Crystal Structure of USP7 Ubiquitin-like Domains with an ICP0 Peptide Reveals a Novel Mechanism Used by Viral and Cellular Proteins to Target USP7

Pfoh, Roland; Lacdao, Ira Kay; Georges, Anna; Capar, Adam; Zheng, Hong; Frappier, Lori; Saridakis, V.

doi:10.1371/journal.ppat.1004950

Cited by 58 publications

(63 citation statements)

References 55 publications

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“…There has been a surge of interest in elucidating the complex structures of the USP7 UBL domain with its interacting proteins (39,(41)(42)(43)(44). Among them, two crystal structures detailing USP7 UBL1-3 domains with peptides derived from the proteins ICP0 and UHRF1 were recently reported (39,42). In these structures, the two lysine residues from ICP0 and UHRF1 peptides were anchored on the acidic patch across the surface of the UBL1 and UBL2 domains via hydrogen bonds and electrostatic attractions, undertaking major tasks for their mutual recognition.…”

Section: Discussionmentioning

confidence: 99%

Dual-utility NLS drives RNF169-dependent DNA damage responses

Jiang

et al. 2017

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

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Loading of p53-binding protein 1 (53BP1) and receptor-associated protein 80 (RAP80) at DNA double-strand breaks (DSBs) drives cell cycle checkpoint activation but is counterproductive to high-fidelity DNA repair. ring finger protein 169 (RNF169) maintains the balance by limiting the deposition of DNA damage mediator proteins at the damaged chromatin. We report here that this attribute is accomplished, in part, by a predicted nuclear localization signal (NLS) that not only shuttles RNF169 into the nucleus but also promotes its stability by mediating a direct interaction with the ubiquitin-specific protease USP7. Guided by the crystal structure of USP7 in complex with the RNF169 NLS, we uncoupled USP7 binding from its nuclear import function and showed that perturbing the USP7-RNF169 complex destabilized RNF169, compromised high-fidelity DSB repair, and hypersensitized cells to poly (ADP-ribose) polymerase inhibition. Finally, expression of USP7 and RNF169 positively correlated in breast cancer specimens. Collectively, our findings uncover an NLS-mediated bipartite mechanism that supports the nuclear function of a DSB response protein.ells respond to DNA double-strand breaks (DSBs) by mounting a series of signal transduction events that culminate in cell arrest and DNA repair (1). Signal transduction entails the coordinated assembly of a cohort of DNA damage mediator proteins, including p53-binding protein 1 (53BP1) and receptor-associated protein 80 (RAP80), at the damaged chromatin, which, in turn, enforces checkpoint control and cell tolerance to DNA damage (2, 3). Paradoxically, although DSB loading of 53BP1 and RAP80 underlies robust activation of DNA damage responses (DDRs), they operate at the expense of high-fidelity DNA repair, because their productive accumulation at DSB-flanking chromatin blocks DNA end resection and suppresses RAD51-dependent homologous recombination (HR) repair (4-11). Exactly how cells achieve a balance between robust DSB signal transduction and HR repair remains to be defined, but several lines of evidence converge on the idea that limiting the extent of DSB ubiquitylation may selectively promote HR repair (12-15), highlighting the need for cell-intrinsic mechanisms to restrain chromatin responses arising from DSBs (16).Intriguingly, one such cell-intrinsic strategy involves ring finger protein 169 (RNF169), a paralog of the RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties) syndrome protein RNF168 (17,18). In stark contrast to RNF168, which plays positive roles in amplifying ubiquitin-dependent DSB signals (18,19), RNF169 is endowed with antagonistic properties in the DSB signal transduction pathway. Indeed, RNF169 competes for RNF168-catalyzed ubiquitin adducts and displaces 53BP1 and RAP80 from DSBs (14,20,21). Despite its putative role as an integral component of the DSB signal transduction cascade, how RNF169 is regulated mechanistically and how it contributes to the multipartite self-restraining mechanisms in DNA damage surveillance and...

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Section: Discussionmentioning

confidence: 99%

Dual-utility NLS drives RNF169-dependent DNA damage responses

Jiang

et al. 2017

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

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“…Members of the herpesvirus family such as herpes simplex virus type 1, Epstein-Barr virus, Kaposi sarcoma herpesvirus, and cytomegalovirus have evolved to interfere with the USP7-p53-Hdm2 pathway by competitively binding to USP7 and hindering its interaction with cellular substrates or binding proteins (14,15,25,34,35,41). HSV-1 ICP0 is an E3 ubiquitin ligase that ubiquitinates and causes degradation of host proteins (42).…”

Section: Discussionmentioning

confidence: 99%

“…His-tagged USP7-CTD was expressed from p15TV-L vector (25). Fulllength His-USP7 in pFastBac was expressed in Spodoptera frugiperda (Sf9) cells as described previously (26).…”

Section: Methodsmentioning

confidence: 99%

Identification of Kaposi Sarcoma Herpesvirus (KSHV) vIRF1 Protein as a Novel Interaction Partner of Human Deubiquitinase USP7

Chavoshi

Egorova

Lacdao

et al. 2016

Journal of Biological Chemistry

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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 SPG-EGPAGTG 53 . Fluorescence polarization analysis indicated that the vIRF1 peptide interacted with USP7-NTD with a K d of 2.0 M. The crystal structure of the USP7-NTD⅐vIRF1 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. Human herpesviruses (HHVs)2 are double-stranded DNA viruses, classified into ␣, ␤, or ␥ subfamilies (1). To establish latency, HHVs suppress the host immune response to evade the immune system (2). In the latent state, herpesviruses express only a small number of proteins essential for suppressing the host immune system (2). HHVs have evolved various mechanisms for host immune evasion including inhibition of cellular senescence and apoptosis as well as promoting cell proliferation (3). Another strategy to evade host immune surveillance is through expression of viral homologues of genes that are the host's first line of defense against viral infection such as interferons and interferon regulatory factors (IRFs) and therefore sabotage the function and regulation of the cellular proteins (4). Deregulation of cellular proteins involved in growth control by HHVs has led to recognition of some HHVs as underlying agents of cancer (3, 5).Kaposi sarcoma herpesvirus (KSHV), HHV-8, is the causative agent of Kaposi sarcoma, primary effusion lymphoma, and multicentric Castleman disease, which are especially prevalent in immunocompromised patients (4, 6). The KSHV vIRF1 protein is encoded by ORF K9 and is believed to have been acquired through molecular piracy (7). vIRF1 contains two domains, an N-terminal DNA binding domain (DBD) and a C-terminal IRF interaction domain (see Fig. 1A) (8). The vIRF1 DBD has ϳ40% sequence similarity to the DBDs of human IRF3 and IRF7 and contains a helix-turn-helix motif, which is common in I...

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“…Some host USPs (with a fold similar to the CoV PL pro ) also include one or more Ub-like domain(s), which is/are used to regulate the catalytic activity as well as to interact with partners (Komander et al, 2009;Faesen et al, 2012;Pfoh et al, 2015). For example, the Nterminal Ubl domain of USP14 is critical for its recruitment at the proteasome, thereby enhancing its catalytic activity (Hu et al, 2005;Faesen et al, 2012).…”

Section: Ubiqutin-like Domain 2 (Ubl2)mentioning

confidence: 99%

Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein

2018

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The multi-domain non-structural protein 3 (Nsp3) is the largest protein encoded by the coronavirus (CoV) genome, with an average molecular mass of about 200 kD. Nsp3 is an essential component of the replication/transcription complex. It comprises various domains, the organization of which differs between CoV genera, due to duplication or absence of some domains. However, eight domains of Nsp3 exist in all known CoVs: the ubiquitin-like domain 1 (Ubl1), the Glu-rich acidic domain (also called "hypervariable region"), a macrodomain (also named "X domain"), the ubiquitin-like domain 2 (Ubl2), the papain-like protease 2 (PL2), the Nsp3 ectodomain (3Ecto, also called "zinc-finger domain"), as well as the domains Y1 and CoV-Y of unknown functions. In addition, the two transmembrane regions, TM1 and TM2, exist in all CoVs. The three-dimensional structures of domains in the N-terminal two thirds of Nsp3 have been investigated by X-ray crystallography and/or nuclear magnetic resonance (NMR) spectroscopy since the outbreaks of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) in 2003 as well as Middle-East Respiratory Syndrome coronavirus (MERS-CoV) in 2012. In this review, the structures and functions of these domains of Nsp3 are discussed in depth.

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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

Cited by 58 publications

References 55 publications

Dual-utility NLS drives RNF169-dependent DNA damage responses

Dual-utility NLS drives RNF169-dependent DNA damage responses

Identification of Kaposi Sarcoma Herpesvirus (KSHV) vIRF1 Protein as a Novel Interaction Partner of Human Deubiquitinase USP7

Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein

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