Saturday, February 18, 2012
Exhibit Hall A-B1 (VCC West Building)
The spontaneous degradation of aspartyl and asparaginyl protein amino acids to isoaspartyl residues is one of the most common types of protein damage in aging organisms. Although the protein isoaspartyl methyltransferase (PIMT) repairs this detrimental modification and is essential to many organisms, no gene homolog or equivalent enzymatic activity is present in Saccharomyces cerevisiae. We therefore used various biochemical and radiochemical approaches to quantify isoaspartyl damage and elucidate how isoaspartyls are repaired and/or degraded in a system absent of PIMT. Surprisingly, cytosolic isoaspartyl damage quantified by base labile volatility assays was strikingly low (~70 pmol isoaspartyls/mg total protein) compared to cells with PIMT, and decreased as the cells aged to stationary phase. Inhibition of the proteasome and autophagy by MG-132 and 3-methyladenine, respectively, did not significantly alter the number of isoaspartyl-damaged proteins. Yeast lacking the key catalytic and regulatory proteasome proteins Rpn4p, Ubr2p, Pre3p and Pup1p, or the autophagy Atg8p protein, also did not exhibit elevated damage and ruled out the involvement of these main degradation pathways in removing isoaspartyls. However, depleting cells of ATP with iodoacetic acid did increase isoaspartyl damage 5-fold. Perturbing the intracellular pH homeostasis, an ATP-dependent process, by incubating exponentially growing yeast in water buffered to pH 3 or 9 resulted in significant isoaspartyl accumulation (>200 pmol/mg of total protein). Further analysis under these extreme pH conditions using pH-sensitive plasma membrane and vacuolar H+-ATPase knockout cells revealed additional increases in protein isoaspartyls, particularly in the vacuole, suggesting that pH is a vital factor controlling damage. Finally, enzymological approaches with synthetic peptides have potentially identified an isoaspartyl-targeted protease. Future investigations will identify and characterize this isoaspartyl-directed proteolytic activity, in addition to elucidating the role of cytosolic and vacuolar pH on isoaspartyl protein damage. Understanding which proteins are most susceptible to damage, and the proteolytic pathways that prevent their accumulation, may aid in the development of therapeutic interventions that enhance repair and/or degradation pathways and thus the healthspan of organisms.