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Protein repair and Aging: A major interest of my laboratory is understanding the biochemistry of the aging process. We are particularly interested in the generation of age-damaged proteins by spontaneous chemical reactions and the physiological role of cellular enzymes that can reverse at least some portion of the damage. We have focused our efforts on the degradation of aspartic acid and asparagine residues and the subsequent metabolism of their racemized and isomerized derivatives. We are presently determining the biological role of protein methyltransferases that can initiate the conversion of D-aspartyl residues to the L-configuration as well as the conversion of isopeptide linkages to normal peptide bonds. Such "repair" reactions may greatly increase the useful lifetime of cellular proteins and may help insure organismal survival. The clear test of the repair hypothesis is whether it functions in vivo. In the last few years, we have taken a genetic approach where we have looked at the consequences of the genetic elimination of the methyltransferase in a variety of organisms. This work has now come to fruition, and we have recently published our work on methyltransferase-deficient bacteria (Visick and Clarke, 1998; Visick et al., 1998), nematode worms (Kagan et al., 1997, Niewmierzycka et al., 1999), and mice (Kim et al., 1997; Kim et al., 1999). We found that both the mutant bacteria and worms were more sensitive to the type of environmental stresses that can damage proteins and reduce life span. However, the most dramatic effects were seen in the repair-enzyme deficient mice where there was a large accumulation of proteins containing L-isoaspartyl residues coupled with a fatal seizure defect! This latter work (done in collaboration with Dr. Stephen Young at the Gladstone Institute in San Francisco) provided the first clear evidence for the operation of the repair pathway in animals and suggested that it was of fundamental importance to complex tissues such as brain. These discoveries have now set the stage for understanding how the accumulation of spontaneous chemical damage to proteins can affect higher mental functions. This novel pathway that has let us know that macromolecular repair is not just for DNA, but for proteins as well! Although it has been established that proteins are subject to spontaneous processes that modify their covalent structure (just like DNA is), it has been previously assumed that the only fate available for a covalently damaged protein is proteolytic degradation back to the free amino acids. This process certainly occurs, but it is now clear that for many cells such degradation would not come without cost. For example, human memory appears to be largely dependent upon the state of phosphorylation of signaling proteins in the brain. Degradation would wipe the slate clean! These studies have not only given us a new window to view protein "life" but also suggest that the biological aging process may be closely linked to how well one can keep polypeptides free of spontaneous damage. Identification of novel types of methyltransferases: In the last several years, we have been involved in approaches to identify new types of methyltransferases in nature. As genomic sequencing projects provide us with an almost overwhelming number of potential new proteins, we have developed methodology for analyzing this output to identify the members of a large family of enzymes that use S-adenosylmethionine to catalyze methyltransfer reactions. We have demonstrated sequence motifs conserved in a variety of procaryotic and eucaryotic S-adenosylmethionine-dependent methyltransferases and have recently used these motifs to initially identify 33 genes encoding putative methyltransferases from the complete genome of the yeast S. cerevisiae (Niewmierzycka and Clarke, 1999). We found that seven of the sequences represent known methyltransferases and 26 are either proteins of unknown function or proteins not previously associated with methylation. We constructed yeast disruption mutants of an initial group of seven genes in the latter group and have initiated biochemical studies to characterize the substrates and function played by each enzyme. The first identification of new proteins from this work has been very exciting (Zobel-Thropp et al., 1998, Niewmierzycka and Clarke, 1999). Through our concurrent interest in protein methylation, we have been able to link one of these gene products with a novel catalytic reaction that results in a previously unknown modification of arginine residues in which the delta nitrogen atom is methylated (Zobel-Thropp et al., 1998). Screening the knockout putative yeast methyltransferase genes revealed one (now designated RMT2 for the second type of protein arginine methyltransferase) where this modification was lacking (Niewmierzycka and Clarke, 1999). We showed that the methylation of susceptible residues in Rmt2 substrates is likely to take place on nascent polypeptide chains and that these substrates exist in the cell as fully methylated species. Interestingly, Rmt2 has 27% sequence identity over 138 amino acids to the mammalian guanidinoacetate N-methyltransferase, an enzyme responsible for methylating the delta-nitrogen of the small molecule guanidinoacetate in creatine synthesis (Niewmierzycka and Clarke, 1999). The completion of the DNA sequence for yeast has spawned worldwide efforts to uncover the function of each protein, including systematic studies of yeast gene knockouts. However, it is significant to note the importance of biochemical approaches to this problem. In the work described above, the rmt2 disruption mutant did not show an obvious phenotype and the amino acid sequence was not closely related to any known proteins. The low degree of homology with guanidinoacetate methyltransferase provided a clue about the substrate, but only in context about what was already known about protein arginine methylation. The actual identification of the Rmt2 methyltransferase required using cells already disrupted in RMT1 gene, previously identified by us in collaboration with Harvey Herschman here at UCLA. Because the Rmt1 methyltransferase is more active, the loss of Rmt2 activity would not have been detected when single mutants were analyzed en masse clearly demonstrating the power of the combination of genomic and biochemical approaches developed in our laboratory to study methylation pathways. We have also recently identified
a novel type of small molecule carboxyl methyltransferase that may be involved
in the regulation of central metabolism in the citric acid cycle. This
enzyme catalyzes the methyl esterification of trans-aconitate in
yeast and bacterial cells (Hui and Clarke, 1999). We have purified the
enzyme to homogeneity and the N-terminal amino acid sequence was found
to match that expected for an open reading frame at 34.57 min on the E.
coli genomic sequence. We have generated a knockout strain of E.
coli lacking this activity and demonstrate the endogenous formation
of trans-aconitate methyl ester in extracts of wild-type but not
tam- mutant cells indicating that trans-aconitate is present in
E. coli. Since trans-aconitate does not appear to be a metabolic
intermediate in these cells but forms spontaneously from the key citric
acid cycle intermediate cis-aconitate, we suggest that its methylation
may limit its potential interference in normal metabolic pathways. We have
also detected trans-aconitate methyltransferase activity in extracts
of the yeast Saccharomyces cerevisiae and are presently searching
for the gene that encodes it.
126. Mudgett, M. B., and Clarke, S. (1996) "A Distinctly-Regulated Protein Repair L-Isoaspartyl Methyltransferase from Arabidopsis thaliana" Plant Mol. Biol. 30, 723-737. 127. Gary, J. D., Lin, W.-J., Yang, M. C., Herschman, H., and Clarke, S. (1996) "The Predominant Protein Arginine Methyltransferase from Saccharomyces cerevisiae" J. Biol. Chem. 271, 12585-12594. 128. Lin, W.-J., Gary, J. D., Yang, M. C., Clarke, S., and Herschman, H. R. (1996) "The Mammalian Immediate-Early TIS21 Protein and the Leukemia- Associated BTG1 Protein Interact with a Protein Arginine N- Methyltransferase" J. Biol. Chem. 271, 15034-15044. 129. MacLaren, D. C., and Clarke, S. (1996) "Rapid Mapping of Genomic P1 Clones: The Mouse L-Isoaspartyl/D-Aspartyl Methyltransferase Gene" Genomics, 35, 299-307. 130. Radkiewicz, J. L., Zipse, H., Clarke, S., and Houk, K. N. (1996) "Accelerated Racemization of Aspartic Acid and Asparagine Residues via Succinimide Intermediates: An ab Initio Theoretical Explanation of Mechanism" J. Amer. Chem. Soc. 118, 9148-9155. 131. DeVry, C. G., Tsai, W., and Clarke, S. (1996) "Structure of the Human Gene Encoding the Protein Repair L-Isoaspartyl (D-Aspartyl) O- Methyltransferase" Arch. Biochem. Biophys. 335, 321-332. 132. Perna, A. F., D'Aniello, A., Lowenson, J. D., Clarke, S., De Santo, N. G., and Ingrosso, D. (1997) "D-Aspartate Content of Erythrocyte Membrane Proteins is Decreased in Uremia: Implications for the Repair of Damaged Proteins" J. Amer. Soc. Nephrol. 8, 95-104. 133. Kim, E., Lowenson, J. D., MacLaren, D. C., Clarke, S., and Young, S. G. (1997) "Deficiency of a Protein-Repair Enzyme Results in the Accumulation of Altered Proteins, Retardation of Growth, and Fatal Seizures in Mice" Proc. Natl. Acad. Sci. U. S. A. 94, 6132-6137. 134. Visick, J. E., and Clarke, S. (1997) "RpoS- and OxyR-Independent Induction of HPI Catalase at Stationary Phase in Escherichia coli and Identification of rpoS Mutations in Common Laboratory Strains" J. Bacteriol. 179, 4158- 4163. 135. Kagan, R. M., McFadden, H. J., McFadden, P. N., O'Connor, C. M., and Clarke, S. (1997) "Molecular Phylogenetics of a Protein Repair Methyltransferase" Comp. Biochem. Physiol. 117B, 379-385. 136. David, C. L., Szumlanski, C. L., DeVry, C. G., Park-Hah, J. O., Clarke, S., Weinshilboum, R. M., and Aswad, D. W. (1997) "Human Erythrocyte Protein L- Isoaspartyl Methyltransferase: Heritability of Basal Activity and Genetic Polymorphism for Thermal Stability" Arch. Biochem. Biophys. 346, 277-286. 137. Mudgett, M. B., Lowenson, J. D., and Clarke, S. (1997) "Protein Repair L- Isoaspartyl Methyltransferase in Plants: Phylogenetic Distribution and the Accumulation of Substrate Proteins in Aged Barley Seeds" Plant Physiol. 115, 1481-1489. 138. Kagan, R. M., Niewmierzycka, A., and Clarke, S. (1997) "Targeted Gene Disruption of the Caenorhabditis elegans L-Isoaspartyl Protein Repair Methyltransferase Impairs Survival of Dauer Stage Nematodes" Arch. Biochem. Biophys. 348, No. 2, Dec. 15, 320- 328. 139. Visick, J. E., Cai, H., and Clarke, S. (1998) "The L-Isoaspartyl Protein Repair Methyltransferase Enhances Survival of Aging Escherichia coli Subjected to Secondary Environmental Stresses" J. Bacteriol. 180, 2623-2629. 140. Gary, J. D., and Clarke, S. (1998) "b-Aspartyl Dipeptidase" in Handbook of Proteolytic Enzymes, (eds. Woessner, G., Rawlings, N., and Barrett, A. J.), Academic Press, New York, pp. 1461-1465. 141. Tang, J, Gary, J. D., Clarke, S., and Herschman, H. R. (1998) "PRMT3, a Type I Protein Arginine N-Methyltransferase that Differs from PRMT1 in its Oligomerization, Subcellular Localization, Substrate Specificity and Regulation" J. Biol. Chem. 273, 16935-16945. 142. Gary, J. D., and Clarke, S. (1998) "RNA and Protein Interactions Modulated by Protein Arginine Methylation" Prog. Nucl. Acid Res. Mol. Biol. 61, 65-130. 143. Visick, J. E., Ichikawa, J. K., and Clarke, S. (1998) "Mutations in the Escherichia coli surE Gene Increase Isoaspartyl Accumulation in a Strain Lacking the pcm Repair Methyltransferase but Suppress Stress-survival Phenotypes" FEMS Microbiol. Lett. 167, 19-25. 144. Ichikawa, J. K., and Clarke, S. (1998) "A Highly Active Protein Repair Enzyme from an Extreme Thermophile: The L-Isoaspartyl Methyltransferase from Thermotoga maritima" Arch. Biochem. Biophys. 358, 222-231. 145. Zobel-Thropp, P. Gary, J. D., and Clarke, S. (1998) "d-N-Methylarginine Is A Novel Posttranslational Modification of Arginine Residues in Yeast Proteins" J. Biol. Chem. 273, 29283-29286. 146. Niewmierzycka, A., and Clarke, S. (1999) "S-Adenosylmethionine-dependent Methylation in Saccharomyces cerevisiae: Identification of a Novel Protein Arginine Methyltransferase" J. Biol. Chem. 274, 814-824. 147. Yan, S.-D., Shi, Y., Zhu, A., Fu, J., Zhu, H., Zhu, Y., Gibson, L., Stern, E., Collison, K., Al-Mohanna, F., Ogawa, S., Roher, A., Clarke, S. G., and Stern, D. (1999) "Role of ERAB/L-3-Hydroxyacyl-Coenzyme A Dehydrogenases Type II Activity in Ab-Induced Cytotoxicity" J. Biol. Chem. 274, 2145-2156. 148. Niewmierzycka, A., and Clarke, S. (1999) "Do Damaged Proteins Accumulate in Caenorhabditis elegans L-Isoaspartate Methyltransferase (pcm-1) Deletion Mutants?" Arch. Biochem. Biophys. 364, 209-218. 149. Cai, H., and Clarke, S. (1999) "A Novel Methyltransferase Catalyzes the Methyl Esterification of trans-Aconitate in Escherichia coli" J. Biol. Chem. 274, 13470-13479. 150. DeVry, C. G., and Clarke, S. (1999) "Assignment of the Protein L- Isoaspartate (D-Aspartate) O-Methyltransferase gene (PCMT1) to human chromosome bands 6q24-q25 with radiation hybrid mapping" Cytogenet. Cell. Gen. 84, 130-131. 151. Frankel, A., and Clarke, S. (1999) "RNase Treatment of Yeast and Mammalian Cell Extracts Affects In Vitro Substrate Methylation by Type I Protein Arginine N-Methyltransferases" Biochem. Biophys. Res. Commun. 259, 391-400. 152. Kim, E., Lowenson, J. D., Clarke, S., and Young, S. G. (1999) "Phenotypic Analysis of Seizure-Prone Mice Lacking L-Isoaspartate (D-Aspartate) O- Methyltransferase" J. Biol. Chem. 274, 20671-20678. 153. Clarke, S. (1999) "A Protein Carboxyl Methyltransferase that Recognizes Age- Damaged Peptides and Proteins and Participates in their Repair" in S- Adenosylmethionine-dependent Methyltransferases: Structures and Function" (Cheng, X, and Blumenthal, R. M., eds) World Scientific Publishing Company, Singapore, pp. 123-140. 154. DeVry, C. G., and Clarke, S. (1999) "Polymorphic Forms of the Protein L- Isoaspartate (D-Aspartate) Methyltransferase Involved in the Repair of Age- Damaged Proteins" J. Hum. Genetics 44, 275-288. 155. Kalhor, H. R., Niewmierzycka, A., Faull, K. F., Yao, X., Grade, S., Clarke, S., and Rubenstein, P. A. (1999) "A Highly Conserved 3-Methylhistidine Modification is Absent in Yeast Actin" Arch. Biochem. Biophys. 370, 105-111. 156. Lowenson, J. D., Clarke, S., and Roher, A. E. (1999) "Chemical Modifications of Deposited Ab Peptides" Methods Enzymol. 309, 89-105. 157. Tang, J., Frankel, A., Cook, R. J., Kim S., Paik, W. K., Williams, K. R., Clarke, S., and Herschman, H. R. (2000) "PRMT1 is the Predominant Type I Protein Arginine Methyltransferase in Mammalian Cells" J. Biol. Chem. 275, 7723-7730. 158. Bedford, M. T., Frankel, A., Yaffe, M. B., Clarke, S., Leder, P. and Richard, S. (2000) "Arginine Methylation Inhibits the Binding of Proline- rich Ligands to Src Homology 3, but not WW Domains" J. Biol. Chem. 275, 16030-16036. 159. Young, S. G., Ambroziak, P., Kim, E., and Clarke, S. (2000) "Post- isoprenylation Protein Processing: CXXX (CaaX) Endoproteases and Isoprenylcysteine Carboxyl Methyltransferase" in Protein Lipidation, a volume of The Enzymes, 3rd edition (Tamanoi, F., and Sigman, D. S., eds), 21, 155-213. 160. Yan, S. D., Zhu, Y., Zhu, H., Stern, E. D., Hwang, Y. C., Hori, O., Ogawa, S., Frosch, M. P., Connolly, E. S., Jr., McTaggert, R., Pinsky, D. J., Clarke, S., Stern, D. M., and Ramasamy, R. (2000) "Amyloid Beta-Peptide Binding Alcohol Dehydrogenase is a Component of the Cellular Response to Nutritional Stress" J. Biol. Chem. 275, 27100-27109. 161. Frankel, A., and Clarke, S. (2000) "PRMT3 is a Distinct Member of the Protein Arginine N-Methyltransferase Family: Conferral of Substrate Specificity by a Zinc-Finger Domain" J. Biol. Chem. 275, 32974-32982. 162. Thapar, N., and Clarke, S. (2000) "Expression, Purification, and Characterization of the Protein Repair L-Isoaspartyl Methyltransferase from Arabidopsis thaliana" Protein Expression Purification 20, 237-251. 163. Zobel-Thropp, P., Yang, M. C., Machado, L., and Clarke, S. (2000) "A Novel Post-translational Modification of Yeast Elongation Factor 1A: Methylesterification at the C-Terminus" J. Biol. Chem. 275, 37150-37158. 164. Thapar, N., Kim, A.-K., and Clarke, S. (2001) "Distinct Patterns of Expression but Similar Biochemical Properties of Protein L-Isoaspartyl Methyltransferases in Plants" Plant Physiol. 125, 1023-1035. 165. Cai, H., Strouse, J., Dumlao, D., Jung, M. E., and Clarke, S. (2001) "Distinct Reactions Catalyzed by Bacterial and Yeast trans-Aconitate Methyltransferases" Biochemistry 40, 2210-2219. 166. Radkiewicz, J. L., Zipse, H., Clarke, S., and Houk, K. N. (2001) "Neighboring Side Effects on Asparaginyl and Aspartyl Degradation: An Ab Initio Study of the Relationship Between Peptide Conformation and Backbone NH Acidity" J. Amer. Chem. Soc. 123, 3499-3506. 167. Lowenson, J. D., Kim, E., Young, S. G., and Clarke, S. (2001) "Limited Accumulation of Damaged Proteins in L-Isoaspartyl (D-Aspartyl) O- Methyltransferase-deficient Mice" J. Biol. Chem. 276, in press. 168. Clarke, S., and Banfield, K. (2001) "S-Adenosylmethionine-dependent Methyltransferases: Potential Targets in Homocysteine-linked Pathology" in Homocysteine in Health and Disease (Carmel, R., and Jacobsen, D., eds), in press. 169. Branscombe, T. L., Frankel, A., Lee, J.-H., Cook, J. R., Pestka, S., and Clarke, S. (2001) "The Janus Kinase Binding Protein 1 (PRMT5) Catalyzes the Formation of Symmetric Dimethylarginine Residues in Proteins" 170. Cai, H., Dumlao, D., Katz, J., and Clarke, S. (2001) "Identification of the Gene and Characterization of the Activity of the trans-Aconitate Methyltransferase from Saccharomyces cerevisiae" (in preparation).
Department
of Chemistry & Biochemistry
Current members of Clarke lab Not yet available. Former members of Clarke lab Not yet available.
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