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Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains.

ABSTRACT: The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGF? signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal "domain present in USPs" (DUSP) and "ubiquitin-like" (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened ?-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys(63)-, Lys(6)-, Lys(33)-, and Lys(11)-linked chains over Lys(27)-, Lys(29)-, and Lys(48)-linked and linear chains consistent with USP11's function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.

SUBMITTER: Harper S

PROVIDER: S-EPMC4020902 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains.

Harper Stephen S Gratton Hayley E HE Cornaciu Irina I Oberer Monika M Scott David J DJ Emsley Jonas J Dreveny Ingrid I

Biochemistry 20140429 18

The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal "domain present in USPs" (DUSP) and "ubiquitin-like" (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a sho ...[more]

PMID: 24724799

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Project description:BackgroundIn myocardial ischemia-reperfusion injury, myocardial damage is aggravated when blood perfusion is restored in myocardial infarction. Ubiquitin-specific protease 11 (USP11), a deubiquitinating enzyme, could remove the ubiquitination of substrate proteins and regulate protein stability, thereby affecting multiple pathological processes.AimsTo investigate the potential function of USP11 in myocardial ischemia-reperfusion injury and its underlying mechanisms.Study designIn vivo and in vitro experimental study.MethodsThe ischemia-reperfusion rat model in vivo was evolved, wherein the left anterior descending coronary artery was ligated for 30 min, followed by ligature release for 120 min. Meanwhile, H9C2 cells were brought to hypoxia for 6 h and then reoxygenated for 18 h to establish a cell hypoxia-reoxygenation (H/R) injury in vitro. Then, the loss-of-function experiments of USP11 were performed. Triphenyltetrazolium chloride and hematoxylin and eosin staining were performed to observe myocardial injury. The MTT assay was utilized to detect H9C2 cell viability. Pyroptosis was analyzed by TUNEL staining and flow cytometry. Pyroptosis-related protein expression and TRAF3 were analyzed via Western blot. The content of inflammatory factors was examined by enzyme-linked immunoassay. Co-immunoprecipitation and ubiquitination assays were performed to analyze for USP11 interacting with TRAF3.ResultsUSP11 was upregulated in the ischemic heart tissue. Ischemia-reperfusion and H/R injuries increased USP11 expression. USP11 loss-of-function assays showed that USP11 knockdown alleviated ischemia-reperfusion- and H/R-induced myocardial cell damage, pyroptosis, pro-inflammatory factor secretion, and IKKβ/NF-κB pathway activation. In H9C2 cells, USP11 stabilized TRAF3 by deubiquitination. Furthermore, rescue experiments revealed that TRAF3 overexpression reversed the protection of silencing USP11 on H/R-induced H9C2 cell injury.ConclusionThis study confirmed that USP11 knockdown ameliorated myocardial ischemia-reperfusion injury by downregulating TRAF3, suggesting that USP11 silencing can be a novel target of myocardial infarction.

Dataset Information

Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains.

Publications

Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains.

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