Proteome Changes Induced by Knock-Down of the Deubiquitylating Enzyme HAUSP/USP7

Kessler, Benedikt M.; Fortunati, Elisabetta; Melis, Monique; Pals, Cornelieke E.G.M.; Clevers, Hans; Maurice, Madelon M.

doi:10.1021/pr0702161

Cited by 43 publications

(31 citation statements)

References 44 publications

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“…HAUSP, on the contrary, probably has a selective set of substrates (64). Indeed its removal using RNA interferencebased strategies does not lead to global changes in ubiquitination (64) even if it has major implications on cellular growth through action on the p53 pathway (65).…”

Section: The Affinity Purification Procedures Described Is Efficient Fmentioning

confidence: 99%

Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins

Bousquet

Baudelet

Guèrin

et al. 2009

Molecular & Cellular Proteomics

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An affinity purification strategy was developed to characterize human proteasome complexes diversity as well as endogenous proteasome-interacting proteins (PIPs). This single step procedure, initially used for 20 S proteasome purification, was adapted to purify all existing physiological proteasome complexes associated to their various regulatory complexes and to their interacting partners. The method was applied to the purification of proteasome complexes and their PIPs from human erythrocytes but can be used to purify proteasomes from any human sample as starting material. The benefit of in vivo formaldehyde cross-linking as a stabilizer of protein-protein interactions was studied by comparing the status of purified proteasomes and the identified proteins in both protocols (with or without formaldehyde cross-linking). Subsequent proteomics analyses identified all proteasomal subunits, known regulators, and recently assigned partners. Moreover other proteins implicated at different levels of the ubiquitin-proteasome system were also identified for the first time as PIPs. One of them, the ubiquitin-specific protease USP7, also known as HAUSP, is an important player in the p53-HDM2 pathway. The specificity of the interaction was further confirmed using a complementary approach that consisted of the reverse immunoprecipitation with HAUSP as a bait. Altogether we provide a valuable tool that should contribute, through the identification of partners likely to affect proteasomal function, to a better understanding of this complex proteolytic machinery in any living human cell and/or organ

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Section: The Affinity Purification Procedures Described Is Efficient Fmentioning

confidence: 99%

Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins

Bousquet

Baudelet

Guèrin

et al. 2009

Molecular & Cellular Proteomics

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“…USP7 (HAUSP) functions as a deubiquitylase that regulates the stability of both p53 and MDM2 (9,10) as well as a number of other proteins (11)(12)(13). We found that USP7 physically interacts with UHRF1.…”

mentioning

confidence: 92%

M phase phosphorylation of the epigenetic regulator UHRF1 regulates its physical association with the deubiquitylase USP7 and stability

Chen

Guo

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

105

144

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UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) plays an important role in DNA CpG methylation, heterochromatin function and gene expression. Overexpression of UHRF1 has been suggested to contribute to tumorigenesis. However, regulation of UHRF1 is largely unknown. Here we show that the deubiquitylase USP7 interacts with UHRF1. Using interaction-defective and catalytic mutants of USP7 for complementation experiments, we demonstrate that both physical interaction and catalytic activity of USP7 are necessary for UHRF1 ubiquitylation and stability regulation. Mass spectrometry analysis identified phosphorylation of serine (S) 652 within the USP7-interacting domain of UHRF1, which was further confirmed by a UHRF1 S652 phosphor (S652ph)-specific antibody. Importantly, the S652ph antibody identifies phosphorylated UHRF1 in mitotic cells and consistently S652 can be phosphorylated by the M phase-specific kinase CDK1-cyclin B in vitro. UHRF1 S652 phosphorylation significantly reduces UHRF1 interaction with USP7 in vitro and in vivo, which is correlated with a decreased UHRF1 stability in the M phase of the cell cycle. In contrast, UHRF1 carrying the S652A mutation, which renders UHRF1 resistant to phosphorylation at S652, is more stable. Importantly, cells carrying the S652A mutant grow more slowly suggesting that maintaining an appropriate level of UHRF1 is important for cell proliferation regulation. Taken together, our findings uncovered a cell cycle-specific signaling event that relieves UHRF1 from its interaction with USP7, thus exposing UHRF1 to proteasome-mediated degradation. These findings identify a molecular mechanism by which cellular UHRF1 level is regulated, which may impact cell proliferation.deubiquitination | phosphorylation UHRF1 S652 E pigenetic regulation has emerged as an important mechanism that regulates many chromatin template-based processes, including transcription, DNA replication, and repair. An important component of epigenetic regulation is DNA CpG methylation, which is mediated by DNA methyltransferases such as DMNT1 and DNMT3a/b and an accessory factor DNMT3L (1, 2). Recent studies demonstrate that maintenance of DNA methylation patterns requires UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) (also called Np95 and ICBP90). UHRF1 binds hemimethylated CpG and recruits DNMT1 to ensure faithful propagation of the DNA methylation patterns through DNA replication (3, 4). UHRF1 is also localized to euchromatic regions where it regulates transcription possibly by impacting DNA methylation and histone modifications (5, 6). UHRF1 has been shown to regulate cell proliferation, and its loss has been implicated in the mis-regulation of both G1 and G2/M phases of the cell cycle, respectively (7). However, very little is known how this important epigenetic regulator itself is regulated. To address this question, we have recently undertaken a proteomics approach and identified a cell cycle signaling-regulated physical interaction of UHRF1 with the deubiquitylase USP7 (HAUSP) (8,9) ...

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“…A number of USP7 substrates have been identified, and USP7 knockdown was shown to affect cellular levels of at least 36 proteins in LS174T colon carcinoma cells (4). Among a variety of substrates, the USP7 deubiquitinates both p53 tumor suppressor and its main regulator E3 ligase Hdm2 (human double minute 2) and thus plays a central role in maintaining an appropriate p53 level in the cell (5)(6)(7)(8).…”

mentioning

confidence: 99%

Structural Characterization of Interaction between Human Ubiquitin-specific Protease 7 and Immediate-Early Protein ICP0 of Herpes Simplex Virus-1

Pozhidaeva

Mohni

Dhe‐Paganon

et al. 2015

Journal of Biological Chemistry

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Background: Herpesvirus infection is dependent upon the viral E3 ligase ICP0 and its interaction with the human deubiquitinating enzyme USP7. Results: The interaction between USP7 and ICP0 was structurally characterized using solution NMR. Conclusion: ICP0 is recognized by ubiquitin-like domains 1 and 2 (UBL12) of the USP7. Significance: Details of the USP7/ICP0 interaction provide new insights into function of the USP7 ubiquitin-like domains.

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Proteome Changes Induced by Knock-Down of the Deubiquitylating Enzyme HAUSP/USP7

Cited by 43 publications

References 44 publications

Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins

Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins

M phase phosphorylation of the epigenetic regulator UHRF1 regulates its physical association with the deubiquitylase USP7 and stability

Structural Characterization of Interaction between Human Ubiquitin-specific Protease 7 and Immediate-Early Protein ICP0 of Herpes Simplex Virus-1

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