Regulation of Protein Turnover by Ubp6 and UBE3B

Abstract

Protein homeostasis is maintained by selective control of degradation. Deubiquitinating enzymes and ubiquitin ligases are principal players in this regulation. The deubiquitinating activity of Ubp6 acts on substrates bearing multiple ubiquitin chains, suppressing their degradation through kinetic competition with the proteasome. Catalytically inactive Ubp6 can also suppress proteasome activity – the noncatalytic effect. Ubiquitin loading within the active site of Ubp6 promotes catalytic domain docking to the proximal proteasome subunit Rpt1 as well as the noncatalytic effect. We report that a I329A L330A mutant in the BL1 active-site crossover loop of Ubp6 prevents activation by the proteasome; notably, this mutation also abrogates the noncatalytic effect of Ubp6 on proteasome activity. The specific role of these residues in Ubp6 activation is conserved in the orthologous mammalian protein, USP14. We also identified a S164R T166K mutant in Rpt1, which phenocopies a ubp6 loss of function. Rpt1-S164R-T166K proteasomes fail to activate Ubp6-mediated deubiquitination and are resistant to the noncatalytic effect. Our results identify a highly specific and conserved switch that exerts simultaneous control over the activity of both Ubp6 and the proteasome. The results suggest that the active states of Ubp6 and the proteasome may be antagonistic and mutually exclusive, with only one of these enzymes assuming an “ON” state at any given time. Ubiquitin ligase UBE3B is implicated in both Kaufman Oculocerebrofacial Syndrome (KOS) and Autism Spectrum Disorders (ASD). However, there is as yet no understanding of how ubiquitination events directed by E3B relate to these diseases. Using quantitative global proteomics, we uncovered scavenger receptor SR-B1 as a presumptive substrate of E3B. SR-B1 has a critical function in mediating cholesterol uptake from the blood. Because KOS patients and E3B null mice also displayed a cholesterol-related phenotype, SR-B1 has a potential link to the disease pathology. We defined by mutagenesis a highly conserved, five-residue hydrophobic element, CYLFW, within the C-terminal cytoplasmic tail of SR-B1 as a signal for E3B-dependent receptor turnover. Two lysines immediately adjacent to the hydrophobic element likely serve as ubiquitination sites. We propose that E3B ubiquitinates SR-B1 on the cell surface, therefore inducing endocytosis and its degradation through the endo-lysosomal pathway.