Asunto(s)
Antirreumáticos/farmacología , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Agammaglobulinemia Tirosina Quinasa , Secuencia de Aminoácidos , Animales , Antirreumáticos/química , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/enzimología , Sitios de Unión , Modelos Animales de Enfermedad , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/metabolismo , Roedores , Relación Estructura-ActividadAsunto(s)
Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/aislamiento & purificación , Animales , Liasas de Carbono-Nitrógeno/metabolismo , Cisteína Endopeptidasas/aislamiento & purificación , Endopeptidasas/aislamiento & purificación , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismoRESUMEN
SUMO, or Smt3 in Saccharomyces cerevisiae, is a ubiquitin-like protein that is post-translationally attached to multiple proteins in vivo. Many of these substrate modifications are cell cycle-regulated, and SUMO conjugation is essential for viability in most eukaryotes. However, only a limited number of SUMO-modified proteins have been definitively identified to date, and this has hampered study of the mechanisms by which SUMO ligation regulates specific cellular pathways. Here we use a combination of yeast two-hybrid screening, a high copy suppressor selection with a SUMO isopeptidase mutant, and tandem mass spectrometry to define a large set of proteins (>150) that can be modified by SUMO in budding yeast. These three approaches yielded overlapping sets of proteins with the most extensive set by far being those identified by mass spectrometry. The two-hybrid data also yielded a potential SUMO-binding motif. Functional categories of SUMO-modified proteins include SUMO conjugation system enzymes, chromatin- and gene silencing-related factors, DNA repair and genome stability proteins, stress-related proteins, transcription factors, proteins involved in translation and RNA metabolism, and a variety of metabolic enzymes. The results point to a surprisingly broad array of cellular processes regulated by SUMO conjugation and provide a starting point for detailed studies of how SUMO ligation contributes to these different regulatory mechanisms.
Asunto(s)
Proteómica/métodos , Proteína SUMO-1/química , Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Núcleo Celular/metabolismo , Reparación del ADN , Genotipo , Espectrometría de Masas , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteoma , Proteínas Recombinantes de Fusión/química , Proteínas de Saccharomyces cerevisiae/química , Homología de Secuencia de Aminoácido , Programas Informáticos , Temperatura , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismoRESUMEN
Protein modification by the ubiquitin-like SUMO protein contributes to many cellular regulatory mechanisms. In Saccharomyces cerevisiae, both sumoylating and desumoylating activities are essential for viability. Of its two known desumoylating enzymes, Ubl-specific protease (Ulp)1 and Ulp2/Smt4, Ulp1 is specifically required for cell cycle progression. A approximately 200-residue segment, the Ulp domain (UD), is conserved among Ulps and includes a core cysteine protease domain that is even more widespread. Here we demonstrate that the Ulp1 UD by itself can support wild-type growth rates and in vitro can cleave SUMO from substrates. However, in cells expressing only the UD of Ulp1, many SUMO conjugates accumulate to high levels, indicating that the nonessential Ulp1 NH2-terminal domain is important for activity against a substantial fraction of sumoylated targets. The NH2-terminal domain also includes sequences necessary and sufficient to concentrate Ulp1 at nuclear envelope sites. Remarkably, NH2-terminally deleted Ulp1 variants are able, unlike full-length Ulp1, to suppress defects of cells lacking the divergent Ulp2 isopeptidase. Thus, the NH2-terminal regulatory domain of Ulp1 restricts Ulp1 activity toward certain sumoylated proteins while enabling the cleavage of others. These data define key functional elements of Ulp1 and strongly suggest that subcellular localization is a physiologically significant constraint on SUMO isopeptidase specificity.