title: The E3 ubiquitin ligase UBE3C enhances proteasome processivity by ubiquitinating partially proteolyzed substrates.
abstract: To maintain protein homeostasis, cells must balance protein synthesis with protein degradation. Accumulation of misfolded or partially degraded proteins can lead to the formation of pathological protein aggregates. Here we report the use of destabilizing domains, proteins whose folding state can be reversibly tuned using a high affinity ligand, as model substrates to interrogate cellular protein quality control mechanisms in mammalian cells using a forward genetic screen. Upon knockdown of UBE3C, an E3 ubiquitin ligase, a reporter protein consisting of a destabilizing domain fused to GFP is degraded more slowly and incompletely by the proteasome. Partial proteolysis is also observed when UBE3C is present but cannot ubiquitinate substrates because its active site has been mutated, it is unable to bind to the proteasome, or the substrate lacks lysine residues. UBE3C knockdown also results in less substrate polyubiquitination. Finally, knockdown renders cells more susceptible to the Hsp90 inhibitor 17-AAG, suggesting that UBE3C protects against the harmful accumulation of protein fragments arising from incompletely degraded proteasome substrates.
authors.affiliation: From the Department of Chemical and Systems Biology, Stanford University, Stanford, California 94305.
authors.forename: Bernard W
authors.initials: BW
authors.lastname: Chu
authors.forename: Kyle M
authors.initials: KM
authors.lastname: Kovary
authors.forename: Johan
authors.initials: J
authors.lastname: Guillaume
authors.forename: Ling-chun
authors.initials: LC
authors.lastname: Chen
authors.forename: Mary N
authors.initials: MN
authors.lastname: Teruel
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Wandless
mesh_terms: D016227:Benzoquinones / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics; D017510:Protein Folding; D059748:Proteolysis / Q000187:drug effects; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects / Q000235:genetics*
chemical_list: D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D049452:Green Fluorescent Proteins; C112765:tanespimycin; C555759:UBE3C protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex
keywords: E3 Ubiquitin Ligase; Proteasome; Protein Degradation; Protein Processing; Protein Stability