OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin conjugating enzymes including UBC13 and UBCH5. OTUB1 binds to E2~Ub thioester intermediates and prevent ubiquitin transfer, thereby non-catalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin, and that this stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1-E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin.

Cell-to-cell transmission of misfolded proteins propagates proteotoxic stress in multicellular organisms when transmitted polypeptides serve as a seeding template to cause protein misfolding in recipient cells, but how misfolded proteins are released from cells to initiate this process is unclear. Misfolding-associated protein secretion (MAPS) is an unconventional protein-disposing mechanism that specifically exports misfolded cytosolic proteins including various neurodegenerative disease-causing proteins. Here we establish the HSC70 co-chaperone DNAJC5 as an essential mediator of MAPS. USP19, a previously uncovered MAPS regulator binds HSC70 and acts upstream of HSC70 and DNAJC5. We further show that as a membrane-associated protein localized preferentially to late endosomes and lysosomes, DNAJC5 can chaperone MAPS client proteins to the cell exterior. Intriguingly, upon secretion, misfolded proteins can be taken up through endocytosis and eventually degraded in the lysosome. Collectively, these findings suggest a transcellular protein quality control regulatory pathway in which a deubiquitinase-chaperone axis forms a “triaging hub”, transferring aberrant polypeptides from stressed cells to healthy ones for disposal.

Members of the RING E3 ubiquitin ligase family bind to both substrate and ubiquitin-charged E2 enzyme, promoting transfer of ubiquitin from the E2 to substrate. Either a single ubiquitin or one of several types of polyubiquitin chains can be conjugated to substrate proteins, with different types of ubiquitin modifications signaling distinct outcomes. E2 enzymes play a central role in governing the nature of the ubiquitin modification, although the essential features of the E2 that differentiate mono- versus polyubiquitinating E2 enzymes remain unclear. RNF4 is a compact RING E3 ligase that directs ubiquitination of polySUMO chains in concert with several different E2 enzymes. RNF4 monoubiquitinates polySUMO substrates in concert with RAD6B and polyubiquitinates substrates together with UBCH5B, a promiscuous E2 that can function with a broad range of E3 ligases. We find that the divergent ubiquitination activities of RAD6B and UBCH5B are governed by differences at the RING-binding surface of the E2. By mutating the RAD6B RING-binding surface to resemble that of UBCH5B, RAD6B can be transformed into a highly active UBCH5B-like E2 that polyubiquitinates SUMO chains in concert with RNF4. The switch in RAD6B activity correlates with increased affinity of the E2 for RNF4. These results point to an important role of the affinity between an E3 and its cognate E2 in governing the multiplicity of substrate ubiquitination.

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