RESUMEN
The linear ubiquitin chain assembly complex (LUBAC) regulates immune signaling, and its function is regulated by the deubiquitinases OTULIN and CYLD, which associate with the catalytic subunit HOIP. However, the mechanism through which CYLD interacts with HOIP is unclear. We here show that CYLD interacts with HOIP via spermatogenesis-associated protein 2 (SPATA2). SPATA2 interacts with CYLD through its non-canonical PUB domain, which binds the catalytic CYLD USP domain in a CYLD B-box-dependent manner. Significantly, SPATA2 binding activates CYLD-mediated hydrolysis of ubiquitin chains. SPATA2 also harbors a conserved PUB-interacting motif that selectively docks into the HOIP PUB domain. In cells, SPATA2 is recruited to the TNF receptor 1 signaling complex and is required for CYLD recruitment. Loss of SPATA2 increases ubiquitination of LUBAC substrates and results in enhanced NOD2 signaling. Our data reveal SPATA2 as a high-affinity binding partner of CYLD and HOIP, and a regulatory component of LUBAC-mediated NF-κB signaling.
Asunto(s)
FN-kappa B/química , Proteínas/química , Proteínas Supresoras de Tumor/química , Ubiquitina-Proteína Ligasas/química , Ubiquitina/química , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Enzima Desubiquitinante CYLD , Endopeptidasas/química , Endopeptidasas/genética , Endopeptidasas/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunidad Innata , Cinética , Simulación del Acoplamiento Molecular , FN-kappa B/genética , FN-kappa B/inmunología , Proteína Adaptadora de Señalización NOD2/química , Proteína Adaptadora de Señalización NOD2/genética , Proteína Adaptadora de Señalización NOD2/inmunología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas/genética , Proteínas/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Especificidad por Sustrato , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/inmunología , Ubiquitina/genética , Ubiquitina/inmunología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/inmunologíaRESUMEN
The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is a sequence-specific DNA-binding protein that plays an essential role in viral episome replication and segregation, by recruiting the cellular complex of DNA replication onto the origin (oriP) and by tethering the viral DNA onto the mitotic chromosomes. Whereas the mechanisms of viral DNA replication are well documented, those involved in tethering EBNA1 to the cellular chromatin are far from being understood. Here, we have identified regulator of chromosome condensation 1 (RCC1) as a novel cellular partner for EBNA1. RCC1 is the major nuclear guanine nucleotide exchange factor for the small GTPase Ran enzyme. RCC1, associated with chromatin, is involved in the formation of RanGTP gradients critical for nucleo-cytoplasmic transport, mitotic spindle formation and nuclear envelope reassembly following mitosis. Using several approaches, we have demonstrated a direct interaction between these two proteins and found that the EBNA1 domains responsible for EBNA1 tethering to the mitotic chromosomes are also involved in the interaction with RCC1. The use of an EBNA1 peptide array confirmed the interaction of RCC1 with these regions and also the importance of the N-terminal region of RCC1 in this interaction. Finally, using confocal microscopy and Förster resonance energy transfer analysis to follow the dynamics of interaction between the two proteins throughout the cell cycle, we have demonstrated that EBNA1 and RCC1 closely associate on the chromosomes during metaphase, suggesting an essential role for the interaction during this phase, perhaps in tethering EBNA1 to mitotic chromosomes.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Mitosis , Proteínas Nucleares/metabolismo , Dominios y Motivos de Interacción de Proteínas , Secuencias de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Cromosomas Humanos/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/química , Antígenos Nucleares del Virus de Epstein-Barr/genética , Transferencia Resonante de Energía de Fluorescencia , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Células HeLa , Humanos , Metafase , Microscopía Confocal , Proteínas Nucleares/química , Proteínas Nucleares/genética , Análisis por Matrices de Proteínas , Mapeo de Interacción de Proteínas , Huso Acromático/metabolismoRESUMEN
Non-degradative ubiquitin chains and phosphorylation events govern signaling responses by innate immune receptors. The deubiquitinase CYLD in complex with SPATA2 is recruited to receptor signaling complexes by the ubiquitin ligase LUBAC and regulates Met1- and Lys63-linked polyubiquitin and receptor signaling outcomes. Here, we investigate the molecular determinants of CYLD activity. We reveal that two CAP-Gly domains in CYLD are ubiquitin-binding domains and demonstrate a requirement of CAP-Gly3 for CYLD activity and regulation of immune receptor signaling. Moreover, we identify a phosphorylation switch outside of the catalytic USP domain, which activates CYLD toward Lys63-linked polyubiquitin. The phosphorylated residue Ser568 is a novel tumor necrosis factor (TNF)-regulated phosphorylation site in CYLD and works in concert with Ser418 to enable CYLD-mediated deubiquitination and immune receptor signaling. We propose that phosphorylated CYLD, together with SPATA2 and LUBAC, functions as a ubiquitin-editing complex that balances Lys63- and Met1-linked polyubiquitin at receptor signaling complexes to promote LUBAC signaling.
Asunto(s)
Enzima Desubiquitinante CYLD/metabolismo , Línea Celular Tumoral , Cristalografía por Rayos X , Enzima Desubiquitinante CYLD/antagonistas & inhibidores , Enzima Desubiquitinante CYLD/genética , Endopeptidasas/química , Endopeptidasas/genética , Endopeptidasas/metabolismo , Humanos , Proteína Adaptadora de Señalización NOD2/genética , Proteína Adaptadora de Señalización NOD2/metabolismo , Fosforilación , Poliubiquitina/metabolismo , Unión Proteica , Dominios Proteicos , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitina/metabolismoRESUMEN
Innate immune signaling relies on the deposition of non-degradative polyubiquitin at receptor-signaling complexes, but how these ubiquitin modifications are regulated by deubiquitinases remains incompletely understood. Met1-linked ubiquitin (Met1-Ub) is assembled by the linear ubiquitin assembly complex (LUBAC), and this is counteracted by the Met1-Ub-specific deubiquitinase OTULIN, which binds to the catalytic LUBAC subunit HOIP. In this study, we report that HOIP also interacts with the deubiquitinase CYLD but that CYLD does not regulate ubiquitination of LUBAC components. Instead, CYLD limits extension of Lys63-Ub and Met1-Ub conjugated to RIPK2 to restrict signaling and cytokine production. Accordingly, Met1-Ub and Lys63-Ub were individually required for productive NOD2 signaling. Our study thus suggests that LUBAC, through its associated deubiquitinases, coordinates the deposition of not only Met1-Ub but also Lys63-Ub to ensure an appropriate response to innate immune receptor activation.