associate professor of biology
Ph.D., Purdue University
Fields of Interest
Protein methylation and the repair of age-damaged proteins.
The goal of our research is to understandthe physiological significance of the reactions catalyzed by a protein carboxyl methyltransferase (PCMT) with a specificity for unusual isoaspartyl residues in proteins. Isoaspartyl residues are not incorporated into proteins during translation, but arise spontaneously from structural changes in proteins that accompany the aging process. The presence of an isoaspartyl residue in a protein would be expected to put a "kink" in the protein backbone, leading to a loss of proper protein function. Biochemical studies with the purified PCMT and isoaspartyl-containing substrates have suggested that PCMT initiates the structural repair of the damaged substrate, thus preventing the accumulation of damaged proteins in cells. Judging from the nearly ubiquitous distribution of PCMT activity in living organisms, PCMT function may be a fundamental component of cellular protein metabolism.
Our laboratory has developed a microinjection model involving Xenopus laevis oocytes to define the biochemical pathway initiated by carboxyl methylation. In these experiments, oocytes are microinjected with isoaspartyl substrates for the PCMT, and at various times after injection, substrates and metabolites are characterized in extracts prepared from the oocytes. These experiments have shown isoaspartyl substrates are either degraded or methylated by competing biochemical pathways following their microinjection into Xenopus oocytes. Degradation of one substrate, an isoaspartyl-containing variant of calmodulin, is catalyzed directly by the 26S proteasome in an unusual pathway which does not require ubiquitination of the isoaspartyl substrate. Thus, cells appear to have redundant mechanisms for preventing the accumulation of potentially dysfunctional isoaspartyl-containing proteins. Based on these results, we hypothesize that PCMT function is especially important during physiological situations characterized by an accumulation of abnormal proteins, such as stress and aging.
We are also using the fruitfly Drosophila melanogaster as a model for studying the effects of PCMT overexpression and depletion in a living organism. Drosophila makes a particularly suitable model for these studies because of the reproducible phenotypes associated with the aging process, which occurs over a period of about six weeks. In addition, many of the fundamental mechanisms underlying development and neural function are conserved from Drosophila to mammals. We have recently shown that overexpression of PCMT in adult flies causes a dramatic extension in the adult lifespan at 29 degrees C, but not at 25 C. The results suggest that protein repair is important in determining the length of the lifespan. PCMT function may be particularly important at slightly elevated temperatures, because the conformational flexibility of proteins is greater at higher temperatures. This increase in flexibility should promote the formation of damaged isoaspartyl residues in proteins. We are now using P elements to generate loss-of-function mutants to determine if loss of PCMT function has a negative effect on longevity. We are also interested in determining the cell types that are most important in the regulation of lifespan and if a quantitative relationship exists between the level of PCMT expression and the length of the lifespan.
O'Connor, C. M. 2006. Protein L-isoaspartyl/D-aspartyl O-methyltransferases: Catalysts for protein repair. In Protein Methyltransferases, Chapter 13 of The Enzymes, Vol. 24, pp. 383-431 (Eds. Tamanoi, F. and Clarke, S.), Elsevier.
Bennett, E.J., Bjerregaard, J., Knapp, J.E., Chavous, D.A., Friedman, A.M., Royer, W.E., Jr., and O'Connor, C.M. 2003. Catalytic implications from the Drosophila protein L-isoaspartyl methyltransferase structure and site-directed mutagenesis. Biochemistry 42: 12844–53.
Chavous, D.A., Jackson, F.R., and O'Connor, C.M. 2001. Extension of the Drosophila lifespan by overexpression of a protein repair methyltransferase. Proceedings of the National Academy of Sciences of the USA 98: 14814–18.
Tarcsa, E., Szymanska, G., Lecker, S., O'Connor, C.M., and Goldberg, A.L. 2000. Ca2+-free calmodulin and calmodulin damaged by in vitro aging are degraded by 26S proteasomes without ubiquitination. Journal of Biological Chemistry 275: 20295–20301.
Chavous, D.A., Hake, L.E., Lynch, R.J., and O'Connor, C.M. 2000. Translation of a unique transcript for protein isoaspartyl methyltransferase in haploid spermatids: Implications for protein storage and repair. Molecular Reproduction and Development 56: 139–144.
Szymanska, G., Leszyk, J.D., and O'Connor, C.M. 1998. Carboxyl methylation of deamidated calmodulin increases its stability in Xenopus oocyte cytoplasm: Implications for protein repair. Journal of Biological Chemistry 273: 28516–28523.
O'Connor, M.B., and O'Connor, C.M. 1998. Complex interactions of the protein L-isoaspartyl methyltransferase with calmodulin detected using the yeast two-hybrid system. Journal of Biological Chemistry 273: 12909–12913.
Szymanska, G., O'Connor, M.B., and O'Connor, C.M. 1997. Construction of an epitope-tagged calmodulin useful for the analysis of calmodulin-binding proteins. Analytical Biochemistry 252: 96–105.
O'Connor, M.B., Galus, A., Hartenstine, M., Magee, M., Jackson, F.R., and O'Connor, C.M. 1997. Structural organization and developmental expression of the protein isoaspartyl methyltransferase gene from Drosophila melanogaster. Insect Biochemistry and Molecular Biology 27: 49–54.