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.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2013
Total Cost
$497,996
Indirect Cost
City
State
Country
Zip Code
Christianson, John C; Ye, Yihong (2014) Cleaning up in the endoplasmic reticulum: ubiquitin in charge. Nat Struct Mol Biol 21:325-35
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; 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
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
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
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