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A 519 OULU 2008 U N I V E R S I T Y O F O U L U P. O. B . 7 5 0 0 F I - 9 0 0 1 4 U N I V E R S I T Y O F O U L U F I N L A N D U N I V E R S I TAT I S S E R I E S SCIENTIAE RERUM NATURALIUM Professor Mikko Siponen HUMANIORA University Lecturer Elise Kärkkäinen TECHNICA Professor Hannu Heusala MEDICA Professor Olli Vuolteenaho SCIENTIAE RERUM SOCIALIUM ACTA UN NIIVVEERRSSIITTAT ATIISS O OU ULLU UEEN NSSIISS U Zhijun Chen E D I T O R S Zhijun Chen A B C D E F G O U L U E N S I S ACTA A C TA A 519 CHARACTERIZATION OF THE 2-ENOYL THIOESTER REDUCTASE OF MITOCHONDRIAL FATTY ACID SYNTHESIS TYPE II IN MAMMALS Senior Researcher Eila Estola SCRIPTA ACADEMICA Information officer Tiina Pistokoski OECONOMICA University Lecturer Seppo Eriksson EDITOR IN CHIEF Professor Olli Vuolteenaho PUBLICATIONS EDITOR Publications Editor Kirsti Nurkkala ISBN 978-951-42-8979-8 (Paperback) ISBN 978-951-42-8980-4 (PDF) ISSN 0355-3191 (Print) ISSN 1796-220X (Online) FACULTY OF SCIENCE, DEPARTMENT OF BIOCHEMISTRY, UNIVERSITY OF OULU; BIOCENTER OULU, UNIVERSITY OF OULU A SCIENTIAE RERUM RERUM SCIENTIAE NATURALIUM NATURALIUM ACTA UNIVERSITATIS OULUENSIS A Scientiae Rerum Naturalium 519 ZHIJUN CHEN CHARACTERIZATION OF THE 2-ENOYL THIOESTER REDUCTASE OF MITOCHONDRIAL FATTY ACID SYNTHESIS TYPE II IN MAMMALS Academic dissertation to be presented, with the assent of the Faculty of Science of the University of Oulu, for public defence in Auditorium GO101, Linnanmaa, on December 4th, 2008, at 12 noon O U L U N Y L I O P I S TO, O U L U 2 0 0 8 Copyright © 2008 Acta Univ. Oul. A 519, 2008 Supervised by Professor Kalervo Hiltunen Reviewed by Docent Peter Mattjus Docent Sami Väisänen ISBN 978-951-42-8979-8 (Paperback) ISBN 978-951-42-8980-4 (PDF) http://herkules.oulu.fi/isbn9789514289804/ ISSN 0355-3191 (Printed) ISSN 1796-220X (Online) http://herkules.oulu.fi/issn03553191/ Cover design Raimo Ahonen OULU UNIVERSITY PRESS OULU 2008 Chen, Zhijun, Characterization of the 2-enoyl thioester reductase of mitochondrial fatty acid synthesis type II in mammals Faculty of Science, Department of Biochemistry, University of Oulu, P.O.Box 3000, FI-90014 University of Oulu, Finland; Biocenter Oulu, University of Oulu, P.O. Box 5000, FI-90014 University of Oulu, Finland Acta Univ. Oul. A 519, 2008 Oulu, Finland Abstract A data base search using the amino acid sequence of Saccharomyces cerevisiae Etr1p, the last enzyme of mitochondrial fatty acid synthesis type II (FAS II), revealed a highly similar human protein, NRBF-1. Expression of NRBF-1 in a yeast etr1? strain rescued its respiratory deficiency. NRBF-1 resides in mitochondria in cultured HeLa cells. The recombinant NRBF-1 is enzymatically active, reducing 2E-enoyl-CoAs to acyl-CoAs in an NADPH-dependent manner. Altogether, our data showed that NRBF-1 is a mitochondrial 2-enoyl-CoA reductase/2-enoyl thioester reductase (MECR/ETR1), the human functional counterpart of yeast Etr1p. In addition, MECR was also isolated from bovine heart. It turns out that mammals contain a mitochondrial FAS II pathway, in addition to cytoplasmic FAS I. To investigate the functional mechanism of MECR/ETR1 at the molecular level, the protein was crystallized and the crystal structure determined. The apo-structure of MECR/ETR1 contains two sulfates in the nucleotide binding site and the domain arrangement resembles the NADPHcontaining holo-structure of yeast Etr1p. The predicted mode of NADPH-binding and kinetic data suggest that Tyr94 and Trp311 play critical roles in catalysis. A pocket was found in the structure extending away from the catalytic site that can accommodate fatty acyl chains up to 16 carbons. An acyl carrier protein (ACP) binding site was also suggested. To study the physiological function of mouse Mecr, two lines of transgenic mice overexpressing Mecr were generated. The Mecr transgenic mice developed cardiac and mitochondrial abnormalities. The phenotyping was carried out using echocardiography, heart perfusion, histology, and endurance testing. Our results suggest Mecr plays a role in mitochondrial and heart function. Therefore, inappropriate expression of the genes of FAS II may result in the development of cardiomyopathy. Keywords: biochemistry, lipid, MECR/ETR1, mitochondria, structural enzymology, transgenic mice Acknowledgements The work has been carried out at Biocenter Oulu and Department of Biochemistry, University of Oulu, during the years 2001–2008. I would like to express my deepest gratitude to my supervisor Professor Kalervo Hiltunen for his excellent guidance and constant support during this study. He shared with me his hand-on experiences from protein purification from animal tissues to communication skills. He has also provided numerous opportunities for me to learn different methods and to use multiple techniques to tackle biological questions. I have also learned from him to be optimistic and persistent. I am grateful to Professor Rik Wierenga for helping me to understand structural enzymology and for guidance in the structure related projects. I would like to thank Professor Yari Ylänne for his invaluable comments during and after evaluations by the thesis committee, influencing not only my projects but also my career. I wish to thank Professor Seppo Vainio for his kind advice and numerous great ideas concerning my mouse projects as well as potential future topics. I also wish to express my special thanks to Professor Raili Myllylä for her encouragement and her very constructive suggestions. I wish to thank Professor Lloyd Ruddock for sharing with me skills in biochemistry and tips in molecular biology and cell biology. My special thanks also to Dr. Ilkka Miinalainen and Dr. Antti Haapalainen for their excellent work during collaboration through this study. I am very grateful to Dr. Sami Väisänen and Dr. Peter Mattjus for acting as reviewers of this thesis and providing constructive comments and Dr. Deborah Kaska for the revision of the language. I wish to warmly acknowledge Dr. Raija Soininen in the Biocenter Oulu mouse facility, Professor Ilmo Hassinen in the department of medical biochemistry, Professor Heikki Roskoho in department of pharmacology and toxicology, Professor Petri Kovanen in the Wihuri Research Institute, and Professor Walter Koch in Thomas Jefferson University for their important guidance. I would like to acknowledge Dr. Hanna Leskinen, Dr. Raija Sormunen, and Dr. Pasi Tavi for teaching me how to study the cardiac phenotype of transgenic mice. I wish to thank Dr. Alexander Kastaniotis for sharing with me the essence of yeast genetics and kind suggestions in writing. I would like to thank Dr. Hanna-Marja Voipio in the Laboratory Animal Centre University of Oulu, Professor Timo Nevalainen in the National Animal Center Kuopio University for teaching me how to handle animals. I wish to thank Professor Aner Gurvitz at the Medical University of Vienna for important comments on scientific 5 writing. I wish to thank Dr. Erkki Liimatta, Dr. Regina Pudas, and Satyan Sharma, Professor Vasily Antonenkov, Dr. Tuomo Glumoff, Dr. Pekka Kilpeläinen, Dr. Ulrich Bergmann, Dr. Päivi Pirila, Dr. Steffen Ohlmeier, Dr. Sakari Kellokumpu, Dr. Aki Manninen, Dr. André Juffer and Dr. Apaja Pirjo for their help and suggestions. I wish to thank all the current and former members of the KH group, as well as colleagues in Oulu University: Professor Yongmei Qin, Petri Lackman, Dr. Mari Ylianttila, Eija Selkälä, Aare Rokka, François Pujol, Dr. Tiina Kotti, Dr. Juha Torkko, Samuli Kursu, Dr. Kaija Autio, Dr. Kristian Koski, Tatu Haataja, Miia Vapola, Dr. Silke Grunau, Maija Malin, Ville Ratas, Leena Ollitervo, Marika Kamps, Virpi Hannas, Johanna Moilanen, Katri Näppä, Fumi Suomi, Antonina Shvetsova, Antti Isomursu, Dr. Kalle Savolainen, Dr. Prasenjit Bhaumik, Dr. Jukka Taskinen, Dr. Tiila-Riikka Kiema, Dr. Ari-pekka Kvist, Kyösti Keränen, Jaakko Keskitalo, Pia Askonen, Anneli Kaattari, Tuula Koret, Anne Vainionpää, Jonna Ranta, Sanna Kauppinen, Eija Nissinen, Tanja Sankala, Sanna Haapalainen, Tarja Huhta, Vivii Majava, Antti Salo, Parasad Kasaragod, Mirva Peltoniemi, Van Dat Nguyen, Heli Alanen, Marco Casteleijn, Ilkka Pietilä, and many others. I also wish to thank Chinese friends in Biocenter Oulu: Chunguang, Weidong, Hongmin, Jindong, Yanfeng, Shaobing, and Lijun for their help. I wish to acknowledge my M.Sc. mentors Professor Yi Li, Professor Zhangliang Chen, Dr. Xiaotian Ming and Professor Changfa Xu and Professor Lanxian Wang at Peking University for their important guidance. I am deeply grateful to my father Weimiao Chen, my mother Judan Jin for their great love and great support. I wish also to express the gratitude to my father-in-law Professor Desheng Wang, and mother-in-law Xiuzheng Hu for their enormous support. My warmest thanks belong to my dear wife Hui Wang, and my dear children, Zhuotong, Zhuokun. The thesis could not have been possible without their great love, great understanding and great support. This work was financially supported by grants from the Sigrid Jusélius Foundation, the Academy of Finland, the NordForsk under the Nordic Centres of Excellence programme in Food, Nutrition and Health, Project (070010) MitoHealth”, CIMO and scholarships from the Finnish Cultural Foundation, and the Oulu University Scholarship Foundation. Oulu, 2008 6 Zhijun Chen Abbreviations ACC ACP ACPS AMPK ATP CoA CPT Cr CrP DAPI DTP EF ETR FAO FAS GFP HSD HTD IEM IVS KAS KAR kDa LVEDD LVESD LVFS MCAT MCD mDa MDR MECR MFE-2 αMHC MRF MT-I acetyl-CoA carboxylase acyl carrier protein holo-ACP synthase 5'-adenosine monophosphate-activated protein kinase adenosine triphosphate coenzyme A carnitine palmitoyltransferse creatine creatine phosphate 4', 6-diamidino-2'-phenylindole dihydrochloride dicarboxylate transport protein ejection fraction enoyl thioester reductase fatty acid oxidation fatty acid synthesis green fluorescent protein hydroxysteroid dehydrogenase 3R-hydroxyacyl-ACP dehydratase immunoelectron microscopy interventricular septum β-ketoacyl synthase β-ketoacyl reductase kilo Dalton left ventricular end-diastolic left ventricular end-systolic LV fractional shorting malonyl-CoA:ACP transacylase (malonyltransferase) malonyl-CoA decarboxylase mega Dalton medium-chain dehydrogenase/reductase mitochondrial 2-enoyl-CoA reductase multifunctional enzyme type 2 alpha myosin heavy chain mitochondrial respiratory function metallothionein-I 7 MTF-I NAD(P) NR NRBF-1 PBS PCR PCC PDC PPT PW RT-PCR SDR SDS-PAGE TCA TEM TG tg wt 8 metal-regulatory factor-1 nicotinamide adenine dinucleotide (phosphate) nuclear receptor nuclear receptor binding factor 1 phosphate buffered saline polymerase chain reaction propionyl-CoA carboxylase pyruvate dehydrogenase complex phosphopantetheine:protein transferase posterior wall reverse template-PCR short-chain dehydrogenase/reductase sodium dodecyl sulfate polyacrylamide gel electrophoresis tricarboxylic acid transmission electron microscopy triacylglycerol transgenic wild type List of original articles This thesis is based on the following articles, which are referred to in the text by their Roman numerals: I Miinalainen IJ*, Chen ZJ, Torkko JM, Pirilä PL, Sormunen RT, Bergmann U, Qin YM & Hiltunen JK (2003) Characterization of 2-enoyl thioester reductase from mammals – an ortholog of Ybr026p/Mrf1’p of the yeast mitochondrial FAS type II. J Biol Chem 278: 20154–20161. II Chen ZJ, Pudas R, Sharma S, Smart O, Juffer A, Hiltunen JK, Wierenga RK & Haapalainen AM (2008) Structure and enzymology of 2-enoyl thioester reductase of human FAS II: New insights into substrate binding and catalysis. J Mol Biol 379: 830–844. III Chen ZJ, Leskinen H, Liimatta E, Sormunen RT, Miinalainen IJ, Hassinen IE & Hiltunen JK (2008) Overexpression of Mecr, a gene of mitochondrial FAS II in heart leads to cardiac dysfunction in mouse. Manuscript. *Publication I has been used as part of the dissertation of Ilkka Miinalainen (ISBN 9514282442). 9 10 Contents Abstract Acknowledgements 5 Abbreviations 7 List of original articles 9 Contents 11 1 Introduction 15 2 Review of the literature 17 2.1 Polyunsaturated fatty acids (PUFA), branched-chain fatty acids, and trans fatty acids ................................................................................ 17 2.2 The transcriptional regulation of lipid metabolism in mammals............. 18 2.2.1 Retinoid X receptors (RXRs) ....................................................... 19 2.2.2 SREBP.......................................................................................... 19 2.2.3 Carbohydrate responsive element-binding protein (ChREBP)..................................................................................... 19 2.2.4 X-box binding protein-1 (XBP1).................................................. 20 2.2.5 Peroxisome proliferator-activated receptors (PPARs) .................. 20 2.2.6 Peroxisome proliferator-activated receptor γ coactivator (PGC) ........................................................................................... 21 2.2.7 RIP140.......................................................................................... 21 2.2.8 Other regulators in lipid metabolism ............................................ 22 2.3 The organelles in mammalian cells......................................................... 22 2.3.1 The single membrane organelles .................................................. 22 2.3.2 The double membrane organelles................................................. 23 2.3.3 Mitochondria ................................................................................ 23 2.4 The components of mitochondrial FAS II............................................... 25 2.4.1 Acyl carrier protein....................................................................... 25 2.4.2 Phosphopantetheine:protein transferase (PPTase) ........................ 25 2.4.3 Malonyl-CoA:ACP transacylase (malonyltransferase; MCAT) ......................................................................................... 26 2.4.4 β-Ketoacyl synthase (KAS) .......................................................... 27 2.4.5 β-Ketoacyl reductase (KAR) ........................................................ 27 2.4.6 3R-Hydroxyacyl-ACP dehydratases (HTD)................................. 28 2.4.7 2-Enoyl thioester reductase (ETR1) ............................................. 28 2.4.8 The FAS II components of Caenorhabditis elegans...................... 29 2.5 Where is malonyl-CoA generated in mammalian mitochondria? ........... 30 11 2.5.1 Acetyl-CoA carboxylase 2 (ACC2) .............................................. 31 2.5.2 Propionyl-CoA carboxylase (PCC)............................................... 32 2.5.3 Putative malonyl-CoA synthetase................................................. 32 2.5.4 Putative malonyl-CoA:ACP transacylase ..................................... 34 2.6 Structural aspects of FAS II components ................................................ 34 2.6.1 Short chain dehydrogenase/reductase (SDR) ............................... 34 2.6.2 Medium-chain dehydrogenase/reductase (MDR) ......................... 36 2.6.3 Interactions of FAS II enzymes and ACP ..................................... 37 2.6.4 Cofactor specificity....................................................................... 37 2.6.5 Substrate binding pocket .............................................................. 38 2.7 Myocardial metabolism and cardiomyopathies....................................... 38 2.7.1 Mitochondrial energy metabolism and heart failure..................... 38 2.7.2 Energy metabolism in ischemic cardiomyopathy ......................... 39 2.7.3 Energy metabolism in dilated cardiomyopathy and hypertrophic cardiomyopathy....................................................... 39 2.7.4 Energy metabolism in diabetic cardiomyopathy and other cardiomyopathies.......................................................................... 40 3 Outlines of the present study 41 4 Materials and methods 43 4.1 Northern blot analysis (I) ........................................................................ 43 4.2 Cloning and mutagenesis (I, II, III)......................................................... 43 4.3 Protein expression and purification (I, II) ............................................... 43 4.4 Enzymatic assay and determination of kinetic parameters for MECR/ETR1 and its mutated forms (I, II).............................................. 44 4.5 Reaction product analysis (I)................................................................... 44 4.6 Fluorescence microscopy (I) ................................................................... 44 4.7 TEM analysis of ybr026c∆ cells expressing MECR (I) .......................... 45 4.8 Crystallization of MECR/ETR1 (II)........................................................ 45 4.9 Data collection, structure determination and refinement (II) .................. 45 4.10 Modeling of NADPH and fatty acyl thioester in the active site of MECR/ETR1 (II) .................................................................................... 46 4.11 Animal care (III) ..................................................................................... 46 4.12 Generation of metallothionein-I (MT-I)-Mecr overexpressing mice (III) ................................................................................................. 46 4.13 Southern blot genotyping (III)................................................................. 46 4.14 Reverse template PCR and real time PCR (III)....................................... 47 4.15 Histological staining (III) ........................................................................ 47 12 4.16 Echocardiographic analysis (III) ............................................................. 47 4.17 Heart perfusion (III) ................................................................................ 47 4.18 Metabolite assays (III) ............................................................................ 48 4.19 Exercise performance test (III)................................................................ 48 4.20 Isolation of heart mitochondria (I, III) .................................................... 48 4.21 Statistical analysis (III) ........................................................................... 48 5 Results 51 5.1 Biochemical properties of MECR/ETR1 ................................................ 51 5.1.1 Yeast etr1 mutant complementation ............................................. 51 5.1.2 Protein purification....................................................................... 51 5.1.3 End product analysis..................................................................... 51 5.1.4 Subcellular localization ................................................................ 52 5.1.5 Tissue distribution ........................................................................ 52 5.2 Structural enzymology of MECR/ETR1 ................................................. 52 5.2.1 The overall structure..................................................................... 52 5.2.2 Substrate binding pocket .............................................................. 53 5.2.3 Binding of ACP ............................................................................ 53 5.2.4 Kinetics......................................................................................... 53 5.3 Mecr transgenic mice .............................................................................. 54 5.3.1 Transgenic mice............................................................................ 54 5.3.2 Generation of Mecr oxerexpressing mice..................................... 55 5.3.3 Echocardiography......................................................................... 56 5.3.4 Heart perfusion ............................................................................. 56 5.3.5 Exercise endurance test ................................................................ 56 6 Discussion 57 6.1 2-Enoyl thioester reductases (ETRs)....................................................... 57 6.2 Lipoic acid and other products................................................................ 58 6.3 CoA and ACP .......................................................................................... 59 6.4 FAS II enzymes exist at low levels in mammalian mitochondria ........... 60 6.5 FAS II and its physiological function...................................................... 61 7 Conclusions 63 References 65 Original articles 83 13 14 1 Introduction In living cells, two distinct fatty acid synthesis systems have been revealed so far, namely, fatty acid synthesis types I (FAS I) and II (FAS II) (Rock & Cronan 1996). In bacteria and in the plastids (chloroplasts) of plants, FAS II serves as the major route for generating cellular fatty acids with reactions catalyzed by a series of discrete enzymes (White et al. 2005). The bacterial FAS II system produces the β-hydroxy fatty acyl groups for lipid A of lipopolysaccharides, intermediates used in vitamin (biotin, lipoic acid, etc) synthesis, fatty acyl moieties used for protein modifications and acyl groups for the synthesis of cellular triacylglycerol (TG) and phospholipids (Rock & Cronan 1996). Progress has been made recently in the development of antibacterials using bacterial FAS II proteins as targets (Heath & Rock 2004). In contrast, in the cells of fungi, mammals, certain mycobacteria, and Corynebacterium ammoniagenes, the vast majority of endogenously derived fatty acids are synthesized in the cytosol through a large polyfunctional enzyme (FAS I). This enzyme is heterododecameric (α6β6) in fungi and homodimeric (α2) in mammals, and in which each of the reactions is catalyzed by distinct domains in the enzyme (Tsukamoto et al. 1983, Smith 1994, Rock & Cronan 1996, Schweizer & Hofmann 2004). An additional biosynthetic process, also conserved in mammals, is the recently identified mitochondrial fatty acid synthesis (Torkko et al. 2001, Joshi et al. 2003, Zhang et al. 2003a, Kastaniotis et al. 2004, Cronan et al. 2005, Zhang et al. 2005, Autio et al. 2007). In this system, the required reactions are catalyzed by separate enzymes and, therefore, the system resembles the well-characterized bacterial FAS II (White et al. 2005). The endoplasmic reticulum (ER) of eukaryotes also contains a fatty acid chain elongation system, producing longchain and very long-chain fatty acids (Cinti et al. 1992, Das et al. 2000, Moon & Horton 2003). The last enzyme of mammalian FAS II is represented by mitochondrial enoylCoA reductase/2-enoyl thioester reductase (MECR/ETR1; EC 1.3.1.38). This enzyme catalyzes the reaction (Torkko et al. 2001): 2E-enoyl-(ACP/CoA) + NADPH + H+ → acyl-(ACP/CoA) + NADP+ The present thesis is concerned with the characterization of this enzyme in humans and mice with reference to fatty acid biosynthesis. To gain more insight 15 into the function of mammalian mitochondrial FAS II, biochemical, structural, and genetic characterizations of the mammalian MECR/ETR1 were undertaken. 16 2 Review of the literature 2.1 Polyunsaturated fatty acids (PUFA), branched-chain fatty acids, and trans fatty acids Fatty acids (FAs) are among the key components of lipids and over 1000 different FAs have been found in nature to date. Mammalian tissues contain some 250 different FAs, 20–30 of which are major FAs. Notably, the concentration of free FAs in human blood serum is on a micromolar level (Jungling & Kammermeier 1988). Most FAs are straight-chain compounds, the simplest of which is saturated FA. However, many straight-chain FAs contain double bonds, ranging from 1 to 6 or more, with a free methyl between two double bonds. The double bonds have significant influence on the physical properties (e.g., the melting point) of FAs. Moreover, the positions of double bonds in FAs also affect the biochemical properties of FAs (Michaud et al. 2002). When counting from the ω-end of FAs, the first double bond which mammals can introduce into the existing covalent chain is in the ∆9 position. Therefore, mammals are completely dependent on dietary intake of ∆3 and ∆6 (poly)-unsaturated FAs, which, however, can be remodified in the body to longer chain FAs with a higher degree of unsaturation through a retroconversion pathway. The retroconversion requires participation of enzymes in both the ER and peroxisomes using unsaturated FAs such as 18:3n-3 as substrates. For instance, docosahexaenoic acid (22:6n-3), which is critical for maintaining the function of the nervous system (Salem et al. 2001), arises either exogenously or from eicosapentaenoic acid (EPA, C20:5n-3) through chain elongation and desaturation in the ER followed by chain contraction in peroxisomal β-oxidation. Moreover, PUFAs can suppress glycolytic and lipogenic genes by inhibiting the translocation of the carbohydrate responsive elementbinding protein (ChREBP) from the cytosol to the nucleus (Dentin et al. 2005). Notably, diet affects the n-3 PUFA/n-6 PUFA ratio of cell membranes. Additionally, exogenous PUFA supply regulates endogenous monounsaturated fatty acid (MUFA) production through modulation of ∆9-desaturase activity by interfering with the function of sterol regulatory element binding proteins (SREBP) (Tabor et al. 1999). Another group of FAs is branched-chain FAs which can be found from microbial and animal lipids. In mammals, branched-chain FAs may act as bound 17 components of hair fibers for maintaining hair hydrophobicity (Jones & Rivett 1997) or esterified to cholesterol, binding to certain enzymes of protein biosynthesis (Tuhackova & Hradec 1985, Hradec & Dufek 1994). The branchedchain FAs can be associated also with brain function and act as an inhibitor of the growth of cancer (Yang et al. 2000, Kniazeva et al. 2004). Trans fatty acids (TFAs) are special unsaturated straight-chain compounds. Most of the trans fatty acids (TFAs) of tissues originate from processed vegetable fats or from milk. TFAs sometimes will raise LDL and lower HDL in serum and also raise the risk of cardiovascular disease by an unclear mechanism (Gebauer et al. 2007). 2.2 The transcriptional regulation of lipid metabolism in mammals Cellular lipid homeostasis is tightly regulated at both the transcriptional and posttranscriptional levels and is connected closely to glucose metabolism. Many metabolic genes are transcribed at a basic level but can be activated or repressed in response to changes in the nutritional or physiological state (White et al. 2008). Many transcription factors in lipid metabolism belong to the superfamily of nuclear receptors (NRs), which consist of an N-terminal domain, a DNA binding domain, a hinge region, a ligand binding domain, and a C-terminal domain (Chawla et al. 2001) (Fig. 1). Most of the known NRs are mainly regulated through ligand interaction. Upon ligand binding, these NRs are dissociated from heat shock proteins, translocated into the nucleus and bind to hormone response elements. The ligands can be either endogenous or exogenous, and can act as agonists or antagonists. NRs also recruit co-activators to the promoter or enhancer regions of target genes. In contrast to this, the apo form of NRs recruits corepressors and thus inhibits gene expression. Notably, in the nucleus, regardless of ligand binding, NRs form heterodimers or homodimers. Furthermore, NRs together with co-activators/repressors can activate histone (de)acetylases, and other chromatin remodeling complexes, hence change chromatin structure to promote or inhibit RNA polymerase II access to the target promoter. DNA binding NH2 Fig. 1. Schematic structure of a typical NR. 18 ligand binding COOH 2.2.1 Retinoid X receptors (RXRs) Specifically, retinoid X receptors (RXRs) can regulate liver X receptors (LXRs), peroxisome proliferators-activated receptors (PPARs), and farnesoid X receptors (FXR). LXR forms heterodimers with RXR where both subunits can be stimulated by ligands (Willy et al. 1995, Svensson et al. 2003). LXR regulates sterol adsorption in the intestine, cholesterol efflux from peripheral cells, cholesterol transport in the bloodstream, and cholesterol secretion into bile. LXR also enhances lipogenesis and increases serum triacylglycerol (TG) levels. Bile acids can activate FXR, and thus regulate many metabolic pathways (Lee et al. 2006). 2.2.2 SREBP Another group of transcription factors is the basic helix-loop-helix leucine zipper proteins (bHLH/LZ), such as, SREBP and upstream stimulating factors (USF) (Goldstein et al. 2006). SREBPs are master regulators of lipid homeostasis through binding to the sterol regulatory element (SRE) in the promoter of target genes. Fourteen enzymes in the cholesterol synthesis pathway are more or less regulated by SREBP (mainly -1a and -2). Via interaction with the SREBP complex, both cholesterol and its oxidized forms can inhibit the transport of the SREBP cleavage-activating protein/SREBP (SCAP/SREBP) to the Golgi (Nohturfft et al. 1999). Specifically, SREBP-1a regulates both cholesterol and fatty acid synthesis (FAS); SREBP-1c regulates hepatic and adipose tissue FAS and glucose metabolism; moreover, SREBP-2 responds to cellular cholesterol levels (Shimano et al. 1997, Joseph et al. 2002). 2.2.3 Carbohydrate responsive element-binding protein (ChREBP) The ChREBP is another bHLH/LZ transcription factor (Yamashita et al. 2001), which can directly regulate glycolytic and lipogenic gene expression through binding to their promoters sequences (Ishii et al. 2004). Interestingly, like many other transcription factors, ChREBP forms heterodimers with the Max-like protein X (Mlx) to generate a functional complex (Stoeckman et al. 2004, Ma et al. 2005, Ma et al. 2006). The roles of ChREBP/Mlx in different tissues remain to be identified (Postic et al. 2007). 19 2.2.4 X-box binding protein-1 (XBP1) XBP1 is a key regulator of the unfolded protein response in the ER in mammals (Ron & Walter 2007). Upon ER stress in mammals, an unconventional spliced version of XBP1 mRNA induced by the proximal sensor and endoribonuclease IRE1α generates an active transcription factor, binding to the promoter of ER chaperone genes (Lee et al. 2003, Shaffer et al. 2004, Acosta-Alvear et al. 2007). Most recently, XBP1 was identified as a novel transcription factor governing hepatic lipogenesis by activating the transcription of key lipogenic genes in the liver, and can be induced by a high-carbohydrate diet. XBP1 liver conditional knock-out mice have a significant decrease in de novo hepatic lipid synthesis, which further leads to decreases in serum TG, cholesterol, and free FAs (Lee et al. 2008). 2.2.5 Peroxisome proliferator-activated receptors (PPARs) Like many other NRs, PPARs can respond to dietary lipids and form heterodimers with the RXRs and act as transcription factors. PPARs are modulated through ligand/coactivator/corepressor binding, which can stimulate or inhibit their function (Yu & Reddy 2007). There are three PPAR isoforms. PPARα is highly expressed in liver, heart and kidney and acts as a mediator of FAs metabolism by upregulating enzymes of fatty acid oxidation (FAO), or downregulating enzymes of FAS such as acetyl-CoA carboxylase 1 (ACC1) (Lee et al. 2002). For instance, when ACC1 is down-regulated, the concentration of malonyl-CoA decreases, resulting in the activation of carnitine palmitoyltransferase I (CPT-1), and consequently more FAs are transported into mitochondria and used for FAO. PPARγ is highly expressed in adipose tissue and the immune system. Expression of PPARγ in fibroblasts and muscle cells leads to adipocyte differentiation (Tontonoz et al. 1994, Hu et al. 1995), which may be achieved through interaction with PPARγ/RXR, C/EBPs and ADD-1/SREBP-1 and result in the expression of adipocyte-specific genes such as aP2, leptin, and lipoprotein lipase (Auwerx 1999, Rosen & Spiegelman 2000). PPARγ knockout mice show a phenotype of early embryonic lethality, and the embryos are devoid of adipose tissue (Barak et al. 1999), suggesting that PPARγ plays a role in the development of multiple tissues. The ligands of PPARγ have not yet been identified conclusively, but the reported ligands include the thiazolidinediones (TZDs) (Kletzien et al. 1992), PUFAs (Xu et al. 1999), eicosanoids (Yu et al. 1995), and 20 many others. PPARγ is associated with both lipid and glucose metabolism and is regarded as a potential drug target for the treatment of multiple diseases including diabetes, cancer and inflammation (Houseknecht et al. 2002). PPARδ is ubiquitously expressed, but not as well-characterized as the other two PPARs. Wang and coauthors generated PPARδ transgenic mice using an aP-2 promoter which is adipocyte-specific (Wang et al. 2004). Notably, this form of PPARδ contains a VP-tag that converts the PPARδ into an active transcription factor even when a ligand is not present. The PPARδ transgenic mice show a lean phenotype and a depletion of serum lipids due to activation of a set of target genes required for FAO and energy dissipation in brown and white adipose tissues. In contrast to this, PPARδ knockout mice show reduced energy uncoupling and are prone to obesity (Wang et al. 2003). These transgenic studies suggest that PPARδ serves as a key regulator of fat burning. 2.2.6 Peroxisome proliferator-activated receptor γ coactivator (PGC) PGC-1s can enhance the transcriptional activity of transcription factors like PPARs by directing protein-protein interaction (Finck & Kelly 2007). Specifically, PGC-1α is a coactivator of PPARγ and other NRs (Wu et al. 1999) and a regulator of mitochondrial biogenesis, thermogenesis, gluconeogenesis, and other catabolic processes (Wu et al. 1999). In particular, PGC-1α is capable of promoting cardiac mitochondrial biogenesis (Lehman et al. 2000). PGC-1α functions as an adaptor or scaffold to recruit other coactivators/proteins that remodel chromatin or modify DNA polymerase II (Finck & Kelly 2007). Recently, the new PGC-1 family members, PGC-1β and a PGC-related coactivator have also been identified (Andersson & Scarpulla 2001, Lin et al. 2002) and they are actively expressed in high-capacity mitochondria tissues such as heart, muscle and brown adipose tissue (Puigserver et al. 1998, Lin et al. 2002). 2.2.7 RIP140 RIP140 is actively expressed mainly in metabolic tissues such as adipose tissue, liver, and muscle (Leonardsson et al. 2004). RIP140 is a ligand-dependent corepressor of energy expenditure such as FAO, oxidative phosphorylation, mitochondrial biogenesis, and glucose uptake (Feige & Auwerx 2007), and functions as a scaffold protein via interaction with NRs such as ERRα (Powelka et al. 2006). Furthermore, through interaction with a dehydrogenase CtBp, 21 RIP140 can respond to changes in levels of NADH/NAD+ (Zhang et al. 2002), by which it senses the cellular redox status. Interestingly, RIP140 itself is heavily modulated by post-translational modifications such as phosphorylation (Gupta et al. 2005), methylation (Mostaqul Huq et al. 2006), and acetylation (Vo et al. 2001). RIP140 shares many target genes with PGC-1, but may have opposing effects (White et al. 2008). RIP140 is a negative regulator of cellular respiration and mitochondrial biogenesis (Powelka et al. 2006). 2.2.8 Other regulators in lipid metabolism Many other factors are also regulators of lipid metabolism. For instance, steroid/thyroid hormones and vitamins A/D can act as ligands of NRs. Other intracellular regulators are also actively involved in lipid metabolism, such as SRC-2/3, CBP, Med1, etc. However, these factors will not be addressed here. 2.3 The organelles in mammalian cells Mammalian cells contain cytoplasm and organelles, separated by either a single or double membrane. 2.3.1 The single membrane organelles The majority of organelles within mammalian cells contain a single membrane. These include the ER, peroxisomes, endosomes, lysosomes, and Golgi complex. The ER is a network of interconnected membranes. The major lipid fraction of the ER membrane is formed by glycerophospholipids (van Meer 2005). The main function of the ER is the synthesis of lipids including FAs, TG, cholesterol, and phospholipids, membrane proteins, and secreted proteins. For example, long chain and very long chain FAs are produced by the ER elongation system. In mammals, the ER is also the place where ceramide is synthesized (Hoetzl et al. 2007). Further, one of the protein quality-control systems also resides in the ER, thus dysfunction of this system leads to the accumulation of misfolded proteins in the lumen of the ER, and ultimately contributes to vascular, cardiac and other diseases (Glembotski 2007). Peroxisomes are roughly spherical organelles, 0.2–1.0 μm in diameter. One of their main functions is the β-oxidation of FAs (Schrader & Yoon 2007), yielding acetyl groups which then are transported into the mitochondria, while the energy 22 released during this process is partially converted into heat. Peroxisomes can also oxidize (poly)unsaturated enoyl-CoA esters which requires the participation of several auxiliary enzymes such as ∆3, ∆2-enoyl-CoA isomerase, 2,4-dienoyl-CoA reductase, and ∆3,5 ∆2,4-dienoyl-CoA isomerase (Hiltunen et al. 1996). Endosomes contain over 10% sphingolipids and 30–40% cholesterol (van Meer 2005). Notably, late endosomes contain locally produced unique lipids, such as lysobisphosphatidic acid (Matsuo et al. 2004). Lysosomes are the “digestive” machinery within mammalian cells. They digest different kinds of molecules taken up by cells via endocytosis, phagocytosis, and autophagy. Dysfunction of enzymes in lysosomes can result in some 30 different inherited lysosomal storage disorders in humans (von Figura & Hasilik 1986). The Golgi complex picks up the protein-loaded vesicles that bud from the ER. The Golgi complex is responsible for the processing and sorting of membrane components and secreted proteins. Moreover, the ER and Golgi complex are organelles which are responsible for glycosylation, sulfation, and phosphorylation of secretory and membrane-bound proteins, proteoglycans, and lipids (Hirschberg et al. 1998). For instance, ceramide is converted to sphingomyelin and complex glycosphingolipids on the inner, non-cytosolic surface of Golgi cisterns (Hoetzl et al. 2007). The trans Golgi network (TGN) has a lipid composition similar to that of the endosomes (van Meer 2005). 2.3.2 The double membrane organelles The nucleus is the largest organelle in mammalian cells, and the composition of proteins within the outer and inner membrane is different. The two membranes are fused at nuclear pores. The nucleus functions as a container of genomic DNA, and is the site of replication of the genetic material, and the synthesis of RNAs (Lodish H. 2003). Further organelles within mammalian cells containing double membranes are mitochondria (see below). 2.3.3 Mitochondria In many mammalian cells, mitochondria occupy up to 25% of the volume of the cytosol. Cardiac mitochondria range from 0.1 to 20 μm in size (Fig. 2). Again, like in the nucleus, the composition of the two membranes differs significantly. 23 The outer mitochondrial membrane consists of approximately 50% proteins including porins which make it highly permeable, whereas the inner mitochondrial membrane contains around 80% proteins which confer several types of activities: they carry out the oxidation reactions of the respiratory chain as well as ATP synthesis, and function as metabolite transport and proteinimporting. Additionally, the inner membrane is rich in unusual phospholipids, the cardiolipins. Furthermore, the surface area of the inner membrane is much larger than that of the outer membrane due to a number of extensively folded cristae (Lodish H. 2003). In contrast to the outer membrane, there is no porin in the inner mitochondrial membrane. This inner membrane is highly impermeable to ions and/or molecules which cross the barrier through transporters. As a consequence, a membrane potential across the inner membrane is generated. Furthermore, mitochondria may play a role in the cellular homeostasis of Ca2+, similar to the role of the ER (Romagnoli et al. 2007). Mitochondria are also the major location of iron-sulfur clusters biosynthesis (Scheffler 2001). Fig. 2. Ultrastructure of mouse heart mitochondria visualized by transmission electron microscopy (TEM), bar represents 5 μm, arrow points to cristae (picture from Dr. Raija Sormunen). The mitochondrial matrix contains hundreds of enzymes, ribosomes, tRNA, and mitochondrial DNA. Many of the enzymes are involved in intermediary and energy metabolism such as oxidation/synthesis of FAs, the tricarboxylic acid (TCA) cycle, pyruvate oxidation, urea cycle, ketone body formation, and steroid metabolism. Human mitochondrial DNA contains 37 genes, including 24 tRNA or rRNA genes as well as 13 polypeptide-encoding genes (Anderson et al. 1981). 24 Proteomic studies revealed that about half of the mammalian mitochondrial proteins are ubiquitously expressed, whereas the rest are tissue specific (Mootha et al. 2003). 2.4 The components of mitochondrial FAS II Mitochondrial FAS II in mammals consists of multiple components including several enzymes and cofactors (Fig. 3). 2.4.1 Acyl carrier protein The acyl carrier protein (ACP) in mammals is a small acidic protein. ACP transfers acyl groups via a thioester linkage to the sulfhydryl group of 4’-phosphopantetheine (Jordan & Cronan 1997). ACP from mammals was isolated first as a component of mitochondrial complex I (Runswick et al. 1991), but later a soluble ACP pool was also described (Cronan et al. 2005). ACP is needed for assembly of the peripheral arm and the membrane arm of complex I (Schulte 2001). Two-dimensional NMR revealed that Escherichia coli ACP has a defined but flexible tertiary structure dominated by three major parallel α-helices (Flaman et al. 2001). Even though the binding of FAs has little influence on ACP conformation, it stabilizes ACP against denaturation at alkaline pH (Rock & Cronan 1979, Rock et al. 1981, Cronan 1982, Jones et al. 1987, Flaman et al. 2001). ACP is highly expressed in heart and skeletal muscle (Triepels et al. 1999). Unlike the mitochondrial ACP, which functions as a structurally independent component, an ACP-like domain is a part of cytoplasmic FAS I (Stuible et al. 1998). 2.4.2 Phosphopantetheine:protein transferase (PPTase) Activation of ACP is mediated by the holo-ACP synthase (ACPS) or phosphopantetheine:protein transferase (PPTase) (Xu et al. 2001). 4’-Phosphopantetheine is transferred from CoA to the hydroxyl group of a specific serine on apo-ACP in a Mg2+-dependent reaction (Flugel et al. 2000). The addition of 4’-phosphopantetheine to ACP does not result in any observable difference in the folding of the apo and holo forms of the protein (Xu et al. 2001). The 4’-phosphopantetheine contains dual functions: (i) it activates acyl groups by 25 thioester linkage, and (ii) acts as a flexible arm that allows translocation of intermediates between catalytic sites of FAS pathways (Stuible et al. 1998). In bacteria, ACP and the ACP-like domain of multiple-domain polypeptides can be modified by five different PPTases, namely, the enterobactin synthetase of E. coli by EntD (Lambalot et al. 1996, Gehring et al. 1997), the surfactin synthetase of Bacillus subtilis by Sfp (Lambalot et al. 1996), and o195 (Lambalot et al. 1996), type II FAS of E. coli by ACPS (Lambalot & Walsh 1995), and type I FAS enzymes of Brevibacterium ammoniagenes by Ppt1p (Stuible et al. 1997). Notably, the substrate for o195 has not yet been determined. Three classes of PPTases in yeast, namely, Fas2, Ppt2, and Lys5, are responsible for FAS I (Schuller et al. 1992), mitochondrial FAS II (Stuible et al. 1998), or lysine biosynthesis (Borell & Bhattacharjee 1988), respectively. The Ppt2p (YPL148C) has a low molecular mass and is responsible specifically for phosphopantetheinylation of the mitochondrial apo-ACP. It is still unclear whether the event takes place in the mitochondria or in the cytosol prior to entry of the ACP into the mitochondria. Ppt2 contains a relatively weak mitochondrial target signal and unprocessed Ppt2 is enzymatically active (Stuible et al. 1998). It would be worthwhile to design and implement experiments that precisely localize Ppt2p in yeast cells. In mammals, a single PPTase may be responsible for posttranslational modification of carrier proteins in three different metabolic pathways: (i) the cytosolic FAS I, (ii) the mitochondrial FAS II, and (iii) the lysine degradative pathway (Joshi et al. 2003). Moreover, the human PPTase localizes predominantly in the cytoplasm, suggesting that posttranslational modification of carrier proteins takes place in the cytosol, regardless of their ultimate destination within the cell. Therefore, the mitochondrial-apo-ACP may be phosphopantetheinylated prior to importation into the mitochondria (Joshi et al. 2003). 2.4.3 Malonyl-CoA:ACP transacylase (malonyltransferase; MCAT) The yeast gene encoding MCAT has been identified by its homology to the bacterial enzyme (Schneider et al. 1997). Disruption of MCAT led to a respiratory deficient phenotype. Again, there has not been report regarding the biochemical properties of the yeast Mcat1p. However, the kinetic characterization of its human ortholog was done using a recombinant protein expressed in an insect cell system (Zhang et al. 2003a). 26 2.4.4 β-Ketoacyl synthase (KAS/CEM1) A gene CEM1 encoding β-ketoacyl synthase (KAS) (condensing enzyme) was identified from Saccharomyces cerevisiae in 1993. Inactivation of CEM1 leads to a respiratory deficient phenotype (Harington et al. 1993). However, the lipid composition of the CEM1 mutant strain showed no major changes, indicating that FAS II in yeast may produce only specialized lipids instead of synthesizing the bulk of the FAs (Harington et al. 1993). Moreover, the biochemical properties of the yeast Cem1p have not been characterized. Importantly, Zhang et al cloned and purified the human ortholog of Cem1p and studied its kinetics. Their results showed that the human CEM/KAS uses acetyl-ACP as a primer, and accepts C2C14 substrates with a maximal catalytic efficiency with C6 and C10 substrates (Zhang et al. 2005). Surprisingly, mitochondrial KAS from Arabidopsis thaliana utilizes neither acetyl-CoA nor acetyl-ACP as a primer, but prefers malonyl-ACP. Interestingly, this A. thaliana mitochondrial CEM1 showed a bimodal substrate preference with maxima at C-8 and C14-16 (Yasuno et al. 2004). 2.4.5 β-Ketoacyl reductase (KAR) E. coli β-ketoacyl reductase (FabG) is a tetramer with a monomeric molecular mass of 25.5 kDa. The N-terminally His-tagged recombinant FabG was purified and showed KAR activity in an NADPH-dependent manner (Heath & Rock 1995). The apo and liganded crystal structures of FabG have been solved. The NADPH binding causes a conformational change, which leads to reorganization of the active site and promotes binding of the ACP substrate. This property is not characteristic of most of the other known short-chain dehydrogenase/reductase (SDR) enzymes (Price et al. 2001), although, like many other SDRs, FabG contains a conserved triad of Ser138-Tyr151-Lys155 residues in its catalytic site (Price et al. 2001). Another KAR ortholog, NodG in Rhizobium meliloti is a component of a FAS II-like pathway that is critical for acylation of a “host-specific” signaling molecule – oligosaccharide (Hopwood & Sherman 1990), which restricts the infectivity of the bacterium (Lerouge et al. 1990, Spaink et al. 1991, Slabas et al. 1992). S. cerevisiae Oar1 (Oar1p) is homologous to FabG and has been characterized based on the analysis of the respiratory deficient phenotype of its mutant strain, whereas the biochemical properties of Oar1p have not yet been determined (Schneider et al. 1997). However, neither a homology-based cloning 27 nor a large scale yeast genetic complemention screening using human cDNA libraries was successfully in cloning of the human counterpart of Oar1p, HsKAR. The leaves of antisense Kar treated plants were curled and slightly smaller at maturity than controls (O'Hara et al. 2000). The plant KAR can also catalyze reduction of acetoacetyl-CoA in a NADPH-dependent manner. The crystal structure of Brassica napus KAR has been determined at 2.3 Å resolution in a binary complex with an NADP+ cofactor. Each subunit of the homotetramer has a dinucleotide-binding fold. Like other SDRs, the BnKAR has a classical conserved triad of Ser154-Tyr167-Lys171 residues in the catalytic site and conformation changes are induced by substrate binding. The structure is similar to another SDR enzyme, the bacterial enoyl-ACP reductase (FabI) (Fisher et al. 2000). 2.4.6 3R-Hydroxyacyl-ACP dehydratases (HTD) The first eukaryotic 3R-hydroxyacyl-ACP dehydratase (HTD) identified using genetic tools was the yeast mitochondrial HTD. This protein is a component of the mitochondrial FAS II in yeast (Kastaniotis et al. 2004). In human mitochondria, a bicistronic gene RPP14 can be transcribed into a single mRNA but it encodes two proteins, namely, ribonuclease P (RNase P) and 3-hydroxyacyl thioester hydratase (HTD2) (Autio et al. 2007). The data suggested a link between mitochondrial FAS and RNA processing in vertebrates. Interestingly, Mycobacterium tuberculosis contains a gene cluster encompassing HadA, HadB, and HadC. These proteins can form two types of heterodimers, HadA–HadB and HadB–HadC, in which HadB apparently is the catalytic subunit (Sacco et al. 2007). The heterodimers (HadA–HadB and HadB–HadC) act as 3R-hydroxyacylACP dehydratases and show distinct chain length specificity in FAS II. The interaction between proteins was essential for stabilizing HadB and potentially confers the specificity of the catalysis. 2.4.7 2-Enoyl thioester reductase (MECR/ETR1) In E. coli, the last step of FAS II is catalyzed by a SDR enzyme, FabI (Heath & Rock 1995, Bergler et al. 1996, Heath et al. 1999). In yeast, the corresponding reaction of the mitochondrial FAS II is catalyzed by Etr1p which is a MDR enzyme (Torkko et al. 2001, Airenne et al. 2003). Interestingly, Candida tropicalis contains two isoforms of Etr1p, which are encoded by two different genes but differ by only three amino acids (Torkko et al. 2003). As the last 28 enzyme of FAS II in mammals, MECR/ETR1 was identified on the basis of similarities to the fungal protein, Etr1p. It is also known as nuclear receptor binding factor 1, Nrbf1’. MECR/ETR1 was localized on 1p36.1-p35.1. The murine protein is encoded by Mecr, at CCDS18715 on chromosome 4. 2.4.8 The FAS II components of Caenorhabditis elegans Y56A3A.19 encodes a mitochondrial ACP in C. elegans. In the larval stage, it is mainly expressed in the intestine, whereas in the adult worm, it is predominantly expressed in the pharynx (McKay et al. 2003, Hunt-Newbury et al. 2007). The RNAi phenotype of Y56A3A.19 includes early larval arrest/lethality (Simmer et al. 2003, Sonnichsen et al. 2005) and slow growth (Kamath et al. 2003). F37C12.3 potentially encodes another mitochondrial ACP in C. elegans. The RNAi phenotype of F37C12.3 includes slow growth (Kamath et al. 2003, Simmer et al. 2003), embryonic lethality (Maeda et al. 2001, Kamath et al. 2003), larval arrest/lethality (Maeda et al. 2001, Rual et al. 2004, Sonnichsen et al. 2005), as well as small (Maeda et al. 2001) and sterile progeny (Maeda et al. 2001). C50D2.9, F10G8.9, dhs-11/25, and Y48A6B.9 potentially encode malonylCoA:ACP transacylase, β-ketoacyl ACP synthase, β-ketoacyl reductase, and MECR/ETR1 of mitochondrial FAS, respectively. However, no phenotype has been observed from high throughput knock-down studies (Fraser et al. 2000, Gonczy et al. 2000, Kamath et al. 2003, Rual et al. 2004, Sonnichsen et al. 2005). Interestingly, W09H1.5 potentially encodes an additional mitochondrial 2-enoyl thioester reductase. In the larval stage, it is expressed in the pharynx, intestine, body wall muscle, nerve ring, head neurons, tail neurons, as well as in unidentified cells in the tail; in the adult stage, it is detectable in pharynx, intestine, vulval muscle, body wall muscle, nerve ring; head neurons, tail neurons, and similarly in unidentified cells in the tail (McKay et al. 2003, Hunt-Newbury et al. 2007). RNAi of W09H1.5 showed an anti-aging phenotype, namely, an extended life span (Hamilton et al. 2005). 29 Fig. 3. Mitochondrial FAS spiral in mammals. A question mark means either the enzymes responsible for the steps have not been identified or the sources/products are unknown. Notably, C8 is one product of FAS II used for the synthesis of lipoic acid, however, another possible product(s) of this pathway is unknown. β-Ketoacyl thioester reductase (KAR) is also missing. The source of mitochondrial malonylACP/CoA is unknown. 2.5 Where is malonyl-CoA generated in mammalian mitochondria? Analysis of the enzymatic properties of FAS II enzymes suggests that mitochondrial FAS can synthesize fatty acyl groups in a malonyl-CoA/ACPdependent manner (Zhang et al. 2003a, Yasuno et al. 2004, Zhang et al. 2005). However, it is not yet known where the malonyl-CoA/ACP in mitochondria is 30 generated, and whether malonyl-CoA/ACP is necessary for de novo mitochondrial FAS (Fig. 3). In mammals, malonyl-CoA is used for the synthesis of FAs within the mitochondria, peroxisomes, and cytosol. Malonyl-CoA is not only an intermediate in the de novo FA biosynthesis or elongation, but also an important cellular signal through inhibition of carnitine palmitoyltransferase 1 (CPT1), which regulates mitochondrial β-oxidation (Saggerson 2008). Several enzymes are described below that are putative candidates for synthesizing malonyl-CoA/ACP within mitochondria in mammals. 2.5.1 Acetyl-CoA carboxylase 2 (ACC2) Biotin-dependent carboxylases include pyruvate carboxylase, propionyl-CoA carboxylase (PCC), β-methylcrotonyl-CoA carboxylase and acetyl-CoA carboxylases (ACCs) (Cole et al. 1994, Schrick & Lingrel 2001). Among them, ACC1 is the catalytic enzyme responsible for the first reaction of FAS, producing malonyl-CoA and is tightly regulated by phosphorylation-de-phosphorylation and cellular metabolites (Kim et al. 1989). Overproduction of ACC activity results in a significant increase in the rate of FAS in E. coli (Davis et al. 2000), indicating its important regulatory role in FAS. The carnitine palmitoyltransferases (CPTs)/malonyl-CoA/ACCs system in mammals is also involved in the sensing of mitochondrial FAs via CPTs and ACCs, which can be further regulated by insulinstimulated protein kinase (Alam & Saggerson 1998, Heesom et al. 1998). Mammalian ACC1 is a 265 kDa protein highly expressed in white adipose tissue and lactating mammary gland (Bianchi et al. 1990, Widmer et al. 1996), whereas ACC2 in mammals is a 280 kDa protein enriched in heart, skeletal muscle and brown adipose tissue (Abe et al. 1998). ACC2 knockout mice show a higher FAO rate and reduced fat content (Abu-Elheiga et al. 2003). ACC2, a mitochondria associated protein, is 75% identical in sequence to the cytosolic ACC1 except for the N-terminal region, where ACC2 has an extra ~200 amino acids (Ha et al. 1996). Interestingly, the N-terminus of ACC2 acts as a mitochondrial targeting signal and is immediately followed by a 100-aa hydrophilic stretch. It was suggested that the fuction of the leader sequence is solely to anchor ACC2 in the membrane of mitochondria, with the bulk of the polypeptide facing the cytosol (Abu-Elheiga et al. 2000). Moreover, it is also possible that ACC2 is associated with the contact sites of the outer and inner mitochondrial membranes. This association can be readily reversed, which may be the reason why previous attempts to establish a subcellular localization of ACC2 have been difficult, as the 31 isolation procedure should not include a harsh treatment. Notably, native ACC2 can be purified using avidin-sepharose chromatography from a heart nonmitochondria-homogenate (after mitochondria were removed from the homogenate) (Thampy 1989). This evidence also indicates that ACC2 resides primarily in the in cytosol rather than in the mitochondrial matrix or membrane. This localization argues against the role of ACC2 as a generator of intramitochondrial malonyl-CoA. 2.5.2 Propionyl-CoA carboxylase (PCC) PCCs from Myxococcus xanthus, plant and Yarrowia lipolytica can also accept the acetyl-CoA substrate with a Km of 0.25 mM for M. xanthus, 0.11–0.26 mM for plant and 0.26 mM for Y. lipolytica, respectively (Mishina et al. 1976, Nikolau & Hawke 1984, Kimura et al. 1998). Notably, MxPCC has a much higher affinity toward propionyl-CoA (Km = 0.032 mM) (Kimura et al. 1998). The relative carboxylation rates of MxPCC were 100% for propionyl-CoA and 14.3% for acetyl-CoA (Kimura et al. 1998). In mammals, PCC is abundant in the heart and also accepts acetyl-CoA as a substrate in addition to propionyl-CoA (Scholte et al. 1986). In certain species, a single enzyme with dual-substrate specificity catalyzes the carboxylation of both acetyl- and propionyl-CoA. The dcm-1 mutant cells of M. xanthus with a deficiency in propionyl-CoA carboxylase activity had decreased levels of long-chain FAs (C16 to C18) compared to wild-type cells (Kimura et al. 1997). PCCs from Mycobacterium smegmatis, Streptomyces erythreus, and Turbatrix aceti also catalyze the carboxylation of acetyl-CoA to yield malonyl-CoA (Meyer & Meyer 1978, Henrikson & Allen 1979, Hunaiti & Kolattukudy 1982). 2.5.3 Putative malonyl-CoA synthetase Even though the possible source of malonyl-CoA in mammalian mitochondria might be carboxylation of acetyl-CoA by ACCs or PCC, there exists another mechanism that might produce malonyl-CoA or malonyl-ACP in mammalian mitochondria. In Rhizobium leguminosarum, a mat operon consisting of three consecutive genes (matABC) has been described. The mat encodes enzymes involved in the uptake and conversion of malonate to acetyl-CoA through malonyl-CoA (An et al. 32 2002). The matB encodes malonyl-CoA synthetase. A human protein (LOC197322) related to MatB in amino acid sequence (identity 31%), containing a putative mitochondrial signal, can be found from databases. The human homolog might function as a malonyl-CoA synthetase like MatB in R. leguminosarum. The matA encodes a malonyl-CoA decarboxylase that is homologous to the human malonyl-CoA decarboxylase (MCD) (identity 30%). Although some studies suggested that human MCD is mainly localized in peroxisomes and cytoplasm (Sacksteder KA, 1999), the rat MCD apparently contains a mitochondrial targeting sequence in the N-terminus and a peroxisomal (Ser-LysLeu) targeting signal in the C-terminus (Voilley et al. 1999). This is consistent with the notion that the rat MCD is mainly localized in the mitochondrial matrix but a small fraction of the activity is extra-mitochondrial (Hamilton & Saggerson 2000, Kerner & Hoppel 2002). Moreover, further studies suggested that human MCD is localized in both mitochondria and peroxisomes (FitzPatrick et al. 1999). Recent studies showed that the rat MCD is present in three subcellular compartments: cytosol, mitochondria and peroxisomes (Joly et al. 2005). The activities of MCD as well as ACCs are important determinants of the cytosolic concentration of malonyl-CoA. The cytosolic localization of the MCD suggests that MCD by its effect on the concentration of malonyl-CoA can function as a regulator of fat oxidation and lipid partitioning. MatC encodes a putative dicarboxylate carrier protein in R. leguminosarum, however, no homologous protein can be found in the human genome of either human or that of any other organism so far, except for Xanthomonas axonopodis, Xanthomonas campestris, Streptomyces coelicolor, and Streptomyces hygroscopicus. The next question concerns the origin of malonate in mitochondria. One study on the origin of free malonate in the brain suggests that it could be the result of the following sequential reactions: acetyl-CoA → malonyl-CoA → malonate. The first reaction could be catalyzed by the cytosolic ACC, and the final step might occur by transfer of the CoA-group from malonyl-CoA to succinate and/or acetoacetate (Riley et al. 1991), although the enzyme catalyzing this reaction is still unknown (Succinyl-CoA:acetoacetate-CoA transferase is supposed to be a mitochondrial enzyme). Malonate can enter the mitochondrial matrix via the dicarboxylate transport protein (DTP). DTP is located in the inner mitochondrial membrane and is capable of taking up malonate, malate and succinate. The 33 mitochondrial DTPs from yeast, plants and animals show high similarity (Kaplan & Pedersen 1985, Vivekananda et al. 1988, Kakhniashvili et al. 1997). In addition, Wada and Baldet suggested that pea mitochondria do not possess ACC (Wada et al. 1997). Instead, they take up and utilize malonate but not acetate or malonyl-CoA from outside the mitochondria (Wada et al. 1997). This indicates that mitochondria may contain a different system to produce malonyl-CoA compared to FAS in the cytosol and chloroplasts that uses ACC to generate malonyl-CoA (Sasaki et al. 1997). Recently, Stephens and coauthors described a FAS in vitro reconstitution system by incubating isolated mitochondria from Trypanosoma brucei with radiolabeled FAs and [14C]malonyl-CoA or [14C]malonic acid (Stephens et al. 2007). The data suggested cytosolic malonyl-CoA instead of malonate is the source of mitochondrial FAS in T. brucei, in contrast to the plant mitochondrial FAS system (Gueguen et al. 2000). 2.5.4 Putative malonyl-CoA:ACP transacylase The initiating steps of FAS II in Plasmodium falciparum are catalyzed by pfACP, pfMCAT, and pfKASIII (Prigge et al. 2003). MCAT, also called FabD, produces malonyl-ACP from malonyl-CoA and ACP. β-Ketoacyl-ACP synthase III (KASIII, also called FabH) catalyzes the condensation of malonyl-ACP and acyl-CoA (typically acetyl-CoA) forming a 3-ketoacyl-ACP product (Prigge et al. 2003). In 2002, we picked up a human MCAT candidate (CAB94798) with an identity of 31% via database searching. This human protein is targeted to mitochondria and contains a GXSXG and some other consensus sequences common to various acyl hydrolases. In 2003, Zhang and coworkers showed that the human recombinant protein indeed contains malonyltransferase activity in vitro (Zhang et al. 2003a) 2.6 Structural aspects of FAS II components 2.6.1 Short chain dehydrogenase/reductase (SDR) SDR enzymes were originally found in lower life forms, and subsequent studies indicated that a number of human proteins also belong to the SDR family. So far around 3000 members of this group have been defined including 63 human 34 proteins (Jornvall et al. 2003). The “short” refers to the length of the polypeptide chain of SDRs rather than the substrates. SDRs catalyze NAD(P)(H)-dependent oxidation/reductions. Although they are divergent in terms of substrate specificities and sequence identities, SDRs possess highly similar α/β folding patterns with a central β-sheet, typical of the Rossmann-fold (Oppermann et al. 2003). SDRs can be further divided into two families, namely, “classical” (~250 residues) and “extended” (~350 residues) (Jornvall et al. 1995). SDRs contain a common GXXXGXG pattern in the N-terminal region, which is characteristic of NAD(P)(H) dependent enzymes, while the C-terminus constitutes the substrate binding site (Kallberg et al. 2002). Recent studies proposed a catalytic tetrad of Asn, Ser, Tyr, Lys residues, probably forming a proton relay system (Oppermann et al. 2003). Two examples of SDRs are described below. 3S-Hydroxyacyl-CoA dehydrogenases (HAD) or 17β-hydroxysteroid dehydrogenase type 10 (17βHSD10) 3S-Hydroxyacyl-CoA dehydrogenase type I (HAD) of the FAO system in mammals consists of two identical monomers with a molecular mass of ~35 kDa. Another mitochondrial enzyme that contains similar activity is the 3S-hydroxyacyl-CoA dehydrogenase type II (HADII), which is a homotetramer with a molecular mass of ~27 kDa for each subunit (Kobayashi et al. 1996). Both proteins show broad tissue distribution. The HADII gene is localized in chromosome Xp11.2, where a multigene domain escapes X inactivation (He et al. 1998, Miller & Willard 1998). Unlike type I and most other FAO matrix enzymes, HADII does not contain a cleavable mitochondrial signal (Furuta et al. 1997). This is similar to another mitochondrial enzyme, 3-ketoacyl-CoA thiolase (Arakawa et al. 1990). As the first mitochondrial enzyme found to catalyze the oxidoreduction of steroid hormones, HADII was also called 17β-hydroxysteroid dehydrogenase type 10 (17βHSD10). Nevertheless, the HADII/17βHSD10 is different from other 17βHSDs, in that it is unable to catalyze the reduction of either estrone or androstenedione by NAD(P)H (He et al. 1999). HADII shows broad substrate specificity, whereas its enzymatic activities are solely based on NAD(H), which can not be replaced by NADP(H). The ratio of the kcat of alcohol dehydrogenase, 17βHSD and 3S-HAD was 1:0.3:1028 (He et al. 2000b), suggesting it has a major role in FAO. It is noteworthy that 3α-adiol is the best steroid substrate for HADII, 35 which means HADII preferably acts in oxidization rather than reduction, regulating 5α-DHT levels in tissues (He et al. 2000a). However, there is no clear sequence similarity between HADII and other cytosolic NADP(H) dependent 3α-HADs. Interestingly, inactivation of scully, the Drosophila counterpart of HADII, leads to the presence of cytoplasmic lipid inclusions and scarce mitochondria in spermatocytes. Furthermore, photoreceptors of the scully mutant fly contain aberrant mitochondria and accumulations of membranous material (Torroja et al. 1998). Also significant is the fact that the amino acid sequence of HADII is identical to the endoplasmic reticulum amyloid β-peptide-binding protein (ERAB), which is associated with Alzheimer’s disease (He et al. 1998). A recent study shows that the reduced expression of HADII in human caused by a C→A mutation, leads to mental retardation, choreoathetosis, and abnormal behavior (Lenski et al. 2007). 17β-hydroxysteroid dehydrogenase type 4 (17βHSD4) or peroxisomal multifunctional enzyme type 2 (MFE-2) The peroxisomal multifunctional enzyme type 2 (MFE-2; EC1.1.1.62) catalyzes the second and third reactions of the peroxisomal spiral of FAO (Hiltunen et al. 1992). MFE-2 contains 3R-hydroxyacyl-CoA dehydrogenase activity as well as 2E-enoyl-CoA hydratase-2 activities. Mammalian MFE-2 shows broad substrate specificity. It is also called 17β-hydroxysteroid dehydrogenase type 4 (17βHSD4), oxidizing 17β-estradiol, 17β-diol, and 5-androstene-3β (Leenders et al. 1994, Adamski et al. 1995). Moreover, MFE-2 also oxidizes 24 hydroxylated intermediates in bile acid synthesis, and the 3-hydroxyl on 2-methyl branched chain fatty acyl-CoAs (Dieuaide-Noubhani et al. 1996, Novikov et al. 1997). 2.6.2 Medium-chain dehydrogenase/reductase (MDR) The MDR family has approximately 1000 members including 23 human proteins (Jornvall et al. 2003). The biological roles of the MDRs include participating in metabolic pathways, the defense system against toxic compounds, and regulatory functions (Jornvall et al. 2003). Like SDRs, MDRs also utilize NAD(P)(H), and some members have one or two zinc ions either at the catalytic or at another site-stabilizing the structure 36 (Nordling et al. 2002). There are two types of MDRs, with/without Zn2+ at the active site (Jornvall et al. 2003). MDRs can be further divided into eight families. The zinc-containing MDRs are as follows: alcohol dehydrogenase (ADH), cinnamyl alcohol dehydrogenase (CAD), polyol dehydrogenases (PDH), and yeast alcohol dehydrogenase (YADH). The non-zinc-containing MDRs are the following: acyl-CoA reductase (ACR), leukotriene B4 dehydrogenase (LTD), mitochondrial response proteins (MRF), and quinone oxidoreductases (QOR) (Nordling et al. 2002). Yeast Etr1p and human MECR/ETR1 belong to the mitochondrial respiratory function (MRF) family. MDR enzymes are more closely addressed in the attached papers and manuscript. 2.6.3 Interactions of FAS II enzymes and ACP FAS II enzymes in bacteria do not contain any sequence similarity that defines a common ACP binding site, but rather an electropositive/hydrophobic surface to interact with helix α2 of ACP. Notably, this special patch is a ubiquitous feature of bacterial FAS II enzymes (Zhang et al. 2003b). In the structure of FabG (the 3-ketoacyl-ACP reductase of FAS II), two conserved residues Arg129 and Arg172, which are located in the hydrophobic patch adjacent to the catalytic site play a role in the interaction with α2 of ACP (Zhang et al. 2003b). 2.6.4 Cofactor specificity Most NAD(P) dependent dehydrogenases contain a Rossmann fold for binding of NAD(P)H (Smiley et al. 1971). 17βHSD1 is a SDR member preferring NADP(H) over NAD(H). Mutagenesis replacements showed that Ser12 and Leu36 are important for selection of the cofactor (Huang et al. 2001). Replacement of Leu36 with aspartic acid altered the preference to NAD(H). Some SDRs show dual cofactor acceptance and therefore may use different coenzymes in different tissues/organelles (Kallberg et al. 2002). Moreover, bovine retinol dehydrogenase (RDH) prefers NAD(H), whereas RDH from rat is NADP(H) dependent (Simon et al. 1995, Peralba et al. 1999). Modeling experiments suggested that a polar residue Thr61 in the bovine protein and a corresponding basic residue Lys64 in the rat enzyme may be responsible for the different preferences (Tsigelny & Baker 1996). 37 The numbers of NAD(H) dependent and NADP(H) dependent SDR members are similar in plant, mouse, and human; however, in yeast, worm and fly, the majority of SDRs is NADP(H) dependent (Kallberg et al. 2002). 2.6.5 Substrate binding pocket Many enzymes contain a substrate binding pocket, which can be classified according to its shape, such as crevice-like, funnel-like and tunnel-like (Yang et al. 2002). The shape, size, and hydrophobicity of the pocket may determine the substrate preference (e.g., chain length) of enzymes. Mutagenesis can be applied to alter the chain length specificities of the enzymes and may result in engineered enzymes with new features (Cahoon et al. 1997, Klein et al. 1997, Whittle & Shanklin 2001, Schmitt et al. 2002, Stinnett et al. 2007). 2.7 Myocardial metabolism and cardiomyopathies Cardiomyopathy is the deterioration of the function of the myocardium, leading to a high risk of arrhythmia and/or sudden cardiac death. Cardiomyopathies can be categorized into two groups: extrinsic and intrinsic cardiomyopathies (Richardson et al. 1996). The most common cause of extrinsic cardiomyopathy is ischemia, which is a weakness in the heart muscle due to inadequate oxygen delivery to the myocardium. Moreover, many systemic diseases (e.g., diabetes) can lead to cardiomyopathy. Intrinsic cardiomyopathies have mainly two forms. In dilated cardiomyopathy, the heart (especially the left ventricle) is enlarged and the systolic function is reduced. In hypertrophic cardiomyopathies, the left ventricle wall thickens, which results in reduced blood flow and in heart dysfunction. Cardiomyopathies arise from different genetic and environmental conditions, and are an important contributor to heart failure. Mutations in a number of metabolic enzymes and contractile proteins can generate cardiomyopathies. In the case of dilated cardiomyopathy, the genetic causes are still poorly understood. 2.7.1 Mitochondrial energy metabolism and heart failure Mitochondria oxidize FAs and carbohydrates and generate the reducing equivalents, NADH and reduced flavin adenine dinucleotide (FADH2). These reducing equivalents transfer electrons into the respiratory chain, linked to the generation of ATP. The cardiac energy metabolism is elaborately coupled to ATP 38 production/hydrolysis, and any imbalance of this coupling could results in cardiac diseases. The ATP requirement of mouse heart is the highest among mouse organs, and ATP insufficiency is frequently associated with impairment of contractile function (Nascimben et al. 1996). For instance, studies on human patients and genemodified mice suggested causal links between derangements in mitochondrial energy metabolism and cardiac dysfunction (Finck & Kelly 2007). Thus, improving the efficiency of ATP production could have therapeutic benefit (Taha 2007). 2.7.2 Energy metabolism in ischemic cardiomyopathy Ischemic cardiomyopathy is associated with insufficiency of oxygen delivery to meet the energy demands of the heart during physical or psychological stress (Taha 2007). The outcome of this shortage of oxygen supply is an inhibition of respiratory function, forcing the heart to rely more heavily on anaerobic glycolysis (Stanley et al. 1997). Consequently, glucose uptake is stimulated and the ratios of mitochondrial acetyl-CoA:CoA and NADH:NAD+ are raised, inhibiting the pyruvate dehydrogenase complex (PDC). Therefore, pyruvate stays in the cytoplasm and is converted to lactate, leading to intracellular acidosis (Liu et al. 1996). A consequence of the intracellular acidosis is that the Na+/H+ exchanger, and the Na+-HCO3− transporter are activated, resulting in a rise in intracellular Na+ level. This leads to activation of the Na+/Ca2+ exchanger, leading to a rise in the intracellular [Ca2+], which may be detrimental to the cell. This finally is translated into myocardial ATP being used for the maintenance of intracellular Ca2+ homeostasis rather than for contraction (Taha 2007). Modulation of the balance between FAs and glucose metabolism has been a target of intensive research concerning the treatment of ischemic cardiomyopathy. 2.7.3 Energy metabolism in dilated cardiomyopathy and hypertrophic cardiomyopathy Dilated cardiomyopathy means that the heart becomes enlarged and weakened and the heart work output is decreased, consequently triggering a cascade of compensatory physiological responses. Patients show reduced respiratory function, higher levels of cardiac glucose uptake and a decrease in FA uptake. 39 In hypertrophic cardiomyopathy patients, mitochondrial respiratory function is reduced. This mimics the metabolic profile of the fetal heart, resulting in intracellular acidosis similar to ischemic cardiomyopathy. 5’-Adenosine monophosphate-activated protein kinase (AMPK) detects changes in the cellular energy status through monitoring the rise and fall in ratios of ATP:AMP, and CrP:Cr. AMPK can be activated by AMP or via phosphorylation by other kinases. The activated AMPK then activates ATP-generating processes and inhibits energy consuming processes (Hardie & Carling 1997). Mutation of AMPK may lead to the Wolff-Parkinson-White (WPW) syndrome (Blair et al. 2001). 2.7.4 Energy metabolism in diabetic cardiomyopathy and other cardiomyopathies A possible cause of diabetic cardiomyopathy is that insulin resistance leads to impaired glucose uptake and enhanced FAs uptake, making it the substrate of choice (Taha 2007). The increased level of FAs in myocytes results in activation of PPARα, facilitating FAO and inhibiting glucose oxidation. This shift in the “substrate preference” causes the oxidative capacity of the heart to be exceeded, potentially increasing the production of reactive oxygen species, and/or causing lipotoxicity. Restrictive cardiomyopathy is characterized by “stiffening” of the myocardium, which leads to impaired diastolic function (Richardson et al. 1996). The genetic causes of this disease have yet to be clarified. Arrhythmogenic right ventricular dysplasia is other type of cardiomyopathy caused by genetic mutations (dominant/recessive), leading to apoptotic loss of cardiac myocytes which are replaced by fatty or fibro-fatty tissue in the right ventricle (Corrado et al. 2000). 40 3 Outlines of the present study The purpose of this study was to identify and characterize a mammalian enoyl thioester reductase, MECR/ETR1. More specifically the aims were: 1. 2. 3. Characterization of the biochemical properties of MECR/ETR1, with focus on its existence and localization in mammalian tissues and organelles, and on its enzymatic activity. Investigation of the crystal structure of MECR/ETR1 and to provide insight into its substrate binding and catalytic mechanism. To shed a light on the physiological function of MECR/ETR1, through generation of Mecr overexpressing mice, and characterization of their phenotype. 41 42 4 Materials and methods The materials and methods have been described in more detail in the original articles. 4.1 Northern blot analysis (I) A human Multiple Tissue Northern BlotTM from various human tissues was hybridized with 32P-labeled MECR cDNA. Human β-actin cDNA was used as a control sample. Hybridized fragments were visualized by autoradiography at −70 °C for 24 h using X-OmatTM AR imaging film. 4.2 Cloning and mutagenesis (I, II, III) cDNAs were amplified from isolated RNA with the RobustRT-PCR kit, and the PCR products ligated into the Acceptor Vector. The DNA fragments were further subcloned into pBluescript SK(+) (pSK), pET3a, pET23, or pYE352 vectors. For making fusion constructs, the DNA fragment was digested and subcloned inframe with the 5' end of GFP/His tag cDNAs. The pET23::MECR construct was used as a template for generating mutant variants using a site-directed mutagenesis kit. 4.3 Protein expression and purification (I, II) The expression plasmids were transformed into BL21 (DE3) pLysS E. coli cells. After induction, the cells were washed with 1 × phosphate buffered saline (PBS) and stored at −20 ºC until used. Cells were resuspended, sonicated in breaking buffer and the disrupted cell suspension centrifuged. For non-tagged protein, the supernatant was applied to a Q-Sepharose column, followed by 2',5'-ADPSepharose 4B, Resource S and Superdex 200. For His-tagged proteins, the supernatant was loaded onto a Ni-NTA column. The column was washed first with 1x binding buffer and then with washing buffer. Bound proteins were eluted with a linear imidazole gradient. Fractions containing MECR/ETR1 or its mutant forms were pooled, concentrated and applied to Superdex 200. The peak fractions were combined, concentrated and stored at −70 ºC until used. For isolation of wt proteins from bovine tissues, the soluble extract of purified mitochondria was prepared using sonication, followed by ultracentrifugation. The supernatant was 43 dialyzed and applied to an ADP-Sepharose column. The fractions containing positive signals were pooled, dialyzed, and subjected to immunoisolation. A suspension of protein A-coated Dynabeads was washed and incubated with antiHsMECRAb. The antibody-loaded beads were washed and transferred to the dialyzed sample containing BtMECR. The beads were then separated, washed, and transferred to SDS sample buffer. The beads were removed after incubation, and the sample was acetylamidated. After electrophoresis, bands were excised, destained, and dried. The gel pieces were rehydrated with trypsin solution and incubated overnight. The digested peptides were recovered by extraction. Extracts were pooled, dried, and dissolved, and subjected to matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (MALDI-TOF MS). 4.4 Enzymatic assay and determination of kinetic parameters for MECR/ETR1 and its mutated forms (I, II) The kinetic constants were determined by monitoring the decrease of absorbance of NADPH at 340 nm minus 385 nm. The substrates from 2E-C4-CoA to 2E-C16CoA were used. In particular, 2E-octenoyl-CoA was chosen to determine the kinetic values of mutated variants. The kinetic data were transferred to Lineweaver-Burk plots using GraFit computer software. 4.5 Reaction product analysis (I) MECR/ETR1 was incubated in the presence of 2E-hexenoyl-CoA and NADPH. Aliquots were acidified, and samples from the aliquots were subjected to reversed phase chromatography. The column was eluted with an isocratic flow of 0.2 ml/min with buffers A (25 mM CH3COONH4, pH 5.5) and B (80% CH3CN and 25 mM CH3COONH4, pH 5.5) at an 83:17 ratio. Metabolites in the eluted peaks were identified by MALDI-TOF MS. 4.6 Fluorescence microscopy (I) Attached HeLa cells were grown on glass coverslips in Dulbecco's modified Eagle's media. Transfection of the HeLa cells was done using the FuGENE 6 reagent and the transfected cells were treated with a mitochondria-selective fluorescent dye and fixed with paraformaldehyde and permeabilized. Nuclear 44 DNA was counterstained with 1 µg/ml 4',6-diamidino-2'-phenylindole dihydrochloride (DAPI), followed by analysis with a fluorescence microscope. 4.7 TEM analysis of ybr026c∆ cells expressing MECR (I) S. cerevisiae ybr026c∆ cells overexpressing MECR or a truncated form of MECR were transferred from overnight cultures in synthetic medium and grown for 17 h. BJ1991 wild-type cells and ybr026c∆ cells were grown overnight and then transferred to oleic acid medium. The cells were fixed with 2.5% glutaraldehyde and immersed in 2% agar. Yeast cells in agar blocks were further post-fixed, dehydrated in acetone, and embedded in Embed 812. Thin sections of 80 nm were cut and examined using TEM. 4.8 Crystallization of MECR/ETR1 (II) The solution for crystallization contained NADP+ and CdCl2. Crystals were obtained at 295 K in three to four weeks by the hanging drop vapor diffusion method in 2 M ammonium sulfate and 100 mM Tris, pH 8.5. 4.9 Data collection, structure determination and refinement (II) X-ray diffraction data was collected in EMBL/DESY, Hamburg, Germany. The measurements were made on a single crystal in a nitrogen stream. The data were processed using the iMOSFLM, SCALA, CHAINSAW, DM of the CCP4 program package and PHASER. Initial phases were determined from molecular replacement solution obtained with the program PHASER using CtEtr1p as a model. The resulting model was subjected to simulated annealing with CNS followed by the refinement cycles with REFMAC. The quality of the maps improved considerably when the BUSTER-TNT software system was implemented into refinement steps with NCS restraints; this allowed further construction of some of the problematic loops with fragmented electron density. The final structure was analyzed with PROCHECK and WHATIF. 45 4.10 Modeling of NADPH and fatty acyl thioester in the active site of MECR/ETR1 (II) NADPH was modeled in the active site of MECR/ETR1. The complex was energy minimized using the steepest descent followed by conjugate gradient minimization procedures. The ligand was docked. The complexes were then subjected to 2 ns of molecular dynamics simulations to equilibrate the complexes. The MD simulations were carried out with the Gromacs 3.2.1 package using a ffG43a1 force field. The topology of the ligand was obtained from the PRODRG server. The LINCS algorithm was employed to constrain bonds. After energy minimizations, a short (10 ps) position restraining run for further equilibation and subsequently a full MD run were carried out. 4.11 Animal care (III) The animal experiments were carried out in the barrier and in the animal facility of the Department of Biochemistry, University of Oulu. All the experiments were performed in accordance with the Animal Care and Use Committee of Oulu University. 4.12 Generation of metallothionein-I (MT-I)-Mecr overexpressing mice (III) A cDNA of mouse Mecr with the poly A signal of Mecr was fused to a vector containing the mouse MT-1 promoter followed by a fragment of the untranslated region of the human ornithine decarboxylase (ODC) gene. The construct was linearized and microinjected into the pronuclei of fertilized eggs that were then transferred to C57Bl/6J recipient mothers. Transgenic founders and their progeny were identified by genomic PCR and/or southern blot. 4.13 Southern blot genotyping (III) Mouse genomic DNA was digested by BamHI. The probe (1 kb) was made by digestion of the construct with PvuII. The intensity of the 6kb band is proportional to the copy number of the transgene. 46 4.14 Reverse template PCR and real time PCR (III) The cDNAs were produced with a First Strand cDNA Synthesis Kit using total RNA isolated from tissues. Semiquantitative real time PCR was performed, GAPDH was used as an internal control for normalizing the gene expression. 4.15 Histological staining (III) The heart tissue samples were fixed and embedded in paraffin, and sections were stained with hematoxylin-eosin/TUNEL. The sections were examined using light microscopy. For TEM, tissue samples from mouse heart were fixed, in 1% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2, dehydrated in acetone and embedded Embed 812. Thin sections of 80 nm were cut and examined by TEM. 4.16 Echocardiographic analysis (III) The dimensions of the cardiac chambers and left ventricular function were assessed using transthoracic echocardiography. A short-axis view of the left ventricle at the level of the papillary muscles was obtained by using 2D imaging for the M-mode recording. Left ventricular end-systolic (LVESD) and enddiastolic (LVEDD) dimensions as well as the thickness of the interventricular septum (IVS) and posterior wall (PW) were measured from the M-mode tracings. LV fractional shortening (LVFS) and ejection fraction (EF) were estimated from the M-mode LV dimensions by applying the following formulas: LVFS (%) = [(LVEDD − LVESD)/LVEDD] × 100 and EF (%) = [(LVEDD3 − LVESD3)/LVEDD3] × 100. 4.17 Heart perfusion (III) Perfusion of isolated hearts was performed retrogradely with a Krebs-Henseleit perfusion medium followed by the Langendorff procedure (Chain et al. 1969). Left ventricular pressure was monitored through the ventricular wall. The pressure wave signal was fed to a Lab-PC+ data acquisition card and heart function parameters (heart rate, peak systolic pressure, diastolic pressure and peak 47 pressure development) were calculated on-line with custom designed software and stored on a computer at 4-s intervals. Coronary flow was measured during the experiments by using a drop counter with an analog frequency output. Oxygen consumption was calculated from the arteriovenous concentration difference multiplied by coronary flow. 4.18 Metabolite assays (III) The heart was quick-frozen with aluminum clamps precooled in liquid nitrogen, and stored in liquid N2 until processed. The frozen heart tissue was immersed in 8% (wt/vol) HClO4 in 40% (vol/vol) ethanol precooled to −20 °C, and homogenized immediately with an Ultra-Turrax homogenizer. After centrifugation, the supernatant was neutralized with 3.75 mol/L K2CO3 containing 5 mol/L triethanolamine hydrochloride. Creatine phosphate (CrP), creatine (Cr), ATP, ADP, AMP and inorganic phosphate levels were measured in the supernatant by enzymatic assays. 4.19 Exercise performance test (III) Mice were familiarized with running on a motorized running belt two days prior to the test. The treadmill was started at a low speed with low slope. The speed and inclination were raised gradually. Exercise was stopped when the mice were exhausted. Performance was assessed based on three parameters: the duration of the run, the distance run, and vertical work performance. 4.20 Isolation of heart mitochondria (I, III) Crude mitochondria were isolated from heart and were purified further by isopycnic density gradient centrifugation on a Percoll gradient. The gradient was unloaded from the bottom of the tube, and marker enzyme activities for mitochondria, microsomes, and cytosol were measured. 4.21 Statistical analysis (III) Values are expressed as mean ± standard deviation (SD) and analyzed by the Student’s t test. The homodynamic variables were analyzed with 2-way repeatedmeasures ANOVA. Echocardiographic measurements were analyzed using 48 normalized values with 1-way ANOVA followed by the Student-Newman-Keul post hoc test. A value of P < 0.05 was considered statistically significant. 49 50 5 Results 5.1 Biochemical properties of MECR/ETR1 5.1.1 Yeast etr1 mutant complementation Yeast etr1∆ strains display a petite phenotype and can not grow on nonfermentable carbon sources such as glycerol. Expression of full length MECR can rescue the phenotype, whereas expression of the truncated form, without the mitochondrial targeting signal, can not restore the respiratory function of the etr1∆ strains (Fig. 7 in I). Further, TEM shows that etr1∆ cells contain only rudimentary mitochondria, whereas MECR overexpressing cells contain enlarged mitochondria compared to the mitochondria from wild-type cells (Fig. 8 in I). 5.1.2 Protein purification The recombinant truncated forms of MECR/ETR1 and its bovine counterpart were purified to apparent homogeneity using a bacterial overexpression system and multiple types of columns (Fig. 3 in I). Further, truncated forms of MECR/ETR1 and their mutant variants with C-terminal histidine tags were also purified to apparent homogeneity using Ni-NTA and gel filtration columns. In addition, the wild-type protein was also isolated from bovine heart mitochondria using a 2’,5’-ADP-Sepharose affinity column and immunoprecipitation (Fig. 6 in I), expanding the list of mitochondrial FAS components found in mammals, even though they are apparently not abundant. 5.1.3 End product analysis MECR/ETR1 was incubated with 2E-hexenoyl-CoA and NADPH. At various time points, samples were taken from the reaction for HPLC analysis. The disappearance of 2E-hexenoyl-CoA was accompanied by the oxidation of NADPH with the appearance of a metabolite. MALDI-TOF/MS analysis showed that the accumulating metabolite (865.7 m/z) was 2 mass units larger than 2E−hexenoyl-CoA (863.7 m/z), consistent with the prediction that the product is hexanoyl-CoA (Fig. 4 in I). The result indicates that MECR/ETR1 is able to reduce 2E-enoyl-CoA to its saturated counterpart. 51 5.1.4 Subcellular localization Previously it was suggested that mammalian MECR/ETR1 is a nuclear localized protein (Masuda et al. 1998). To reinvestigate the subcellular localization of the protein, the full length cDNA of MECR/ETR1 carrying a cDNA encoding EGFP at the 3’end was expressed in HeLa cells. The mitochondria were stained with MitoTracker Red, and nuclear DNA was stained with DAPI. A punctuated pattern of green fluorescence was superimposed on the MitoTracker staining (Fig. 5A and D in I), indicating that the chimeric MECR/ETR1-EGFP was targeted into mitochondria. Notably, the cells expressing EGFP alone showed a diffuse uniform fluorescence pattern, which was not superimposed on the MitoTracker staining (Fig. 5E and H in I). 5.1.5 Tissue distribution The expression levels of MECR/ETR1 in different human tissues were analyzed by Northern blot. The signal of MECR/ETR1 (1.4 kb) was observed in multiple samples, namely, heart, brain, placenta, liver, skeletal muscle, kidney and pancreas. The mRNA of β-actin was used as a control. Importantly, the highest expression levels of MECR/ETR1 were observed in the high energy demanding tissues such as heart and skeletal muscle, suggesting a role for MECR/ETR1 in energy metabolism in these tissues (Fig. 2A in I). 5.2 Structural enzymology of MECR/ETR1 5.2.1 The overall structure The structure of MECR/ETR1 was refined at 2.41 Å resolution. Like most other MDR enzymes, the apo structure of MECR/ETR1 forms a dimer. Each monomer is composed of two domains, namely, the N-terminal (catalytic, Arg42-Val166) and the C-terminal (cofactor binding, Asn167-Trp311) domains, with the exception of the last 61 residues (after W311) which fold back to the catalytic domain (Fig. 3 in II). The two domains are separated by a deep cleft. The cofactor binding domain contains βαβαβ motifs and is arranged into a Rossmann fold that is a classical nucleotide binding motif. Two sulfates from the crystallization buffer bound to the groove of the NADPH binding site, making the structure adopt a ligand-bound form (a closed conformation) (Fig. 4 in II). The 52 191 AlaSerAsnSerGlyValGly197 motif interacts with the pyrophosphate moiety of NADPH. Simulation of the MECR/ETR1–NADPH complex suggested that Arg216 and Arg218 are important for the interaction and for cofactor specificity. 5.2.2 Substrate binding pocket A substrate binding pocket was found in the structure of MECR/ETR1 (Fig. 5 in II). Three residues lining the fatty acyl binding pocket were studied by mutagenesis and kinetic characterization. Ile129 is a conserved residue localized at the end of the substrate binding pocket. Gly165 and Phe324 are residues in the middle of the pocket. Three mutant variants, namely, Ile129Met, Gly165Ser, and Phe324Tyr display a similar level of activity toward the C8 substrate compared to the wild type protein. Interestingly, they show a much lower activity toward long chain substrates such as C14 and C16 compared to the wild type MECR (Table 4 in II), confirming that the observed pocket is real and can be manipulated. 5.2.3 Binding of ACP In the fatty acyl–ACP substrate, the fatty acyl moiety is covalently bound to the pantetheine moiety of ACP. The fatty acyl moiety can enter the catalytic site of MECR/ETR1 via a narrow cleft (Fig. 7 in II). The length of the cleft is approximately equal to the length of an extended pantetheine moiety (~14 Å). Further, a groove is formed near the entrance of the cleft. The ~15 Å wide groove contains a few basic residues (Lys248, Lys252, Arg274, Arg278 and Lys303), which may interact with the acidic residues of the α2 helix of ACP. 5.2.4 Kinetics Bimodal distribution of catalytic efficiency of wild-type MECR/ETR1 MECR/ETR1 showed a high Km for short chain substrates (C4 and C6) and a low Km for medium and long chain substrates (C8–C16) (Table 2 in II). Specifically, the Km values decrease from 74.2 to 3.6 μM when the chain length of the substrate increases from C4 to C12, although the Km values for C14 and C16 are slightly higher than that for C12. Furthermore, MECR/ETR1 shows higher turnover for the C8 and C12 substrates than for C6, C10, and C14 and other 53 substrates as well. Altogether, MECR/ETR1 shows a bimodal distribution of catalytic efficiencies and a higher affinity and turnover of MECR/ETR1 for medium- and long-chain substrates than for short-chain substrates. Catalytic residues To probe the role of several residues in the active site (Fig. 6 in II), the mutated forms (Ser85Ala, Tyr94Phe, Thr170Ala, Trp311Ala, and Trp311Leu) were purified and their kinetic properties were determined (Table 3 in II). Tyr94Phe and Trp311Ala showed a substantial increase in Km (10.2 μM for wild-type, 78.5 μM for Tyr94Phe, and 56.5 μM for Trp311Ala) and decrease in kcat (3.0 s−1 for wild-type, 0.011 s−1 for Tyr94Phe, and 0.31 s−1 for Trp311Ala), suggesting that they are important for substrate binding and catalysis. Further, Trp311Leu showed a higher kcat/Km than that of Trp311Ala, suggesting that the size of the Trp311 side chain is relevant for the catalysis. The mutagenesis of Ser85 and Thr170 to alanines reduced the catalytic efficiency to approximately 30% of the wild-type, suggesting they are not very critical for the catalysis. 5.3 Mecr transgenic mice 5.3.1 Transgenic mice Gordon and coauthors succeeded in genetic transformation of mouse embryos by microinjection of DNA (Gordon et al. 1980). Soon after that, a number of transgenic mice were generated using pronuclear microinjection. Transgenic and knockout mouse models can be used to study gene expression and human diseases making them essential tools for biomedical research. Furthermore, gene trapped mice can be used to find new genes (Ward et al. 2000). A transgenic construct usually forms a transgene array with multiple copies before integration into a chromosome, where it is attacked by an exonuclease resulting in single strand exposure. The complementary regions of the exposed strands hybridize to and form duplexes with genomic DNA, and then a repair process mediates integration via DNA synthesis and ligation. Integration often occurs through illegitimate recombination between poorly matched sequences. The strands in the region are covalently closed by well characterized repair reactions involving further nuclease action, ligation and probably DNA synthesis. 54 A replication “eye”, which is formed by the replication forks diverging from a chromosomal origin of replication, might be invaded by the transgenic construct. The frequency of mosaicism among transgenic mice produced by embryo microinjection is around 65%. Many of the embryos develop normally after microinjection and are born alive, normally between 5 and 25% of injected embryos incorporate the foreign DNA into their chromosomes (Bishop 1999). 5.3.2 Generation of Mecr oxerexpressing mice Transcription of the mammalian MT-I genes is activated by heavy metal load via the metal-regulatory transcription factor-I (MTF-I), which is expressed at similar levels in all tissues, except in the testes where the mRNA level is apparently higher than in other tissues (Auf der Maur et al. 2000). The current model suggests that the zinc-finger domain of MTF-1 directly (and reversibly) binds to zinc (Andrews et al. 2001). The protein then adopts a DNA-binding conformation and translocates to the nucleus, where it binds to metal-response elements in a few gene promoters (e.g., MT-I), leading to increased transcription. Ralph L. Brinster is the pioneer of using mouse MT-I promoter to generate transgenic animals. In one of his publications, 70% of transgenic mice for overexpression of a growth hormone gene showed high levels of expression, and the expression level is influenced by the site of integration (Palmiter et al. 1983). Brinster also tried to overexpress müllerian inhibiting substance (MIS) using the mouse MT-I promoter, and therefore generated nine transgenic founders (7 male and 2 female) (Behringer et al. 1990). Two male transgenic mice lines showed no overexpression, while all the other transgenic mouse lines overexpressed the target gene, and thus have disorders in their reproduction systems. Miller and coauthors used the mouse MT-I promoter for expression of the growth hormone gene in swine (Miller et al. 1989). Eight of nine transgenic animals showed overexpression of the target genes with significant variation in the expression level. To study the function of Mecr in a physiological context in mouse, a construct was prepared under the control of the MT-I promoter, which is ubiquitously active at low levels in tissues. Two transgenic founders (tg60 and tg62) were generated (Fig. 1a and 1b in III). Transgenic line tg60 showed lower level of expression (1.72) in terms of mRNA than transgenic line tg62 (13.3) (Fig. 1c in III). 55 5.3.3 Echocardiography Both Mecr transgenic lines had significantly reduced cardiac systolic function (Fig. 3 in III). The ejection fraction was 95.4% in wild-type, 88.1% in tg60 and 85.3% in tg62; (P < 0.01, wt vs. both tg groups). The fractional shortening was 66.8% in wild-type, 52.2% in tg60 and 48.5% in tg62 (P < 0.01, wt vs. both tg groups). The thickness of the left ventricular posterior wall was 1.2 mm in wildtype vs. 0.8 mm in tg62; P < 0.05. In line tg62, left ventricular dilatation was seen, as the diameter of the left ventricle was 2.9 mm in wild-type, 3.1 mm in tg60, p = 0.47, and 3.4 mm in tg62, P < 0.05. 5.3.4 Heart perfusion The heart perfusion experiments were monitored mainly in three levels (Fig. 4 in III). At the endogenous frequency (3.2–3.8 Hz), no significant difference in the work output was found between Mecr transgenic hearts (36.2 ± 11.6) and wildtype (38.3 ± 5.9), although the mean value of the heart rate for transgenic mice is somewhat slower than that of wild-type mice. At 5 Hz, the work output was lower in the Mecr transgenic hearts (55.3 ± 24.6) than in wild-type (65.6 ± 11.8). At 7.5 Hz, the difference in the work output between two groups was increased (58.0 ± 25.1 for transgenic vs 81.9 ± 10.2 for wild-type). Moreover, in the wildtype hearts a linear correlation (P < 0.005) between heart rate and work output was observed, which is not the case for the transgenic hearts. When the pacing was increased to 10 Hz, the wild-type hearts responded, but the Mecr transgenic hearts did not. After pacing at 7.5 Hz the transgenic hearts show a lower mean value of the CrP/Pi ratio (1.26 ± 0.17 for transgenic vs 1.40 ± 0.43 for wild-type). 5.3.5 Exercise endurance test When the mice were challenged to exercise on a treadmill, the Mecr tg62 mice and wild-type mice differed significantly in three parameters (Fig. 5 in III), the running distance (396 ± 85 m for tg62 vs. 740 ± 150 m for wt, P < 0.005), vertical distance (118 ± 43 m for tg62 vs. 301 ± 81 m for wt, P < 0.005) and vertical work performance (3.62 ± 1.62 kg·m for tg62 vs. 8.45 ± 1.85 kg·m for wt, P < 0.005). 56 6 Discussion 6.1 2-Enoyl thioester reductases (ETRs) The last step of mitochondrial FAS is catalyzed by 2-enoyl thioester reductase (ETR), producing saturated FAs which can enter the next round of FAS reactions or can be used for final function and further processing. There are several types of ETRs found in nature, which differ between organisms as well as between cellular compartments. In bacteria, there are at least four ETRs, namely, FabI (E. coli and Bacillus subtilis), FabK (Streptococcus pneumoniae), FabL (B. subtilis) and FabV (Vibrio cholerae). FabI belongs to the SDR superfamily and uses NADH as a cofactor (Rock & Cronan 1996). A chlorinated bisphenol called triclosan can inhibit FabI by forming a FabI–NAD+–triclosan ternary complex (Heath et al. 1999), whereas FabK, FabL and FabV are all triclosan-resistant (Massengo-Tiasse & Cronan 2008). FabK is distinct from FabI in amino acid sequence and uses flavin mononucleotide (FMN) and NADH as cofactors (Marrakchi et al. 2002). Moreover, the overall structure of FabK is similar to ETR of the ER fatty acid elongation system in fungi (Saito et al. 2008). Each monomer of dimeric FabK has a triose phosphate isomerase (TIM) barrel fold. AG205, a phenylimidazole derivative inhibitor binds to a hydrophobic pocket in the catalytic site of FabK probably competing with NADH for binding to FabK (Saito et al. 2008). Interestingly, B. subtilis contains both FabI and FabL, which share a low level of sequence similarity (Heath & Rock 2000). FabV is 60% larger than the typical SDRs in length, and shows a strong preference for NADH over NADPH as a cofactor (Massengo-Tiasse & Cronan 2008). Fungal cytosolic FAS is a 2.6-megadalton barrel-shaped α6β6 complex, containing two separated reaction chambers, each chamber having three sets of active sites (Jenni et al. 2006). This organization may be more suitable for the coordinated biosynthesis of FAs compared to that of bacterial type II FAS. The ETR domain of fungal FAS contains binding sites for both FMN and NADPH as cofactors (Fox & Lynen 1980). Interestingly, the FMN-binding part consists of a TIM-barrel fold; and the NADPH-binding part is predominantly formed by a α-helical domain, clearly different from a Rossmann fold (Jenni et al. 2006). The yeast mitochondrial Etr1p is a member of MDR family. Interestingly, Candida tropicalis contains two mitochondrial ETRs, forming both homodimers and 57 heterodimers (Torkko et al. 2003). It is noteworthy that the structure of the yeast Etr1p is very similar to that of its mammalian functional counterpart MECR/ETR1, an MDR enzyme. The ETR of the ER-elongation system in yeast is Tsc13p (Kohlwein et al. 2001), which apparently bears no similarity to ETRs of other FAS systems in yeast. Tsc13p contains six membrane-spanning domains, and the putative catalytic residues of Tsc13p may lie on the cytosolic face of the ER, partially embedded within the ER membrane (Paul et al. 2007). In addition, Tsc13p and other enzymes in the elongation pathway are probably organized in a complex (Tehlivets et al. 2007). In mammals, the cytosolic FAS I is formed by a 0.54 mDa X-shaped intertwined α2 homodimer of a single 270 kDa polypeptide. The ETR domain of mammalian FAS I belongs to the MDR superfamily rather than to the bacterial type SDR; and it is structurally very similar to a zinc-free bacterial quinone reductase (QORTt) from Thermus thermophilus HB8 (Maier et al. 2006). The ETR domain of mammalian FAS I contains a NADPH-binding Rossmann fold as well as a substrate binding domain (Maier et al. 2008). The mitochondrial ETR (MECR/ETR1) in mammals is the most extensively studied. It forms a dimer with a molecular mass of 37 kDa for each monomer. MECR/ETR1 shows a bimodal distribution of catalytic specificity with maxima at C8 and C12. The ETR in mammalian ER shows the highest activity with 2-hexenoyl-CoA (Podack et al. 1974), although its molecular and biochemical nature has not yet been determined. The peroxisomal ETR in mammals is a monomeric SDR with a molecular mass of 32.5 kDa, and displays a maximum activity with the medium chain length substrate (C10) (Das et al. 2000). Interestingly, the respiratory deficient phenotype of yeast deficient in Etr1p, a dimeric member of MDR family, can be rescued by mitochondrially targeted FabI, a tetrameric member of SDR family (Torkko et al. 2001). This observation suggested that the enzyme activity per se is more important than the structural properties of the protein complexes in vivo. 6.2 Lipoic acid and other products Structural and kinetic studies show that HsmtKAS and MECR/ETR1 have broad substrate acceptance (from C4 to C16) (Zhang et al. 2005, Christensen et al. 2007, Chen et al. 2008). This suggests that the physiological role of the mitochondrial FAS may not be limited to the previously proposed role – making octanoic acid, the lipoic acid precursor (Harington et al. 1993, Brody et al. 1997, Wada et al. 58 1997, Witkowski et al. 2007). In fact, some reports also claimed that mitochondrial FAS may produce longer chain products, which might be linked to the remodeling of cardiolipins (Zensen et al. 1992, Schneider et al. 1995) or mitochondrial phospholipids (Schneider et al. 1995, Guler et al. 2008). Lipoic acid is a critical component of the E2 subunit of the mitochondrial α-ketoacid dehydrogenase complexes or linked to the H-protein of the glycine cleavage system (Schonauer et al. 2008). In mammals, in addition to the dietary source, there is a lipoic acid synthase which is indispensable for embryonic development (Yi & Maeda 2005). Deletion of S. cerevisiae Etr1 resulted in respiration deficiency (Torkko et al. 2001), which may be at least partially due to the insufficiency of lipoic acid production. Altogether, one of the main products of mitochondrial FAS is apparently C8 which is used for making lipoic acid. Nevertheless, another longer chain product is possible even though it has not yet been determined conclusively, especially in mammals. It is noteworthy that the catalytic efficiency of mammalian enzymes of mitochondrial FAS is much higher toward longer chain substrates (> C8) compared to that of the yeast counterparts (Torkko et al. 2003, Chen et al. 2008), suggesting a possibility that the function of the mammalian pathway may be different from the yeast one. An RNAi knockdown experiment would provide some information about the role of mammalian mitochondria on lipoic acid synthesis. 6.3 CoA and ACP During FA metabolism, fatty acyls frequently form thiol esters with 4'-phosphopantetheine moiety of coenzymes (CoA or ACP). Fatty acyl CoA/ACPs attach to enzymes transiently and transfer the covalent fatty acyl tails between distinct catalytic sites via a phosphopantethiene chain, the flexibility and length of which allows the intermediate transfer to process efficiently. CoA is synthesized in five steps from pantothenic acid (vitamin B5), which is also the source of 4’-phosphopantethiene, the prosthetic group of ACP (Leonardi et al. 2005). Prokaryotic and eukaryotic CoA synthetic enzymes (CoaA, CoaB, CoaC, CoaD, CoaE) are different in their primary sequences although they display the same catalytic activities. For instance, CoaD and CoaE are two separated enzymes in prokaryotes whereas they are fused to form a bifunctional protein in mammals. The concentration of CoAs is tightly regulated. In the mammalian heart, CoA can reach ~100 nmol/g tissue, which is lower than in liver (~130–430 nmol/g) but higher than in most of other tissues (Tahiliani & Beinlich 59 1991). Interestingly, tumors have low CoA levels (McAllister et al. 1988). Within a mammalian cell, the concentration of CoA is around 0.08 mM in the cytosol, 3.5 mM in mitochondria, 0.7 mM in peroxisomes (Leonardi et al. 2005). In FAS systems, the fatty acyl-ACPs are the substrates, which are different from FAO systems where only fatty acyl-CoAs are accepted as substrates. In the organelles such as mitochondria where both FAO and FAS present, the nature of substrates becomes very important to separate the two pathways. Notably, even though FAS enzymes apparently contain recognition sites for the interaction with ACP, many of them can also accept fatty acyl-CoA substrates at least in vitro. It is to be determined if these FAS enzymes can use fatty acyl-CoAs substrates in vivo. If this is the case, the mitochondrial FAS may also play a role in the regulation of FAO in addition to its primary role in the synthesis of specialized FAs. 6.4 FAS II enzymes exist at low levels in mammalian mitochondria The isolation of ACP from bovine heart mitochondria was the first piece of evidence suggesting the existence of the FAS II enzyme in mammals (Runswick et al. 1991). In agreement with this suggestion, we were able to isolate MECR/ETR1 from bovine heart mitochondria. It is important to note that FAS II components in mammals are probably of low abundance compared to β-oxidation enzymes in mitochondria (unpublished observation). The fact that many of the proteomic studies do not reveal any components of mitochondrial FAS II supports this hypothesis (Ohlmeier et al. 2004, Rezaul et al. 2005). Carroll et al revealed an ACP which is the substrate shuttling component of mitochondrial FAS, through analysis of the subunit composition of complex I from bovine heart mitochondria (Carroll et al. 2003). Most recently, Pagliarini et al identified several mammalian FAS II components (e.g., MECR/ETR1, KAS/CEM1, malonyl CoA:ACP acyltransferase), during the investigation of 1098 putative mitochondrial proteins from mouse tissues using multiple advanced techniques (Pagliarini et al. 2008). Nevertheless, in bovine heart mitochondria, the amount of BtMECR/ETR1 protein (and most likely other components as well) is much less than that of 3-hydroxyacyl-CoA dehydrogenase or other β-oxidation enzymes (data not shown). This is line with the notion that the end products of mitochondrial FAS are most likely certain specialized lipids, instead of the bulk of the FAs within the cells. 60 6.5 FAS II and its physiological function Inactivation of each of the yeast FAS II genes results in loss of cytochromes and defects in mitochondrial respiration (Harington et al. 1993, Schneider et al. 1995, Schneider et al. 1997, Torkko et al. 2001, Kastaniotis et al. 2004), indicating that eukaryotic mitochondrial FAS is important for maintaining the function of these organelles. Moreover, mitochondrial FAS members are mainly expressed in mitochondria-enriched tissues, such as heart and skeletal muscle (Joshi et al. 2003, Zhang et al. 2003a, Zhang et al. 2005, Autio et al. 2007). Mitochondrial FAS is linked genetically to RNA processing in vertebrates (Autio et al. 2007) and functionally to RNA processing in yeast (Schonauer et al. 2008). In addition, yeast mutants with compromised mitochondrial FAS function are synthetically petite (synthetically lethal on non-fermentable carbon source) with mutations in the mitochondrial RNA processing and translation apparatus (Kursu et al, personal communication). Overexpression of Mecr leads to a change in mitochondrial morphology including the increased density of cristae. The average size of the mitochondria from Mecr transgenic mice was also enlarged, partially resembles the ETR1 overexpressing phenotype in yeast, although the difference between transgenic and wild-type groups maybe not be as large as that in yeast groups. The consequence of overexpression of Mecr in mice ultimately leads to the impairment of heart function. In particular, systolic function was significantly reduced. It will be interesting to know if aberrant expression of other components of mitochondrial FAS can result in heart dysfunction in mice. Furthermore, recent studies showed that Mecr is linked to kidney development in Xenopus (Kyuno et al. 2008). Therefore, it is also important to investigate the physiological roles of FAS II in other mitochondria-containing tissues/organs. 61 62 7 Conclusions This study aimed to characterize 2E-enoyl thioester reductase of mitochondrial FAS II in mammals. To achieve this goal, three major approaches were used. The mammalian protein MECR/ETR1, previously called NRFB-1, was purified, and localized in mammalian mitochondria using biochemical approaches. The protein displays 2E-enoyl thioester reductase activity. Using structure enzymological approaches, MECR/ETR1 was crystallized and the structure of MECR/ETR1 was solved. A substrate binding pocket of this enzyme was revealed based on the results of structural, mutagenesis and kinetic analysis. An ACP binding site was suggested in this study. In addition, a bimodal distribution of activity was shown based on the kinetic properties of the wild type enzyme, supporting the possibility of two major products of human mitochondrial FAS. To study the physiological significance of mitochondrial FAS in mammals, Mecr transgenic mice were generated. The Mecr-overexpressing mice showed an impairment of heart systolic function and damaged mitochondrial ultrastructure, as demonstrated using histology, echocardiography, heart perfusion, and an endurance performance test. It was proposed that FAS II in mammals may play a role in the function of mitochondria and the heart. Thus a new set of genes that may be involved in cardiomyopathy was suggested. Finally, we provided the first mammalian model for studying mitochondrial FAS II. 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III Chen ZJ, Leskinen H, Liimatta E, Sormunen RT, Miinalainen IJ, Hassinen IE & Hiltunen JK (2008) Overexpression of Mecr, a gene of mitochondrial FAS II in heart leads to cardiac dysfunction in mouse. Manuscript. Reprinted with permission from the American Society for Biochemistry and Molecular Biology (I) and Elsevier (II). Original publications are not included in the electronic version of the dissertation. 83 84 ACTA UNIVERSITATIS OULUENSIS SERIES A SCIENTIAE RERUM NATURALIUM 502. Asikkala, Janne (2008) Application of ionic liquids and microwave activation in selected organic reactions 503. Rinta-aho, Marko (2008) On monomial exponential sums in certain index 2 cases and their connections to coding theory 504. Sallinen, Pirkko (2008) Myocardial infarction. Aspects relating to endogenous and exogenous melatonin and cardiac contractility 505. Xin, Weidong (2008) Continuum electrostatics of biomolecular systems 506. Pyhäjärvi, Tanja (2008) Roles of demography and natural selection in molecular evolution of trees, focus on Pinus sylvestris 507. Kinnunen, Aulis (2008) A Palaeoproterozoic high-sulphidation epithermal gold deposit at Orivesi, southern Finland 508. Alahuhta, Markus (2008) Protein crystallography of triosephosphate isomerases: functional and protein engineering studies 509. Reif, Vitali (2008) Birds of prey and grouse in Finland. Do avian predators limit or regulate their prey numbers? 510. Saravesi, Karita (2008) Mycorrhizal responses to defoliation of woody hosts 511. Risteli, Maija (2008) Substrate specificity of lysyl hydroxylase isoforms and multifunctionality of lysyl hydroxylase 3 512. Korsu, Kai (2008) Ecology and impacts of nonnative salmonids with special reference to brook trout (Salvelinus fontinalis Mitchill) in North Europe 513. Laitinen, Jarmo (2008) Vegetational and landscape level responses to water level fluctuations in Finnish, mid-boreal aapa mire – aro wetland environments 514. Viljakainen, Lumi (2008) Evolutionary genetics of immunity and infection in social insects 515. Hurme, Eija (2008) Ecological knowledge towards sustainable forest management. Habitat requirements of the Siberian flying squirrel in Finland 516. Kaukonen, Risto (2008) Sulfide-poor platinum-group element deposits. A mineralogical approach with case studies and examples from the literature 517. Rokka, Aare (2008) Solute traffic across the mammalian peroxisomal membrane—the role of Pxmp2 518. Sharma, Satyan (2008) Computational Studies on Prostatic Acid Phosphatase Book orders: OULU UNIVERSITY PRESS P.O. Box 8200, FI-90014 University of Oulu, Finland Distributed by OULU UNIVERSITY LIBRARY P.O. Box 7500, FI-90014 University of Oulu, Finland A 519 OULU 2008 U N I V E R S I T Y O F O U L U P. O. B . 7 5 0 0 F I - 9 0 0 1 4 U N I V E R S I T Y O F O U L U F I N L A N D U N I V E R S I TAT I S S E R I E S SCIENTIAE RERUM NATURALIUM Professor Mikko Siponen HUMANIORA University Lecturer Elise Kärkkäinen TECHNICA Professor Hannu Heusala MEDICA Professor Olli Vuolteenaho SCIENTIAE RERUM SOCIALIUM ACTA UN NIIVVEERRSSIITTAT ATIISS O OU ULLU UEEN NSSIISS U Zhijun Chen E D I T O R S Zhijun Chen A B C D E F G O U L U E N S I S ACTA A C TA A 519 CHARACTERIZATION OF THE 2-ENOYL THIOESTER REDUCTASE OF MITOCHONDRIAL FATTY ACID SYNTHESIS TYPE II IN MAMMALS Senior Researcher Eila Estola SCRIPTA ACADEMICA Information officer Tiina Pistokoski OECONOMICA University Lecturer Seppo Eriksson EDITOR IN CHIEF Professor Olli Vuolteenaho PUBLICATIONS EDITOR Publications Editor Kirsti Nurkkala ISBN 978-951-42-8979-8 (Paperback) ISBN 978-951-42-8980-4 (PDF) ISSN 0355-3191 (Print) ISSN 1796-220X (Online) FACULTY OF SCIENCE, DEPARTMENT OF BIOCHEMISTRY, UNIVERSITY OF OULU; BIOCENTER OULU, UNIVERSITY OF OULU A SCIENTIAE RERUM RERUM SCIENTIAE NATURALIUM NATURALIUM
