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Lori Frappier - One of the best experts on this subject based on the ideXlab platform.
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Identification and Characterization of USP7 Targets in Cancer Cells.
Scientific reports, 2018Co-Authors: Anna A. Georges, Edyta Marcon, Jack Greenblatt, Lori FrappierAbstract:The ubiquitin specific protease, USP7, regulates multiple cellular pathways relevant for cancer through its ability to bind and sometimes stabilize specific target proteins through deubiquitylation. To gain a more complete profile of USP7 interactions in cancer cells, we performed affinity purification coupled to mass spectrometry to identify USP7 binding targets in gastric carcinoma cells. This confirmed reported associations of USP7 with USP11, PPM1G phosphatase and TRIP12 E3 ubiquitin ligase as well as identifying novel interactions with two DEAD/DEAH-box RNA helicases, DDX24 and DHX40. Using USP7 binding pocket mutants, we show that USP11, PPM1G, TRIP12 and DDX24 bind USP7 through its TRAF domain binding pocket, while DHX40 interacts with USP7 through a distinct binding pocket in the Ubl2 domain. P/A/ExxS motifs in USP11 and DDX24 that are critical for USP7 binding were also identified. Modulation of USP7 expression levels and inhibition of USP7 catalytic activity in multiple cells lines showed that USP7 consistently stabilizes DDX24, DHX40 and TRIP12 dependent on its catalytic activity, while USP11 and PPM1G levels were not consistently affected. Our study better defines the mechanisms of USP7 interaction with known targets and identifies DDX24 and DHX40 as new targets that are specifically bound and regulated by USP7.
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crystal structure of USP7 ubiquitin like domains with an icp0 peptide reveals a novel mechanism used by viral and cellular proteins to target USP7
PLOS Pathogens, 2015Co-Authors: Roland Pfoh, Lori Frappier, Ira Kay Lacdao, Anna Georges, Adam Capar, Hong Zheng, Vivian SaridakisAbstract:Herpes simplex virus-1 immediate-early protein ICP0 activates viral genes during early stages of infection, affects cellular levels of multiple host proteins and is crucial for effective lytic infection. Being a RING-type E3 ligase prone to auto-ubiquitination, ICP0 relies on human deubiquitinating enzyme USP7 for protection against 26S proteasomal mediated degradation. USP7 is involved in apoptosis, epigenetics, cell proliferation and is targeted by several herpesviruses. Several USP7 partners, including ICP0, GMPS, and UHRF1, interact through its C-terminal domain (CTD), which contains five ubiquitin-like (Ubl) structures. Despite the fact that USP7 has emerged as a drug target for cancer therapy, structural details of USP7 regulation and the molecular mechanism of interaction at its CTD have remained elusive. Here, we mapped the binding site between an ICP0 peptide and USP7 and determined the crystal structure of the first three Ubl domains bound to the ICP0 peptide, which showed that ICP0 binds to a loop on Ubl2. Sequences similar to the USP7-binding site in ICP0 were identified in GMPS and UHRF1 and shown to bind USP7-CTD through Ubl2. In addition, co-immunoprecipitation assays in human cells comparing binding to USP7 with and without a Ubl2 mutation, confirmed the importance of the Ubl2 binding pocket for binding ICP0, GMPS and UHRF1. Therefore we have identified a novel mechanism of USP7 recognition that is used by both viral and cellular proteins. Our structural information was used to generate a model of near full-length USP7, showing the relative position of the ICP0/GMPS/UHRF1 binding pocket and the structural basis by which it could regulate enzymatic activity.
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A role for USP7 in DNA replication.
Molecular and cellular biology, 2013Co-Authors: Madhav Jagannathan, Vivian Saridakis, Tin Nguyen, David Gallo, Niharika Luthra, Grant W. Brown, Lori FrappierAbstract:The minichromosome maintenance (MCM) complex, which plays multiple important roles in DNA replication, is loaded onto chromatin following mitosis, remains on chromatin until the completion of DNA synthesis, and then is unloaded by a poorly defined mechanism that involves the MCM binding protein (MCM-BP). Here we show that MCM-BP directly interacts with the ubiquitin-specific protease USP7, that this interaction occurs predominantly on chromatin, and that MCM-BP can tether USP7 to MCM proteins. Detailed biochemical and structure analyses of the USP7-MCM-BP interaction showed that the (155)PSTS(158) MCM-BP sequence mediates critical interactions with the TRAF domain binding pocket of USP7. Analysis of the effects of USP7 knockout on DNA replication revealed that lack of USP7 results in slowed progression through late S phase without globally affecting the fork rate or origin usage. Lack of USP7 also resulted in increased levels of MCM proteins on chromatin, and investigation of the cause of this increase revealed a defect in the dissociation of MCM proteins from chromatin in mid- to late S phase. This role of USP7 mirrors the previously described role for MCM-BP in MCM complex unloading and suggests that USP7 works with MCM-BP to unload MCM complexes from chromatin at the end of S phase.
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the herpesvirus associated ubiquitin specific protease USP7 is a negative regulator of pml proteins and pml nuclear bodies
PLOS ONE, 2011Co-Authors: Feroz Sarkari, Tin Nguyen, Xueqi Wang, Lori FrappierAbstract:The PML tumor suppressor is the founding component of the multiprotein nuclear structures known as PML nuclear bodies (PML-NBs), which control several cellular functions including apoptosis and antiviral effects. The ubiquitin specific protease USP7 (also called HAUSP) is known to associate with PML-NBs and to be a tight binding partner of two herpesvirus proteins that disrupt PML NBs. Here we investigated whether USP7 itself regulates PML-NBs. Silencing of USP7 was found to increase the number of PML-NBs, to increase the levels of PML protein and to inhibit PML polyubiquitylation in nasopharyngeal carcinoma cells. This effect of USP7 was independent of p53 as PML loss was observed in p53-null cells. PML-NBs disruption was induced by USP7 overexpression independently of its catalytic activity and was induced by either of the protein interaction domains of USP7, each of which localized to PML-NBs. USP7 also disrupted NBs formed from some single PML isoforms, most notably isoforms I and IV. CK2α and RNF4, which are known regulators of PML, were dispensable for USP7-associated PML-NB disruption. The results are consistent with a novel model of PML regulation where a deubiquitylase disrupts PML-NBs through recruitment of another cellular protein(s) to PML NBs, independently of its catalytic activity.
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USP7/HAUSP Promotes the Sequence-Specific DNA Binding Activity of p53
PloS one, 2010Co-Authors: Feroz Sarkari, Yi Sheng, Lori FrappierAbstract:The p53 tumor suppressor invokes cellular responses to stressful stimuli by coordinating distinct gene expression programs. This function relies heavily on the ability of p53 to function as a transcription factor by binding promoters of target genes in a sequence specific manner. The DNA binding activity of the core domain of p53 is subject to regulation via post-translational modifications of the C-terminal region. Here we show that the ubiquitin specific protease, USP7 or HAUSP, known to stabilize p53, also regulates the sequence-specific DNA binding mediated by the core domain of p53 in vitro. This regulation is contingent upon interaction between USP7 and the C-terminal regulatory region of p53. However, our data suggest that this effect is not mediated through the N-terminal domain of USP7 previously shown to bind p53, but rather involves the USP7 C-terminal domain and is independent of the deubiquitylation activity of USP7. Consistent with our in vitro observations, we found that overexpression of catalytically inactive USP7 in cells promotes p53 binding to its target sequences and p21 expression, without increasing the levels of p53 protein. We also found that the USP7 C-terminal domain was sufficient for p21 induction. Our results suggest a novel mode of regulation of p53 function by USP7, which is independent of USP7 deubiquitylating activity.
Altaf A. Wani - One of the best experts on this subject based on the ideXlab platform.
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USP7 mediated deubiquitination differentially regulates csb but not uvssa upon uv radiation induced dna damage
Cell Cycle, 2020Co-Authors: Qianzheng Zhu, Gulzar Wani, Nan Ding, Shengcai Wei, Altaf A. WaniAbstract:Cockayne syndrome group B (CSB) protein participates in transcription-coupled nucleotide excision repair. The stability of CSB is known to be regulated by ubiquitin-specific protease 7 (USP7). Yet, whether USP7 acts as a deubiquitinating enzyme for CSB is not clear. Here, we demonstrate that USP7 deubiquitinates CSB to maintain its levels after ultraviolet (UV)-induced DNA damage. While both CSB and UV-stimulated scaffold protein A (UVSSA) exhibit a biphasic decrease and recovery upon UV irradiation, only CSB recovery depends on USP7, which physically interacts with and deubiquitinates CSB. Meanwhile, CSB overexpression stabilizes UVSSA, but decrease UVSSA's presence in nuclease-releasable/soluble chromatin, and increase the presence of ubiquitinated UVSSA in insoluble chromatin alongside CSB-ubiquitin conjugates. Remarkably, CSB overexpression also decreases CSB association with USP7 and UVSSA in soluble chromatin. UVSSA exists in several ubiquitinated forms, of which mono-ubiquitinated form and other ubiquitinated UVSSA forms are detectable upon 6xHistidine tag-based purification. The ubiquitinated UVSSA forms, however, are not cleavable by USP7 in vitro. Furthermore, USP7 disruption does not affect RNA synthesis but decreases the recovery of RNA synthesis following UV exposure. These results reveal a role of USP7 as a CSB deubiquitinating enzyme for fine-tuning the process of TC-NER in human cells.
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USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing RNF168*
Cell cycle (Georgetown Tex.), 2015Co-Authors: Qianzheng Zhu, Nidhi Sharma, Gulzar Wani, Altaf A. WaniAbstract:During DNA damage response (DDR), histone ubiquitination by RNF168 is a critical event, which orchestrates the recruitment of downstream DDR factors, e.g. BRCA1 and 53BP1. Here, we report USP7 deubiquitinase regulates the stability of RNF168. We showed that USP7 disruption impairs H2A and ultraviolet radiation (UVR)-induced γH2AX monoubiquitination, and decreases the levels of pBmi1, Bmi1, RNF168 and BRCA1. The effect of USP7 disruption was recapitulated by siRNA-mediated USP7 depletion. The USP7 disruption also compromises the formation of UVR-induced foci (UVRIF) and ionizing radiation-induced foci (IRIF) of monoubiquitinated H2A (uH2A) and polyubiquitinated H2AX/A, and subsequently affects UVRIF and IRIF of BRCA1 as well as the IRIF of 53BP1. USP7 was shown to physically bind RNF168 in vitro and in vivo. Overexpression of wild-type USP7, but not its interaction-defective mutant, prevents UVR-induced RNF168 degradation. The USP7 mutant is unable to cleave Ub-conjugates of RNF168 in vivo. Importantly, ectopic expression of RNF168, or both RNF8 and RNF168 together in USP7-disrupted cells, significantly rescue the formation of UVRIF and IRIF of polyubiquitinated H2A and BRCA1. Taken together, these findings reveal an important role of USP7 in regulating ubiquitin-dependent signaling via stabilization of RNF168.
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USP7 modulates UV-induced PCNA monoubiquitination by regulating DNA polymerase eta stability
Oncogene, 2014Co-Authors: Jiang Qian, Kyle Pentz, Qi-en Wang, Qianzheng Zhu, Amit Kumar Srivastava, Altaf A. WaniAbstract:DNA polymerase eta (Polη) has unique and pivotal functions in several DNA damage-tolerance pathways. Steady-state level of this short-lived protein is tightly controlled by multiple mechanisms including proteolysis. Here, we have identified the deubiquitinating enzyme (DUB), ubiquitin-specific protease 7 (USP7), as a novel regulator of Polη stability. USP7 regulates Polη stability through both indirect and direct mechanisms. Knockout of USP7 increased the steady-state level of Polη and slowed down the turnover of both Polη and p53 proteins through destabilizing their E3 ligase murine double minute 2 (Mdm2). Also, USP7 physically binds Polη in vitro and in vivo. Overexpression of wild-type USP7 but not its catalytically-defective mutants deubiquitinates Polη and increases its cellular steady-state level. Thus, USP7 directly serves as a specific DUB for Polη. Furthermore, ectopic expression of USP7 promoted the UV-induced proliferating cell nuclear antigen (PCNA) monoubiquitination in Polη-proficient but not in Polη-deficient XPV (Xeroderma pigmentosum variant) cells, suggesting that USP7 facilitates UV-induced PCNA monoubiquitination by stabilizing Polη. Taken together, our findings reveal a modulatory role of USP7 in PCNA ubiquitination-mediated stress-tolerance pathways by fine-tuning Polη turnover.
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Abstract 2391: USP7 deubiquitinates XPC in response to ultraviolet light irradiation
Molecular and Cellular Biology, 2014Co-Authors: Qianzheng Zhu, Jiang Qian, Kyle Pentz, Qi-en Wang, Nidhi Sharma, Gulzar Wani, Chunhua Han, Altaf A. WaniAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ultraviolet light (UV)-induced Xeroderma pigmentosum complementation group C (XPC) protein ubiquitination is mediated by an E3 ubiquitin ligase complex containing UV damaged-DNA binding protein. Here, we report that ubiquitin specific protease 7 (USP7) deubiquitinates XPC during NER. We have demonstrated that transiently compromising cellular USP7, by siRNA or specific inhibitor HBX41108, leads to accumulation of ubiquitinated forms of XPC. However, complete USP7 disruption causes an ubiquitin-mediated XPC degradation upon cellular irradiation. We show that USP7 interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further showed that valosin-containing protein (VCP)/p97 is required for UV-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and co-localizes with XPC. Inhibition of VCP/p97 causes an accumulation of ubiquitinated XPC on DNA damaged chromatin. Moreover, USP7 disruption severely impairs the repair of cyclobutane pyrimidine dimers (CPD) and, to a lesser extent, affects the repair of 6-4 photoproducts (6-4PP). Taken together, our findings have uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis (This work was supported by grants from NIH). Citation Format: Jinshan He, Qianzheng Zhu, Nidhi Sharma, Gulzar Wani, Chunhua Han, Jiang Qian, Kyle Pentz, Qi-en Wang, Altaf A Wani. USP7 deubiquitinates XPC in response to ultraviolet light irradiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2391. doi:10.1158/1538-7445.AM2014-2391
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ubiquitin specific protease 7 regulates nucleotide excision repair through deubiquitinating xpc protein and preventing xpc protein from undergoing ultraviolet light induced and vcp p97 protein regulated proteolysis
Journal of Biological Chemistry, 2014Co-Authors: Qianzheng Zhu, Jiang Qian, Kyle Pentz, Qi-en Wang, Nidhi Sharma, Gulzar Wani, Chunhua Han, Altaf A. WaniAbstract:Ubiquitin specific protease 7 (USP7) is a known deubiquitinating enzyme for tumor suppressor p53 and its downstream regulator, E3 ubiquitin ligase Mdm2. Here we report that USP7 regulates nucleotide excision repair (NER) via deubiquitinating xeroderma pigmentosum complementation group C (XPC) protein, a critical damage recognition factor that binds to helix-distorting DNA lesions and initiates NER. XPC is ubiquitinated during the early stage of NER of UV light-induced DNA lesions. We demonstrate that transiently compromising cellular USP7 by siRNA and chemical inhibition leads to accumulation of ubiquitinated forms of XPC, whereas complete USP7 deficiency leads to rapid ubiquitin-mediated XPC degradation upon UV irradiation. We show that USP7 physically interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces the ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further show that valosin-containing protein (VCP)/p97 is involved in UV light-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and colocalizes with XPC. Chemical inhibition of the activity of VCP/p97 ATPase causes an increase in ubiquitinated XPC on DNA-damaged chromatin. Moreover, USP7 deficiency severely impairs the repair of cyclobutane pyrimidine dimers and, to a lesser extent, affects the repair of 6-4 photoproducts. Taken together, our findings uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis.
Vivian Saridakis - One of the best experts on this subject based on the ideXlab platform.
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Identification of Kaposi Sarcoma Herpesvirus (KSHV) vIRF1 Protein as a Novel Interaction Partner of Human Deubiquitinase USP7
The Journal of biological chemistry, 2016Co-Authors: Sara Chavoshi, Yi Sheng, Olga Egorova, Ira Kay Lacdao, Sahar Farhadi, Vivian SaridakisAbstract:Viral interferon regulatory factor 1 (vIRF1), a Kaposi sarcoma herpesvirus protein, destabilizes p53 by inhibiting p53 acetylation and Hdm2 phosphorylation. This leads to increased ubiquitination and degradation of p53 by Hdm2, which cripples the cellular p53-mediated antiviral response. Ubiquitin-specific protease 7 (USP7) deubiquitinates p53 and Hdm2 and regulates their stability. We identified an EGPS consensus sequence in vIRF1, which is identical to that found in Epstein-Barr virus nuclear antigen 1 (EBNA1) that interacts with the N-terminal domain of USP7 (USP7-NTD). GST pulldown assays demonstrated that vIRF1 interacts with USP7-NTD via its EGPS motif. NMR heteronuclear single quantum correlation (HSQC) analysis revealed chemical perturbations after titration of USP7-NTD with vIRF1 (44)SPGEGPSGTG(53) peptide. In contrast, these perturbations were reduced with a mutant vIRF1 peptide, (44)SPGEGPAGTG(53). Fluorescence polarization analysis indicated that the vIRF1 peptide interacted with USP7-NTD with a Kd of 2.0 μm. The crystal structure of the USP7-NTD·vIRF1 peptide complex revealed an identical mode of binding as that of the EBNA1 peptide to USP7-NTD. We also showed that USP7 interacts with vIRF1 in U2OS cells. Decreased levels of p53, but not Hdm2 or ataxia telangiectasia-mutated (ATM), were seen after expression of vIRF1, but not with a vIRF1 mutant protein. Our results support a new role for vIRF1 through deregulation of the deubiquitinating enzyme USP7 to inhibit p53-mediated antiviral responses.
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crystal structure of USP7 ubiquitin like domains with an icp0 peptide reveals a novel mechanism used by viral and cellular proteins to target USP7
PLOS Pathogens, 2015Co-Authors: Roland Pfoh, Lori Frappier, Ira Kay Lacdao, Anna Georges, Adam Capar, Hong Zheng, Vivian SaridakisAbstract:Herpes simplex virus-1 immediate-early protein ICP0 activates viral genes during early stages of infection, affects cellular levels of multiple host proteins and is crucial for effective lytic infection. Being a RING-type E3 ligase prone to auto-ubiquitination, ICP0 relies on human deubiquitinating enzyme USP7 for protection against 26S proteasomal mediated degradation. USP7 is involved in apoptosis, epigenetics, cell proliferation and is targeted by several herpesviruses. Several USP7 partners, including ICP0, GMPS, and UHRF1, interact through its C-terminal domain (CTD), which contains five ubiquitin-like (Ubl) structures. Despite the fact that USP7 has emerged as a drug target for cancer therapy, structural details of USP7 regulation and the molecular mechanism of interaction at its CTD have remained elusive. Here, we mapped the binding site between an ICP0 peptide and USP7 and determined the crystal structure of the first three Ubl domains bound to the ICP0 peptide, which showed that ICP0 binds to a loop on Ubl2. Sequences similar to the USP7-binding site in ICP0 were identified in GMPS and UHRF1 and shown to bind USP7-CTD through Ubl2. In addition, co-immunoprecipitation assays in human cells comparing binding to USP7 with and without a Ubl2 mutation, confirmed the importance of the Ubl2 binding pocket for binding ICP0, GMPS and UHRF1. Therefore we have identified a novel mechanism of USP7 recognition that is used by both viral and cellular proteins. Our structural information was used to generate a model of near full-length USP7, showing the relative position of the ICP0/GMPS/UHRF1 binding pocket and the structural basis by which it could regulate enzymatic activity.
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Structural-Functional Analysis of USP7
Acta Crystallographica Section A Foundations and Advances, 2014Co-Authors: Vivian SaridakisAbstract:Ubiquitin Specific Protease 7 (USP7) catalyzes the deubiquitination of several substrate proteins including p53, Mdm2 and UbE2E1. Deubiquitination results in their rescue from 26S proteasome mediated degradation thus USP7 is critical in maintaining the cellular steady state levels of its substrates. USP7 is also known to interact with several viral proteins including herpes simplex virus Infected Cell Protein 0 (ICP0), Epstein Barr Nuclear Antigen 1 (EBNA1) and Kaposi's sarcoma-associated viral interferon regulatory factor 4 (vIRF4). USP7 possesses several domains including an N-terminal TRAF, a catalytic and five ubiquitin-like domains. The crystal structures of several complexes revealed that most substrate proteins interact with the N-terminal domain of USP7 which folds into an eight-stranded antiparallel beta sandwich. EBNA1 and MCM-BP also interact with USP7 through its N-terminal domain in an identical manner to its substrates p53, Mdm2 and UbE2E1 however these proteins are not deubiquitinated by USP7. Substrates that interact with USP7 through its N-terminal domain possess a P/AxxS binding motif. The identification, characterization and structure determination of novel USP7 substrates is ongoing.
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A role for USP7 in DNA replication.
Molecular and cellular biology, 2013Co-Authors: Madhav Jagannathan, Vivian Saridakis, Tin Nguyen, David Gallo, Niharika Luthra, Grant W. Brown, Lori FrappierAbstract:The minichromosome maintenance (MCM) complex, which plays multiple important roles in DNA replication, is loaded onto chromatin following mitosis, remains on chromatin until the completion of DNA synthesis, and then is unloaded by a poorly defined mechanism that involves the MCM binding protein (MCM-BP). Here we show that MCM-BP directly interacts with the ubiquitin-specific protease USP7, that this interaction occurs predominantly on chromatin, and that MCM-BP can tether USP7 to MCM proteins. Detailed biochemical and structure analyses of the USP7-MCM-BP interaction showed that the (155)PSTS(158) MCM-BP sequence mediates critical interactions with the TRAF domain binding pocket of USP7. Analysis of the effects of USP7 knockout on DNA replication revealed that lack of USP7 results in slowed progression through late S phase without globally affecting the fork rate or origin usage. Lack of USP7 also resulted in increased levels of MCM proteins on chromatin, and investigation of the cause of this increase revealed a defect in the dissociation of MCM proteins from chromatin in mid- to late S phase. This role of USP7 mirrors the previously described role for MCM-BP in MCM complex unloading and suggests that USP7 works with MCM-BP to unload MCM complexes from chromatin at the end of S phase.
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Further Insight into Substrate Recognition by USP7: Structural and Biochemical Analysis of the HdmX and Hdm2 Interactions with USP7
Journal of molecular biology, 2010Co-Authors: Feroz Sarkari, Lori Frappier, Yi Sheng, Anthony La Delfa, Cheryl H. Arrowsmith, Vivian SaridakisAbstract:Ubiquitin-specific protease 7 (USP7) catalyzes the deubiquitination of several substrate proteins including p53 and Hdm2. We have previously shown that USP7, and more specifically its amino-terminal domain (USP7-NTD), interacts with distinct regions on p53 and Hdm2 containing P/AxxS motifs. The ability of USP7 to also deubiquitinate and control the turnover of HdmX was recently demonstrated. We utilized a combination of biochemistry and structural biology to identify which domain of USP7 interacts with HdmX as well as to identify regions of HdmX that interact with USP7. We showed that USP7-NTD recognized two of six P/AxxS motifs of HdmX ((8)AQCS(11) and (398)AHSS(401)). The crystal structure of the USP7-NTD:HdmX(AHSS) complex was determined providing the molecular basis of complex formation between USP7-NTD and the HdmX(AHSS) peptide. The HdmX peptide interacted within the same residues of USP7-NTD as previously demonstrated with p53, Hdm2, and EBNA1 peptides. We also identified an additional site on Hdm2 ((397)PSTS(400)) that interacts with USP7-NTD and determined the crystal structure of this complex. Finally, analysis of USP7-interacting peptides on filter arrays confirmed the importance of the serine residue at the fourth position for the USP7-NTD interaction and showed that phosphorylation of serines within the binding sequence prevents this interaction. These results lead to a better understanding of the mechanism of substrate recognition by USP7-NTD.
Qianzheng Zhu - One of the best experts on this subject based on the ideXlab platform.
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USP7 mediated deubiquitination differentially regulates csb but not uvssa upon uv radiation induced dna damage
Cell Cycle, 2020Co-Authors: Qianzheng Zhu, Gulzar Wani, Nan Ding, Shengcai Wei, Altaf A. WaniAbstract:Cockayne syndrome group B (CSB) protein participates in transcription-coupled nucleotide excision repair. The stability of CSB is known to be regulated by ubiquitin-specific protease 7 (USP7). Yet, whether USP7 acts as a deubiquitinating enzyme for CSB is not clear. Here, we demonstrate that USP7 deubiquitinates CSB to maintain its levels after ultraviolet (UV)-induced DNA damage. While both CSB and UV-stimulated scaffold protein A (UVSSA) exhibit a biphasic decrease and recovery upon UV irradiation, only CSB recovery depends on USP7, which physically interacts with and deubiquitinates CSB. Meanwhile, CSB overexpression stabilizes UVSSA, but decrease UVSSA's presence in nuclease-releasable/soluble chromatin, and increase the presence of ubiquitinated UVSSA in insoluble chromatin alongside CSB-ubiquitin conjugates. Remarkably, CSB overexpression also decreases CSB association with USP7 and UVSSA in soluble chromatin. UVSSA exists in several ubiquitinated forms, of which mono-ubiquitinated form and other ubiquitinated UVSSA forms are detectable upon 6xHistidine tag-based purification. The ubiquitinated UVSSA forms, however, are not cleavable by USP7 in vitro. Furthermore, USP7 disruption does not affect RNA synthesis but decreases the recovery of RNA synthesis following UV exposure. These results reveal a role of USP7 as a CSB deubiquitinating enzyme for fine-tuning the process of TC-NER in human cells.
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USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing RNF168*
Cell cycle (Georgetown Tex.), 2015Co-Authors: Qianzheng Zhu, Nidhi Sharma, Gulzar Wani, Altaf A. WaniAbstract:During DNA damage response (DDR), histone ubiquitination by RNF168 is a critical event, which orchestrates the recruitment of downstream DDR factors, e.g. BRCA1 and 53BP1. Here, we report USP7 deubiquitinase regulates the stability of RNF168. We showed that USP7 disruption impairs H2A and ultraviolet radiation (UVR)-induced γH2AX monoubiquitination, and decreases the levels of pBmi1, Bmi1, RNF168 and BRCA1. The effect of USP7 disruption was recapitulated by siRNA-mediated USP7 depletion. The USP7 disruption also compromises the formation of UVR-induced foci (UVRIF) and ionizing radiation-induced foci (IRIF) of monoubiquitinated H2A (uH2A) and polyubiquitinated H2AX/A, and subsequently affects UVRIF and IRIF of BRCA1 as well as the IRIF of 53BP1. USP7 was shown to physically bind RNF168 in vitro and in vivo. Overexpression of wild-type USP7, but not its interaction-defective mutant, prevents UVR-induced RNF168 degradation. The USP7 mutant is unable to cleave Ub-conjugates of RNF168 in vivo. Importantly, ectopic expression of RNF168, or both RNF8 and RNF168 together in USP7-disrupted cells, significantly rescue the formation of UVRIF and IRIF of polyubiquitinated H2A and BRCA1. Taken together, these findings reveal an important role of USP7 in regulating ubiquitin-dependent signaling via stabilization of RNF168.
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USP7 modulates UV-induced PCNA monoubiquitination by regulating DNA polymerase eta stability
Oncogene, 2014Co-Authors: Jiang Qian, Kyle Pentz, Qi-en Wang, Qianzheng Zhu, Amit Kumar Srivastava, Altaf A. WaniAbstract:DNA polymerase eta (Polη) has unique and pivotal functions in several DNA damage-tolerance pathways. Steady-state level of this short-lived protein is tightly controlled by multiple mechanisms including proteolysis. Here, we have identified the deubiquitinating enzyme (DUB), ubiquitin-specific protease 7 (USP7), as a novel regulator of Polη stability. USP7 regulates Polη stability through both indirect and direct mechanisms. Knockout of USP7 increased the steady-state level of Polη and slowed down the turnover of both Polη and p53 proteins through destabilizing their E3 ligase murine double minute 2 (Mdm2). Also, USP7 physically binds Polη in vitro and in vivo. Overexpression of wild-type USP7 but not its catalytically-defective mutants deubiquitinates Polη and increases its cellular steady-state level. Thus, USP7 directly serves as a specific DUB for Polη. Furthermore, ectopic expression of USP7 promoted the UV-induced proliferating cell nuclear antigen (PCNA) monoubiquitination in Polη-proficient but not in Polη-deficient XPV (Xeroderma pigmentosum variant) cells, suggesting that USP7 facilitates UV-induced PCNA monoubiquitination by stabilizing Polη. Taken together, our findings reveal a modulatory role of USP7 in PCNA ubiquitination-mediated stress-tolerance pathways by fine-tuning Polη turnover.
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Abstract 2391: USP7 deubiquitinates XPC in response to ultraviolet light irradiation
Molecular and Cellular Biology, 2014Co-Authors: Qianzheng Zhu, Jiang Qian, Kyle Pentz, Qi-en Wang, Nidhi Sharma, Gulzar Wani, Chunhua Han, Altaf A. WaniAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ultraviolet light (UV)-induced Xeroderma pigmentosum complementation group C (XPC) protein ubiquitination is mediated by an E3 ubiquitin ligase complex containing UV damaged-DNA binding protein. Here, we report that ubiquitin specific protease 7 (USP7) deubiquitinates XPC during NER. We have demonstrated that transiently compromising cellular USP7, by siRNA or specific inhibitor HBX41108, leads to accumulation of ubiquitinated forms of XPC. However, complete USP7 disruption causes an ubiquitin-mediated XPC degradation upon cellular irradiation. We show that USP7 interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further showed that valosin-containing protein (VCP)/p97 is required for UV-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and co-localizes with XPC. Inhibition of VCP/p97 causes an accumulation of ubiquitinated XPC on DNA damaged chromatin. Moreover, USP7 disruption severely impairs the repair of cyclobutane pyrimidine dimers (CPD) and, to a lesser extent, affects the repair of 6-4 photoproducts (6-4PP). Taken together, our findings have uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis (This work was supported by grants from NIH). Citation Format: Jinshan He, Qianzheng Zhu, Nidhi Sharma, Gulzar Wani, Chunhua Han, Jiang Qian, Kyle Pentz, Qi-en Wang, Altaf A Wani. USP7 deubiquitinates XPC in response to ultraviolet light irradiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2391. doi:10.1158/1538-7445.AM2014-2391
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ubiquitin specific protease 7 regulates nucleotide excision repair through deubiquitinating xpc protein and preventing xpc protein from undergoing ultraviolet light induced and vcp p97 protein regulated proteolysis
Journal of Biological Chemistry, 2014Co-Authors: Qianzheng Zhu, Jiang Qian, Kyle Pentz, Qi-en Wang, Nidhi Sharma, Gulzar Wani, Chunhua Han, Altaf A. WaniAbstract:Ubiquitin specific protease 7 (USP7) is a known deubiquitinating enzyme for tumor suppressor p53 and its downstream regulator, E3 ubiquitin ligase Mdm2. Here we report that USP7 regulates nucleotide excision repair (NER) via deubiquitinating xeroderma pigmentosum complementation group C (XPC) protein, a critical damage recognition factor that binds to helix-distorting DNA lesions and initiates NER. XPC is ubiquitinated during the early stage of NER of UV light-induced DNA lesions. We demonstrate that transiently compromising cellular USP7 by siRNA and chemical inhibition leads to accumulation of ubiquitinated forms of XPC, whereas complete USP7 deficiency leads to rapid ubiquitin-mediated XPC degradation upon UV irradiation. We show that USP7 physically interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces the ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further show that valosin-containing protein (VCP)/p97 is involved in UV light-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and colocalizes with XPC. Chemical inhibition of the activity of VCP/p97 ATPase causes an increase in ubiquitinated XPC on DNA-damaged chromatin. Moreover, USP7 deficiency severely impairs the repair of cyclobutane pyrimidine dimers and, to a lesser extent, affects the repair of 6-4 photoproducts. Taken together, our findings uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis.
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Identification of Kaposi Sarcoma Herpesvirus (KSHV) vIRF1 Protein as a Novel Interaction Partner of Human Deubiquitinase USP7
The Journal of biological chemistry, 2016Co-Authors: Sara Chavoshi, Yi Sheng, Olga Egorova, Ira Kay Lacdao, Sahar Farhadi, Vivian SaridakisAbstract:Viral interferon regulatory factor 1 (vIRF1), a Kaposi sarcoma herpesvirus protein, destabilizes p53 by inhibiting p53 acetylation and Hdm2 phosphorylation. This leads to increased ubiquitination and degradation of p53 by Hdm2, which cripples the cellular p53-mediated antiviral response. Ubiquitin-specific protease 7 (USP7) deubiquitinates p53 and Hdm2 and regulates their stability. We identified an EGPS consensus sequence in vIRF1, which is identical to that found in Epstein-Barr virus nuclear antigen 1 (EBNA1) that interacts with the N-terminal domain of USP7 (USP7-NTD). GST pulldown assays demonstrated that vIRF1 interacts with USP7-NTD via its EGPS motif. NMR heteronuclear single quantum correlation (HSQC) analysis revealed chemical perturbations after titration of USP7-NTD with vIRF1 (44)SPGEGPSGTG(53) peptide. In contrast, these perturbations were reduced with a mutant vIRF1 peptide, (44)SPGEGPAGTG(53). Fluorescence polarization analysis indicated that the vIRF1 peptide interacted with USP7-NTD with a Kd of 2.0 μm. The crystal structure of the USP7-NTD·vIRF1 peptide complex revealed an identical mode of binding as that of the EBNA1 peptide to USP7-NTD. We also showed that USP7 interacts with vIRF1 in U2OS cells. Decreased levels of p53, but not Hdm2 or ataxia telangiectasia-mutated (ATM), were seen after expression of vIRF1, but not with a vIRF1 mutant protein. Our results support a new role for vIRF1 through deregulation of the deubiquitinating enzyme USP7 to inhibit p53-mediated antiviral responses.
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USP7/HAUSP Promotes the Sequence-Specific DNA Binding Activity of p53
PloS one, 2010Co-Authors: Feroz Sarkari, Yi Sheng, Lori FrappierAbstract:The p53 tumor suppressor invokes cellular responses to stressful stimuli by coordinating distinct gene expression programs. This function relies heavily on the ability of p53 to function as a transcription factor by binding promoters of target genes in a sequence specific manner. The DNA binding activity of the core domain of p53 is subject to regulation via post-translational modifications of the C-terminal region. Here we show that the ubiquitin specific protease, USP7 or HAUSP, known to stabilize p53, also regulates the sequence-specific DNA binding mediated by the core domain of p53 in vitro. This regulation is contingent upon interaction between USP7 and the C-terminal regulatory region of p53. However, our data suggest that this effect is not mediated through the N-terminal domain of USP7 previously shown to bind p53, but rather involves the USP7 C-terminal domain and is independent of the deubiquitylation activity of USP7. Consistent with our in vitro observations, we found that overexpression of catalytically inactive USP7 in cells promotes p53 binding to its target sequences and p21 expression, without increasing the levels of p53 protein. We also found that the USP7 C-terminal domain was sufficient for p21 induction. Our results suggest a novel mode of regulation of p53 function by USP7, which is independent of USP7 deubiquitylating activity.
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USP7 hausp promotes the sequence specific dna binding activity of p53
PLOS ONE, 2010Co-Authors: Feroz Sarkari, Yi Sheng, Lori FrappierAbstract:The p53 tumor suppressor invokes cellular responses to stressful stimuli by coordinating distinct gene expression programs. This function relies heavily on the ability of p53 to function as a transcription factor by binding promoters of target genes in a sequence specific manner. The DNA binding activity of the core domain of p53 is subject to regulation via post-translational modifications of the C-terminal region. Here we show that the ubiquitin specific protease, USP7 or HAUSP, known to stabilize p53, also regulates the sequence-specific DNA binding mediated by the core domain of p53 in vitro. This regulation is contingent upon interaction between USP7 and the C-terminal regulatory region of p53. However, our data suggest that this effect is not mediated through the N-terminal domain of USP7 previously shown to bind p53, but rather involves the USP7 C-terminal domain and is independent of the deubiquitylation activity of USP7. Consistent with our in vitro observations, we found that overexpression of catalytically inactive USP7 in cells promotes p53 binding to its target sequences and p21 expression, without increasing the levels of p53 protein. We also found that the USP7 C-terminal domain was sufficient for p21 induction. Our results suggest a novel mode of regulation of p53 function by USP7, which is independent of USP7 deubiquitylating activity.
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Further Insight into Substrate Recognition by USP7: Structural and Biochemical Analysis of the HdmX and Hdm2 Interactions with USP7
Journal of molecular biology, 2010Co-Authors: Feroz Sarkari, Lori Frappier, Yi Sheng, Anthony La Delfa, Cheryl H. Arrowsmith, Vivian SaridakisAbstract:Ubiquitin-specific protease 7 (USP7) catalyzes the deubiquitination of several substrate proteins including p53 and Hdm2. We have previously shown that USP7, and more specifically its amino-terminal domain (USP7-NTD), interacts with distinct regions on p53 and Hdm2 containing P/AxxS motifs. The ability of USP7 to also deubiquitinate and control the turnover of HdmX was recently demonstrated. We utilized a combination of biochemistry and structural biology to identify which domain of USP7 interacts with HdmX as well as to identify regions of HdmX that interact with USP7. We showed that USP7-NTD recognized two of six P/AxxS motifs of HdmX ((8)AQCS(11) and (398)AHSS(401)). The crystal structure of the USP7-NTD:HdmX(AHSS) complex was determined providing the molecular basis of complex formation between USP7-NTD and the HdmX(AHSS) peptide. The HdmX peptide interacted within the same residues of USP7-NTD as previously demonstrated with p53, Hdm2, and EBNA1 peptides. We also identified an additional site on Hdm2 ((397)PSTS(400)) that interacts with USP7-NTD and determined the crystal structure of this complex. Finally, analysis of USP7-interacting peptides on filter arrays confirmed the importance of the serine residue at the fourth position for the USP7-NTD interaction and showed that phosphorylation of serines within the binding sequence prevents this interaction. These results lead to a better understanding of the mechanism of substrate recognition by USP7-NTD.
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ebna1 mediated recruitment of a histone h2b deubiquitylating complex to the epstein barr virus latent origin of dna replication
PLOS Pathogens, 2009Co-Authors: Feroz Sarkari, Teresa Sanchezalcaraz, Melissa N Holowaty, Shan Wang, Yi Sheng, Lori FrappierAbstract:The EBNA1 protein of Epstein-Barr virus (EBV) plays essential roles in enabling the replication and persistence of EBV genomes in latently infected cells and activating EBV latent gene expression, in all cases by binding to specific recognition sites in the latent origin of replication, oriP. Here we show that EBNA1 binding to its recognition sites in vitro is greatly stimulated by binding to the cellular deubiquitylating enzyme, USP7, and that USP7 can form a ternary complex with DNA-bound EBNA1. Consistent with the in vitro effects, the assembly of EBNA1 on oriP elements in human cells was decreased by USP7 silencing, whereas assembly of an EBNA1 mutant defective in USP7 binding was unaffected. USP7 affinity column profiling identified a complex between USP7 and human GMP synthetase (GMPS), which was shown to stimulate the ability of USP7 to cleave monoubiquitin from histone H2B in vitro. Accordingly, silencing of USP7 in human cells resulted in a consistent increase in the level of monoubquitylated H2B. The USP7-GMPS complex formed a quaternary complex with DNA-bound EBNA1 in vitro and, in EBV infected cells, was preferentially detected at the oriP functional element, FR, along with EBNA1. Down-regulation of USP7 reduced the level of GMPS at the FR, increased the level of monoubiquitylated H2B in this region of the origin and decreased the ability of EBNA1, but not an EBNA1 USP7-binding mutant, to activate transcription from the FR. The results indicate that USP7 can stimulate EBNA1-DNA interactions and that EBNA1 can alter histone modification at oriP through recruitment of USP7.
