A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild-type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

• MeSH
• alanin genetika   MeSH
• cystein genetika   MeSH
• deubikvitinasy chemie   genetika   MeSH
• endopeptidasy chemie   genetika   MeSH
• katalýza MeSH
• konformace proteinů MeSH
• lidé MeSH
• mutace genetika   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• specifické proteázy ubikvitinu chemie   genetika   MeSH
• substituce aminokyselin genetika   MeSH
• trans-aktivátory chemie   genetika   MeSH
• transportní proteiny chemie   genetika   MeSH
• ubikvitin chemie   genetika   MeSH
• ubikvitinace genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH