Protein modification by ubiquitin (Ub) is a critical regulatory process for virtually all aspects of cell biology. Substrate proteins can be modified with single ubiquitin on one (monoubiquitylation) or multiple sites (multi-ubiquitylation). Alternatively, several rounds of ubiquitination can occur on ubiquitin itself, leading to the formation of a polyubiquitin chain. Any of the seven lysines, or the amino terminus, of ubiquitin can be used to polymerize ubiquitin (Peng et al., 2003), so there are a huge number of differently linked polyubiquitin signals that can be formed. Ub signals are reversible as ubiquitin can be removed from substrates by deubiquitinating enzymes. The diverse Ub signals are recognized in cells by a myriad of receptors that carry distinct ubiquitin binding motifs recognizing mono- or polyubiquitinated substrates (Hicke et al., 2005). The mechanisms that regulate deubiquitinases in the cells are unclear. Here we characterize 34 human DUBs including 25 USP, 4 OTU, 1 Josephin and 4 UCHL subfamily members. We show that many of these enzymes are reversibly inactivated when oxidized by reactive oxygen species (ROS) in vitro and in the cell. Oxidation occurs preferentially on the catalytic cysteine, abrogating the isopeptide-cleaving activity without affecting these enzymes affinity to ubiquitin. Sensitivity to oxidative inhibition is associated with the activation of the DUBs wherein the active site cysteine is converted to a deprotonated state prone to oxidation. We further demonstrate that this redox-dependent regulation is essential for mono-ubiquitination of PCNA to occur in response to oxidative DNA damage, which initiates a DNA damage tolerance program. These findings establish a novel mechanism of DUB regulation that may be integrated with other redox-dependent signaling circuits to govern cellular adaptation to oxidative stress, a process intimately linked to aging and cancer. We recently characterized the function and regulation of USP19. We identify Hsp90 as a specific partner that binds the catalytic domain of USP19 to promote substrate association. Intriguingly, although overexpressed USP19 interacts with Derlin-1 and other ERAD machinery factors in the membrane, endogenous USP19 is mostly in the cytosol where it binds Hsp90. Accordingly, we detect neither interaction of endogenous USP19 with Derlin-1 nor significant effect on ERAD by USP19 depletion. The USP19 transmembrane domain appears to be partially stabilized in the cytosol by an interaction with its own catalytic domain, resulting in auto-inhibition of its deubiquitinating activity. These results clarify the role of USP19 in ERAD and suggest a novel DUB regulation that involves chaperone association and membrane integration. We also discover a new cellular process that is regulated by USP19. Specifically, we report a pathway termed Misfolding-Associated Protein Secretion (MAPS), which uses the endoplasmic reticulum (ER)-associated deubiquitinase USP19 to preferentially export aberrant cytosolic proteins. Intriguingly, the catalytic domain of USP19 possesses an unprecedented chaperone activity, allowing recruitment of misfolded proteins to the ER surface for deubiquitination. Deubiquitinated cargos are encapsulated into ER-associated late endosomes and secreted to cell exterior. USP19 deficient cells cannot efficiently secrete unwanted proteins and grow more slowly than wild-type cells upon exposure to a proteasome inhibitor. Together, our findings delineate a protein quality control (PQC) pathway, which unlike degradation-based PQC mechanisms, promotes protein homeostasis by exporting misfolded proteins through an unconventional protein secretion process.

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Support Year
6
Fiscal Year
2016
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U.S. National Inst Diabetes/Digst/Kidney
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Lee, Jin-Gu; Takahama, Shokichi; Zhang, Guofeng et al. (2016) Unconventional secretion of misfolded proteins promotes adaptation to proteasome dysfunction in mammalian cells. Nat Cell Biol 18:765-76
Liu, Yanfen; Soetandyo, Nia; Lee, Jin-Gu et al. (2014) USP13 antagonizes gp78 to maintain functionality of a chaperone in ER-associated degradation. Elife 3:e01369
Christianson, John C; Ye, Yihong (2014) Cleaning up in the endoplasmic reticulum: ubiquitin in charge. Nat Struct Mol Biol 21:325-35
Huang, Chih-Hsiang; Chu, Yue-Ru; Ye, Yihong et al. (2014) Role of HERP and a HERP-related protein in HRD1-dependent protein degradation at the endoplasmic reticulum. J Biol Chem 289:4444-54
Lee, Jin-Gu; Kim, Woong; Gygi, Steven et al. (2014) Characterization of the deubiquitinating activity of USP19 and its role in endoplasmic reticulum-associated degradation. J Biol Chem 289:3510-7
Zhang, Ting; Ye, Yihong (2014) The final moments of misfolded proteins en route to the proteasome. DNA Cell Biol 33:477-83
Huang, Chih-Hsiang; Hsiao, Hui-Ting; Chu, Yue-Ru et al. (2013) Derlin2 protein facilitates HRD1-mediated retro-translocation of sonic hedgehog at the endoplasmic reticulum. J Biol Chem 288:25330-9
Lee, Jin-Gu; Ye, Yihong (2013) Bag6/Bat3/Scythe: a novel chaperone activity with diverse regulatory functions in protein biogenesis and degradation. Bioessays 35:377-85
Ramanathan, Harish N; Ye, Yihong (2012) Cellular strategies for making monoubiquitin signals. Crit Rev Biochem Mol Biol 47:17-28
Liu, Yanfen; Ye, Yihong (2012) Roles of p97-associated deubiquitinases in protein quality control at the endoplasmic reticulum. Curr Protein Pept Sci 13:436-46