RESUMEN
Protein ubiquitination is a key post-translational modification that regulates diverse cellular processes in eukaryotic cells. The specificity of ubiquitin (Ub) signalling for different bioprocesses and pathways is dictated by the large variety of mono-ubiquitination and polyubiquitination events, including many possible chain architectures. Deubiquitinases (DUBs) reverse or edit Ub signals with high sophistication and specificity, forming an integral arm of the Ub signalling machinery, thus impinging on fundamental cellular processes including DNA damage repair, gene expression, protein quality control and organellar integrity. In this review, we discuss the many layers of DUB function and regulation, with a focus on insights gained from budding yeast. Our review provides a framework to understand key aspects of DUB biology.
Asunto(s)
Enzimas Desubicuitinizantes/química , Enzimas Desubicuitinizantes/metabolismo , Saccharomycetales/enzimología , Ciclo Celular , Segregación Cromosómica , Enzimas Desubicuitinizantes/genética , Exoftalmia , Regulación de la Expresión Génica , Familia de Multigenes , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteolisis , Proteostasis , Saccharomycetales/genética , Transducción de Señal , Relación Estructura-Actividad , Especificidad por Sustrato , Ubiquitina/química , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
We applied a yeast-two-hybrid (Y2H) analysis to screen for ubiquitin variant (UbV) inhibitors of a human deubiquitinase (DUB), ubiquitin-specific protease 2 (USP2). The Y2H screen used USP2 as the bait and a prey library consisting of UbVs randomized at four specific positions, which were known to interact with USP2 from phage display analysis. The screen yielded numerous UbVs that bound to USP2 both as a Y2H interaction in vivo and as purified proteins in vitro. The Y2H-derived UbVs inhibited the catalytic activity of USP2 in vitro with nanomolar-range potencies, and they bound and inhibited USP2 in human cells. Mutational and structural analysis showed that potent and selective inhibition could be achieved by just two substitutions in a UbV, which exhibited improved hydrophobic and hydrophilic contacts compared to the wild-type ubiquitin interaction with USP2. Our results establish Y2H as an effective platform for the development of UbV inhibitors of DUBs in vivo, providing an alternative strategy for the analysis of DUBs that are recalcitrant to phage display and other in vitro methods.
Asunto(s)
Enzimas Desubicuitinizantes/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina/metabolismo , Enzimas Desubicuitinizantes/antagonistas & inhibidores , Células HEK293 , Humanos , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Técnicas del Sistema de Dos Híbridos , Ubiquitina Tiolesterasa/antagonistas & inhibidoresRESUMEN
We generated a global genetic interaction network for Saccharomyces cerevisiae, constructing more than 23 million double mutants, identifying about 550,000 negative and about 350,000 positive genetic interactions. This comprehensive network maps genetic interactions for essential gene pairs, highlighting essential genes as densely connected hubs. Genetic interaction profiles enabled assembly of a hierarchical model of cell function, including modules corresponding to protein complexes and pathways, biological processes, and cellular compartments. Negative interactions connected functionally related genes, mapped core bioprocesses, and identified pleiotropic genes, whereas positive interactions often mapped general regulatory connections among gene pairs, rather than shared functionality. The global network illustrates how coherent sets of genetic interactions connect protein complex and pathway modules to map a functional wiring diagram of the cell.