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[This corrects the article DOI: 10.1371/journal.pgen.1003007.].
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Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with-and regulation of its ubiquitination and stability by-RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5(-/-) MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5(-/-) using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5(-/-) mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5(-/-) macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.
Asunto(s)
Autofagia , Infecciones Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Infecciones Bacterianas/genética , Infecciones Bacterianas/mortalidad , Caenorhabditis elegans/metabolismo , Línea Celular , Membrana Celular/metabolismo , Estabilidad de Enzimas , Predisposición Genética a la Enfermedad , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Fagosomas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Transporte de Proteínas , Proteolisis , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
BACKGROUND: Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation. METHODS #ENTITYSTARTX00026; FINDINGS: We describe here a versatile system for reconstituting protease activation networks in yeast and assaying the activity of these pathways using a cleavable transcription factor substrate in conjunction with reporter gene read-outs. The utility of these versatile assay components and their application for screening strategies was validated for all ten human Caspases, a family of intracellular proteases involved in cell death and inflammation, including implementation of assays for high throughput screening (HTS) of chemical libraries and functional screening of cDNA libraries. The versatility of the technology was also demonstrated for human autophagins, cysteine proteases involved in autophagy. CONCLUSIONS: Altogether, the yeast-based systems described here for monitoring activity of ectopically expressed mammalian proteases provide a fascile platform for functional genomics and chemical library screening.