RESUMEN
Alpha-1 antitrypsin deficiency (AATD) arises due to inherited variants in SERPINA1, the AAT gene that impairs the production or secretion of this hepatocellular protein and leads to a gain-of-function liver proteotoxicity. Homozygous Pi*Z pathogenic variant (Pi*ZZ genotype) is the leading cause of severe AATD. It manifests in 2 to 10% of carriers as neonatal cholestasis and 20 to 35% of adults as significant liver fibrosis. Both children and adults may develop an end-stage liver disease requiring liver transplantation. Heterozygous Pi*Z pathogenic variant (Pi*MZ genotype) constitutes an established disease modifier. Our review summarizes the natural history and management of subjects with both pediatric and adult AATD-associated liver disease. Current findings from a phase 2 clinical trial indicate that RNA silencing may constitute a viable therapeutic approach for adult AATD. In conclusion, AATD is an increasingly appreciated pediatric and adult liver disorder that is becoming an attractive target for modern pharmacologic strategies.
Asunto(s)
Colestasis , Deficiencia de alfa 1-Antitripsina , Adulto , Humanos , Niño , Recién Nacido , Deficiencia de alfa 1-Antitripsina/complicaciones , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/terapia , Cirrosis Hepática/genética , Cirrosis Hepática/complicaciones , Genotipo , Colestasis/complicacionesRESUMEN
ABSTRACT: The presence of modifier genes is now well recognized in severe liver disease outcome associated with alpha-1-antitrypsin deficiency (A1ATD) but their identification remains to be fully elucidated. To address this goal, we performed a candidate gene study with the SORL1 gene, already identified as risk gene in early-onset Alzheimer Disease families. A particular SORL1 micro-haplotype constituted with 3 SNPs (wild-type form TTG) was genotyped on 86 ZZ A1ATD children issued from 66 families. Interestingly, the mutated forms of this micro-haplotype (CAT most of the time) were associated with lower occurrence of severe liver disease and in cellulo studies showed that SORL1 influences Z-A1ATD cellular toxicity and biogenesis. These data suggest that the mutated CAT form of SORL1 micro-haplotype may partly prevent from severe liver disease in A1ATD children. Overall, these findings support a replication study on an independent cohort and additional in cellulo studies to confirm these promising results.
Asunto(s)
Proteínas Relacionadas con Receptor de LDL , Hepatopatías/genética , Proteínas de Transporte de Membrana , Deficiencia de alfa 1-Antitripsina , alfa 1-Antitripsina , Niño , Estudios de Cohortes , Francia , Haplotipos , Humanos , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas de Transporte de Membrana/genética , Polimorfismo de Nucleótido Simple , alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/genéticaRESUMEN
The maintenance of proteome homeostasis, or proteostasis, is crucial for preserving cellular functions and for cellular adaptation to environmental challenges and changes in physiological conditions. The capacity of cells to maintain proteostasis requires precise control and coordination of protein synthesis, folding, conformational maintenance, and clearance. Thus, protein degradation by the ubiquitin-proteasome system (UPS) or the autophagy-lysosomal system plays an essential role in cellular functions. However, failure of the UPS or the autophagic process can lead to the development of various diseases (aging-associated diseases, cancer), thus both these pathways have become attractive targets in the treatment of protein conformational diseases, such as alpha 1-antitrypsin deficiency (AATD). The Z alpha 1-antitrypsin (Z-AAT) misfolded variant of the serine protease alpha 1-antitrypsin (AAT) is caused by a structural change that predisposes it to protein aggregation and dramatic accumulation in the form of inclusion bodies within liver hepatocytes. This can lead to clinically significant liver disease requiring liver transplantation in childhood or adulthood. Treatment of mice with autophagy enhancers was found to reduce hepatic Z-AAT aggregate levels and protect them from AATD hepatotoxicity. To date, liver transplantation is the only curative therapeutic option for patients with AATD-mediated liver disease. Therefore, the development and discovery of new therapeutic approaches to delay or overcome disease progression is a top priority. Herein, we review AATD-mediated liver disease and the overall process of autophagy. We highlight the role of this system in the regulation of Z-variant degradation and its implication in AATD-medicated liver disease, including some open questions that remain challenges in the field and require further elucidation. Finally, we discuss how manipulation of autophagy could provide multiple routes of therapeutic benefit in AATD-mediated liver disease.
Asunto(s)
Autofagia , Hepatocitos , Hepatopatías , Trasplante de Hígado , Hígado , Agregación Patológica de Proteínas , alfa 1-Antitripsina , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Hígado/metabolismo , Hígado/patología , Hepatopatías/metabolismo , Hepatopatías/patología , Hepatopatías/cirugía , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Agregación Patológica de Proteínas/cirugía , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/patologíaRESUMEN
Inflammation is a major contributor to tubular epithelium injury in kidney disorders, and the involvement of blood platelets in driving inflammation is increasingly stressed. CD154, the ligand of CD40, is one of the mediators supporting platelet proinflammatory properties. Although hypoxia is an essential constituent of the inflammatory reaction, if and how platelets and CD154 regulate inflammation in hypoxic conditions remain unclear. Here, we studied the control by CD154 of the proinflammatory cytokine interleukin- (IL-) 6 secretion in short-term oxygen (O2) deprivation conditions, using the HK-2 cell line as a kidney tubular epithelial cell (TEC) model. IL-6 secretion was markedly stimulated by CD154 after 1 to 3 hours of hypoxic stress. Both intracellular IL-6 expression and secretion were stimulated by CD154 and associated with a strong upregulation of IL-6 mRNA and increased transcription. Searching for inhibitors of CD154-mediated IL-6 production by HK-2 cells in hypoxic conditions, we observed that chloroquine, a drug that has been repurposed as an anti-inflammatory agent, alleviated this induction. Therefore, CD154 is a potent early stimulus for IL-6 secretion by TECs in O2 deprivation conditions, a mechanism likely to take part in the deleterious inflammatory consequences of platelet activation in kidney tubular injury. The inhibition of CD154-induced IL-6 production by chloroquine suggests the potential usefulness of this drug as a therapeutic adjunct in conditions associated with acute kidney injury.
Asunto(s)
Ligando de CD40/farmacología , Hipoxia de la Célula/fisiología , Cloroquina/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Interleucina-6/metabolismo , Túbulos Renales/citología , Apoptosis , Western Blotting , Línea Celular , Proliferación Celular , Supervivencia Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Microscopía Fluorescente , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Human cells express large amounts of different proteins continuously that must fold into well-defined structures that need to remain correctly folded and assemble in order to ensure their cellular and biological functions. The integrity of this protein balance/homeostasis, also named proteostasis, is maintained by the proteostasis network (PN). This integrated biological system, which comprises about 2000 proteins (chaperones, folding enzymes, degradation components), control and coordinate protein synthesis folding and localization, conformational maintenance, and degradation. This network is particularly challenged by mutations such as those found in genetic diseases, because of the inability of an altered peptide sequence to properly engage PN components that trigger misfolding and loss of function. Thus, deletions found in the ΔF508 variant of the Cystic Fibrosis (CF) transmembrane regulator (CFTR) triggering CF or missense mutations found in the Z variant of Alpha 1-Antitrypsin deficiency (AATD), leading to lung and liver diseases, can accelerate misfolding and/or generate aggregates. Conversely to CF variants, for which three correctors are already approved (ivacaftor, lumacaftor/ivacaftor, and most recently tezacaftor/ivacaftor), there are limited therapeutic options for AATD. Therefore, a more detailed understanding of the PN components governing AAT variant biogenesis and their manipulation by pharmacological intervention could delay, or even better, avoid the onset of AATD-related pathologies.
Asunto(s)
Agregación Patológica de Proteínas/metabolismo , Pliegue de Proteína , Deficiencias en la Proteostasis/metabolismo , Proteostasis , Deficiencia de alfa 1-Antitripsina/metabolismo , Humanos , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/patología , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/patologíaRESUMEN
BACKGROUND & AIMS: To identify prognostic factors for liver disease in children with alpha-1 antitrypsin deficiency, irrespective of phenotype, using the DEFI-ALPHA cohort. METHODS: Retrospective, then prospective from 2010, multicentre study including children known to have alpha-1 antitrypsin blood concentration below 0.8 g/L, born in France since 1989. Clinical and biological data were collected. Liver disease was classified as "severe" (portal hypertension, liver failure, liver transplantation or death); "moderate" (persistent abnormal liver biology without portal hypertension); and "mild/none" (normal or almost normal liver biology and native liver). Prognostic factors for severe liver disease were evaluated using a Cox semiparametric model. RESULTS: In January 2017, 153 patients from 19 centres had been included; genotypes were PIZZ in 81.9%, PISZ in 8.1%, other in 10.0%. Mean ± SD follow-up was 4.7 ± 2.1 years. Half of patients had moderate liver disease. Twenty-eight children (18.3%) had severe liver disease (mean age 2.5 years, range: 0-11.6): diagnosis of alpha-1 antitrypsin deficiency was made before two months of age in 65.4%, genotypes were PIZZ in 25 (89.3%), PISZ in 2, PIMlike Z in 1, 15 children underwent liver transplantation, 1 child died at 3 years of age. Neonatal cholestasis was significantly associated with severe liver disease (P = 0.007). CONCLUSION: Alpha-1 antitrypsin-deficient patients presenting with neonatal cholestasis were likely to develop severe liver disease. Some patients with non-homozygous ZZ genotype can develop severe liver disease, such as PISZ and M variants, when associated with predisposing factors. Further genetic studies will help to identify other factors involved in the development of liver complications.
Asunto(s)
Hepatopatías/sangre , Deficiencia de alfa 1-Antitripsina/sangre , alfa 1-Antitripsina/sangre , Niño , Preescolar , Colestasis/sangre , Colestasis/etiología , Colestasis/patología , Femenino , Francia , Genotipo , Humanos , Lactante , Recién Nacido , Hepatopatías/etiología , Hepatopatías/patología , Pruebas de Función Hepática , Modelos Logísticos , Masculino , Fenotipo , Estudios Prospectivos , Estudios Retrospectivos , Deficiencia de alfa 1-Antitripsina/complicaciones , Deficiencia de alfa 1-Antitripsina/patologíaRESUMEN
Diseases of protein folding arise because of the inability of an altered peptide sequence to properly engage protein homeostasis components that direct protein folding and function. To identify global principles of misfolding disease pathology we examined the impact of the local folding environment in alpha-1-antitrypsin deficiency (AATD), Niemann-Pick type C1 disease (NPC1), Alzheimer's disease (AD), and cystic fibrosis (CF). Using distinct models, including patient-derived cell lines and primary epithelium, mouse brain tissue, and Caenorhabditis elegans, we found that chronic expression of misfolded proteins not only triggers the sustained activation of the heat shock response (HSR) pathway, but that this sustained activation is maladaptive. In diseased cells, maladaptation alters protein structure-function relationships, impacts protein folding in the cytosol, and further exacerbates the disease state. We show that down-regulation of this maladaptive stress response (MSR), through silencing of HSF1, the master regulator of the HSR, restores cellular protein folding and improves the disease phenotype. We propose that restoration of a more physiological proteostatic environment will strongly impact the management and progression of loss-of-function and gain-of-toxic-function phenotypes common in human disease.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/etiología , Proteínas de Unión al ADN/genética , Deficiencias en la Proteostasis/genética , Factores de Transcripción/genética , Animales , Antineoplásicos Alquilantes/uso terapéutico , Caenorhabditis elegans , Línea Celular , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Proteínas de Unión al ADN/metabolismo , Diterpenos/uso terapéutico , Evaluación Preclínica de Medicamentos , Compuestos Epoxi/uso terapéutico , Silenciador del Gen , Factores de Transcripción del Choque Térmico , Humanos , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Ratones Transgénicos , Organoides , Fenantrenos/uso terapéutico , Prostaglandina-E Sintasas , Pliegue de Proteína , Mucosa Respiratoria/metabolismo , Estrés Fisiológico , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND & AIMS: Fifteen to twenty percent of alpha-1 antitrypsin deficiency patients (A1ATD) have a severe liver outcome (portal hypertension - PHT) during childhood. Since they all share the same ZZSERPINA1 genotype and that environmental factors such as alcohol cannot be advanced, the presence of modifier genes is now well recognized. SNPs located on the SERPINA1 and MAN1B1 genes have already been tested in very few studies with contradictory or not replicated results. METHODS: Our genotype-phenotype correlation study, performed on 92 ZZ children, aimed at determining once and for all if SERPINA1 and MAN1B1 polymorphisms may be implied in the onset of PHT. To do so, we also performed for the first time a complete haplotype reconstruction for data analysis. RESULTS: The two genetic associations with severe liver disease that had been suspected previously (one SNP for SERPINA1 and another for MAN1B1) were not confirmed in our cohort. Moreover, the haplotype analysis identified only one major genetic background for the SERPINA1 Z-allele, allowing us to exclude the presence of a frequent modifier SNP within. For MAN1B1, four major haplotypes were identified but the prevalence of PHT did not significantly differ between them. CONCLUSION: We conclude that genetic polymorphisms in these two genes probably do not influence the onset of severe liver disease in A1ATD.
Asunto(s)
Hipertensión Portal/genética , Manosidasas/genética , Deficiencia de alfa 1-Antitripsina/complicaciones , alfa 1-Antitripsina/genética , Alelos , Niño , Preescolar , Estudios de Cohortes , Femenino , Francia , Estudios de Asociación Genética , Haplotipos , Humanos , Lactante , Masculino , Polimorfismo de Nucleótido SimpleAsunto(s)
COVID-19/patología , Elastasa de Leucocito/sangre , Metaloproteinasa 12 de la Matriz/sangre , alfa 1-Antitripsina/sangre , Adulto , Anciano , Líquido del Lavado Bronquioalveolar/química , COVID-19/virología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Unidades de Cuidados Intensivos , Masculino , Persona de Mediana Edad , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Índice de Severidad de la EnfermedadRESUMEN
The endoplasmic reticulum (ER) is an organelle specialized for the folding and assembly of secretory and transmembrane proteins. ER homeostasis is often perturbed in tumor cells because of dramatic changes in the microenvironment of solid tumors, thereby leading to the activation of an adaptive mechanism named the unfolded protein response (UPR). The activation of the UPR sensor IRE1α has been described to play an important role in tumor progression. However, the molecular events associated with this phenotype remain poorly characterized. In the present study, we examined the effects of IRE1α signaling on the adaptation of glioma cells to their microenvironment. We show that the characteristics of U87 cell migration are modified under conditions where IRE1α activity is impaired (DN_IRE1). This is linked to increased stress fiber formation and enhanced RhoA activity. Gene expression profiling also revealed that loss of functional IRE1α signaling mostly resulted in the upregulation of genes encoding extracellular matrix proteins. Among these genes, Sparc, whose mRNA is a direct target of IRE1α endoribonuclease activity, was in part responsible for the phenotypic changes associated with IRE1α inactivation. Hence, our data demonstrate that IRE1α is a key regulator of SPARC expression in vitro in a glioma model. Our results also further support the crucial contribution of IRE1α to tumor growth, infiltration and invasion and extend the paradigm of secretome control in tumor microenvironment conditioning.
Asunto(s)
Comunicación Autocrina , Neoplasias Encefálicas/patología , Movimiento Celular , Endorribonucleasas/metabolismo , Glioma/patología , Osteonectina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Citoesqueleto de Actina/metabolismo , Comunicación Autocrina/genética , Neoplasias Encefálicas/genética , Adhesión Celular/genética , Movimiento Celular/genética , Proliferación Celular , Regulación hacia Abajo/genética , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Humanos , Modelos Biológicos , Osteonectina/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/genética , Esferoides Celulares/patología , Células Tumorales Cultivadas , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Alpha-1-antitrypsin (α1AT) deficiency is a genetic disorder that manifests as pulmonary emphysema and liver cirrhosis. α1AT deficiency is the most common genetic cause of liver disease in children and also an underappreciated cause of liver disease in adults. The prevalence in the general population in Western Europe is approximately 1 in 2,000. The most common and severe deficiency allele is the Z variant (two alleles mutated). This variant is characterized by the accumulation of Z-α1AT polymers in the endoplasmic reticulum of hepatocytes leading to cell death and to a severe reduction of α1AT in the serum. The latter results in a loss of its antiprotease activity and its ability to protect lung tissue. Thus far, there are only very limited therapeutic options in α1AT deficiency. A more detailed understanding of the biology governing α1AT biogenesis is required in order to identify new pharmacological agents and biomarkers. This review will present current knowledge on α1AT deficiency and focus on recent discoveries and new strategies in the treatment of this disease.
Asunto(s)
Deficiencia de alfa 1-Antitripsina/genética , Adulto , Animales , Niño , Humanos , Mutación , alfa 1-Antitripsina/fisiología , Deficiencia de alfa 1-Antitripsina/complicaciones , Deficiencia de alfa 1-Antitripsina/patología , Deficiencia de alfa 1-Antitripsina/terapiaRESUMEN
α1-Antitrypsin (α1AT) deficiency (α1ATD) is a consequence of defective folding, trafficking, and secretion of α1AT in response to a defect in its interaction with the endoplasmic reticulum proteostasis machineries. The most common and severe form of α1ATD is caused by the Z-variant and is characterized by the accumulation of α1AT polymers in the endoplasmic reticulum of the liver leading to a severe reduction (>85%) of α1AT in the serum and its anti-protease activity in the lung. In this organ α1AT is critical for ensuring tissue integrity by inhibiting neutrophil elastase, a protease that degrades elastin. Given the limited therapeutic options in α1ATD, a more detailed understanding of the folding and trafficking biology governing α1AT biogenesis and its response to small molecule regulators is required. Herein we report the correction of Z-α1AT secretion in response to treatment with the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA), acting in part through HDAC7 silencing and involving a calnexin-sensitive mechanism. SAHA-mediated correction restores Z-α1AT secretion and serpin activity to a level 50% that observed for wild-type α1AT. These data suggest that HDAC activity can influence Z-α1AT protein traffic and that SAHA may represent a potential therapeutic approach for α1ATD and other protein misfolding diseases.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Deficiencia de alfa 1-Antitripsina/prevención & control , alfa 1-Antitripsina/metabolismo , Calnexina/genética , Calnexina/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/metabolismo , Expresión Génica/efectos de los fármacos , Células HCT116 , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Immunoblotting , Hígado/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutación , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Pliegue de Proteína , Transporte de Proteínas/efectos de los fármacos , Deficiencias en la Proteostasis/sangre , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/prevención & control , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vorinostat , alfa 1-Antitripsina/sangre , alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/sangre , Deficiencia de alfa 1-Antitripsina/genéticaRESUMEN
Inositol-requiring enzyme 1 (IRE1) is a proximal endoplasmic reticulum (ER) stress sensor and a central mediator of the unfolded protein response. In a human glioma model, inhibition of IRE1alpha correlated with down-regulation of prevalent proangiogenic factors such as VEGF-A, IL-1beta, IL-6, and IL-8. Significant up-regulation of antiangiogenic gene transcripts was also apparent. These transcripts encode SPARC, decorin, thrombospondin-1, and other matrix proteins functionally linked to mesenchymal differentiation and glioma invasiveness. In vivo, using both the chick chorio-allantoic membrane assay and a mouse orthotopic brain model, we observed in tumors underexpressing IRE1: (i) reduction of angiogenesis and blood perfusion, (ii) a decreased growth rate, and (iii) extensive invasiveness and blood vessel cooption. This phenotypic change was consistently associated with increased overall survival in glioma-implanted recipient mice. Ectopic expression of IL-6 in IRE1-deficient tumors restored angiogenesis and neutralized vessel cooption but did not reverse the mesenchymal/infiltrative cell phenotype. The ischemia-responsive IRE1 protein is thus identified as a key regulator of tumor neovascularization and invasiveness.
Asunto(s)
Endorribonucleasas/metabolismo , Glioma/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias Experimentales/metabolismo , Neovascularización Patológica/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/metabolismo , Membrana Corioalantoides/patología , Endorribonucleasas/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioma/sangre , Glioma/patología , Humanos , Inmunohistoquímica , Interleucina-6/genética , Interleucina-6/metabolismo , Estimación de Kaplan-Meier , Proteínas de la Membrana/genética , Ratones , Ratones Desnudos , Microscopía Fluorescente , Microscopía por Video , Invasividad Neoplásica , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/patología , Neovascularización Patológica/patología , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante HeterólogoRESUMEN
The protein-disulfide isomerase (PDI) family member anterior gradient 2 (AGR2) is reportedly overexpressed in numerous cancers and plays a role in cancer development. However, to date the molecular functions of AGR2 remain to be characterized. Herein we have identified AGR2 as bound to newly synthesized cargo proteins using a proteomics analysis of endoplasmic reticulum (ER) membrane-bound ribosomes. Nascent protein chains that translocate into the ER associate with specific ER luminal proteins, which in turn ensures proper folding and posttranslational modifications. Using both imaging and biochemical approaches, we confirmed that AGR2 localizes to the lumen of the ER and indirectly associates with ER membrane-bound ribosomes through nascent protein chains. We showed that AGR2 expression is controlled by the unfolded protein response and is in turn is involved in the maintenance of ER homeostasis. Remarkably, we have demonstrated that siRNA-mediated knockdown of AGR2 significantly alters the expression of components of the ER-associated degradation machinery and reduces the ability of cells to cope with acute ER stress, properties that might be relevant to the role of AGR2 in cancer development.
Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Retículo Endoplásmico/enzimología , Regulación de la Expresión Génica/fisiología , Homeostasis/fisiología , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Respuesta de Proteína Desplegada/fisiología , Animales , Células COS , Chlorocebus aethiops , Perros , Retículo Endoplásmico/genética , Células HEK293 , Humanos , Ratones , Mucoproteínas , Proteínas Oncogénicas , Proteínas/genética , Proteínas Proto-Oncogénicas/genéticaRESUMEN
The endoplasmic reticulum (ER) is an essential organelle whose major functions are to ensure proper secretory protein folding and trafficking. These mechanisms involve the activation of specific ER-resident molecular machines, which might be regulated by their membranous environments. Based on this observation, we aimed to characterize the proteome of ER-membrane microdomains to identify new components of the ER that have a role in secretory pathway-associated functions. Using this approach with dog pancreatic rough microsomes, we found that mitochondrial Bcl-2 inhibitor of transcription (BIT1) localized in the early secretory pathway and accumulated in the Golgi complex. Using both a chimeric protein of the luminal and transmembrane domains of ER-resident TRAPalpha and the cytosolic domain of BIT1, and silencing of BIT1 expression, we perturbed endogenous BIT1 oligomerization and localization to the Golgi. This led to enhanced ERK signaling from the Golgi complex, which resulted in improved stress resistance. This work provides the first evidence for the existence of ER microdomains that are involved in the regulation of BIT1 structure and trafficking, and identifies BIT1 as a negative regulator of the ERK-MAPK signaling pathway in the Golgi.
Asunto(s)
Fosfatasa Ácida/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Aparato de Golgi/metabolismo , Isoenzimas/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Fosfatasa Ácida/genética , Animales , Hidrolasas de Éster Carboxílico/genética , Membrana Celular/metabolismo , Perros , Retículo Endoplásmico Rugoso/metabolismo , Ingeniería Genética , Isoenzimas/genética , Sistema de Señalización de MAP Quinasas/genética , Microsomas/metabolismo , Microsomas/ultraestructura , Mitocondrias/metabolismo , Páncreas/ultraestructura , Transporte de Proteínas/genética , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/genética , Estrés Fisiológico , Fosfatasa Ácida TartratorresistenteRESUMEN
Activation of the bifunctional kinase/RNase enzyme IRE1α is part of an adaptive response triggered on accumulation of misfolded proteins in the endoplasmic reticulum (ER). To facilitate recovery of ER homeostasis, IRE1α molecules oligomerize, allowing for their transautophosphorylation and endoribonuclease activation. These, in turn, induce the activation of specific transcriptional and post-transcriptional programs. To identify novel and selective modulators of IRE1α activity, we investigated IRE1α oligomerization properties using IRE1α-derived peptides identified through an activity-based in vitro assay. We then used these peptides to probe IRE1α activity in vitro and in vivo using both cultured human hepatocellular carcinoma-derived HuH7 cells and Caenorhabditis elegans experimental systems. We identified a peptide derived from the kinase domain of human IRE1α, which promoted IRE1α oligomerization in vitro, enhanced its Xbp1 mRNA cleavage activity in vitro (1.7×) in cell culture (1.8×) and in vivo (1.3×), and attenuated both ER stress-mediated JNK activation and regulated IRE1-dependent mRNA decay (RIDD). This was accompanied by a 2.5-fold increase in survival on tunicamycin-induced ER stress and reduced apoptosis by 1.4-fold in cells expressing this peptide. Hence, targeted and selective activation of the catalytic properties of IRE1α may consequently define new strategies to protect cells from deleterious effects of ER stress signaling.
Asunto(s)
Retículo Endoplásmico/metabolismo , Endorribonucleasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Caenorhabditis elegans , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endorribonucleasas/genética , Regulación de la Expresión Génica , Humanos , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Modelos Moleculares , Péptidos , Conformación Proteica , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción del Factor Regulador X , Transducción de Señal , Estrés Fisiológico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-BoxRESUMEN
BACKGROUND & AIMS: A single point mutation in the Z-variant of alpha 1-antitrypsin (Z-AAT) alone can lead to both a protein folding and trafficking defect, preventing its exit from the endoplasmic reticulum (ER), and the formation of aggregates that are retained as inclusions within the ER of hepatocytes. These defects result in a systemic AAT deficiency (AATD) that causes lung disease, whereas the ER-retained aggregates can induce severe liver injury in patients with ZZ-AATD. Unfortunately, therapeutic approaches are still limited and liver transplantation represents the only curative treatment option. To overcome this limitation, a better understanding of the molecular basis of ER aggregate formation could provide new strategies for therapeutic intervention. METHODS: Our functional and omics approaches here based on human hepatocytes from patients with ZZ-AATD have enabled the identification and characterisation of the role of the protein disulfide isomerase (PDI) A4/ERP72 in features of AATD-mediated liver disease. RESULTS: We report that 4 members of the PDI family (PDIA4, PDIA3, P4HB, and TXNDC5) are specifically upregulated in ZZ-AATD liver samples from adult patients. Furthermore, we show that only PDIA4 knockdown or alteration of its activity by cysteamine treatment can promote Z-AAT secretion and lead to a marked decrease in Z aggregates. Finally, detailed analysis of the Z-AAT interactome shows that PDIA4 silencing provides a more conducive environment for folding of the Z mutant, accompanied by reduction of Z-AAT-mediated oxidative stress, a feature of AATD-mediated liver disease. CONCLUSIONS: PDIA4 is involved in AATD-mediated liver disease and thus represents a therapeutic target for inhibition by drugs such as cysteamine. PDI inhibition therefore represents a potential therapeutic approach for treatment of AATD. LAY SUMMARY: Protein disulfide isomerase (PDI) family members, and particularly PDIA4, are upregulated and involved in alpha 1-antitrypsin deficiency (AATD)-mediated liver disease in adults. PDI inhibition upon cysteamine treatment leads to improvements in features of AATD and hence represents a therapeutic approach for treatment of AATD-mediated liver disease.
RESUMEN
The cellular receptor Notch1 is a central regulator of T-cell development, and as a consequence, Notch1 pathway appears upregulated in > 65% of the cases of T-cell acute lymphoblastic leukemia (T-ALL). However, strategies targeting Notch1 signaling render only modest results in the clinic due to treatment resistance and severe side effects. While many investigations reported the different aspects of tumor cell growth and leukemia progression controlled by Notch1, less is known regarding the modifications of cellular metabolism induced by Notch1 upregulation in T-ALL. Previously, glutaminolysis inhibition has been proposed to synergize with anti-Notch therapies in T-ALL models. In this work, we report that Notch1 upregulation in T-ALL induced a change in the metabolism of the important amino acid glutamine, preventing glutamine synthesis through the downregulation of glutamine synthetase (GS). Downregulation of GS was responsible for glutamine addiction in Notch1-driven T-ALL both in vitro and in vivo. Our results also confirmed an increase in glutaminolysis mediated by Notch1. Increased glutaminolysis resulted in the activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, a central controller of cell growth. However, glutaminolysis did not play any role in Notch1-induced glutamine addiction. Finally, the combined treatment targeting mTORC1 and limiting glutamine availability had a synergistic effect to induce apoptosis and to prevent Notch1-driven leukemia progression. Our results placed glutamine limitation and mTORC1 inhibition as a potential therapy against Notch1-driven leukemia.
Asunto(s)
Glutamato-Amoníaco Ligasa/genética , Glutamina/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Animales , Línea Celular Tumoral , Regulación hacia Abajo/genética , Regulación Enzimológica de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Glutamato-Amoníaco Ligasa/metabolismo , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transducción de Señal/genéticaRESUMEN
Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disease caused by mutations in the SERPINA1 gene and is associated with a decreased level of circulating alpha-1 antitrypsin (AAT). Among all the known mutations in the SERPINA1 gene, homozygous for the Z allele is well-known to result in both lung and liver disease. Unlike the lung injury that occurs in adulthood with the environment (notably, tobacco) as a co-factor, the hepatic damage is more complicated. Despite a common underlying gene mutation, the liver disease associated with AATD presents a considerable variability in the age-of-onset and severity, ranging from transient neonatal cholestasis (in early childhood) to cirrhosis and liver cancer (in childhood and adulthood). Given that all the cofactors- genetics and/or environmental- have not been fully identified, it is still impossible to predict which individuals with AATD may develop severe liver disease. The discovery of these modifiers represents the major challenge for the detection, diagnosis, and development of new therapies to provide alternative options to liver transplantation. The aim of this current review is to provide an updated overview of our knowledge on why some AATD patients associated with liver damage progress poorly.
RESUMEN
Upon accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER), a specific adaptive response, named the Unfolded Protein Response (UPR) is activated in order to protect cells from this stress. In metazoans, the UPR is mediated by three transmembrane ER resident proteins: PERK, ATF6 and IRE1. Among these, only IRE1 is found to be conserved from yeasts to mammals. IRE1 is a type I transmembrane protein which bears two enzymatic activities serine/threonine kinase and endoribonuclease. Crystal structures of S. cerevisiae luminal and cytosolic domains allowed a better understanding of its activation mode. However, IRE1 regulatory mechanisms and IRE1-dependent signalling pathways still remain to be fully explored. This review will present current knowledge on IRE1 protein and focus on its roles in physiological and pathophysiological processes.