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Deleterious Deoxyguanosine Kinase Double Destruction Westosha Central SMART Team: Julia Alberth, Nicholas Bielski, Dylan Clements, Jared Hollway, Evan Kirsch, Mitchell Kirsch Becca Lawrence, Julia Mellor, Maddie Murphy, Adam Papendick, Sean Quist, AJ Reeves, Zachary Wermeling, Julia Williams Teacher: Jonathan Kao Mentor: Jason Kowalski, Ph.D., University of Wisconsin Parkside I. Abstract IV. mtDNA and Central Dogma Mitochondrial Deficiency Syndrome (MDS) is characterized by a deficient amount of mitochondrial DNA (mtDNA). Without sufficient copies of mtDNA, the mitochondria cannot manufacture an adequate amount of ATP, leading to complications in energy expensive tissues such as the brain and liver, ultimately causing death in early infancy. Deoxyguanosine kinase (dGK), an enzymatic protein, plays a role in regulation of mtDNA by attaching a phosphate to a sugar/nitrogen-base nucleoside. Once phosphorylated, the assembly of mtDNA proceeds. Mutations in dGK prevent the phosphorylation of mtDNA and lead to a decrease in mitochondrial function. Two point mutations have been shown to have a deleterious impact on dGK: the R142K mutation is 0.2% active when compared to the wild type, and the E227K mutation is 5.5% active when compared to the wild type. The 3D model printed by the Westosha Central SMART (Students Modeling A Research Topic) team displays these two specific mutations and additional mutations reported in MDS patients. Screening for mutations in dGK may be a potential method for quickly diagnosing MDS, saving precious time for those affected. II. Mitochondrial Deficiency Syndrome Mitochondrial Deficiency Syndrome (MDS) is a disorder that arises from insufficient copying of mitochondrial DNA (mtDNA) resulting in a lack of ATP production. Symptoms include muscle malfunction, along with liver, muscle, kidney, and lung failure. These are energy expensive tissues, therefore ATP deficiency has the greatest effect on them. MDS is inherited through an autosomal recessive gene and exists in multiple forms. Various symptoms are present in different forms, as shown in Figure 1, but a commonality between all forms of MDS is insufficient production of ATP. Most forms of MDS have early onset and result in death during early infancy, however some forms of MDS have a later onset and symptoms may occur within five years of birth. Cases are extremely rare and specific to the individual; few cases exist globally at one time, because death occurs in the first few years. Through studying MDS, scientists have concluded that organ transplants and gene therapy are possible approaches in the quest for a cure. Figure 1. Symptoms of MDS. Affected organs and tissues display III. Biological Significance symptoms due to inadequate ATP3 Figure 2: A) EPR spectrum of mouse liver comprised of at least 17 overlapping metallo-protein spectra. B) A model of individual metalloprotein spectra simulations to model the experimental spectrum in 2A. C) A hypothetical model of a mouse liver affected by a mutation in dGK6. Screening for MDS can be done with whole tissue Electron Paramagnetic Resonance (EPR). EPR data has demonstrated it is possible to resolve up to 17 different metallo-protein spectra in mouse organs, Figure 2A. Of these 17 proteins, 11 are found in the mitochondria. It is possible to model individual spectra of all 17 proteins into a simulation of the experimental data, Figure 2B. A mouse with MDS would have a noticeable decrease in the EPR signal of mitochondrial proteins, Figure 2C. This is just one of many protein specific forms of MDS that EPR of whole tissue may be able diagnose more quickly than traditional methods. The story of mitochondrial proteins is interesting because they involve two iterations of the central dogma. The central dogma of molecular biology states that DNA codes for proteins using RNA as an intermediate as shown in Figure 3. Nuclear proteins, such as dGK, are responsible for the assembly of mtDNA. As expected, dGK is coded for by chromosome 2 and transcribed to mRNA and then is produced by a ribosome in the cytoplasm. The new protein is then transported to a mitochondrion where it constructs mtDNA through the phosphorylation of a guanosine monomer as shown in Figure 4. This is where the second iteration of the central dogma begins. When dGK is properly functioning, mitochondrial DNA (mtDNA) is assembled correctly and efficiently. Improper dGK function leads to misassembled and mutated mtDNA. Mitochondrial DNA codes for proteins and ribosomes that are necessary for the proper formation of ATP as shown in Figure 5. A) B) C) D) Figure 3. The Central Dogma A) DNA is transcribed to mRNA in the nucleus. B) Translation from a nucleic acid sequence to an amino acid sequence occurs at the ribosome. C) Amino acid sequences fold spontaneously based on amino acid properties. An error in the process can cause mutations. D) The final tertiary structure of a protein determines function. V. Point Mutations and dGK Function Several mutations have been reported in dGK. Almost all of these mutations have a deleterious effect on the function of dGK. Mutations discussed in Eriksson (2003) are highlighted in Figure 6. The E227K mutation results when the nucleotide 679 interchanges A G. The resulting codon mutates a Glutamic Acid into a Lysine. This reduces the enzyme’s ability to phosphorylate deoxyguanosine, a pre-cursor to the monomer of mitochondrial DNA. The E227K mutation is only 2.8%-5.5% efficient when compared to wild type. This efficiency drop creates insufficient copies of mtDNA, leading to ATP deficiency. Fig. 7 E227K Mutation This mutation is the result of a Glutamic Acid mutating to a Lysine. This mutation occurs outside the active site but drops the efficiency of dGK. A) B) C) D) Figure 4. Location of Mitochondrial DNA A) All Eukaryotic cells contain mitochondria in the cytoplasm1. B) Mitochondria produce ATP, the molecular currency of energy4. C) mtDNA is a circular piece of DNA specific to the mitochondria which codes for proteins necessary to produce ATP. D) dGK phosphorylates a guanosine monomer creating guanine, a nitrogen base found in mtDNA. Figure 5. Mitochondrial DNA Almost the entirety of mtDNA is devoted to coding for proteins necessary for ATP synthesis. Incorrectly assembled mtDNA may result in proteins incapable of producing ATP5. Misassembled mtDNA cannot code for proteins involved in ATP synthesis resulting in insufficient ATP production by the mitochondria. In this case, the central dogma has failed as mtDNA is not coding for mitochondrial proteins accurately. Lack of ATP leads to muscle malfunction, along with liver, kidney, and lung failure which can be broadly characterized as MDS. This sequence of events necessitates that dGK does not have deleterious mutations such that it will not mutate or inhibit mtDNA. Proteins coded for by mtDNA act as if downstream from the dGK gene; any earlier mutation will result in a cascade of unwanted consequences. The R142K mutation results when nucleotide 425 interchanges A G. The resulting codon mutates an Arginine into a Lysine. Alteration of this specific dGK amino acid leads to a lethal mutation in the active site. While Arginine and Lysine are both positively charged amino acids, Arginine is a stronger base and will donate hydrogen more readily than Lysine. The R142K mutant is still active but has a decreased phosphorylation rate, slowing it to 0.2% of the wild type, effectively terminating the protein’s function. If phosphorylation is terminated, then the mitochondria will be inhibited from producing ATP and the infant with the R142K mutation will die. Figure 8. R142K Mutation This mutation is the result of an Arginine mutating to a Lysine. This mutation affects the active site of dGK. Despite a mutation in the active site, the similar properties of Arginine and Lysine allow phosphorylation to continue, albeit at a drastically slower rate. These are truncation mutations that are caused by a change in nucleotide sequence such that a premature stop codon is coded for, ending synthesis of the protein by the ribosome. This results in an incomplete protein that has an improperly folded active site, or no active site at all. As a result, these mutations usually result in completely inactive proteins. Figure 6. A Model of dGK based on file 2OCP.pdb VI. References 1. Animal cell clipart. (n.d.). Clker.com. Retrieved February 27, 2014, from http://www.clker.com/clipart-animal-cell.html 2. Eriksson, S. (2003). Mitochondrial deoxyguanosine kinase mutations and mitochondrial DNA depletion syndrome. FEBS Letters, 554(3), 319-322. http://www.sciencedirect.com/science/article/pii/S0014579303011815 3. Hesterlee, S. (n.d.). Mitochondrial Disease in Perspective Symptoms, Diagnosis and Hope for the Future. Mitochondria Research Society. Retrieved March 4, 2014, from http://www.mitoresearch.org/treatmentdisease.html Fig 9. Truncation Mutations Mutations in the amino acid sequence that result in the early termination of the protein have been reported at positions 105, 213, and 255. 4. Mari, M., Tooze, S. A., & Reggiori, F. (2012, February 1). The Enigmatic Membrane. The Scientist. Retrieved February 27, 2014, from http://www.the-scientist.com/?articles.view/articleNo/31626/title/The-Enigmatic-Membrane/ 5. Mitochondrial DNA Map. (n.d.). Mitochondrial DNA Map. Retrieved March 10, 2014, from http://lslab.lscore.ucla.edu/MTDNA/mtDNAMap.htm 6. Unpublished data from the lab of Dr. Brian Bennett, Medical College of Wisconsin SMART Teams are supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR000055. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH