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
Intracellular nucleotide binding and oligomerization domain (NOD) receptors recognize antigens including bacterial peptidoglycans and initiate immune responses by triggering the production of pro-inflammatory cytokines through activating NF-κB and MAP kinases. Receptor interacting protein kinase 2 (RIPK2) is critical for NOD-mediated NF-κB activation and cytokine production. Here we develop and characterize a selective RIPK2 kinase inhibitor, WEHI-345, which delays RIPK2 ubiquitylation and NF-κB activation downstream of NOD engagement. Despite only delaying NF-κB activation on NOD stimulation, WEHI-345 prevents cytokine production in vitro and in vivo and ameliorates experimental autoimmune encephalomyelitis in mice. Our study highlights the importance of the kinase activity of RIPK2 for proper immune responses and demonstrates the therapeutic potential of inhibiting RIPK2 in NOD-driven inflammatory diseases.
Asunto(s)
Citocinas/metabolismo , Inflamación/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Adenosina Trifosfato/química , Animales , Cromatografía Liquida , Encefalomielitis Autoinmune Experimental/genética , Femenino , Humanos , Sistema Inmunológico , Concentración 50 Inhibidora , Interferón gamma/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , FN-kappa B/metabolismo , Unión Proteica , Conformación Proteica , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/antagonistas & inhibidores , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Transducción de Señal , Espectrometría de Masas en Tándem , Ubiquitina/metabolismoRESUMEN
N-terminal methionine-linked ubiquitin (Met1-Ub), or linear ubiquitin, has emerged as a central post-translational modification in innate immune signalling. The molecular machinery that assembles, senses and, more recently, disassembles Met1-Ub has been identified, and technical advances have enabled the identification of physiological substrates for Met1-Ub in response to activation of innate immune receptors. These discoveries have significantly advanced our understanding of how nondegradative ubiquitin modifications control proinflammatory responses mediated by nuclear factor-κB and mitogen-activated protein kinases. In this review, we discuss the current data on Met1-Ub function and regulation, and point to some of the questions that still remain unanswered.