RESUMEN
Programmed cell death is regulated by the balance between activating and inhibitory signals. Here, we have identified RECS1 (responsive to centrifugal force and shear stress 1) [also known as TMBIM1 (transmembrane BAX inhibitor motif containing 1)] as a proapoptotic member of the TMBIM family. In contrast to other proteins of the TMBIM family, RECS1 expression induces cell death through the canonical mitochondrial apoptosis pathway. Unbiased screening indicated that RECS1 sensitizes cells to lysosomal perturbations. RECS1 localizes to lysosomes, where it regulates their acidification and calcium content, triggering lysosomal membrane permeabilization. Structural modeling and electrophysiological studies indicated that RECS1 is a pH-regulated calcium channel, an activity that is essential to trigger cell death. RECS1 also sensitizes whole animals to stress in vivo in Drosophila melanogaster and zebrafish models. Our results unveil an unanticipated function for RECS1 as a proapoptotic component of the TMBIM family that ignites cell death programs at lysosomes.
RESUMEN
Endoplasmic reticulum (ER) stress is a common feature of several physiological and pathological conditions affecting the function of the secretory pathway. To restore ER homeostasis, an orchestrated signaling pathway is engaged that is known as the unfolded protein response (UPR). The UPR has a primary function in stress adaptation and cell survival; however, under irreversible ER stress a switch to pro-apoptotic signaling events induces apoptosis of damaged cells. The mechanisms that initiate ER stress-dependent apoptosis are not fully understood. Several pathways have been described where we highlight the participation of the BCL-2 family of proteins and ER calcium release. In addition, recent findings also suggest that microRNAs and oxidative stress are relevant players on the transition from adaptive to cell death programs. Here we provide a global and integrated overview of the signaling networks that may determine the elimination of a cell under chronic ER stress. This article is part of a Special Section entitled: Cell Death Pathways.
Asunto(s)
Estrés del Retículo Endoplásmico , Animales , Muerte Celular , Humanos , Modelos Biológicos , Respuesta de Proteína DesplegadaRESUMEN
Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER) stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO) cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-X(L) overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD) expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.
Asunto(s)
Apoptosis/fisiología , Ciclofilinas/fisiología , Proteína Destructora del Antagonista Homólogo bcl-2/fisiología , Proteína X Asociada a bcl-2/fisiología , Animales , Sangre , Caspasa 9/metabolismo , Peptidil-Prolil Isomerasa F , Citocromos c/metabolismo , Retículo Endoplásmico/metabolismo , Ratones , Respuesta de Proteína DesplegadaRESUMEN
Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1α (IRE1α), which functions as an endoribonuclease that splices the mRNA of the transcription factor XBP-1 (X-box-binding protein-1). Through a global proteomic approach we identified the BCL-2 family member PUMA as a novel IRE1α interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1α with BIM, another BH3-only protein. BIM and PUMA double-knockout cells failed to maintain sustained XBP-1 mRNA splicing after prolonged ER stress, resulting in early inactivation. Mutation in the BH3 domain of BIM abrogated the physical interaction with IRE1α, inhibiting its effects on XBP-1 mRNA splicing. Unexpectedly, this regulation required BCL-2 and was antagonized by BAD or the BH3 domain mimetic ABT-737. The modulation of IRE1α RNAse activity by BH3-only proteins was recapitulated in a cell-free system suggesting a direct regulation. Moreover, BH3-only proteins controlled XBP-1 mRNA splicing in vivo and affected the ER stress-regulated secretion of antibodies by primary B cells. We conclude that a subset of BCL-2 family members participates in a new UPR-regulatory network, thus assuming apoptosis-unrelated functions.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Endorribonucleasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Respuesta de Proteína Desplegada , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Técnicas de Inactivación de Genes , Inmunoprecipitación , Proteínas de la Membrana/genética , Ratones , Unión Proteica , Mapeo de Interacción de Proteínas , Proteoma/análisis , Proteínas Proto-Oncogénicas/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Both autophagy and apoptosis are tightly regulated processes playing a central role in tissue homeostasis. Bax inhibitor 1 (BI-1) is a highly conserved protein with a dual role in apoptosis and endoplasmic reticulum (ER) stress signalling through the regulation of the ER stress sensor inositol requiring kinase 1 α (IRE1α). Here, we describe a novel function of BI-1 in the modulation of autophagy. BI-1-deficient cells presented a faster and stronger induction of autophagy, increasing LC3 flux and autophagosome formation. These effects were associated with enhanced cell survival under nutrient deprivation. Repression of autophagy by BI-1 was dependent on cJun-N terminal kinase (JNK) and IRE1α expression, possibly due to a displacement of TNF-receptor associated factor-2 (TRAF2) from IRE1α. Targeting BI-1 expression in flies altered autophagy fluxes and salivary gland degradation. BI-1 deficiency increased flies survival under fasting conditions. Increased expression of autophagy indicators was observed in the liver and kidney of bi-1-deficient mice. In summary, we identify a novel function of BI-1 in multicellular organisms, and suggest a critical role of BI-1 as a stress integrator that modulates autophagy levels and other interconnected homeostatic processes.
Asunto(s)
Autofagia/genética , Endorribonucleasas/metabolismo , Proteínas de la Membrana/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Respuesta de Proteína Desplegada/genética , Ácidos/metabolismo , Animales , Supervivencia Celular/genética , Células Cultivadas , Drosophila/genética , Endorribonucleasas/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Organismos Modificados Genéticamente , Fagosomas/genética , Fagosomas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Saccharomyces cerevisiae/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Inanición/metabolismo , Vesículas Transportadoras/metabolismo , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
Apoptosis is essential for maintenance of tissue homeostasis and its deregulation results in a variety of disease conditions. The BCL-2 family of proteins is a group of evolutionarily conserved regulators of cell death that comprises both anti- and pro-apoptotic members, that operate at the mitochondrial membrane to control caspase activation. Different BCL-2-related proteins are also located in the endoplasmic reticulum (ER), where important roles in organelle physiology are proposed. Adaptation to ER stress is mediated by the activation of a complex signal transduction pathway known as the unfolded protein response (UPR). Recent reports indicate that the ER stress sensor IRE1alpha, signals through the formation of a protein complex platform at the ER membrane, here termed the "UPRosome". Alternatively, BCL-2 family members are contained in other multiprotein complexes at the ER that are involved in the control of diverse cellular processes including calcium homeostasis, autophagy and ER morphogenesis. Here we describe the emerging concept that BCL-2 family members are important regulators of essential cellular processes beyond apoptosis.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis/fisiología , Autofagia/fisiología , Calcio/metabolismo , Retículo Endoplásmico/ultraestructura , Pliegue de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/genética , Estrés Fisiológico , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
Endoplasmic reticulum (ER) stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR). Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.
Asunto(s)
Apoptosis , Citoprotección , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/patología , Endorribonucleasas/metabolismo , Proteínas del Choque Térmico HSP72/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Estrés Fisiológico , Factores de Transcripción/metabolismo , Empalme Alternativo/genética , Animales , Supervivencia Celular , Citocromos c/metabolismo , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/metabolismo , Proteínas del Choque Térmico HSP72/química , Humanos , Potencial de la Membrana Mitocondrial , Ratones , Modelos Biológicos , Células PC12 , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Factores de Transcripción del Factor Regulador X , Transducción de Señal , Factores de Transcripción/genética , Proteína 1 de Unión a la X-BoxRESUMEN
Adaptation to endoplasmic reticulum (ER) stress depends on the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). Bax inhibitor-1 (BI-1) is an evolutionarily conserved ER-resident protein that suppresses cell death. Here we have investigated the role of BI-1 in the UPR. BI-1 expression suppressed IRE1alpha activity in fly and mouse models of ER stress. BI-1-deficient cells displayed hyperactivation of the ER stress sensor IRE1alpha, leading to increased levels of its downstream target X-box-binding protein-1 (XBP-1) and upregulation of UPR target genes. This phenotype was associated with the formation of a stable protein complex between BI-1 and IRE1alpha, decreasing its ribonuclease activity. Finally, BI-1 deficiency increased the secretory activity of primary B cells, a phenomenon regulated by XBP-1. Our results suggest a role for BI-1 in early adaptive responses against ER stress that contrasts with its known downstream function in apoptosis.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Retículo Endoplásmico/fisiología , Endorribonucleasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Linfocitos B/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endorribonucleasas/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Inmunoglobulina M/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/genética , Empalme del ARN , Factores de Transcripción del Factor Regulador X , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-BoxRESUMEN
The unfolded protein response (UPR) is a conserved adaptive reaction that increases cell survival under conditions of endoplasmic reticulum (ER) stress. The UPR controls diverse processes such as protein folding, secretion, ER biogenesis, protein quality control and macroautophagy. Occurrence of chronic ER stress has been extensively described in neurodegenerative conditions linked to protein misfolding and aggregation, including Amyotrophic lateral sclerosis, Prion-related disorders, and conditions such as Parkinson's, Huntington's, and Alzheimer's disease. Strong correlations are observed between disease progression, accumulation of protein aggregates, and induction of the UPR in animal and in vitro models of neurodegeneration. In addition, the first reports are available describing the engagement of ER stress responses in brain post-mortem samples from human patients. Despite such findings, the role of the UPR in the central nervous system has not been addressed directly and its contribution to neurodegeneration remains speculative. Recently, however, pharmacological manipulation of ER stress and autophagy - a stress pathway modulated by the UPR - using chemical chaperones and autophagy activators has shown therapeutic benefits by attenuating protein misfolding in models of neurodegenerative disease. The most recent evidence addressing the role of the UPR and ER stress in neurodegenerative disorders is reviewed here, along with therapeutic strategies to alleviate ER stress in a disease context.
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Autofagia , Enfermedades Neurodegenerativas/metabolismo , Pliegue de Proteína , Enfermedad de Alzheimer/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Degeneración Nerviosa/metabolismo , Enfermedad de Parkinson/metabolismo , Estrés Fisiológico/metabolismoRESUMEN
Neurotrophins are trophic factors that regulate important neuronal functions. They bind two unrelated receptors, the Trk family of receptor-tyrosine kinases and the p75 neurotrophin receptor (p75). p75 was recently identified as a new substrate for gamma-secretase-mediated intramembrane proteolysis, generating a p75-derived intracellular domain (p75-ICD) with signaling capabilities. Using PC12 cells as a model, we studied how neurotrophins activate p75 processing and where these events occur in the cell. We demonstrate that activation of the TrkA receptor upon binding of nerve growth factor (NGF) regulates the metalloprotease-mediated shedding of p75 leaving a membrane-bound p75 C-terminal fragment (p75-CTF). Using subcellular fractionation to isolate a highly purified endosomal fraction, we demonstrate that p75-CTF ends up in endosomes where gamma-secretase-mediated p75-CTF cleavage occurs, resulting in the release of a p75-ICD. Moreover, we show similar structural requirements for gamma-secretase processing of p75 and amyloid precursor protein-derived CTFs. Thus, NGF-induced endocytosis regulates both signaling and proteolytic processing of p75.