Influence of the protein cross-linker diethyl acetylenedicarboxylate on 20s core particle degradation of candidates for the 26s proteasome
Proteins containing highly reactive cysteine residues can undergo electrophilic modification which often results in altered function and, in some cases, inactivation. Tsa1 is a peroxiredoxin involved in yeast oxidative stress response that has been shown to be cross-linked by the electrophile, diethyl acetylenedicarboxylate (DAD), to become inactivated. The fate of the DAD-Tsa1 conjugate is not known, although it is potentially targeted for degradation upon modification. Previous data have shown DAD to be extremely cytotoxic compared to analogous electrophilic molecules. Therefore, we propose that this enhanced toxicity is due to its influence on proteasome-mediated degradation. We hypothesize that modification by DAD inhibits degradation of Tsa1 and other cross-linked products by the 26S proteasome. To this end, we set out to detect increased accumulation of polyubiquitin, proteasomal substrates, and damaged proteins like Tsa1 in the presence of DAD, indicating a disturbance in this cellular protein degradation mechanism by protein cross-linkers. To accomplish this, we treated yeast cells with various concentrations of electrophiles and compared levels of accumulation to cells treated with β-clasto-lactacystin using Western blot analysis. We discovered evidence which indicates that damaged protein degradation facilitated by the catalytic 20S core particle of the 26S proteasome is significantly inhibited upon exposure to DAD and that these proteasomal inhibitory effects induced by DAD modification can further be applied to well characterized proteasomal substrates such as the cell cycle regulatory protein cyclin. Follow up in vitro inhibition studies with purified proteasome and protein targets as well as a more diverse range of substrates are needed to help confirm our findings.
© Copyright 2012 TeQuion M. Brookins