Costanzo, University of Toronto, Toronto, Canada. One of the genes identified by the screen, LPT1, was found to encode for an acyltransferase that uses a variety of lysophospholipid species, including 1-acyl-sn-glycerol-3-phosphate. Deletion of LPT1 had a minimal effect on 1-acyl-sn-glycerol-3-phosphate acyltransferase activity but overexpression increased activity 7-fold. other lysophospholipids and Deletion of LPT1 abrogated the esterification of over-expression increased lysophosphatidylcholine acyltransferase activity 7-fold. The majority of this activity (65%) co-purified with microsomes. To test the putative role for this enzyme in selectively incorporating unsaturated acyl chains into phospholipids, in vitro, substrate concentration series experiments were performed with the four acyl-CoA species commonly found in yeast. 77 While the saturated palmitoyl-CoA and stearoyl-CoA showed a lower apparent Km, the monounsaturated palmitoleoyl-CoA and oleoyl-CoA showed a higher apparent Vmax. Arachidonyl-CoA, although not abundant in yeast, also had a high apparent Vmax. Pulse-labeling of lpt1∆ strains showed a 30% reduction in [3H] oleate incorporation into phosphatidylcholine only. Therefore, Lpt1, a member of the membrane bound- O- acyltransferase (MBOAT) gene family, seems to work in conjunction with Slc1 to mediate the incorporation of unsaturated acyl-chains into the sn-2 position of phospholipids. These findings identified LPT1, a novel lysophospholipid acyltransferase, as a component of the Lands cycle responsible for acyl chain remodeling at the sn-2 position of phospholipids. LPT1 belongs to the MBOAT family and shares sequence similarity with uncharacterized human MBOAT proteins. Identification of LPT1 provides a molecular tool to identify human homologs with similar function and study phospholipid remodeling and turnover in yeast. 78 3.2 Introduction According to the fluid mosaic model for the structure of biological membranes, phospholipids form a bilayer in which protein molecules are embedded. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phospatidylinositol (PI), cardiolipin, sphingolipids and sterols are major membrane lipids. Each subcellular fraction has a distinct lipid composition for instance mitochondrial membranes have high cardiolipin content while cholesterol is a major component of plasma membrane. The acyl chains in phospholipids are thought to influence the degree of order and motion of phospholipid molecules in lipid bilayer, a property referred to as membrane fluidity. Saturated acyl chains in membrane phospholipids pack tightly in an array forming a gel like phase, while the unsaturated acyl chains cannot be packed as tightly leading to fluid bilayer. The temperature at which membrane transitions from a gel like state to a fluid like state is dependent on the acyl chain composition of the phospholipids (Cronan 1975). In general, phospholipids have saturated acyl groups in the sn-1 position and unsaturated acyl groups in sn-2 position (Hanahan 1960) At temperatures in the physiological range the presence of saturated and unsaturated acyl chains in phospholipids allows the lipid bilayer to be more fluid and simultaneously avoid drastic phase changes with temperature. Changes in acyl chain composition of membrane phospholipids are also associated with changes in cellular functions. In mouse peritoneal macrophages an increase in unsaturation has been shown to result in an increase in phagocytic activity. A reduction in endocytosis was observed in peritoneal macrophages 79 which were enriched in saturated fatty acids (Mahoney 1980). In calf thymus lymphocytes, increase in linoleic acid and arachidonic acid content of the plasma membrane was associated with increase in activities of lecithin acyltransferase, Na+ / K+ ATPase and decrease in Mg2+- ATPase and γ-glutamyl transferase activities (Mavis 1972; Spector 1985). Thus the pathways that establish this positional distribution of acyl chains in phospholipids are of importance to the cell. According to Kennedy pathway acyl chains are introduced at the sn-1 and sn-2 position by de novo synthetic enzymes glycerol 3phosphate acyltransferase (GPAT) and 1-acylglycerol-3-phosphate acyltransferase (AGPAT) to synthesize phosphatidic acid (PA), a precursor to phospholipids and triglycerides (Figure 3.1). One plausible hypothesis is that AGPAT selectively incorporates unsaturated acyl groups in the sn-2 position to establish asymmetry in acyl chain composition in phospholipids. However, human AGPAT1 and AGPAT2 when expressed in baculovirus utilize both saturated as well as monounsaturated acyl-CoAs of 12-18 carbons at a single concentration of 8μM in the presence of 8 μM 33P labeled sn-118:1 lysoposphatidic acid (lysoPA). With varying concentrations of oleoyl-CoA (18:1CoA) and 8mM of 33P lysoPA the apparent Vmax and Km of the human AGPAT2 activity were 210 nmol/min/mg and 0.4 μM respectively. Since single concentration of acyl-CoAs was used to study acyl-CoA specificity, the catalytic efficiency for different acyl-CoAs could not be calculated. However competition experiments between 0.6 μM 14 C-18:1- CoA and 1.2 μM of other unlabeled acyl-CoAs with 6 μM 3H-labeled lysoPA and recombinant human AGPAT2 showed similar preference for 16:0-CoA, 18:0-CoA and 80 18:1-CoA. Other acyl-CoAs were utilized in the order of 18:2-CoA> 12:0-CoA>14:0CoA>20:0-CoA>20:4-CoA ( Hollenback 2006). Acyl-CoA specificity of AGPAT1 and AGPAT2 suggests they might not be responsible for establishing acyl chain asymmetry in phospholipids. AGPAT family currently includes 3 other putative AGPATs that share sequence similarity with AGPAT1 and AGPAT2 (Lu 2005; Agarwal 2007). AGPAT2 mutation is associated with congenital generalized lipodystrophy-1 (CGL-1) and is believed to have a regulatory role in adipocyte differentiation. AGPAT-1 and AGPAT-2 show robust AGPAT activity but the biochemical role of AGPAT 3-5 and their contribution to AGPAT activity is not clear (Agarwal 2002; Gale 2006). In yeast, SLC1 is the only member of AGPAT gene family (Nagiec 1993; Athenstaedt 1997) and encodes for a protein which shares 25% and 20% amino acid sequence identity with human AGPAT1 and AGPAT2. The slc1Δ yeast mutants show no AGPAT activity in lipid particles but microsomes prepared from slc1∆ strain retain 46% of the total AGPAT activity. These results indicate the presence of another microsomal AGPAT in yeast, which contributes to the formation of phosphatidic acid in the absence of Slc1 (Athenstaedt 1997). Phosphatidic acid synthesized by AGPAT can then be used to synthesize glycerophospholipids, phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI) and cardiolipin through cytidinediphosphodiacylglycerol (CDP-DAG) pathway (Figure 3.1). Alternatively, phosphatidic acid can be dephosphorylated to form diacylglycerol, a precursor for synthesis of PE and PC via the CDP-ethanolamine and CDP-choline, pathway (Kennedy 1961). After de novo synthesis, the acyl chain composition of phospholipids can be modulated by phospholipid remodeling (Figure 3.1). 81 Figure 3.1 Pathways for phospholipid synthesis in Saccharomyces cerevisiae. A, de novo synthesis. B. Remodeling/ salvage pathways. 82 This involves deacylation by phospholipase A2 at the sn-2 position and reacylation of resulting lysophospholipid by acyl-CoA dependent lysophospholipid acyltransferase (Lands 1960). The deacylation-reacylation referred to as Lands cycle, has many possible physiological functions. In addition to selectively incorporating unsaturated acyl chains for membrane fluidity, arachidonic acid (20:4), a precursor for eicosanoid synthesis is incorporated into sn-2 position by remodeling of phospholipids. This may prime signal cascades that involve phospholipase A2 and eicosanoids synthesis (Balsinde 2006). Besides maintaining acyl chain asymmetry in phospholipids, remodeling is also involved in preferential incorporation of saturated acyl groups in phosphatidylcholine to synthesize surfactant lipids in lungs (Yamashita 1997). In mammals, lysophospholipid acyltransferase might play an important role in keeping the levels of lysophospholipid low to prevent inflammation caused by lysophospholipids (Kougias 2006). So far only two lysophospholipid acyltransferases have been cloned and designated lysophosphatidylcholine acyltransferase1 (LPCAT1) and lysoplatelet activating factor acyltransferase (lysoPAFAT / LPCAT2) (Chen 2006; Nakanishi 2006; Shindou 2007). Both the LPCATs contain conserved motifs found in members of the AGPAT family and share ~ 50% similarities in their amino acid sequence. LPCAT1 is highly expressed in lungs and has a preference for short to medium chain saturated acylCoAs (C10:0>C8:0>C12:0>C6:0=C14:0=C16:0) at a fixed concentration of 25μM (Nakanishi 2006). LPCAT1 is highly expressed in alveolar type II cells of the rat lung and a two fold LPCAT activity over endogenous activity was seen with lysophosphatidylcholine and palmitoyl-CoA. 83 Based on these observations it was suggested that LPCAT1 might be involved in the production of components of pulmonary surfactant lipids which include 40% dipalmitoylphosphatidylcholine (DPPC) (Nakanishi 2006). However no gene targeting studies were done to confirm the physiological role of LPCAT1 in the production of surfactant lipids. LPCAT2 is expressed in inflammatory cells and is involved in synthesis of platelet activating factor (PAF) from lyso-PAF (Shindou 2007). In yeast, acyl-CoA independent LPCAT activity is mediated by Taz1 (Testet 2005). Acyl-CoA independent enzymes catalyze the transfer of acyl chain from phospholipids to lysophospholipids without requiring the presence of acyl-CoA. The acyl donor is a fatty acid present in the sn-2 position of phospholipids. The coenzyme A independent transacylases are considered to play an important role in the incorporation of plyunsaturated fatty acids (C20-C22) in lysophospholipids with ether linkages (Yamashita 1997). Acyl-CoA dependent activity has been reported in yeast but no enzyme-gene relationship has been established (Richard 1998; Testet 2005). In this study, we established this relationship with LPT1 as the result of a synthetic genetic array analysis (SGA) analysis. SGA involves automated crossing of yeast strain containing a gene deletion of interest to ~4700 viable yeast gene deletion strains. Inviability of haploid double deletion mutants is inferred to be due to synthetic lethal interactions between two genes. Two mutations are considered synthetically lethal if in combination in a haploid cell, result in cell death, but each by itself leads to a viable cell (Tong 2001). Synthetic lethal interactions identify genes acting in the same essential pathway or in parallel redundant pathways, if product of one gene functionally compensates for defect due to another. 84 3.3. Experimental Procedures Material Synthetic defined media and yeast nitrogen base were obtained from Q-biogene (MP Biomedicals). Myristoyl lysophosphatidylethanolamine and oleoyl [1-14C] lysophosphatidylserine were from Avanti Polar Lipids (Alabaster, AL). Palmitoyl–lysoPC (55mCi/mmol) was from Perkin-Elmer Life Sciences. All other chemicals were obtained from either Sigma or Fisher. Yeast strains Molecular biology and yeast genetic procedures were performed according to conventional protocols (Ausubel 1998). Deletion mutant strains for SLC1 and LPT1 were generated by homologous recombination in a W303-1B haploid (MATa ade2–1, can1–1, trp1–1, ura3–1, his3–11, 15, leu2–3, 112) (Thomas 1989) by transformation with PCR derived products containing 50 bp of gene-specific sequence flanking the Kluyveromyces lactis URA3 sequence (Ito 1983; Rothstein 1991): for LPT1, LPT1F, 5' ATGTACAATCCTGTGGACGCTGTTTTAACAAAGATAATTACCAACTATGGAC ATGGCAATTCCCGGGGATCGL and LPT1R, 5’ CTACTCTTCCTTTTTTGAAATAGGCTTTGGTGAGTAACCACTAAAACTCATCA ACATGGTGGTCAGCTGGAATT; for SLC1: SLC1F, 5’ ATGAGTGTGATAGGTAGGTTCTTGTATTACTTGAGGTCCGTGTTGGTCGTACA TGGCAATTCCCGGGGATCG and SLC1R, 5’ TTAATGCATCTTTTTTACAGATGAACCTTCGTTATGGGTATTGACATCGTTCA ACATGGTGGTCAGCTGGAATT; for YBR042, YBR042F, 5’ GATATATCAGGTCCACTGGTATTGAGATTGCTAGCGAGGTCAATAAGCTGCT 85 GTACTTCCTTGTTCATGTGTG and YBR042R, 5’ TCAAAAAATAAAACAATAAAGTTTATAAACTAACCAAATTATTGTTAGAGAC CTGTATTCCTTTACATCCTCC and for YDR018 YDR018F, 5’ GAAATGAACAAACAACCCTACGTACGACTGTGTTTTGCAGTTTAAGAAGGCC TGATTCTTGATCTCCTTTAGC and YDR018R, 5’ TCAATGATTTTTTTTCATCACAAATACAAGAATAAGAAAAGCGAAGAACCGT TCAGAATGACACGTATAGAATG. Successful deletion was confirmed by PCR using gene specific primers. Yeast were grown in YP media (1% yeast extract, 2% peptone) with 2% glucose (YPD) or in synthetic complete (SC) media . Synthetic lethality screen Synthetic genetic array (SGA) analysis was performed by Michael Costanzo and Dr. Charles Boone, University of Toronto, Toronto, Canada. A Mat α slc1∆:: NatMX4 can1∆:: STE2 prSp his5 lyp1∆ strain was crossed to an array containing the set of ~ 5,000 viable deletion mutant strains (Tong 2001). Yeast Expression Plasmid Construction A PCR product including the LPT1 open reading frame, 90 bp of 5’- flanking sequence and 46 bp of 3’-flanking sequence was generated using the primers: 175-1, CAAAATACAGGCACAGGTCAAGC; 175-2, GACAACAAGACTGTGACTTCCAC, and W303-1B genomic DNA. The 2.0-kb PCR product was cloned into pCR 2.1 TOPO (Invitrogen). The LPT1 DNA fragment was digested by NotI / SacI and subsequently subcloned into similarly digested pRS423GP to create pRS423GP-LPT1. This was performed by Dr. Peter Oelkers. 86 Pulse-labeling A dilute inoculation (1/1000 of a saturated culture) of each strain into YP + 2% dextrose was grown overnight at 30°C into logarithmic phase (OD660 = 0.55–0.80). 0.5 μCi of [3H] oleate (58,000 dpm / pmol) was added to 4 ml of cells and incubated at 30° C for 30 min. with shaking (Oelkers 2002). Cells were washed and cellular lipids extracted as described previously. After resolving phospholipids by thin layer chromatography in chloroform: methanol: acetic acid: water (50:25:8:4), each lane was cut according to lipid standards and counted by liquid scintillation (Oelkers 2002). Assays were performed on a minimum of two independent strains of each genotype on two different days. Preparation of cell lysates, mitochondria and microsomes Yeast cells were grown in YP containing either 2% glucose or 3% glycerol at 30°C into early stationary phase (O.D.660 = 1.0). SC-His medium containing 2% galactose was used for yeast harboring a plasmid. The cells were harvested, washed and cell lysates prepared as described earlier (Oelkers 2002). Mitochondria were prepared using a previously published protocol. Briefly, cells were grown in YP media containing 3% glycerol, harvested, lysed and crude mitochondria were precipitated by centrifugation at 12,000 x g. Resuspension of the pellet, followed by sucrose step gradients allowed purified mitochondria to be collected (Meisinger 2000). The microsomes were purified from the 12,000 x g supernatant by precipitation by centrifugation at 100,000 x g for 1 hour (Oelkers 2002). Lowry assays were used to measure protein concentration. 87 AGPAT and LPLAT assays, spectrophotometric method AGPAT activity was measured by the reaction of thiol groups of the released CoA with 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB) resulting in absorbance at 412nm (Yamashita 1981) The reaction mixture contained 100mM Tris-HCl, pH 7.4, 1.5 mM DTNB, 50 μM lysophosphatidic acid (lysoPA), 50μM acyl-CoA, cell lysate (210 - 840 μg) in a total volume of 1 ml. The reaction was monitored in real time for 3 minutes in a Spectronic Genesys 2 spectrophotometer at room temperature. A molar absorbance of 13,600 M-1 cm-1 was used to calculate relative specific activity. Lysophospholipid acyltransferase (LPLAT) activity was measured by replacing lysoPA with lysoPC, lysoPE, or lysoPI. LPCAT assay, radioactive substrate LPCAT activity was measured by the incorporation of [1- 14 C] palmitoyl lysoPC into PC. The reaction contained 100 mM Tris-HCl, pH 7.4, 50 μM [1- 14C] palmitoyl lysoPC (50,000 dpm/nmol), 1 - 110 μM of the respective acyl-CoA, and 3μg of cell lysate protein in a final volume of 100 μl. Fixed time assays were performed for 5 minutes at 28°C. The reactions were stopped by adding chloroform: methanol (2:1) and lipids were extracted and resolved as described elsewhere (Nakanishi 2006) . EZ – fit software was used for nonlinear regression, curve fit analysis to calculate apparent Km and Vmax (Perrella 1988). To calculate Vmax / Km, Vmax was first change to nM / min / mg to remove volume from the units of the ratio. 88 Statistical analysis The statistical analyses involved comparing two means by t tests and greater than two means by employing ANOVA. F test for equality of variance among two samples was conducted before comparing means of two samples by t test. Levene’s test of homogeneity was used to establish equality of variances before analyzing the data by ANOVA. Post hoc tests were conducted after ANOVA analysis to find out which means significantly differed from each other. Tukey’s test was used for post hoc analysis if the variances were equal and Tamhane’s T2 or Dunnett’s T3 were used if the variances were not equal. The statistical analysis was done by using SPSS 16 and Excel software. All the experiments were done at least 3 times on 3 different days with at least two different strains with the same genotype to account for day to day and strain to strain variation. 89 3.4 Results Identification of LPT1 in yeast by SGA analysis To identify the novel AGPAT that mediates phosphatidic acid synthesis in yeast in the absence of SLC1, SGA was carried out using a slc1∆ query strain (Tong 2001). Synthetic-lethal genetic interactions were identified with 52 single-gene deletion strains. Of these 52 genes, 17 genes encode for proteins involved in DNA repair, transcription, translation, and protein transport. 13 of the deleted genes encode for proteins with known function of which only one gene, fatty acid elongase (ELO2) is involved in lipid metabolism (Oh 1997). The remaining 22 uncharacterized genes included YOR175c (from here on referred to as LPT1). This gene, along with PMT5 which encodes for a protein O-mannosyltransferase, was previously shown to be required for the viability of slc1∆ mutants (Schuldiner 2005). To test this further, independent slc1Δ and lpt1Δ haploid strains were generated and crossed to form compound heterozygous diploids. Following meiosis, eighty haploid spores were isolated by tetrad dissection. Twenty spores failed to grow into colonies. Of the sixty viable haploids, genotyping showed that all were normal, slc1Δ or lpt1Δ. In line with Hardy Weinberg equilibrium, ¼ of the progeny were therefore inviable and slc1Δlpt1Δ, confirming the synthetic lethality. In addition to synthetic lethality, a candidate gene approach was used to identify the protein that mediates Slc1-independent AGPAT activity. Two unassigned open reading frames YBR042C and YDR018C were identified by amino acid sequence homology to known AGPATs in eukaryotes. The sequence databases Gene Bank and 90 alignment tool BLASTP was used. YBR042 and YDR018 encoded proteins are ~ 16% identical to characterized AGPATS: Slc1, AGPAT1 and AGPAT2. AGPAT3-5 have not been biochemically characterized and share 23% identity with YBRO42 and YDR018 predicted proteins. YBR042 and YDR018 encoded proteins are 37% identical to each other. The ybr042∆, ydr018 single deletion and ybr042∆ydro18∆ double deletion yeast strains were generated. We also generated ybr042∆, ydr018∆ and ybr042∆ydro18∆ strains in the slc1∆ background. Unlike lpt1∆, ybr042∆, ydr018∆ and ybr042∆ydro18∆ strains did not show any growth defects in slc1∆ background. To find out whether these two candidate genes had any effect on triglyceride and phospholipid synthesis, normal and above mentioned single and multiple deletion strains grown to exponential phase in YPD were pulse labeled with [3H] oleate for 30 minutes (Figure 3.2). Consistent with its role as an AGPAT in yeast, absence of Slc1 resulted in significant reduction in diglyceride and phospholipid levels in comparison to the normal and ybr042∆, ydr018∆ single and double deletions strains. Interestingly the triglyceride levels were significantly increased in slc1∆ strain in comparison to normal and other ydr018∆, ybr042∆ single or double deletions strains. A previous study also reported two fold increase in cellular triglyceride levels in slc1∆ strain in comparison to the normal strain during logarithmic growth phase (Athenstaedt 1997). One would expect, however, that defect in phosphatidic synthesis an intermediate required for both phospholipids and triglyceride synthesis, would affect the phospholipid as well as triglyceride levels. This selective increase in triglyceride over phospholipid levels or vice versa has also been reported in yeast strains deficient in Gat1 and Gat2, the enzymes which catalyze first step in triglyceride and phospholipid synthesis (Zaremberg 2002). 3 30 (% of total lipids) [ H] oleate incorporation into DG,TG,P 91 * 20 * 10 * % DG 0 % TG Normal slc1Δ ydr018Δ ybr042Δ % PL Figure 3.2 Incorporation of [3H] oleate into diglyceride, triglyceride and phospholipids in slc1Δ, ydr018Δ, ybr042Δ. Normal, slc1Δ, ydr018Δ, ybr042Δ yeast strains were grown in YPD to logarithmic phase and pulse-labeled with [3H] oleate for 30 minutes. Diglyceride (DG), triglyceride (TG), phospholipids were measured as described in "Experimental Procedures". The data represent mean ± S.D. n = 3. Asterisk indicates statistically significant difference between normal and slc11Δ (p<0.05) 30 20 10 or m al 0 N 3 [ H] oleate incorporation into DG,TG,PL (% of total lipids) 92 % DG % TG % PL Figure 3.3 Incorporation of [3H] oleate into diglyceride, triglyceride and phospholipids in ybr042∆, ydr018∆ and ybr042∆ydr018∆ in slc1∆ background. Normal and indicated yeast strains were grown in YPD to logarithmic phase and pulselabeled with [3H] oleate for 30 minutes. Diglyceride (DG), triglyceride (TG), phospholipids were measured as described in "Experimental Procedures". The data represent mean ± S.D. n = 3. 93 However, YDR018 and YBR042 gene deletions in conjunction with SLC1 showed similar effects as SLC1 deletion by itself. There was no significant difference in triglyceride, diglyceride levels and phospholipid levels in the ybro42∆ and ydr018∆ single and double deletions in logarithmic phase. This coupled with the fact that no growth defect or lethality was seen in ydr018∆ and ybr042∆ single double and triple deletion strains in slc1 background led us to focus on LPT1. The inviability of lpt1∆slc1∆ double deletion strain and the presence of acyltransferase motif in LPT1 further strengthened the possibility of it being a candidate AGPAT. Studies on the physiological role of YBR042 and YDR018 genes are being conducted by other members of Oelkers lab. Lpt1 is a member of MBOAT family: LPT1 is predicted to encode for a 619 amino acid protein with seven transmembrane domains. The C terminus of Lpt1 has a dilysine motif which functions as an ER retention signal (KKXX) (Vincent 1998) . Analysis of yeast strains expressing green fluorescent protein tagged proteins have indicated that Lpt1 is localized to endoplasmic reticulum (Huh 2003). LPT1 belongs to membrane bound-O-acyltransferase (MBOAT) superfamily of proteins. Members of this superfamily are postulated to encode for enzymes that catalyze transfer of fatty acyl groups to hydroxyl groups (Hofmann 2000). Lpt1 shares 25% sequence similarity to three characterized members of the MBOAT family which utilize diacylglycerol and cholesterol as substrates (Figure 3.4). Lpt1 contains the conserved hydrophobic region with putative catalytic histidine residue typical of an acyltransferase (Hofmann 2000). 94 LPT1 (341 ) ACAT1(418 ) ACAT2(382 ) DGAT1(374 ) WNMN TNKWLK YSVYLR VT.KKG KK PGFRSTLFT FLTSAF WHGTRP GYYLTF A WNVV VHDWLY YYAYKD FLWFFS KR FKSAAMLAV FAVSAV VHEYAL AVCLSF F WNVV VHDWLY SYVYQD GLRLLG AR ARGVAMLGV FLVSAV AHEYIF CFVLGF F WNIP VHKWCI RHFYKP MLR..R GS SKWMARTGV FLASAF FHEYLV SVPLRM F Figure 3.4 Alignment of LPT1 and 3 human members of the MBOAT gene family. The four amino acid sequences are aligned over the MBOAT motif (Pfam PFO3062). Positions where 3 out of 4 amino acids are conserved are boxed. In parentheses is the position of the first amino acid. Acyl-CoA cholesterol acyltransferase (ACAT); acyl-CoA diacylglycerol acyltransferase (DGAT) Determining if AGPAT activity is decreased in lpt1∆ Initial experiments examined whether lpt1∆ deletion affected AGPAT activity. AGPAT activity in cell lysates was measured by a spectrophotometric assay. This assay estimated AGPAT activity by measuring the release of coenzyme from the acyl-CoA added to the reaction mixture containing lysophosphatidic acid. Cell lysates prepared from normal, slc1∆ and lpt1∆ strains were assayed in the presence and absence of lysophosphatidic acid (Figure 3.5). AGPAT activity was calculated by subtracting coenzyme A generation in the absence of lysophosphatidic acid from that in the presence of lysophosphatidic acid. There was no difference in AGPAT independent activity in slc1∆, lpt1∆ and normal yeast strain. AGPAT assays were conducted with saturated and monounsaturated acyl-CoAs. In line with previous studies, slc1∆ cell lysates showed 63% reduction in AGPAT activity in comparison to normal yeast. However, in lpt1∆ cell lysates no statistically significant reduction in AGPAT activity was observed. 95 Since the depletion of Lpt1 did not affect the AGPAT activity, we further studied whether the overexpression of Lpt1 would influence AGPAT activity. Overexpression of LPT1 led to 7-fold increase in AGPAT activity suggesting that Lpt1 can mediate this reaction (Figure 3.5). These results indicate that Slc1 contributes to most of AGPAT activity in the yeast cells grown to early stationary growth phase. Lpt1 mediates esterification of other lysophospholipids LPCAT activity has been described in yeast (Yamada 1977; Richard 1998), and the enzyme mediating this reaction has not been identified. We examined whether Lpt1 can mediate LPCAT activity in yeast. Spectrophotometric assays were conducted with lysophosphatidylcholine (lysoPC) and three different acyl donors: palmitoyl-CoA, oleoylCoA and arachidonoyl-CoA. LPCAT activity with monounsaturated and polyunsaturated acyl-CoAs was 4-7 fold higher than the saturated acyl-CoA at a fixed concentration of 50μM (Figure 3.6). No LPCAT activity was observed in lpt1∆ strain with any of the acyl donors. Similar results were obtained when [14C] lysoPC and oleoyl-CoA were used. Cell lysates of lpt1∆ cell lysates showed no activity while wild type cell lysates showed robust activity (55.6 ± 4.7 nmol/min/mg). These results suggest that Lpt1 is a major LPCAT in yeast. The possibility exists that other yeast LPCATs might not be expressed or active in the growth or assay conditions used in present study or might require acyl-CoAs of particular chain length. 96 A AGPAT activity (nmol/min/mg) 20 15 ∗ 10 5 0 ∗ WT lpt1Δ slc1Δ B ∗ AGPAT activity (nmol/min/mg) 100 80 60 40 20 0 pRS423GP pRS423GP-LPT1 Figure 3.5 In vitro 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT) assays. (A) Cell lysates from wild-type, lpt1Δ and slc1Δ haploids, grown to late logarithmic phase, were assayed for AGPAT activity by the spectrophotometric method described in "Experimental Procedures". The assay contained 50μM 1-oleoyl lysophosphatidic acid, 50μM acyl-CoA, 1.5 mM DTNB, 0.42-0.84 mg cell lysate and 100 mM Tris-HCl, pH 7.4. palmitoyl-CoA (black), oleoyl-CoA (grey). The data represent mean ± S.E. n = 3-5. Asterisk indicates statistically significant difference compared with wild-type strain (p< 0.05). B. Similar assays were performed with cell lysates from wild-type cells transformed with pRS423GP or pRS423GP-LPT1. 97 LPCAT activity nmol/min/mg A LPCAT activity nmol/min/mg B 16 14 12 10 8 6 4 2 0 ∗ WT ∗ ∗ lpt1Δ 200 ∗ 150 100 50 0 pRS423GP pRS423GP-LPT1 Figure 3.6 Lysophosphatidylcholine acyltransferase (LPCAT) activity is absent in lpt1Δ. (A). Cell lysates from wild-type, lpt1Δ and slc1Δ haploids, grown to late logarithmic phase, were assayed for LPCAT activity by the spectrophotometric method described in "Experimental Procedures" except lysoPA was replaced with lysoPC. Palmitoyl-CoA (black), oleoyl-CoA (grey) and arachidonyl-CoA (striped). The data represent mean ± S.E. n = 3-4. Asterisks indicate statistically significant difference compared with wild-type strain (p< 0.01). (B) Similar assays were performed with cell lysates from wild-type cells transformed with pRS423GP or pRS423GP-LPT1 98 To verify that Lpt1 was not an accessory protein required for true LPCAT activity, LPT1 was over expressed in wild type yeast. LPCAT activity was increased by 7-fold in comparison to the wild type yeast transformed with empty vector (Figure 3.6). This correlated with the role of Lpt1 as LPCAT. We further examined substrate specificity of Lpt1 for other lysophospholipid acyl acceptors with palmitoyl-CoA and oleoyl-CoA as acyl-donors (Figure 3.7). Wild type cell lysates showed lysophosphatidylethanolamine (LysoPE), lysophosphatidylinositol (LysoPE) and lysophosphatidylserine (Lyso PS) acyltransferase activities. These activities were 6-12 fold higher with oleoyl-CoA in comparison to palmitoyl-CoA, each at 50μM. In lpt1∆ strain very little to no lyso PI, lysoPS and lysoPE acyltransferase activity was left. Assays with oleoyl-CoA as acyl donor established that lysoPC, lysoPI and lysoPS were better substrates for Lpt1 in comparison to LysoPE. These results suggest that Lpt1 has broad substrate specificity for lysophospholipids and is a lysophospholipid acyltransferase (LPLAT). LPLAT Activity (nmol/min/mg) 99 15 10 5 * 0 Normal lpt1Δ LysoPI * * * Normal lpt1Δ LysoPE Normal lpt1Δ LysoPS Figure 3.7 Lysophospholipid substrate specificity of Lpt1p. Lysophosphatidylethanolamine (lysoPE), lysophosphatidylinositol (lysoPI) and lysophosphatidylserine (lysoPS) were used as acyl acceptors and LPLAT activity was determined by the spectrophotometric method using cell lysates prepared from wild-type yeast. palmitoyl-CoA (black), oleoyl-CoA (textured). The data represents mean ± S.E. n = 3. Asterisk indicates statistically significant difference (p<0.01) between wild-type and lpt1Δ. 100 Acyl-CoA substrate specificity of Lpt1 To determine whether Lpt1 was responsible for establishing acyl chain asymmetry in phospholipids by esterifying lysophospholipids, we determined acyl-CoA substrate specificity of Lpt1 with [14C] lysoPC as acyl acceptor. We predicted that Lpt1 consistent with its role in phospholipid remodeling would use unsaturated acyl-CoAs preferentially. Substrate concentration series experiments were performed with [14C] lysoPC and five acyl-CoA species: palmitoyl-CoA (16:0), palmitoleoyl-CoA (16:1), stearoyl-CoA (18:0), oleoyl-CoA (18:1) and arachidonyl-CoA (20:4) (Figure 3.8.1-3). Except arachidonoylCoA, the other four acyl-CoA substrates are the most abundant acyl chains in yeast phospholipids. The kinetic data was analyzed by EZ – fit software to obtain apparent Km and Vmax by fitting Michaelis Menten curve or sigmoidal curve to data (Table 3.1). There was discordance in Km and Vmax values obtained by these two methods. Then the data was transformed and Lineweaver-Burk and Eadie Hofstee plots were used to determine Vmax and Km. The data could not be analyzed for most of the acyl-CoAs due to the lack of linear relationship and when analyzed gave inflated Km and Vmax values. The kinetic data was then analyzed by Hill plot to obtain apparent K0.5 and Vmax for different acyl-CoAs (Table 3.1). The Hill number ranged from 1.5-2 for all the acylCoAs suggesting that kinetics did not follow the Michaelis Menten equation and were, in fact, cooperative. LPCAT activity showed low apparent K0.5 and Vmax for palmitoyl-CoA (16:0) and stearoyl-CoA (18:0). Higher apparent K0.5 and Vmax for palmitoleoyl-CoA (16:1) and oleoyl-CoA (18:1) was seen for the monounsaturated acyl-CoA substrates while polyunsaturated arachidonoyl-CoA (20:4) showed intermediate values. 101 LPCAT activity (nmol/min/mg) A 10 8 6 4 2 0 0 10 20 30 Palmitoyl - CoA (μM) 40 B 120 LPCAT activity (nmol/min/mg) 100 80 60 40 20 0 0 10 20 30 40 50 Palmitoleoyl - CoA (μM) 60 Figure 3.8.1 Acyl-CoA substrate specificity of Lpt1. Cell lysates from lpt1Δ yeast transformed with pRS423GP / LPT1 were assayed for LPCAT activity by the radioactive substrate method described in "Experiemental Procedures". The reaction contained 50μM [1-14C] palmitoyl lysophosphaitdylcholine, 1 - 50 μM of acyl-CoA, 3μg of cell lysate , and 100 mM Tris-HCl, pH 7.4 and incubated for 5 minutes at 28°C. A. palmitoyl-CoA B. palmitoleoyl-CoA. The data represent mean ± S.E. n = 3 - 5. 102 LPCAT activity (nmol/min/mg) C 14 12 10 8 6 4 2 0 0 5 10 15 20 25 30 Stearoyl-CoA (μM) 35 40 20 40 60 80 100 120 140 160 Oleoyl - CoA (μM) D LPCAT activity (nmol/min/mg) 100 80 60 40 20 0 0 Figure 3.8.2 Acyl-CoA substrate specificity of Lpt1p. Cell lysates from lpt1Δ yeast transformed with pRS423GP / LPT1 were assayed for LPCAT activity by the radioactive substrate method described in "Experiemental Procedures". The reaction contained 50μM [1-14C] palmitoyl lysophosphaitdylcholine, 1 - 140 μM of acyl-CoA, 3μg of cell lysate , and 100 mM Tris-HCl, pH 7.4 and incubated for 5 minutes at 28°C. C. stearoyl-CoA D. oleoyl-CoA. The data represent mean ± S.E. n = 3 - 5. 103 E 40 LPCAT activity (nmol/min/mg) 30 20 10 0 0 20 40 60 Arachidonoyl - CoA (μM) 80 Figure 3.8.3 Acyl-CoA substrate specificity of Lpt1p. Cell lysates from lpt1Δ yeast transformed with pRS423GP / LPT1 were assayed for LPCAT activity by the radioactive substrate method described in "Experiemental Procedures". The reaction contained 50μM [1-14C] palmitoyl lysophosphaitdylcholine, 2.5 - 75 μM of arachidonoyl-CoA, 3μg of cell lysate , and 100 mM Tris-HCl, pH 7.4 and incubated for 5 minutes at 28°C. The data represent mean ± S.E. n = 3 - 5. 104 Table 3.1 Comparison of different methods used to calculate kinetic parameters of Lpt1. Kinetic data from Figure 3.8.1-3 was used to calculate apparent Vmax and Km of Lpt1 for different acyl-CoAs. Acyl-CoA Palmitoyl-CoA (16:0) Palmitoleoyl-CoA (16:1) Stearloyl-CoA (18:0) Oleoyl-CoA (18:1) Arachidonyl-CoA (20:4) Hill Plot Analysis Michaelis Menten (Curve to fit data) Km Vmax (nmol / min / mg) (μM) Km (μM) Vmax (nmol / min / mg) 2.2 6.8 1.8 7.8 11 105 21 142 4.7 9.6 5.9 12 30.5 95.4 49 125 10.5 34.4 11 58 Table 3.2 Kinetic parameters of Lpt1: Endogenous acyl-CoA profile in wild type (S288C background) yeast strain was obtained from literature (Faergeman 2001). * The concentration in (μM) was calculated by assuming a yeast haploid cell volume of 75 μm3. Acyl-CoA K0.5 (μM) In vitro acylCoA conc.* (μM) Palmitoyl-CoA (16:0) Palmitoleoyl-CoA (16:1) Stearloyl-CoA (18:0) Oleoyl-CoA (18:1) Arachidonyl-CoA (20:4) 2.2 11 5.5 30.4 10.5 5.4 7.9 6.2 2.9 - Expected LPCAT Activity at in vitro acyl conc. (nmol/min/mg) 7.2 30 5 5 Vmax (nmol / min / mg) Vmax / K0.5 (min-1 mg-1) 6.8 105 11.6 95.4 34.4 44 95 21 31 33 105 The ratio of Vmax/Km or the efficiency of enzyme can be used to find out what the preferred substrate is (Table 3.2). The value for efficiency of the enzyme was highest for palmitoleoyl-CoA followed by palmitoyl-CoA. The Vmax/Km ratio was in general higher for unsaturated acyl-CoAs in comparison to saturated acyl-CoA, although palmitoyl-CoA was an exception due to its low apparent K0.5. Subcellular distribution of LPLAT activity Acyl-CoA dependent LPCAT activities have been identified in yeast microsomes and mitochondria. Yamada et al., have shown that yeast LPCAT activity was localized mainly in microsomes and that mitochondrial fraction had much lower activity (4%of the microsomal activity). Acyl-CoA dependent LPCAT activity in mitonchodria was reported in yeast, although the percentage recovery of protein and activity in this subcellular fraction was not examined (Yamada 1977; Testet 2005). To verify the location of LPLAT activity we used sucrose gradients to purify mitochondria, microsomes, and cell lysates by differential centrifugation. LPCAT activity (representative of LPLAT activity) was measured in these subcellular fractions (Table 3.3). The microsomal fraction was highly enriched for LPLAT activity and had 10- fold higher activity than cell lysates. The mitochondrial fraction did not show any enrichment in LPLAT activity. No LPLAT activity independent of Lpt1 was detected in lpt1∆ mitochondria. This suggested that there is no acyl-CoA dependent mitochondrial LPCAT. 106 Table 3.3 Subcellular localization of acyl-CoA dependent LPCAT activity. Wild-type yeast were grown in YP with 3% glycerol to O.D.660 = 1. Cell lysates, mitochondria and microsomes were isolated as described under "Experimental Procedures". LPCAT activity was measured using the spectrophotometric method as described in fig. 3. The data represent mean ± S.E. n = 3. Subcellular fraction LPCAT activity (nmol / min / mg) Fold enrichment Percent recovery Cell lysate 10.2 ± 0.5 Mitochondria 8.1 ± 1.9 0.8 ± 0.1 1.9 ± 0.9 Microsomes 99.2 ± 2.7 9.7 ± 0.2 65.1 ± 14.5 107 Counts per minute x 103 A Counts per minute x 103 B 50 40 30 * 20 10 ∗ 0 Normal lpt1Δ 100 80 60 40 20 0 Normal lpt1Δ Figure 3.9 Incorporation of [3H] oleate into phospholipids and neutral lipids. Wildtype and lpt1Δ yeast were grown in YPD to logarithmic phase and pulse-labeled with [3H]oleate for 30 minutes. A. Incorporation into the major phospholipids (PC, black; PE, white; PS/PI, striped) B. neutral lipids (diglyceride, dark gray; triglyceride, light gray; ergosterol ester, hatched) was measured as described in "Experimental Procedures". The data in counts per minute represent mean ± S.E. n = 4. Asterisk indicates statistically significant difference between wild-type and lpt1∆ (p<0.05) 108 Role of Lpt1 in oleate incorporation into glycerolipids We further studied the physiological role of Lpt1 mediated LPLAT activity in yeast. Wild type and lpt1∆ strains were pulse labeled with [3H]oleate for 30 minutes and the incorporation of radioactive label in different phospholipid species and neutral lipids was measured (Figure 3.9). The lpt1∆ strains incorporated 30% less label in PC than the wild type strain. PS, PE, PI and neutral lipids did not differ between lpt1∆ strain and wild type yeast. The effect on PC synthesis may be due to reduction in AGPAT activity or LPLAT activity mediated by Lpt1. AGPAT mediates the synthesis of phosphatidic acid (PA), an important intermediate in the de novo synthesis of phospholipids and triglycerides. Therefore, one would expect that changes in Lpt1 mediated AGPAT activity would decrease the incorporation of oleate in triglycerides and phospholipids. Thus it is more likely that selective reduction in oleate incorporation in PC in lpt1∆ strains is due to the contribution of LPLAT activity mediated by Lpt1 towards PC synthesis. Since Lpt1 has the ability to esterify other lysophospholipids it is difficult to say why the effect was limited to PC. 109 3.5 Discussion AGPAT mediates the acylation of lysophosphatidic acid to phosphatidic acid, an essential intermediate in the de novo triglyceride and phospholipid synthetic pathway. The basic synthetic pathways of triglyceride and phospholipids (with some exceptions) in S. cerevisiae are same as in higher eukaryotes. Human AGPAT-1 and AGPAT-2 were cloned by homology to Slc1, the yeast AGPAT. The slc1∆ mutants are viable and microsomes prepared from slc1∆ strain retain the ability to synthesize phosphatidic acid (Athenstaedt 1997) indicating that alternative pathway or another AGPAT must exist in yeast. We initiated the present study to identify the novel AGPAT in yeast, and potentially identify novel AGPAT members of the existing human AGPAT family or a new family of enzymes with AGPAT activity. Yeast is a versatile model system with well-defined genetic system making it possible to conduct a systematic analysis of lethality and other phenotypes associated with deletion of a gene in yeast. Because of the essential requirement of phosphatidic acid for the synthesis of membrane phospholipids in yeast, and indeed all eukaryotes, absence of enzymes synthesizing PA should prove lethal to cell. Therefore the present study employed synthetic lethal screen to identify candidate AGPAT genes which when deleted in conjunction with SLC1 would lead to cell inviability. SGA analysis utilizing slc1∆ mutant was undertaken and a total of 52 synthetic lethal interactions were identified. Among the 13 synthetic lethal genes with known functions, FEN1 (ELO2) encodes protein required for the synthesis of very long chain fatty acids (VLCFA). 110 ELO2 and ELO3 belong to elongase family of proteins and have been shown to catalyze elongation of C16 to C18 fatty acids to C24-C26 respectively. These VLCFA are required for sphingolipid synthesis. Simultaneous disruption of ELO2 and ELO3 in a haploid cell is synthetically lethal (Dickson 2008). Sphingolipids are made up of long chain base (LCB), very long chain fatty acids (usually C26) and a polar head group. LCBs are required for cell viability; however strains lacking LCBs have been isolated. These lcb1- defective cells can grow because of a suppressor mutation in SLC1. SLC1-1 mutant cells, with Q44L substitution in the Slc1 protein suppresses the defect in sphingolipid synthesis by incorporating long chain acyl groups (C26) in the sn-2 position of inositol containing glycerolipids (Nagiec 1993). This novel lipid is postulated to mimic ceramides required for sphingolipid synthesis. Overexpression studies on SLC1-1 and SLC1 have shown that Slc1 shows AGPAT activity in the presence of 22:1-CoA and 24:0-CoA (Zou 1997). Although this activity is only 21%-33% of that of SLC1-1 encoded mutant protein it might be sufficient to compensate for loss of sphingolipid synthesis in yeast. It is possible that ELO3 in conjunction with SLC1 can compensate for the defect in sphingolipid synthesis caused by the absence of ELO2. A yeast strain with a disruption in SLC1, ELO3 and LCB1 (lcb1∆slc1∆elo3) has been shown to be inviable (Dickson 2008). LPT1, a member of MBOAT superfamily, was also identified by the synthetic lethality screen and further characterized. The MBOAT superfamily which consists of membrane bound acyltransferases is evolutionarily conserved. 5 members of this family that are found in Saccharomyces cerevisiae are sterol acyltransferases Are1, Are2; glycosylphosphatidylinositol anchor remodeling enzyme Gup1, Gup2 and Lpt1. 111 In humans the MBOAT family consists of 16 members of which ACAT1, ACAT2, and DGAT1 are the only members whose biochemical role has been established. Lpt1 does not share sequence homology with Slc1 or any other AGPAT family members including lysophosphatidylcholine acyltransferases, LPCAT1 and LPCAT2. Lpt1 shares 24%, 21% and 22% amino acid sequence identity with human MBOAT1, 2 and 5 respectively. Further studies on the human counterparts of Lpt1 have been described later. To investigate whether Lpt1 was responsible for AGPAT activity in slc1∆ strain, we compared the AGPAT activity in lpt1∆ and slc1∆ cell lysates. AGPAT activity was reduced by 60% in the absence of SLC1, the known AGPAT in yeast. This is consistent with the results of a previous study that showed 54% reduction in microsomal phosphatidic acid synthesis in slc1∆ in comparison to the wild type yeast strain. However, AGPAT activity in lpt1∆ yeast strain was not statistically different from that in wild type strain. It is possible that in the absence of Lpt1, Slc1 is up regulated and hence AGPAT activity is unaffected. However, overexpression of LPT1 in a wild type yeast strain increased AGPAT activity by 7-fold. Also, simultaneous deletion of SLC1 and LPT1 in haploid cell lead to inviability suggesting that LPT1 is the novel AGPAT in yeast responsible for phosphatidic acid synthesis in the absence of SLC1. Additional experiments in the present study demonstrated that LPT1 is the major lysophospholipid acyltransferase in yeast. There is a complete loss of the ability to esterify sn-2 lysophosphatidylcholine upon LPT1 deletion in the assay conditions here employed. This LPCAT activity was mostly associated with microsomes (65%), which was in agreement with previous studies which showed that Lpt1 localizes to ER (Huh 2003). 112 Loss of 1/3 of LPCAT activity might have been due to degradation of enzyme or inefficiency in the process of preparation of subcellular fractions. Acyl-CoA dependent LPCAT activity has been described in mitochondria (Testet 2005) but we found very little LPCAT activity (2%) to be associated with mitochondrial fraction in wild type strain and no activity in mitochondrial fractions prepared from an lpt1∆ strain. The wild type strain showed the ability to esterify other lysophospholipids (lysoPE, lysoPI and lysoPS) by an acyl-CoA dependent reaction. The acylation rate of different lysophospholipids was in the order lysoPC=lysoPI=lysoPS>lysoPE when each was included at 50 μM. The lpt1∆ yeast strain showed very little to no ability to esterify lysoPC, lysoPE and lysoPI. In lpt1∆ yeast strain, around 15% of lysoPS acyltransferase activities with unsaturated aycl doors and 50% activity with saturated acyl-CoAs remained. It is possible that there are yet to be identified lysoPS acyltransferases in yeast. Lysates of slc1∆ yeast strain transformed with yeast expression vector containing coding region of SLC1 (pYEUra3-SLC1) showed no lysophospholipid acyltransferase activity with 45μM lysoPC, lysoPE and lysoPI and 18 μM [14C] 18:1-CoA (Zou 1997). Since in these assays lysoPS was not included as acyl acceptor it is possible that Slc1 is responsible for residual lysoPS acyltransferase activity in the absence of Lpt1. In yeast, the role of lysoPE esterification was proposed in uptake and synthesis of PE from exogenous lysoPE (Riekhof 2006), however the enzyme mediating this esterification was not identified. Microsomal lysoPI acyltransferase activity has been described in rat brain (Baker 1973), pancreas (Rubin 1983) and bovine brain (Sanjanwala 1989). Acyl-CoA dependent conversion of lysoPS to phosphatidylserine is also known to occur in rat liver (Holub 1980). 113 The ability of Lpt1 to transfer acyl groups to sn-2 position of wide variety of lysophospholipids and the absence of activity upon its deletion shows that Lpt1 is the major LPLAT in yeast and may be the first characterized member of a LPLAT branch of the MBOAT gene family. Since Lpt1 esterifies lysophospholipids at the sn-2 position, we examined whether acyl-CoA selectivity of Lpt1 contributes to asymmetrical distribution of acyl groups in phospholipids. Using lysoPC as the acyl acceptor, low Km and Vmax values were obtained with saturated acyl-CoAs while unsaturated acyl-CoA showed higher Km and Vmax values. In comparison to previously described LPCAT activity in yeast (Km-152 μM; Vmax- 10.9 nmol/min/mg), the Km for oleoyl-CoA was lower and the Vmax was higher. Vmax/Km ratio was used to compare the utilization of acyl-CoAs by the enzyme. In general the efficiency was higher for the unsaturated acyl-CoAs compared to the saturated acyl-CoAs. Comparison of LPLAT activity at physiological concentration of each acyl-CoAs would give better idea about the acyl-CoA specificity of Lpt1. There are very few studies which have measured the intracellular acyl-CoA pool in yeast. Endogenous levels of acyl-CoA species in wild type yeast cells grown to exponential phase (O.D 660 0.8) in YEPD media have been measured in nmol/ 2 * 109 cells (Faergemann 2001). The average volume of haploid yeast is about 70 μm3 and this information allows us to calculate the concentration of each acyl-CoA in the cell (Sherman 2002). At these concentrations the Lpt1 will utilize the acyl-CoAs in the following order: 16:1-CoA> 16:0-CoA> 18:1-CoA = 18:0 CoA (Table 3.2). As can be derived from our data, small changes in acyl-CoA species concentration may change the substrate used the most by Lpt1. 114 The physiological role of Lpt1 mediated LPLAT activity on glycerolipid synthesis was studied by pulse labeling wild type and lpt1∆ strain with [3H] oleate. 30% reduction in incorporation of label in PC was seen in lpt1∆ strain. This decrease is less likely to be due to decrease in AGPAT activity that mediates the synthesis of phosphatidic acid, a precursor for all phospholipid species and triglyceride. Since no changes were seen in incorporation of label in any other phospholipids and triglyceride in lpt1∆ strain, it is more likely that the absence of LPLAT activity affects the PC synthesis via a remodeling pathway. Lpt1 can mediate esterification of lysoPE, lysoPI and lysoPS and a lack of effect on these phospholipids might be due to their low abundance or low turn over in cell. Mitochondrial phospholipase A2 activity in yeast has been reported but the preference for phospholipid species is not known (de Kroon 2007). Yeast phospholipase B, Nte1 selectively degrades PC synthesized by CDP-choline pathway, producing glycerolphosphocholine (Zaccheo 2004). Recently, glycerophosphocholine acyltransferase (GPCAT) activity was reported in yeast however, no gene–enzyme relationship was established. It was suggested that glycerophosphocholine is sequentially acylated by GPCAT and Lpt1 to synthesize phosphatidylcholine (Stalberg 2008). Another reaction which produces lyso-PC in yeast is the transfer of an acyl group from PC to diglyceride to synthesize triglycerides (Oelkers 2000). This acyl-CoA independent reaction catalyzed by Lro1, may contribute to substrates for Lpt1. In addition to membrane remodeling Lpt1 might be required to regulate intracellular levels of lysophospholipids in the event of increased rate of phospholipid turnover. The esterification of lysophospholipids to synthesize phospholipids might be energetically favorable for cell in comparison to de novo synthesis of phospholipids. 115 Phospholipids in general have saturated acyl chains in the sn-1 position and unsaturated acyl chains in sn-2 position. This asymmetrical distribution of acyl chains in phospholipids is thought to occur by phospholipid remodeling requiring lysophospholipid acyltransferase activity (Lands 1960). In Saccharomyces cerevisiae, Lpt1 mediates most of the lysophospholipid acyltransferase activity with a preference for unsaturated acylCoAs suggesting its role in phospholipid remodeling. Absence of SLC1 in conjunction with LPT1 leads to cell lethality, suggesting a minor but important role of LPT1 as an AGPAT. After the publication of the present study, Ict1 a novel soluble yeast AGPAT was identified (Ghosh 2008). This AGPAT is expressed in yeast cells upon exposure to organic solvents and perhaps contributes to the phospholipid synthesis in conditions in which requirement of phospholipids for repair is increased. Further studies will be required to examine the contribution of Slc1, Lpt1 and Ict1 in synthesis of PA, an important intermediate for triglyceride and phospholipid synthesis. Parallel studies by other investigators also identified LPT1 by using different genetic approaches (Benghezal 2007; Chen 2007; Riekhof 2007). Identification of LPT1, a member of MBOAT superfamily will allow us to understand the functional role of the mammalian orthologues in establishing phospholipid acyl chain composition. 116 CHAPTER 4: IDENTIFICATION AND BIOCHEMICAL CHARACTERIZATION OF HUMAN LYSOPHOSPHATIDYLCHOLINE ACYLTRANSFERASE-3 (LPCAT3) 4.1. Abstract Phospholipids are important constituents of membranes. They are synthesized by a de novo synthetic pathway and are remodeled by the Lands cycle to achieve the asymmetric distribution of acyl chains commonly found in the phospholipids. The Lands cycle has two components, deacylation by phospholipase to form lysophospholipids and reacylation by lysophospholipid acyltransferases to form phospholipids. Recently, we characterized Lpt1, a lysophospholipid acyltransferase with broad substrate specificity in Saccharomyces cerevisiae. In this study we describe the characterization of lysophosphatidylcholine acyltransferase 3 (LPCAT3), a human homolog of Lpt1 which specifically esterifies lysophosphatidylcholine. LPCAT3, when expressed in insect cells, resulted in 10 fold higher LPCAT activity in cell lysates over endogenous activity. No other acyltransferase activity was detected with a variety of other acyl acceptors including lysophosphatidic acid. The LPCAT3 activity did not display specificity towards acyl chains present in the sn-1 position of lysophosphatidylcholine. Concentration series experiments with saturated, monounsaturated and polyunsaturated acyl-CoAs, showed that LPCAT3 has a low apparent K0.5 (5μM) and Vmax (50 nmol/min/mg) for saturated acyl-CoA and higher K0.5 (46.5 μM) and Vmax (759 nmol/min/mg) with monounsaturated aycl-CoA. Polyunsaturated acyl-CoA showed intermediate values. 117 Electrospray ionization-mass spectrometric analysis of phospholipids in Sf9 cells expressing LPCAT3 showed a relative increase in phosphatidylcholine containing saturated acyl chains and a decrease in phosphatidylcholine containing unsaturated acyl chains. This shift in acyl chain composition of phospholipids agrees with the in vitro preference of LPCAT3 for saturated acyl-CoA when physiological acyl-CoA concentrations are less than or equal to 5 μM. Characterization of molecular nature of human LPCAT3 will enable us to investigate whether other human MBOAT protein acylate lysophospholipids and help us understand the role of this family in phospholipid remodeling. 118 4.2 Introduction Lysophosphatidylcholine (LysoPC) is a major lysophospholipid in plasma with concentrations around 200µM (Murph 2007). It is mainly produced by phospholipase A2 and lecithin:cholesterolacyltransferase (LCAT) (Albers 1976). High levels of lysophosphatidylcholine lead to apoptosis (Takahashi 2002; Han 2008), cell lysis (Weltzien 1979) and inflammation (Kougias 2006). The enzyme systems that utilize this lysophospholipid and might clear it from plasma include lysophospholipases and lysophosphatidylcholine acyltransferases (LPCAT) (Wang 1999). LPCATs are also part of the acylation arm of Lands Cycle to synthesize remodeled phosphatidylcholine (PC) in rat liver (Lands 1960). Remodeling of phospholipids helps to maintain acyl chain composition of membrane lipids and allows for the incorporation of arachidonic acid in the sn-2 position of phosphatidylcholine which when released by cytosolic PLA2 is a substrate for eicosanoids (Funk 2001). The acyl chain composition of the peritoneal macrophage membrane phospholipids, when enriched for saturated fatty acids, showed a reduction in endocytosis and bilayer fluidity (Mahoney 1980). Thus, the acylation of lysophospholipids might regulate their plasma levels and maintain membrane function. The lysophospholipid acyltransferases esterify lysophospholipids by utilizing acyl groups of varying chain length and saturation (Lands 1960). These enzyme systems might catalyze the formation of remodeled phospholipids with specific biological function in a particular tissue. 119 Mouse LPCAT1 is highly expressed in lung and is suggested to catalyze the formation of dipalmitoyl lysophosphatidylcholine, a pulmonary surfactant (Nakanishi 2006). LPCAT2 primarily synthesizes platelet activating factor in inflammatory cells (Shindou 2007). Previous studies have shown that lysoPC in rat liver microsomes is acylated in vitro preferentially by oleoyl-CoA and arachidonoyl Co-A (Yamashita 1997). Similarly, LPCAT activity in guinea pig gall bladder utilizes oleoyl CoA as a preferred acyl donor (Niederhiser 1978). This suggests that there are other yet to be identified LPCATs, which are tissue specific and maintain acyl chain composition of membranes or quench excess lysophospholipids in their environment. We previously cloned LPT1, a novel lysophospholipid acyltransferase (LPLAT) in Saccharomyces cerevisiae with broad substrate specificity for lysophospholipids (Jain 2007). LPT1 is a member of membrane bound O-acyltransferase (MBOAT) super family. Members of this family are membrane proteins and have a conserved histidine in their active site (Hofmann 2000). The human homologues of LPT1 shared no sequence similarity to the LPCAT1 and LPCAT2 family and most of them have not been biochemically characterized. MBOAT1 gene disruption due to translocation was associated with a novel brachydactyly-syndactyly syndrome (Dauwerse 2007). Recently, one of the members of the MBOAT family LENG4, encoded protein was characterized as lysophosphatidylinositol acyltransferase (Lee 2007). In this study, we report the identification and biochemical characterization of human LPCAT3, a LPT1 homolog and a member of MBOAT family. 120 4.3 Experimental Procedures Materials Lysophosphatidylethanolamine (lysoPE), lysophosphatidylserine (lysoPS), lysophosphatidylinositol (lysoPI), 1-O-alkyl lysoPC, and lysoPG were from Avanti Polar Lipids (Alabaster, AL). [1-14C] palmitoyl- lysoPC (55mCi/mmol) was from Perkin-Elmer Life. All other chemicals were obtained from Sigma or Fisher. Protein sequence analysis Full-length sequences were aligned using clustalW2 (Labarga 2007). For percent identity, the conserved amino acids were counted and divided by the length of the shorter of the two proteins compared. Transmembrane domains were predicted by the transmembrane helix prediction hidden Markov model (TMHMM) 2.0 program (Krogh 2001). Insect cell expression A 4.4 kb human LPCAT3 cDNA clone (GenBank Acc. # BC065194) was obtained from the American Type Culture Collection in pCMVsport6. Restriction digest with SmaI, PvuII released the open reading frame with 45 bp of 5'UTR and 121 bp of 3'UTR. This fragment was ligated into StuI digested pFastbac and sequenced. Subsequent baculovirus expression in Sf9 insect cells was performed by Dr. David Schultz, Wistar Institute Protein Expression Core Facility. The pFastbac / LPCAT3 plasmid and a pFastbac / Glutathione S-transferase (GST) control were transposed into DH10Bac cells. 121 Recovered bacmid DNAs were screened by PCR for proper transposition of the transfer vector into the baculovirus genome. Positive bacmid DNAs were transfected into Sf9 cells and passage 1 (P1) virus stocks were recovered 96 hours post-transfection. A hightiter P2 virus stock was generated by infecting Sf9 at a multiplicty of infection (MOI) of ~0.1, followed by incubation for 96-120 hours. For productions, 1 x 106 Sf9 cells/ml in Sf900-II medium (Invitrogen) were infected with viruses at an MOI of 1. Cells were harvested 48 hours post-infection. Generating cell lysates for enzymatic assays Sf9 cells were homogenized in 1 mM EDTA / 200 mM sucrose / 100 mM Tris-HCl, pH 7.4 by 10-15 passages through a 26.5 gauge needle (Yen 2002). Unlysed cells were removed by centrifugation at 1,000 x g for 5 min. at 4° C and the supernatant was stored frozen (-70° C) prior to assays. LPCAT assay, radioactive substrate Lysophosphatidylcholine acyltransferase (LPCAT) activity was measured by the incorporation of [1-14C] palmitoyl lysoPC into phosphatidylcholine (PC). The reaction contained 100 mM Tris-HCl, pH 7.4, 50 μM [1-14C]palmitoyl lysoPC (50,000 dpm / nmol), 1 - 150 μM of the respective acyl-CoA, and 1 μg of cell lysate protein in a total volume of 100μl. Fixed time assays were performed for 7.5 minutes at 28° C. Reactions were stopped by adding chloroform: methanol (2:1) and lipids were extracted, resolved by thin layer chromatography (TLC), and quantified as described elsewhere (Nakanishi 2006). EZ-Fit software was used for non-linear regression, curve fit analysis to calculate apparent K0. and Vmax. To calculate Vmax / Km, Vmax was first changed to nM / min / mg to 122 remove volume from the units of the ratio. Hill plot analysis was done to obtain Hill number. LPLAT assays, spectrophotometric These assays were essentially performed as described in the previous chapter. The reaction mixture contained 100 mM Tris-HCl, pH 7.4, 50 μM of the respective lysoPL, 50 μM oleoyl CoA, 1 mM DTNB, and 2 - 5μg cell lysate protein in a total volume of 1 ml. Real time assays were performed for 5 minutes at room temperature. Bligh-Dyer lipid extraction Cellular phospholipids were extracted using chloroform and methanol according to the method of Bligh and Dyer (Bligh 1959). Following transfection, Sf9 cells were homogenized in 3 mL of methanol: water (2:0.8), transferred to a glass test tube and 1.25 mL chloroform was added. Tubes were vortexed for 30 seconds and allowed to sit for 10 minutes on ice. After centrifugation at 213 x g for 1 minute the chloroform layer was collected. The extraction was repeated and the lipid extracts were combined, dried under argon, reconstituted with 50μL of methanol: chloroform (2:1), and stored at -20°C. Lipid phosphorus assay This was performed by Dr. Brian Cummings, Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, Athens, GA. 10 μL of lipid extract was dried under argon. 200 μL of perchloric acid was added and heated at 130° C for 2-3 hours. To this was added 1 mL of dH2O, 1.5 mL of reagent C (4.2 g ammonium molybdate tetrahydrate in 100 mL 5 N HCl and 0.15 g malachite green (Zhou 1992) oxalate in 300 mL dH20) and 200 μL of 1.5% (v/v) Tween 20. After 25 minutes at room temperature, the A590 was measured. 123 Electrospray ionization-mass spectrometry (ESI-MS) This was performed in collaboration with Dr. Brian Cummings, Department of Pharmaceutical and Biomedical Sciences, The University of Georgia; Athens, GA. Lipid extracts (500 pmol/μl) were prepared by reconstituting in chloroform: methanol (2:1). Mass spectrometry was performed as described previously (Taguchi 2000). Samples were analyzed using a Trap XCT ion-trap mass spectrometer (Agilent Technologies, Santa Clara, CA) equipped with an ESI source. 5 μL of sample was introduced by means of a flow injector into the ESI source at a rate of 0.2 ml/min. The elution solvent was acetonitrile: methanol: water (2:3:1, v/v/v) containing 0.1% (w/v) ammonium formate (pH 6.4). The mass spectrometer was operated in the positive and negative ionization modes. The nitrogen drying gas flow-rate was 8.0 L/min at 350°C, nebulizer pressure was 30 psi, and capillary voltage was 3 kV. As previously described (Taguchi 2000) qualitative identification of individual PL molecular species was based on their calculated theoretical monoisotopic mass values and subsequent MSn fragmentation to identify the polar head groups. Relative quantification was done by comparison to the most abundant phospholipid in each sample, which corresponded to m/z 760 or, 34:1 (16:0-18:1) PC. MassLynx 4.0 software was used for data analysis. MSn fragmentation was performed on the same instrument under similar conditions except that the ion source and ion optic parameters were optimized with respect to the positive molecular ion of interest. Statistical Analysis Data from four separate Sf9 infections underwent analysis of variance using SAS software. Individual means were compared by post hoc tests. 124 4.4 Results Identification of MBOAT5/ C3F as a LPT1 homolog To characterize functional homologs of Lpt1, we performed homology search of amino acid sequence data bases using Lpt1 amino acid sequence as the query. Three human proteins, MBOAT1, MBOAT2 and MBOAT5 (C3F) with approximately 25% identity to Lpt1 were identified. MBOAT5 (also called C3F) was chosen for initial analysis because of the conservation of the KKME motif at the C-terminal as an endoplasmic retention signal (Figure 4.1). Immunofluorescence studies using antibody against a peptide sequence of C3F have shown that C3F has endoplasmic reticulum localization in both HeLa and HEK293 cells (Hodges 2005). MBOAT5/C3F is predicted to encode for 487 amino acid protein. Analysis of the predicted protein sequence by TMHMM 2.0 program identified seven transmembrane domains (Figure 4.2). Biochemical characterization of MBOAT5/C3F encoded Lysophosphatidylcholine acyltransferase activity To determine whether MBOAT5/C3F encoded protein was a functional homolog of Lpt1, a lysophospholipid acyltransferase in Saccharomyces cerevisiae, a putative full length cDNA clone for MBOAT5 was found in the mammalian gene collection and inserted into baculovirus. Insect cells were infected with the recombinant hMBOAT5 baculovirus to express MBOAT5 protein. To account for the endogenous acyltransferase activity, parallel infections with a GST-encoding virus were done. 125 Figure 4.1 Alignment of Lpt1p and MBOAT5 / C3F. ClustalW2 was used to align the yeast Lpt1p (610 amino acids) and human MBOAT5 / C3F (487 amino acids) peptide sequences. Numbers at the ends of lines refer to the location of the last residue. Not shown are the C-terminal 107 amino acids of Lpt1p which terminate in KKEE. Figure 4.2 Predicted transmembrane domains of LPCAT3. Seven transmembrane domains (boxes in dark grey) were predicted by TMHMM 2.0 for LPCAT3 126 Cell lysates prepared from insect cells expressing human MBOAT5 showed 10-fold higher [14C] lysoPC incorporation in PC with oleoyl-CoA over the GST-expressing controls (Figure 4.3). We therefore renamed MBOAT5/C3F as LPCAT3. The yeast ortholog, Lpt1 can utilize a variety of lysophospholipid species therefore we examined the ability of LPCAT3 to esterify other lysophospholipids. Lysophosphatidylserine (lysoPS) and lysophosphatidylethanolamine (lysoPE) were used as substrates but showed 8 fold less activity in comparison to lysoPC at 50μM. Lysophosphatidic acid (lysoPA), lysophosphatidylglycerol (lysoPG), lysophosphatidylinositol (lysoPI) and lyso-platelet activating factor (lysoPAF) were not used as acyl acceptors. LPCAT3 activity was not affected by variation of chain length or by the presence of double bond in the sn-1 acyl groups in lysoPC (Figure 4.4). Kinetic analysis was carried out by examining the LPCAT activity with increasing substrate concentrations of 1-palmitoyl-lysoPC and 50μM oleoyl-CoA (Figure 4.5). The kinetic data was analyzed by EZ – fit software to obtain apparent Km and Vmax of LPCAT3 for different acyl-CoAs by fitting Michaelis Menten curve or sigmoidal curve to data. There was discordance in apparent Km and Vmax values obtained by these two methods (Table 4.1). Then the data was transformed and Lineweaver-Burk and Eadie Hofstee plots were used to determine Vmax and Km. Using Lineweaver-Burk plot the Km and Vmax for lysoPC were 85μM and 1666 nmol/min/mg respectively. By Eadie Hofstee transformation the Km and Vmax for lysoPC were 64.5 μM and 1320 nmol/min/mg respectively. 127 LPCAT activity (nmol/min/mg) 600 * 500 400 300 200 100 0 hLPCAT3 GST Figure 4.3 Lysophosphatidylcholine acyltransferase (LPCAT) activity in infected Sf9 insect cells. Cell lysates from Sf9 cells with baculovirus-mediated expression of MBOAT5 or GST were assayed for LPCAT activity by the radioactive substrate method. 50 μM [1-14C] palmitoyl-lysoPC, 50 μM oleoyl-CoA, 1 μg cell lysate, and 100 mM TrisHCl, pH 7.4 were incubated for 7.5 minutes at 28°C. Radioactive product (PC) was resolved from substrate (lysoPC) by TLC. The data represent mean ± S.E. n = 4. Asterisk indicates statistically significant difference (p< 0.001). 128 LPCAT activity (nmol/min/mg) 1600 1200 800 400 0 14:0 16:0 18:0 18:1 LysoPC 14:0 18:1 LysoPE LysoPS Figure 4.4 Lysophospholipid substrate specificity of LPCAT3. LPLAT activity was measured using 50 μM oleoyl-CoA and 50 μM of lysoPC: 1-myristoyl (C14:0), 1palmitoyl (C16:0), 1-stearoyl (C18:0), 1-oleoyl (C18:1) and lysoPE 1-myristoyl (14:0), lysoPS 1-oleoyl (18:1). The spectrophotometric method was used, as described in Experimental procedures, with cell lysates prepared from LPCAT3 or GST infected Sf9 cells. The data represents mean ± S.E. n = 4. 129 A LPCAT activity (nmol/min/mg) 600 500 400 300 200 100 0 0 10 20 30 40 50 60 LysoPC (μM) 70 80 B Hill Number = 1.8 Log (v/Vmax-v) 2 1 0 0 0.5 1 1.5 2 -1 -2 -3 Log (1-palmitoyl-lysoPC) Figure 4.5 Kinetics of lysoPC utilization by LPCAT3. (A) LPCAT assays were performed as described in Figure 4.2 except a range (5- 75 μM) of [1-14C] palmitoyl lysoPC was used. The data represent the mean of the net LPCAT activity S.E. n = 4 (B) Hill plot for the data in panel A. 130 Analysis of the velocity at varying concentrations of oleoyl-CoA by Hill plot yielded a Hill number of 1.8 and apparent K0.5 of 12.5 μM for lysoPC. The enzyme exhibited positive cooperative kinetics with respect to concentration of lysoPC. Therefore sigmoidal curve was fitted to the data to obtain the apparent Vmax. Next, we analyzed the acyl-CoA selectivity of LPCAT3 using [1-14C] palmitoyllysoPC as an acyl acceptor. Increasing concentrations of saturated (stearoyl-CoA), monounsaturated (oleoyl-CoA), polyunsaturated, omega-6 (arachidonic acid), and polyunsaturated, omega-3 (linolenoyl-CoA) acyl-CoAs were used (Figures 4.6, 4.7, 4.8 4.9). The sigmoidal curve was fitted to the data to obtain the apparent Vmax for each of the acyl-CoAs (Table 4.1). The Michaelis Menten analysis of kinetic data gave higher Vmax and Km values for acyl-CoAs. The data could not be analyzed for all the acyl-CoAs by Lineweaver-Burk and Eadie Hofstee plots due to the lack of linear relationship.Subsequent analysis of the kinetic data by Hill equation yielded a Hill number and Km values. The Hill number for all the acyl-CoAs except linolenoyl-CoA were greater than 1 therefore LPCAT3 shows positive cooperative kinetics with respect to the varying concentration of acyl-CoAs (Table 4.1). Linolenoyl-CoA was the only substrate for which concentrations higher than 110 μM did not inhibit activity and hence a correct estimate of apparent Vmax and Km could not be obtained. According to the apparent Km values of LPCAT3 for acyl-CoAs, stearoyl-CoA was the preferred acyl donor (Table 4.1). Oleoyl-CoA was the most preferred substrate based on the capacity of enzyme (Vmax). 131 A LPCAT activity (nmol/min/mg) 60 40 20 0 0 2 4 6 8 10 12 14 16 18 20 22 Stearoyl-CoA (μM) B Hill Number = 2.7 Log(v/Vmax-v) 2 1 0 0 0.4 0.8 1.2 1.6 -1 -2 Log (Stearoyl-CoA conc.) Figure 4.6 Kinetics of Stearoyl-CoA (18:0-CoA) utilization by LPCAT3. (A) LPCAT assays were performed as described except a range (2- 20 μM) of Stearoyl-CoA was used. For each data point, net LPCAT activity was obtained by subtracting LPCAT activity in Sf9-GST lysates from Sf9-LPCAT3 lysates. The data represent mean ± S.E. n = 4. (B) Hill plot for the data in panel A 132 A LPCAT activity (nmol/min/mg) 800 600 400 200 0 0 20 40 60 80 Oleoyl-CoA (μM) 100 120 B Hill Number = 2 1 Log (v/Vmax-v) 0 0 0.5 1 1.5 2 -1 -2 -3 Log (Oleoyl-CoA conc.) Figure 4.7 Kinetics of Oleoyl-CoA (18:1-CoA) utilization by LPCAT3. (A) LPCAT assays were performed as described except a range (5- 110 μM) of oleoyl-CoA was used. For each data point, net LPCAT activity was obtained by subtracting LPCAT activity in Sf9GST lysates from Sf9-LPCAT3 lysates. The data represent mean ± S.E. n = 4. (B) Hill plot for the data in panel A 133 A LPCAT activity (nmol/min/mg) C 600 400 200 0 0 20 40 60 80 100 Arachidonoyl-CoA (μM) 120 140 B Hill Number = 2 2 Log (v/Vmax-v) 1 0 0 0.5 1 1.5 2 -1 -2 -3 -4 Log ( Arachidonoyl-CoA conc.) Figure 4.8 Kinetics of Arachidonoyl-CoA (20:4-CoA) utilization by LPCAT3. (A) LPCAT assays were performed as described except a range (10-130 μM) of arachidonoyl-CoA was used. For each data point, net LPCAT activity was obtained by subtracting LPCAT activity in Sf9-GST lysates from Sf9-LPCAT3 lysates. The data represent mean ± S.E. n = 4. (B) Hill plot for the data in panel A 134 A LPCAT activity (nmol/min/mg) 160 120 80 40 0 0 20 40 60 80 100 120 140 160 Linolenoyl-CoA (μM) B Hill Number = 1 2 Log(v/Vmax-v) 1 0 0 1 2 3 4 -1 -2 -3 Log (Linolenoyl-CoA conc.) Figure 4.9 Kinetics of Linolenoyl-CoA (18:3-CoA) utilization by LPCAT3. (A) LPCAT assays were performed as described except a range (20-150 μM) of arachidonoyl-CoA was used. For each data point, net LPCAT activity was obtained by subtracting LPCAT activity in Sf9-GST lysates from Sf9-LPCAT3 lysates. The data represent mean ± S.E. n = 4. (B) Hill plot for the data in panel A 135 Table 4.1 Kinetic parameters of human LPCAT3. Kinetic data from figures 4.6-4.9 were used to calculate apparent Km and Vmax using Michaelis Menten analysis and Hill equation. Lyso-PC Stearoyl-CoA (18:0) Oleoyl-CoA (18:1) ArachidonylCoA (20:4) α-linolenyl- CoA (18:3) Hill Plot Analysis Km Vmax (μM) (nmol/min/m g) 13 486 Michaelis Menten Analysis Vmax Vmax / Km Km (nmol/min/mg) (min-1 mg-1) (μM) 44 1010 5.1 50 9.8 19 117 46.5 759 16.3 419 3600 55.8 529 9.5 334 1794 214.1 268 1.3 338 354 136 The ratio of Vmax/Km or the efficiency of enzyme (assuming enzyme concentration is constant for all reactions) can be used to find out which substrate an enzyme is likely to turn into product faster. This value was the highest for oleoyl-CoA. Effect of expressing LPCAT3 on phospholipid species in Sf9 cells This was performed in collaboration with Dr. Brian Cummings, Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA. To begin to examine the physiological role of LPCAT3 in phospholipid metabolism, the effect of expressing LPCAT3 on the phospholipid profile in Sf9 cells was measured. We hypothesized that if LPCAT3 preferentially incorporates unsaturated acyl-chains into phospholipids then the cells expressing LPCAT3 will show abundance of unsaturated aycl chains in phsopholipids in comparison to the cells expressing GST. ESI-MS was performed on four independent cell lysates expressing LPCAT3 and GST (Figure 4.10). To account for alteration in ionization efficiency, the relative abundance of each PL species was expressed relative to the most abundant species (34:1 PC; m/z 760) within the sample. Normalizing to an added standard (28:0 PC, m/z 678) was also performed and showed similar trends (data not shown). After analyzing the abundance of 73 different m/z ratios in the positive mode, 10 showed a significant difference between the LPCAT3 and GST expressing cells. Assigning specific PL species to m/z values was based on their calculated theoretical monoisotopic masses and verified by MS-MS. 137 Relative Phospholipid Abundance . 1.0 0.8 0.6 0.4 0.2 0.0 30:0 32:0 36:0 34:2 36:1 36:2 32:0 32:0 34:1 34:2 PE ePC PC Figure 4.10 Effect of LPCAT3 expression on Sf9 phospholipid species. Lipids were isolated from Sf9 cells expressing GST or LPCAT3. After lipid phosphorous content determination, 250 nmol was analyzed by ESI-MS as described in Experimental Procedures. The results from 4 independent Sf9 infections were averaged. The abundance of each PL species is expressed relative to that of the most abundant within the sample (34:1 PC in each case). Of the 73 m/z values analyzed, all the ones that significantly differed (p<0.05) are shown. ePC = ether PC. 138 The five species that increased with LPCAT3 expression were 32:0 PE, 32:0 PC, 36:0 PC, 32:0 (1-alkyl) PC, and 34:1 (1-alkyl) PC. The five species that decreased were 30:0 PC, 34:2 PC, 36:1 PC, 36:2 PC, and 34:2 (1-alkyl) PC (Fig. 4.10) became less abundant. One thing to note is that when normalized to the added standard, 34:1 PC was about 30% more abundant in the LPCAT3 expressing cells. The overall trend was that the abundance of PC with more saturated chains increased with LPCAT3 expression while PC with more unsaturated chains. No differences between LPCAT3 and GST expressing cells were detected in the negative mode, suggesting that PI and PS species were unaffected. Tissue distribution of human LPCAT3 The expression pattern of LPCAT3 in human tissues was obtained from GNF SymAtlas and GeneNote, databases of human genes and their expression profiles in healthy human tissues. According to the GNF SymAtlas, LPCAT3 was expressed in all the 79 tissues and cell types. LPCAT3 was expression was 7-10 folds higher in adipocytes and bone marrow endothelial cells (CD34+, CD71+, or CD105+) when compared to most other tissues. Modest expression of LPCAT3 (3 fold over the basal) was seen in placenta and prostrate. The GeneNote microarray data found no differences in LPCAT3 expression in 12 tissues including bone marrow but not adipose tissue. 139 4.5. Discussion In this study, we identified and characterized a human LPCAT3. hLPCAT3 was identified by its sequence similarity to previously characterized Lpt1, a lysophospholipid acyltransferase in S.cerevisiae (Benghezal 2007; Jain 2007; Riekhof 2007; Tamaki 2007). Our findings suggest that the LPCAT3 encodes for a protein that catalyzes the LPCAT reaction. Firstly, the cDNA of human LPCAT3 when overexpressed in insect cells confers a 10-fold increase in incorporation of radioactive [1-14C] lysoPC in PC over the endogenous activity. Secondly, the activity was specific for lysophosphatidylcholine. Other acyl acceptors lysoPA, lysoPI, lysoPG, lysoPAF were not used as substrates. The enzyme could use lysoPE and lysoPS as acyl acceptors, but the activity was eight fold less than that for lysoPC suggesting its specificity for lysoPC. However, this preference for acyl acceptors is limited to only one fixed concentration (50μM) of lysophospholipids used in the assay. These findings reaffirm that the hLPCAT3 cDNA encodes for a protein with LPCAT activity. We realize LPCAT3 has been designated in literature as MBOAT5/C3F and changes in enzyme nomenclature make the integration with previous reports troublesome. However, the consistent approach of incorporating the reaction catalyzed in any protein’s name provides clarity. LPCAT3 does not share sequence homology to previously identified LPCAT1 and LPCAT2 genes (Nakanishi 2006; Shindou 2007). It is a member of the MBOAT super family, which includes enzymes responsible for transfer of fatty acids to their substrates (Hofmann 2000). There are 16 proteins that belong to this super family (MBOAT1 and MBOAT2, each has 2 isoforms and Porcupine gives rise to 4 isoforms by alternative splicing) and a conserved histidine residue is thought to be the catalytic site (Figure 4.11). 140 Figure 4.11 Dendogram showing members of human MBOAT family 141 Acyl-CoA cholesterol acyltransferases (ACAT1 and ACAT2) that esterify cholesterol and diacylglycerol acyltransferase-1 (DGAT1) which utilizes diacylglycerol are the only members whose biochemical role has been established (Cases 1998). LPCAT3 encoded protein is 20% identical to MBOAT1 and MBOAT2 proteins. MBOAT1 and MBOAT2 are overall 52% identical. The disruption of MBOAT1 is associated with brachydactyly-syndactyly syndrome (Dauwerse 2007). The protein encoded by Drosophila homolog of LPCAT3 known as nessy, is 32% identical to the human counterpart and is regulated by homeotic (HOX) proteins during embryogenesis. HOX proteins are found in wide variety of organisms including Drosophila, mouse, and humans and are known to regulate morphognetic processes (Zaffran 1999). It is possible that LPCAT3 is a target of human HOX proteins and thus has a regulatory role in developmental processes. MBOAT3, MBOAT4, Porcupine and Leng4 share 15% amino acid sequence similarity with LPCAT3. MBOAT3, shares sequence similarity to mammalian hedgehog acyltransferase (HHAT) but does not show in vitro HHAT activity (Abe 2007). Porcupine has been associated with processing and secretion of Wnt signaling proteins (Tanaka 2000; Caricasole 2002). Besides these studies very little information exists about the biochemical role of by these orthologs. LPT1, the yeast homolog of LPCAT3, encodes for LPLAT activity and utilizes all major lysophospholipids. It is possible that each human homolog utilizes specific lysophospholipid species as substrate and is tissue specific in expression. Recently LENG4 encoded protein was shown to have lysophophotidylinositol acyltransferase 142 (LPIAT) activity (Lee 2007). In the assay conditions used in this study, LPCAT3 showed robust activity with lysoPC. A comparison of LPCAT3 affinities for lysoPC and acylCoAs with that of identified LPCAT1 and LPCAT2 will give us an idea about the likely contribution of each LPCAT at varying substrate concentrations. LPCAT1 has lower apparent Km for lysoPC in comparison to LPCAT3 (2.3 μM vs. 13.1 μM). Inspite of LPCAT3 having a higher Vmax than LPCAT1, both the enzymes seem to saturate with lysoPC at concentrations between 25-35 μM. The kinetic paramters of LPCAT2 are not known. Regarding acyl chain specificity it is expected that LPCATs would preferentially introduce unsaturated acyl groups in the sn-2 position of phospholipids to maintain asymmetrical distribution of acyl chains in phsopholipids. However, LPCAT1 utilizes wide variety of acyl donors at a single concentration of 25 μM (Nakanishi 2006). LPCAT2 has lower apparent Km for arachidonoyl-CoA in comparison to acetyl-CoA (21.1 Μm vs 50.4 μM) (Shindou 2007). Whether or not LPCAT2 utilizes acyl-CoAs other than arachidonoyl-CoA and acetyl-CoA is not known. LPCAT3 showed lower apparent Km for stearoyl-CoA but higher capacity for oleoyl-CoA. At concentrations ≤ 5 μM stearoyl-CoA was a better substrate for LPCAT3 than oleoyl-CoA. However, LPCAT3 showed highest catalytic efficiency for oleoyl-CoA among all acyl-CoAs. The enzyme showed similar affinity for arachidonoyl-CoA and oleoyl-CoA but lesser capacity. LPCAT3 showed higher apparent Km for arachidonoylCoA compared to LPCAT2 (55.8 μM vs. 21.1 μM). Depending on physiological concentrations of arachidonoyl-CoA, LPCAT3 might be responsible for enrichment of this acyl group in the sn-2 position of phosphatidylcholine. Previous studies have shown that lysoPC in rat liver microsomes is acylated in vitro preferentially by oleoyl CoA (4- 143 fold higher) in comparison to the stearoyl-CoA at 15μM (Lands 1963). It is not known whether LPCAT3 is responsible for the LPCAT activity reported in liver. The enzyme activity seems to be not only affected by the presence of a double bond but also by the position of the double bond. Linolenoyl CoA, a n-3 fatty acyl CoA was utilized at higher concentrations compared to other acyl donors and showed activity even at concentrations beyond 150 µM at which the LPCAT activity declined for oleoyl and arachidonoyl CoA. Thus the kinetic parameters for linolenoyl CoA could not be accurately measured. The enzyme did not seem to show robust activity with linolenoyl CoA in comparison to n-9 or n-6 fatty acyl CoA. Perhaps, the position of the double bond hinders in the effective formation of enzyme substrate complex. The ability of the enzyme to discern between the substrates with different position of double bonds might spare linolenic acid as precursor to docosahexaenoic acid and eicosapentaenoic acid. Interestingly, endogenous LPCAT activity in Sf-9 cells had low Km for all the acyl donors. Calcium independent PLA2 has been reported in the Sf-9 cells (Yeh 1997) however, lysophospholipid acyltransferase activity has not been characterized. Identification of this LPCAT activity in insect cells will be the subject of future studies. The physiological role of LPCAT3 mediated activity in the maintenance of acyl chain composition of phospholipids can only be speculated by its expression pattern in various tissues. LPCAT3 is highly expressed in adipose tissue and endothelial cells of bone marrow (CD34+, CD71+, or CD105+). In adipocytes, LPCAT3 might be involved in regulation of the acyl chain composition of the adipocyte membrane phospholipids which in turn might regulate the properties of adipocyte membranes. 144 The binding property of insulin receptor has been shown to be affected by the acyl group composition of surrounding phospholipids in membrane (Gould 1982). Adipocytes from rats fed on diet rich in polyunsaturated fats bound more insulin than the cells from animals fed diets rich in saturated fats (Field 1988). It is possible that LPCAT3 mediated changes in acyl chain composition of membrane phospholipids in adipocytes has a role in modulating the insulin sensitivity. In addition, LPCAT3 introduced arachidonic acid in lysoPC when released by PLA2 might prime the formation of eicosanoids in adipocytes. Arachidonic acid derived eicosanoids PGF2α and PGJ2 have been shown to act as ligands for peroxisome proliferator-activated receptor gamma, a nuclear receptor that plays an important role in adipocyte differentiation (Reginato 1998). Recently, mouse lysocardiolipin acyltransferase (LyCAT) a member of AGPAT gene family, part of cardiolipin remodeling pathway, was shown to be required for hematopoietic and endothelial cell development in mouse embryonic stem cells. High LyCAT mRNA levels were detected in CD31+ and CD45- endothelial cells in adult mouse bone marrow. Overexpression of LyCAT in mouse stem cells increased hematopoietic and endothelial gene expression while siRNA mediated knock down of the gene showed an opposite effect (Wang 2007). The substrates modified by LyCAT that might have mediated this effect were not described. Whether LPCAT3 has similar role in bone marrow endothelial cells is not known. Lysophosphatidylcholine has been shown to be proinflammatory in nature and has been shown to be a ligand for G-protein- coupled receptors expressed in endothelial cells. The lysophospholipid G-protein-coupled receptors regulate a variety of cellular functions: cell proliferation and survival, migration and chemotaxis, cytoskeletal architecture, cell-cell-contacts and adhesion, Ca2+ homoeostasis and Ca2+-dependent 145 functions (Meyer 2007). It would be interesting to study whether LPCAT3 has any role in regulating the levels of ligands for GPCRs. To understand the physiological function of LPCAT3, the abundance of phospholipid species was analyzed in Sf-9 cells expressing LPCAT3. Increased levels of disaturated PC (32:0 and 34:0) and decreased levels of unsaturated acyl chains in PC (34:2, 36:1, 36:2) was seen. To account for this change in acyl chain composition of phospholipids, the endogenous saturated acyl-CoA concentrations in Sf-9 cells proposed to be ≤ 5 μM for preferential incorporation of saturated acyl groups in phosphatidylcholine by LPCAT3. It needs to be established whether similar effect are seen by in vitro or in vivo studies in mammalian system. Finally, the identification of human LPCAT3 will enable us to investigate whether other human MBOAT protein acylate lysophospholipids and help us understand the role of this family in phospholipid remodeling. 146 CHAPTER 5: SUMMARY Triglyceride (TG) molecules allow storage of surplus energy in form of fatty acyl chains that are esterified to glycerol molecule. The TG synthetic pathway shares substrates (acyl-CoA and glycerol 3-phosphate) and initial steps with phospholipid synthesis (Kennedy 1961). The de novo glycerolipid synthetic pathway begins with acylation of glycerol-3-phosphate at the sn-1 and sn-2 position in the first two steps to form first lysophosphatidic acid (1-acyl glycerol 3-phosphate) and then phosphatidic acid (PA) (Figure 1.1).Glycerol 3-phosphate acyltransferase (GPAT) and 1-acyl glycerol-3phosphate (AGPAT) catalyze the first and second reaction respectively. Phosphatidic acid can be either dephosphorylated by phosphatidic acid phosphatase (PAP) to form diacylglycerol (DG) or used as precursor for phospholipid synthesis. The DG formed from this reaction is esterified with a third acyl-CoA at the sn-3 position to form TG. This terminal reaction is unique to TG synthesis and is mediated by diacylglycerol acyltransferase (DGAT). Alternatively, DG can be used by CDP-choline pathway to synthesize phosphatidylethanolamine (PE) and phosphatidylcholine (PC) (Kennedy 1961). Cells must maintain a balance between TG and phospholipid synthesis to allow for energy storage as well as cell growth. Regulation at the branch point of TG and phospholipid synthesis seems likely. Hence we hypothesized that that the rate of TG synthesis is regulated at the step of DG esterification and that the DG esterifying enzymes 147 are regulated. Another hypothesis was that cells unable to esterify DG to TG will increase glycogen synthesis as an alternative to store energy. To understand the intracellular regulation of TG synthesis at the step of diacylglycerol esterification we used Saccharomyces cerevisiae as our model system. Yeast cells synthesize TG and phospholipids by pathways largely homologous to those in mammalian cells (Rajakumari 2008). In yeast the DG esterifying enzymes have been completely characterized. Dga1 (a homologue of mammalian DGAT2), and Lro1 (a homologue of mammalian lecithin cholesterol acyltransferase) contribute to 98% of TG synthesis, and the residual activity is contributed by Are2 (a homologue of mammalian DGAT1) (Oelkers 2002). To address our hypothesis we first identified genetic backgrounds and growth conditions where TG synthesis was altered. Next step was to determine whether in these conditions, changes in the mRNA, protein or activity levels of Dga1 and Lro1 were responsible for altering TG synthesis. We also measured glycogen levels in TG deficient yeast cells to examine any evidence of coordinated regulation of energy stores. Among the genetic backgrounds which were examined, disabling fatty acid hydrolysis was found to increase TG synthesis by 75%. Addition of inositol and choline, water soluble phospholipid precursors to growth media is known to affect the activity of phospholipid synthetic enzymes (McCammon 1982) mainly though the presence of upstream activating sequence known as inositol and choline responsive element (ICRE) or UASINO (Carman 1999). Although ICRE motifs are not found upstream of DGA1 and LRO1, genes encoding for DG esterifying enzymes in yeast (Saccharomyces Genome Database) we hypothesized that perturbations in phospholipid synthetic pathway would 148 affect TG synthesis as these pathways share initial steps and have common intermdiates of glycerolipid synthesis. However addition of inositol and choline did not affect TG synthesis. Absence of glycogen synthesis did not stimulate storage of energy in TG and vice versa. This result established TG and glycogen synthesis are not coordinately regulated in the growth conditions utilized. Glycogen levels in wild type yeast have been shown to increase 8 fold in response to poor nitrogen media (Francois 2001). It would be interesting to see whether yeast cells unable to store glycogen in nitrogen poor media upregulate their TG synthesis. Variability in the measurement of mRNA and protein levels of the DG esterifying enzymes did not allow us to examine the changes at transcriptional or translational level. In vitro microsomal DGAT assays were performed and Dga1 mediated DGAT activity showed cooperative kinetics with varying concentrations of oleoyl-CoA. Results from studies on permeabilized rat hepatocytes in the presence of saturating amounts of glycerol 3-phosphate and DG suggest that the rate of TG synthesis might be determined by acylCoA supply and the affinity of DGAT for acyl-CoA (Stals 1994). Perhaps positive cooperativity with oleoyl-CoA can amplify the response of the enzyme to the substrate. At a fixed concentration of 50μM oleoyl-CoA and palmitoyl-CoA are preferred substrates for Dga1 (Oelkers 2002). To address the possibility of allosteric regulation of DG esterifying enzymes in vitro microsomal DGAT activity assays were performed in the presence of metabolites indicative of cellular energy status, ATP, ADP, AMP, NAD, NADH, acetyl-CoA and malonyl-CoA. No changes in the kinetic parameters of Dga1 were seen by addition of these metabolites. We further addressed the question whether the regulation of TG 149 synthesis occurs at the level of phosphatidic acid (PA), another branch point in the TG and phospholipid synthetic pathway. Another possibility at the time was whether AGPAT which catalyzes the synthesis of PA by introducing acyl chain at sn-2 position might determine asymmetrical distribution of acyl groups (saturated at sn-1 and unsaturated at sn-2 position) in phospholipids. Slc1 was the only known AGPAT in yeast when we began our study. The fact that slc1∆ deletion mutants retained ~ 50% of the total microsomal AGPAT activity indicated the presence of another AGPAT (Athenstaedt 1997). Since synthesis of phosphatidic acid is an essential requirement for de novo synthesis of phospholipids we hypothesized that absence of SLC1 in conjunction with the novel AGPAT gene in a haploid cell would be lethal. A synthetic lethality screen was performed by crossing a slc1∆ strain with each of ~ 5000 viable single deletion haploids in collaboration with Charlie Boone, University of Toronto. Synthetic lethality screen identified LPT1 (YOR175c), a member of membranebound-O-acyltransferase (MBOAT) super family. LPT1 does not share sequence similarity with SLC1. The lpt1∆ cell lysates did not show significant decrease in vitro AGPAT activity, possibly due to up regulation of SLC1 activity. Since the depletion of Lpt1 did not affect the AGPAT activity, we further studied whether the overexpression of Lpt1 would influence AGPAT activity. Overexpression of LPT1 led to 7-fold increase in AGPAT activity suggesting that Lpt1 can mediate this reaction. Subsequent experiments showed that Lpt1 can mediate lysophospholipid acyltransferase (LPLAT) reaction by the introduction of acyl chains in the sn-2 position of lysophospholipids. In lpt1∆ yeast cells there was a complete loss of LPLAT activity suggesting that Lpt1 is the major LPLAT in yeast (Jain 2007). The acyl chain selectivity 150 studies on Lpt1 showed high capacity for unsaturated species but low affinity for saturated species. LPLAT is the reacylation arm of Lands cycle, which involves acyl chain remodeling of de novo synthesized phospholipids at the sn-2 position by phospholipase A2 mediated deacylation followed by reacylation. This phospholipid remodeling is believed to result in positional distribution of saturated acyl groups in sn-1 and unsaturated acyl groups in sn-2 position (Lands 1960). In yeast, LysoPC acyltransferase activity was reported but the enzyme-gene relationship was not established (Yamada 1977; Richard 1998). In lpt1∆ yeast cells there is 30% reduction in incorporation of [3H] oleate in phosphatidylcholine (PC) presumably due to lack of LPLAT activity in yeast. We established that Lpt1 is the novel LPLAT/AGPAT in yeast which might be responsible for selective incorporation of acyl chains in sn-2 position of lysophospholipids (Jain 2007). LPT1 does not share sequence similarity with lysoPC acyltransferase-1 (LPCAT1) and lysoPC acyltransferase-2 (LPCAT2) the only LPCATs which have been characterized so far and is a member of MBOAT family which is conserved across species (Figure 5.1).The amino acid sequence of LPT1 encoded protein is 25% identical to human MBOAT1, MBOAT2 and MBOAT5 proteins (Figure 5.2).To determine whether MBOAT5 possessed LPLAT activity similar to Lpt1 we expressed human MBOAT5 in Sf-9 cells. Cell lysates prepared from insect cells expressing human MBOAT5 showed 10-fold higher [14C] lysoPC incorporation in PC with oleoyl-CoA over the GST-expressing controls. Further experiments showed that MBOAT5 preferentially utilized lysoPC compared to other lysophospholipid species suggesting that it is a LPCAT. We therefore renamed MBOAT5 as LPCAT3. MBOAT5 similar to Lpt1 shows more activity with unsaturated acyl-CoAs but has low affinity for saturated acyl-CoA. 151 Figure 5.1 Evolutionary dendogram showing homologs of Lpt1 in other eukaryotes 152 Figure 5.2 Dendogram showing Lpt1 homologs in human MBOAT family 153 There are 16 human MBOAT proteins of which only 3 have been characterized. These characterized proteins include cholesterol acyltransferases (ACAT1 and ACAT2) and DGAT1. While our study was in progress, MBOAT7 was characterized as a lysophosphatidylinositol acyltransferase (Lee 2007). Perhaps each human MBOAT protein specifically esterifies a lysophospholipid species unlike Lpt1 in yeast which has broad substrate specificity. To elucidate the physiological role of LPCAT3, the abundance of phospholipid species was analyzed in Sf-9 cells expressing LPCAT3. There was an increase in PC species with saturated acyl chains and decrease in unsaturated PC species in cells expressing LPCAT3. Although it needs to be established whether similar effect are seen by in vitro or in vivo studies in mammalian system this finding does suggest that LPCAT3 might be responsible for asymmetric distribution of acyl groups in phosphatidylcholine in tissues where it is highly expressed (adipose and endothelial cells). Significance of the findings: 1. A new genetic background (pox1∆) in which TG synthesis is altered was identified.The kinetic paramters (Km and Vmax) of Dga1, the DG esterifying enzyme in S.cerevisiae were determined and it was shown that the enzyme displays cooperative kinetics for oleoyl-CoA. The enzyme activity was not regulated by AMP, ADP, ATP, NAD+, NADH, acetyl-CoA and malonyl-CoA, the levels of which are sensitive to energy status of cell. No coordinated regulation in triglyceride synthesis and glycogen synthesis was seen in energy rich conditions. 154 2. A novel AGPAT/ LPLAT, distinct from the known AGPAT or LPCAT families was identified in Saccharomyces cerevisiae. Molecular characterization of the novel AGPAT has completed the characterization of almost all of the enzymes in the triglyceride synthetic pathway. 3. Human LPCAT3, a member of MBOAT family was identified by its sequence similarity to LPT1. 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Platelet Activating Factor 176 APPENDIX 2: MISCELLANEOUS EXPERIMENTS 1. Effect of pox1∆, tgl3∆tgl4∆tgl5∆ on glycogen levels Rationale: The pox1∆ shows 75% more incorporation of [3H] oleate in triglyceride (TG) compared to the normal strain. The tgl3∆tgl4∆tgl5∆ was also chosen because Tgl3, Tgl4 and Tgl5 are TG lipases hence absence of the lipases leads to accumulation of TG in yeast cell. Cells store energy in form of TG and glycogen and if there is coordinated regulation between these two energy reserves the glycogen synthesis will be down regulated when TG accumulates in yeast cell. 1 7 Glycogen (mg/10 cells) 1.2 0.8 0.6 0.4 0.2 0 1.4 2 2.6 O.D. 660 nm 3 3.6 Normal tgl3tgl4tgl5 pox1 Figure A1. Glycogen levels in pox1, tgl3∆tgl4∆tgl5∆ yeast strains. Yeast strains were grown in YPD to an O.D.660 of 1.4 to 3.6. The cells were lysed, boiled, treated with amyloglucosidase and glucose oxidase in order to determine the amount of glycogen present. n=1 177 Result: Although the expected trend was seen in pox1∆ strain at O.D. 2.6-3.6 but glycogen levels remain unchanged in tgl3∆tgl4∆tgl5∆ yeast strain for most part of the growth curve. At O.D. 3.6 20% increase in triple deletion was seen which was contrary to what we hypothesized. The experiment was not pursued due to ade2 mutation in pox1∆ strain which caused the strain to grow faster in comparison to other yeast strains. 2. Effect of cerulenin, fatty acid synthase inhibitor on glycogen levels Rationale: Cerulenin specifically inhibits fatty acid synthesis in yeast. This would decrease TG synthesis and would increase glycogen synthesis in case these energy reserves are coordinately regulated. Glycogen (mg/10 7 cells) 1.4 1.2 1 0.8 0.6 0.4 0.2 0 With Cerulenin 0 hr 0.5 hr 1 hr. 3 hrs. Without Cerulenin Figure A2. Effect of cerulenin on glycogen levels in wild type yeast strain Yeast strains were grown in YPD to log phase. The cells were lysed, boiled, treated with amyloglucosidase and glucose oxidase in order to determine the amount of glycogen present. n=1 178 Experimental Set up: Wild type strain was grown to O.D. 3.6. 40 ml of the culture was split into 8 tubes with 5 ml in each tube. 5μl of cerulenin was added to 4 tubes. The cells were harvested at 0 hour, 0.5 hour, 1 hour and 3 hours after the addition of cerulenin. In parallel cells were harvested from culture without cerulenin. Result: No difference in glycogen levels were seen in wild type yeast strain treated with cerulenin in comparison to untreated yeast strain. 3. Effect of inositol and choline on TG synthesis at different growth phases Rationale: Addition of water soluble phospholipid precursors (inositol and choline or both) would affect phospholipid synthetic enzymes (reviewed in chapter 1) and hence [3H]oleate incorporation in TG (% of total lipids) TG synthesis. 40 30 20 10 0 0.6 1.6 O.D. 660 nm 3 Control 75 micromolar Inositol 1mM Choline Inositol and Choline Figure A3. Effect of inositol and choline on TG synthesis in wild type yeast strain at different growth phases, n=1 Experimental Set up: Wild type yeast strain was grown to different growth phases (O.D. 0.6 to 3) in synthetic complete media with 50µM inositol, 1 mM choline or both to log phase and labeled with [3H] oleate. 179 Result: At O.D. 0.6 we have seen earlier that inositol alone or with choline shows decrease in TG synthesis. However this result is not reproducible. At O.D. 1.6 the addition of inositol or choline or both does not have a pronounced effect. At O.D. 3 the addition of inositol does not show any effect on TG synthesis while addition of inositol and choline decreases TG synthesis. According to literature addition of choline to inositol enhances its effects. 180