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.  

Current Members of Clarke Lab

Dr. Steven Clarke Our Leader

Laboratory Phone: (310) 825-3137 Laboratory Fax: (310) 825-1968 UCLA Department of Chemistry & Biochemistry Paul Boyer Hall 628 Box 951569 (post) 607 Charles E. Young Drive East (courier) Los Angeles, CA 90095-1569

Research Staff

Dr. Jonathan Lowenson Ph.D., University of California, Los Angeles

Jon studies the accumulation of damaged aspartyl residues in aging proteins and their repair in mammalian tissues.

Postdoctoral Students

Dr. Carole Linster Ph.D., Université Catholique de Louvain, Belgium

Carole studies the function of a group of HIT proteins, including the VTC enzyme involved in vitamin C synthesis in plants.

Ph.D. Students

	Lital Adler B.Sc., The Hebrew University of Jerusalem, Israel	Lital studies the role of vitamin C as an anti-aging factor in plants and worms.
	Shilpi Khare B.A., Biochemistry & Molecular Biology, University of California, Berkeley	Shilpi is studying the effects of oxidative stress on protein-repair deficient worms and the role of protein arginine methyltransferases in autoimmune diseases.
	Rebecca Lipson B.S., University of California, Santa Barbara	Rebecca is identifying new methyltransferases with SET domains.
	Kennen MacKay B.A., The Colorado College	Kennen studies protein repair and degradation pathways in mammalian tissues.
	Tanya Petrossian B.S., Biochemistry, University of California, Los Angeles	Tanya is identifying novel yeast lipid methyltranferases.
	Christopher Vinci B.S., Biochemistry, University of California, Los Angeles	Christopher studies the cellular responses to the spontaneous generation of potentially harmful metabolites.
	Kristofor Webb B.S., Chemistry, Fort Lewis College, Durango, Colorado	Kristofor is investigating automethylation in protein arginine methytransferase.
	Cecilia Zurita-Lopez B.S., Chemistry, California State University, Los Angeles	Cecilia is investigating novel protein arginine methytransferases.

Research Assistants

Kathy Rea B.A. English, University of California, Berkeley

Research Assistant

Collaborators

Dr. Jonathan Katz B.S., University of California, Los Angeles M.S., University of California, Los Angeles Ph.D., University of California, Los Angeles

Clarke Lab Alumni

   Where are they now? Clarke Lab Alumni