RESUMO
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.
Assuntos
Herpesvirus Humano 8 , Fatores Reguladores de Interferon/química , Ubiquitina Tiolesterase/química , Proteínas Virais/química , Motivos de Aminoácidos , Domínio Catalítico , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Fatores Reguladores de Interferon/fisiologia , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/fisiologia , Peptidase 7 Específica de Ubiquitina , Proteínas Virais/fisiologiaRESUMO
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.
Assuntos
Infecções por Herpesviridae/metabolismo , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/metabolismo , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Sítios de Ligação/fisiologia , Western Blotting , Cristalização , Células HEK293 , Humanos , Imunoprecipitação , Ligação Proteica/fisiologia , Conformação Proteica , Transfecção , Peptidase 7 Específica de UbiquitinaRESUMO
USP7 is a deubiquitinase that regulates many diverse cellular processes, including tumor suppression, epigenetics, and genome stability. Several substrates, including GMPS, UHRF1, and ICP0, were shown to bear a specific KxxxK motif that interacts within the C-terminal region of USP7. We identified a similar motif in Enhancer of Zeste 2 (EZH2), the histone methyltransferase found within Polycomb Repressive Complex 2 (PRC2). PRC2 is responsible for the methylation of Histone 3 Lys27 (H3K27) leading to gene silencing. GST pull-down and coimmunoprecipitation experiments showed that USP7 interacts with EZH2. We determined the structural basis of interaction between USP7 and EZH2 and identified residues mediating the interaction. Mutations in these critical residues disrupted the interaction between USP7 and EZH2. Furthermore, USP7 silencing and knockout experiments showed decreased EZH2 levels in HCT116 carcinoma cells. Finally, we demonstrated decreased H3K27Me3 levels in HCT116 USP7 knockout cells. These results indicate that USP7 interacts with EZH2 and regulates both its stability and function.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Inativação Gênica/fisiologia , Células HCT116 , Humanos , Imunoprecipitação , Complexo Repressor Polycomb 2/genética , Estabilidade Proteica , Peptidase 7 Específica de Ubiquitina/genética , Ubiquitinação/genética , Ubiquitinação/fisiologiaRESUMO
Deubiquitinases (DUBs) play important roles and therefore are potential drug targets in various diseases including cancer and neurodegeneration. In this review, we recapitulate structure-function studies of the most studied DUBs including USP7, USP22, CYLD, UCHL1, BAP1, A20, as well as ataxin 3 and connect them to regulatory mechanisms and their growing protein interaction networks. We then describe DUBs that have been associated with endocrine carcinogenesis with a focus on prostate, ovarian, and thyroid cancer, pheochromocytoma, and adrenocortical carcinoma. The goal is enhancing our understanding of the connection between dysregulated DUBs and cancer to permit the design of therapeutics and to establish biomarkers that could be used in diagnosis and prognosis.