title: Deubiquitination by proteasome is coordinated with substrate translocation for proteolysis in vivo.
abstract: The 26S proteasome mediates degradation of protein substrates labeled with polyUb chains. After recognition by the 19S proteasome regulatory complex, polyUb chains are disassembled and substrates are processed in the 20S core of proteasome. However, the exact relationship of degradation-associated deubiquitination to substrate processing remains unclear. Here, using Ub-based tagging strategies, we provided evidence that removable polyUb chains serve as the signal for proteolytic processing of ubiquitinated substrates. We showed that inhibition of the proteasome by proteasome inhibitor MG132 results in trapping of the substrate in the proteasome. Such a trapping allows proteasomal cleavage of attached non-removable Ub mutant (UbV75,76), which is otherwise a "difficult" deubiquitination substrate. Characterization of deubiquitination and degradation intermediates, generated due to incomplete proteolytic inhibition, revealed changes in proteolytic cleavage sites, within the Gal4-VP16 model substrate, suggesting that the copy number of attached UbV75,76 affects substrate processing. Conversion of lysine48 to arginine48 in UbV75,76 did not have significant effect on in vivo polyubiquitination of multiple Ub-fused substrates, but considerably reduced proteolytic intermediates. Taken together, the results support a model in which deubiquitination process is a crucial event for proteolysis of ubiquitinated substrates and such an event is coordinated with substrate translocation.
authors.affiliation: Department of Radiology, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Qianzheng
authors.initials: Q
authors.lastname: Zhu
authors.forename: Gulzar
authors.initials: G
authors.lastname: Wani
authors.forename: Qi-en
authors.initials: QE
authors.lastname: Wang
authors.forename: Mohamed
authors.initials: M
authors.lastname: El-mahdy
authors.forename: Robert M
authors.initials: RM
authors.lastname: Snapka
authors.forename: Altaf A
authors.initials: AA
authors.lastname: Wani
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008239:Lysine / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D021122:Protein Subunits / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*
chemical_list: D015853:Cysteine Proteinase Inhibitors; C070633:Gal-VP16; D007976:Leupeptins; D009842:Oligopeptides; D010446:Peptide Fragments; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D021122:Protein Subunits; D011506:Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D025801:Ubiquitin; D001120:Arginine; D046988:Proteasome Endopeptidase Complex; D008239:Lysine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; C078846:epoxomicin