76 - Decreased proteasome cleavage rates at nitrotyrosine sites in proteins and peptides

Abstract

Oxidative post-translational modifications to proteins can lead to conformationally altered and/or dysfunctional proteoforms. Removal of moderately oxidized proteins is mainly carried out by the proteasome while highly modified proteins are no longer degradable. However, in the case of proteins modified by nitration of tyrosine residues to 3-nitrotyrosine (NO2Y), the role of the proteasome remains to be established. For this purpose, degradation assays and mass spectrometry analyses were performed using isolated proteasome and purified fractions of native cytochrome c (cyt c) and tyrosine nitrated proteoforms (NO2-cyt c). While cyt c pre-treated under mild conditions with hydrogen peroxide is preferentially degraded by the proteasome, NO2Y74- and NO2Y97-cyt c are less degradable. Peptide mapping analysis confirmed a decreased chymotrypsin-like cleavage at NO2Y sites. Degradation yields by the other two proteasome active sites remained the same in the unmodified and NO2-cyt c. Additionally, studies with the proteasome substrate N-suc-LLVY-AMC (Y-AMC) and its NO2Y-containing analog, N-suc-LLVNO2Y-AMC (NO2Y-AMC) were performed. Comparison of both substrates showed a significantly decreased proteasome activity towards NO2Y-AMC either using the isolated proteasome or cell lysates. Moreover, NO2Y-AMC degradation rates were largely blunted by increasing the reaction pH, suggesting an inhibitory influence of the additional negative charge contained in NO2Y-AMC secondary to nitration. The mechanism of slowing of proteasome activity in NO2Y-contaning peptides was further substantiated in studies using the phenylalanine and nitro-phenylalanine peptide analog substrates. Finally, degradation rates of Y-AMC and NO2Y-AMC with proteinase K were the same, demonstrating the selective inability of the proteasome to readily cleave at nitrotyrosine sites.