RESUMEN
The accumulation of serpin oligomers and polymers within the endoplasmic reticulum (ER) causes cellular injury in patients with the classical form α1-antitrypsin deficiency (ATD). To better understand the cellular and molecular genetic aspects of this disorder, we generated transgenic C. elegans strains expressing either the wild-type (ATM) or Z mutant form (ATZ) of the human serpin fused to GFP. Animals secreted ATM, but retained polymerized ATZ within dilated ER cisternae. These latter animals also showed slow growth, smaller brood sizes and decreased longevity; phenotypes observed in ATD patients or transgenic mouse lines expressing ATZ. Similar to mammalian models, ATZ was disposed of by autophagy and ER-associated degradation pathways. Mutant strains defective in insulin signaling (daf-2) also showed a marked decrease in ATZ accumulation. Enhanced ATZ turnover was associated with the activity of two proteins central to systemic/exogenous (exo)-RNAi pathway: the dsRNA importer, SID-1 and the argonaute, RDE-1. Animals with enhanced exo-RNAi activity (rrf-3 mutant) phenocopied the insulin signaling mutants and also showed increased ATZ turnover. Taken together, these studies allude to the existence of a novel proteostasis pathway that mechanistically links misfolded protein turnover to components of the systemic RNAi machinery.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Interferencia de ARN , Deficiencia de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , Animales , Animales Modificados Genéticamente , Proteínas de Caenorhabditis elegans/genética , Línea Celular , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Degradación Asociada con el Retículo Endoplásmico , Expresión Génica , Genes Reporteros , Humanos , Insulina/metabolismo , Ratones , Ratones Transgénicos , Mutación , Fenotipo , Regiones Promotoras Genéticas , Proteolisis , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serpinas , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/genética , alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/metabolismoRESUMEN
As an experimental system, Caenorhabditis elegans offers a unique opportunity to interrogate in vivo the genetic and molecular functions of human disease-related genes. For example, C. elegans has provided crucial insights into fundamental biologic processes, such as cell death and cell fate determinations, as well as pathologic processes such as neurodegeneration and microbial susceptibility. The C. elegans model has several distinct advantages, including a completely sequenced genome that shares extensive homology with that of mammals, ease of cultivation and storage, a relatively short lifespan and techniques for generating null and transgenic animals. However, the ability to conduct unbiased forward and reverse genetic screens in C. elegans remains one of the most powerful experimental paradigms for discovering the biochemical pathways underlying human disease phenotypes. The identification of these pathways leads to a better understanding of the molecular interactions that perturb cellular physiology, and forms the foundation for designing mechanism-based therapies. To this end, the ability to process large numbers of isogenic animals through automated work stations suggests that C. elegans, manifesting different aspects of human disease phenotypes, will become the platform of choice for in vivo drug discovery and target validation using high-throughput/content screening technologies.
Asunto(s)
Caenorhabditis elegans/genética , ADN de Helmintos , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Modelos Animales , Animales , Animales Modificados Genéticamente , Apoptosis/genética , Secuencia de Bases , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/metabolismo , Secuencia Conservada , Bases de Datos Genéticas , Descubrimiento de Drogas , Regulación de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Humanos , Mutación , Necrosis , Enfermedades Parasitarias/genética , Fenotipo , Reproducibilidad de los Resultados , Especificidad de la EspecieRESUMEN
Extracellular serpins such as antithrombin and alpha1-antitrypsin are the quintessential regulators of proteolytic pathways. In contrast, the biological functions of the intracellular serpins remain obscure. We now report that the C. elegans intracellular serpin, SRP-6, exhibits a prosurvival function by blocking necrosis. Minutes after hypotonic shock, srp-6 null animals underwent a catastrophic series of events culminating in lysosomal disruption, cytoplasmic proteolysis, and death. This newly defined hypo-osmotic stress lethal (Osl) phenotype was dependent upon calpains and lysosomal cysteine peptidases, two in vitro targets of SRP-6. By protecting against both the induction of and the lethal effects from lysosomal injury, SRP-6 also blocked death induced by heat shock, oxidative stress, hypoxia, and cation channel hyperactivity. These findings suggest that multiple noxious stimuli converge upon a peptidase-driven, core stress response pathway that, in the absence of serpin regulation, triggers a lysosomal-dependent necrotic cell death routine.
