RESUMEN
Alcohol dehydrogenase 1B (ADH1B) is a primate-specific enzyme which, uniquely among the ADH class 1 family, is highly expressed both in adipose tissue and liver. Its expression in adipose tissue is reduced in obesity and increased by insulin stimulation. Interference with ADH1B expression has also been reported to impair adipocyte function. To better understand the role of ADH1B in adipocytes, we used CRISPR/Cas9 to delete ADH1B in human adipose stem cells (ASC). Cells lacking ADH1B failed to differentiate into mature adipocytes manifested by minimal triglyceride accumulation and a marked reduction in expression of established adipocyte markers. As ADH1B is capable of converting retinol to retinoic acid (RA), we conducted rescue experiments. Incubation of ADH1B-deficient preadipocytes with 9-cis-RA, but not with all-transretinol, significantly rescued their ability to accumulate lipids and express markers of adipocyte differentiation. A homozygous missense variant in ADH1B (p.Arg313Cys) was found in a patient with congenital lipodystrophy of unknown cause. This variant significantly impaired the protein's dimerization, enzymatic activity, and its ability to rescue differentiation in ADH1B-deficient ASC. The allele frequency of this variant in the Middle Eastern population suggests that it is unlikely to be a fully penetrant cause of severe lipodystrophy. In conclusion, ADH1B appears to play an unexpected, crucial and cell-autonomous role in human adipocyte differentiation by serving as a necessary source of endogenous retinoic acid.
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Adipocitos , Adipogénesis , Alcohol Deshidrogenasa , Humanos , Alcohol Deshidrogenasa/metabolismo , Alcohol Deshidrogenasa/genética , Adipogénesis/genética , Adipocitos/metabolismo , Adipocitos/citología , Tretinoina/metabolismo , Diferenciación Celular , Sistemas CRISPR-Cas , Mutación Missense , Tejido Adiposo/metabolismoRESUMEN
Cytochrome P450 2C9 (CYP2C9) is a major drug-metabolizing enzyme that represents 20% of the hepatic CYPs and is responsible for the metabolism of 15% of drugs. A general concern in drug discovery is to avoid the inhibition of CYP leading to toxic drug accumulation and adverse drug-drug interactions. However, the prediction of CYP inhibition remains challenging due to its complexity. We developed an original machine learning approach for the prediction of drug-like molecules inhibiting CYP2C9. We created new predictive models by integrating CYP2C9 protein structure and dynamics knowledge, an original selection of physicochemical properties of CYP2C9 inhibitors, and machine learning modeling. We tested the machine learning models on publicly available data and demonstrated that our models successfully predicted CYP2C9 inhibitors with an accuracy, sensitivity and specificity of approximately 80%. We experimentally validated the developed approach and provided the first identification of the drugs vatalanib, piriqualone, ticagrelor and cloperidone as strong inhibitors of CYP2C9 with IC values <18 µM and sertindole, asapiprant, duvelisib and dasatinib as moderate inhibitors with IC50 values between 40 and 85 µM. Vatalanib was identified as the strongest inhibitor with an IC50 value of 0.067 µM. Metabolism assays allowed the characterization of specific metabolites of abemaciclib, cloperidone, vatalanib and tarafenacin produced by CYP2C9. The obtained results demonstrate that such a strategy could improve the prediction of drug-drug interactions in clinical practice and could be utilized to prioritize drug candidates in drug discovery pipelines.
Asunto(s)
Biología Computacional/métodos , Citocromo P-450 CYP2C9 , Inhibidores Enzimáticos del Citocromo P-450 , Aprendizaje Automático , Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP2C9/metabolismo , Inhibidores Enzimáticos del Citocromo P-450/análisis , Inhibidores Enzimáticos del Citocromo P-450/química , Inhibidores Enzimáticos del Citocromo P-450/metabolismo , Interacciones Farmacológicas , HumanosRESUMEN
T cells have the potential to maintain immunological memory and self-tolerance by recognizing antigens from pathogens or tumors. In pathological situations, failure to generate de novo T cells causes immunodeficiency resulting in acute infections and complications. Hematopoietic stem cells (HSC) transplantation constitutes a valuable option to restore proper immune function. However, delayed T cell reconstitution is observed compared to other lineages. To overcome this difficulty, we developed a new approach to identify populations with efficient lymphoid reconstitution properties. To this end, we use a DNA barcoding strategy based on the insertion into a cell chromosome of a lentivirus (LV) carrying a non-coding DNA fragment named barcode (BC). These will segregate through cell divisions and be present in cells' progeny. The remarkable characteristic of the method is that different cell types can be tracked simultaneously in the same mouse. Thus, we in vivo barcoded LMPP and CLP progenitors to test their ability to reconstitute the lymphoid lineage. Barcoded progenitors were co-grafted in immuno-compromised mice and their fate analyzed by evaluating the BC composition in transplanted mice. The results highlight the predominant role of LMPP progenitors for lymphoid generation and reveal valuable novel insights to be reconsidered in clinical transplantation assays.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Linfocitos , Animales , Ratones , Linaje de la Célula/genética , Linfocitos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Linfocitos T , Diferenciación CelularRESUMEN
The inactivation of S6 kinases mimics several aspects of caloric restriction, including small body size, increased insulin sensitivity and longevity. However, the impact of S6 kinase activity on cellular senescence remains to be established. Here, we show that the constitutive activation of mammalian target of rapamycin complex 1 (mTORC1) by tuberous sclerosis complex (TSC) mutations induces a premature senescence programme in fibroblasts that relies on S6 kinases. To determine novel molecular targets linking S6 kinase activation to the control of senescence, we set up a chemical genetic screen, leading to the identification of the nuclear epigenetic factor ZRF1 (also known as DNAJC2, MIDA1, Mpp11). S6 kinases phosphorylate ZRF1 on Ser47 in cultured cells and in mammalian tissues in vivo Knock-down of ZRF1 or expression of a phosphorylation mutant is sufficient to blunt the S6 kinase-dependent senescence programme. This is traced by a sharp alteration in p16 levels, the cell cycle inhibitor and a master regulator of senescence. Our findings reveal a mechanism by which nutrient sensing pathways impact on cell senescence through the activation of mTORC1-S6 kinases and the phosphorylation of ZRF1.
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Envejecimiento , Proteínas del Choque Térmico HSP40/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Quinasas S6 Ribosómicas/metabolismo , Animales , Células Cultivadas , Proteínas de Unión al ADN , Ratones , Chaperonas Moleculares , Fosforilación , Proteínas de Unión al ARNRESUMEN
Lymphocyte functions triggered by antigen recognition and co-stimulation signals are associated with a rapid and intense cell division, and hence with metabolism adaptation. The nucleotide cytidine 5' triphosphate (CTP) is a precursor required for the metabolism of DNA, RNA and phospholipids. CTP originates from two sources: a salvage pathway and a de novo synthesis pathway that depends on two enzymes, the CTP synthases (or synthetases) 1 and 2 (CTPS1 with CTPS2); the respective roles of these two enzymes are not known. CTP synthase activity is a potentially important step for DNA synthesis in lymphocytes. Here we report the identification of a loss-of-function homozygous mutation (rs145092287) in CTPS1 in humans that causes a novel and life-threatening immunodeficiency, characterized by an impaired capacity of activated T and B cells to proliferate in response to antigen receptor-mediated activation. In contrast, proximal and distal T-cell receptor (TCR) signalling events and responses were only weakly affected by the absence of CTPS1. Activated CTPS1-deficient cells had decreased levels of CTP. Normal T-cell proliferation was restored in CTPS1-deficient cells by expressing wild-type CTPS1 or by addition of exogenous CTP or its nucleoside precursor, cytidine. CTPS1 expression was found to be low in resting T cells, but rapidly upregulated following TCR activation. These results highlight a key and specific role of CTPS1 in the immune system by its capacity to sustain the proliferation of activated lymphocytes during the immune response. CTPS1 may therefore represent a therapeutic target of immunosuppressive drugs that could specifically dampen lymphocyte activation.
Asunto(s)
Ligasas de Carbono-Nitrógeno/deficiencia , Ligasas de Carbono-Nitrógeno/metabolismo , Activación de Linfocitos , Linfocitos/citología , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Complejo CD3/inmunología , Ligasas de Carbono-Nitrógeno/genética , Proliferación Celular , Preescolar , Citidina Trifosfato/metabolismo , Femenino , Humanos , Síndromes de Inmunodeficiencia/enzimología , Síndromes de Inmunodeficiencia/genética , Lactante , Recién Nacido , Activación de Linfocitos/genética , Linfocitos/inmunología , Linfocitos/metabolismo , Masculino , Mutación/genética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Although a growing body of evidence indicates that phenotypic plasticity exhibited by glioblastoma cells plays a central role in tumor development and post-therapy recurrence, the master drivers of their aggressiveness remain elusive. Here we mapped the changes in active (H3K4me3) and repressive (H3K27me3) histone modifications accompanying the repression of glioblastoma stem-like cells tumorigenicity. Genes with changing histone marks delineated a network of transcription factors related to cancerous behavior, stem state, and neural development, highlighting a previously unsuspected association between repression of ARNT2 and loss of cell tumorigenicity. Immunohistochemistry confirmed ARNT2 expression in cell sub-populations within proliferative zones of patients' glioblastoma. Decreased ARNT2 expression was consistently observed in non-tumorigenic glioblastoma cells, compared to tumorigenic cells. Moreover, ARNT2 expression correlated with a tumorigenic molecular signature at both the tissue level within the tumor core and at the single cell level in the patients' tumors. We found that ARNT2 knockdown decreased the expression of SOX9, POU3F2 and OLIG2, transcription factors implicated in glioblastoma cell tumorigenicity, and repressed glioblastoma stem-like cell tumorigenic properties in vivo. Our results reveal ARNT2 as a pivotal component of the glioblastoma cell tumorigenic signature, located at a node of a transcription factor network controlling glioblastoma cell aggressiveness.
Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias Encefálicas/metabolismo , Cromatina/metabolismo , Glioblastoma/metabolismo , Anciano , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Células Cultivadas , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glioblastoma/genética , Glioblastoma/patología , Código de Histonas , Proteínas de Homeodominio/metabolismo , Humanos , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Invasividad Neoplásica/fisiopatología , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Factores del Dominio POU/metabolismo , Factor de Transcripción SOX9/metabolismoRESUMEN
CEACAM1 expressed by granulocytes and epithelial cells is recognized as a membrane-associated receptor by some Gram-negative pathogens. Here we report a previously unsuspected role of human CEACAM1-4L (hCEACAM1-4L) in polarized epithelial cells. We find that in contrast with non-transfected cells, Madin Darby Canine Kidney strain II (MDCK) engineered for the apical expression of the long cytoplasmic chain protein hCEACAM1-4L showed a serum-independent increase in the phosphorylation of the extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinases (MAPKs) after treatment with lipopolysaccharide (LPS) of wild-type, diffusely adhering Afa/Dr Escherichia coli (Afa/Dr DAEC) strain IH11128. Aggregates of FITC-LPS bind the apical domain of MDCK-hCEACAM1-4L cells colocalizing with the apically expressed hCEACAM1-4L protein and do not bind MDCK-pCEP cells, and surface plasmon resonance analysis shows that LPS binds to the extracellular domain of the CEACAM1-4L protein. We showed that cell polarization and lipid rafts positively control the LPS-IH11128-induced phosphorylation of Erk1/2 in MDCK-hCEACAM1-4L cells. Structure-function analysis using mutated hCEACAM1-4L protein shows that the cytoplasmic domain of the protein is needed for LPS-induced MAPK signalling, and that phosphorylation of Tyr-residues is not increased in association with MAPK signalling. The hCEACAM1-4L-dependent Erk1/2 phosphorylation develops in the presence of lipid A and does not develop in the presence of penta-acylated LPS. Finally, small interfering RNA (siRNA) silencing of canine TLR4 abolishes the hCEACAM1-4L-dependent, LPS-induced phosphorylation of Erk1/2. Collectively, our results support the notion that the apically expressed, full-length hCEACAM1-4L protein functions as a novel LPS-conveying molecule at the mucosal surface of polarized epithelial cells for subsequent MD-2/TLR4 receptor-dependent MAPK Erk1/2 and p38 signalling.
Asunto(s)
Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Riñón/metabolismo , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Receptor Toll-Like 4/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Antígenos CD/genética , Moléculas de Adhesión Celular/genética , Línea Celular , Polaridad Celular , Perros , Escherichia coli/química , Ingeniería Genética , Humanos , Lípido A , Lipopolisacáridos/inmunología , Microdominios de Membrana/metabolismo , Membrana Mucosa/metabolismo , Membrana Mucosa/fisiología , Fosforilación , Isoformas de Proteínas/genética , Interferencia de ARN , ARN Interferente Pequeño , Resonancia por Plasmón de Superficie , Receptor Toll-Like 4/genéticaRESUMEN
The Mineralocorticoid Receptor (MR) mediates the sodium-retaining action of aldosterone in the distal nephron, but mechanisms regulating MR expression are still poorly understood. We previously showed that RNA Binding Proteins (RBPs) regulate MR expression at the post-transcriptional level in response to variations of extracellular tonicity. Herein, we highlight a novel regulatory mechanism involving the recruitment of microRNAs (miRNAs) under hypertonicity. RT-qPCR validated miRNAs candidates identified by high throughput screening approaches and transfection of a luciferase reporter construct together with miRNAs Mimics or Inhibitors demonstrated their functional interaction with target transcripts. Overexpression strategies using Mimics or lentivirus revealed the impact on MR expression and signaling in renal KC3AC1 cells. miR-324-5p and miR-30c-2-3p expression are increased under hypertonicity in KC3AC1 cells. These miRNAs directly affect Nr3c2 (MR) transcript stability, act with Tis11b to destabilize MR transcript but also repress Elavl1 (HuR) transcript, which enhances MR expression and signaling. Overexpression of miR-324-5p and miR-30c-2-3p alter MR expression and signaling in KC3AC1 cells with blunted responses in terms of aldosterone-regulated genes expression. We also confirm that their expression is increased by hypertonicity in vivo in the kidneys of mice treated with furosemide. These findings may have major implications for the pathogenesis of renal dysfunctions, sodium retention, and mineralocorticoid resistance.
Asunto(s)
MicroARNs/metabolismo , Receptores de Mineralocorticoides , Aldosterona/metabolismo , Animales , Riñón/metabolismo , Ratones , MicroARNs/genética , Mineralocorticoides/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Transducción de Señal , Sodio/metabolismoRESUMEN
Cell motility is critical for tumor malignancy. Metabolism being an obligatory step in shaping cell behavior, we looked for metabolic weaknesses shared by motile cells across the diverse genetic contexts of patients' glioblastoma. Computational analyses of single-cell transcriptomes from thirty patients' tumors isolated cells with high motile potential and highlighted their metabolic specificities. These cells were characterized by enhanced mitochondrial load and oxidative stress coupled with mobilization of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase (MPST). Functional assays with patients' tumor-derived cells and -tissue organoids, and genetic and pharmacological manipulations confirmed that the cells depend on enhanced ROS production and MPST activity for their motility. MPST action involved protection of protein cysteine residues from damaging hyperoxidation. Its knockdown translated in reduced tumor burden, and a robust increase in mice survival. Starting from cell-by-cell analyses of the patients' tumors, our work unravels metabolic dependencies of cell malignancy maintained across heterogeneous genomic landscapes.
Asunto(s)
Glioblastoma , Ratones , Animales , Glioblastoma/genética , Cisteína/metabolismo , Sulfurtransferasas/genética , Sulfurtransferasas/metabolismo , Estrés Oxidativo , Movimiento Celular/genéticaRESUMEN
Lentiviruses are among the most promising viral vectors for in vivo gene delivery. To overcome the risk of insertional mutagenesis, integrase-deficient lentiviral vectors (IDLVs) have been developed. We show here that strong and persistent specific cytotoxic T cell (CTL) responses are induced by IDLVs, which persist several months after a single injection. These responses were associated with the induction of mild and transient maturation of dendritic cells (DCs) and with the production of low levels of inflammatory cytokines and chemokines. They were independent of the IFN-I, TLR/MyD88, interferon regulatory factor (IRF), retinoic acid induced gene I (RIG-I), and stimulator of interferon genes (STING) pathways but require NF-κB signaling in CD11c+ DCs. Despite the lack of integration of IDLVs, the transgene persists for 3 months in the spleen and liver of IDLV-injected mice. These results demonstrate that the capacity of IDLVs to trigger persistent adaptive responses is mediated by a weak and transient innate response, along with the persistence of the vector in tissues.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Vectores Genéticos/metabolismo , Integrasas/deficiencia , Lentivirus/enzimología , Proteínas de la Membrana/metabolismo , Animales , Diferenciación Celular , Células Dendríticas/citología , Células HeLa , Humanos , Inmunidad , Integrasas/metabolismo , Interferones/metabolismo , Hígado/metabolismo , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Ovalbúmina/inmunología , Transducción de Señal , Bazo/metabolismo , Linfocitos T Citotóxicos/inmunología , Transcriptoma/genética , TransgenesRESUMEN
The mechanisms driving the development of extracapillary lesions in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN) remain poorly understood. A key question is how parietal epithelial cells (PECs) invade glomerular capillaries, thereby promoting injury and kidney failure. Here we show that expression of the tetraspanin CD9 increases markedly in PECs in mouse models of CGN and FSGS, and in kidneys from individuals diagnosed with these diseases. Cd9 gene targeting in PECs prevents glomerular damage in CGN and FSGS mouse models. Mechanistically, CD9 deficiency prevents the oriented migration of PECs into the glomerular tuft and their acquisition of CD44 and ß1 integrin expression. These findings highlight a critical role for de novo expression of CD9 as a common pathogenic switch driving the PEC phenotype in CGN and FSGS, while offering a potential therapeutic avenue to treat these conditions.
Asunto(s)
Enfermedades Renales/patología , Tetraspanina 29/fisiología , Animales , Movimiento Celular/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Femenino , Glomerulonefritis/genética , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Enfermedades Renales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Tetraspanina 29/genética , Tetraspanina 29/metabolismoRESUMEN
Using the in situ liver model system, we have recently shown that, after cholera toxin binding to hepatic cells, cholera toxin accumulates in a low-density endosomal compartment, and then undergoes endosomal proteolysis by the aspartic acid protease cathepsin-D [Merlen C, Fayol-Messaoudi D, Fabrega S, El Hage T, Servin A, Authier F (2005) FEBS J272, 4385-4397]. Here, we have used a subcellular fractionation approach to address the in vivo compartmentalization and cytotoxic action of cholera toxin in rat liver parenchyma. Following administration of a saturating dose of cholera toxin to rats, rapid endocytosis of both cholera toxin subunits was observed, coincident with massive internalization of both the 45 kDa and 47 kDa Gsalpha proteins. These events coincided with the endosomal recruitment of ADP-ribosylation factor proteins, especially ADP-ribosylation factor-6, with a time course identical to that of toxin and the A subunit of the stimulatory G protein (Gsalpha) translocation. After an initial lag phase of 30 min, these constituents were linked to NAD-dependent ADP-ribosylation of endogenous Gsalpha, with maximum accumulation observed at 30-60 min postinjection. Assessment of the subsequent postendosomal fate of internalized Gsalpha revealed sustained endolysosomal transfer of the two Gsalpha isoforms. Concomitantly, cholera toxin increased in vivo endosome acidification rates driven by the ATP-dependent H(+)-ATPase pump and in vitro vacuolar acidification in hepatoma HepG2 cells. The vacuolar H(+)-ATPase inhibitor bafilomycin and the cathepsin D inhibitor pepstatin A partially inhibited, both in vivo and in vitro, the cAMP response to cholera toxin. This cathepsin D-dependent action of cholera toxin under the control of endosomal acidity was confirmed using cellular systems in which modification of the expression levels of cathepsin D, either by transfection of the cathepsin D gene or small interfering RNA, was followed by parallel changes in the cytotoxic response to cholera toxin. Thus, in hepatic cells, a unique endocytic pathway was revealed following cholera toxin administration, with regulation specificity most probably occurring at the locus of the endosome and implicating endosomal proteases, such as cathepsin D, as well as organelle acidification.
Asunto(s)
Catepsina D/fisiología , Supervivencia Celular/efectos de los fármacos , Toxina del Cólera/farmacología , Endocitosis/fisiología , Endosomas/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Hígado/efectos de los fármacos , Factor 6 de Ribosilación del ADP , Ácidos/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Animales , Hígado/citología , Masculino , Transporte de Proteínas , Ratas , Ratas Sprague-DawleyRESUMEN
To assess glucagon receptor compartmentalization and signal transduction in liver parenchyma, we have studied the functional relationship between glucagon receptor endocytosis, phosphorylation and coupling to the adenylate cyclase system. Following administration of a saturating dose of glucagon to rats, a rapid internalization of glucagon receptor was observed coincident with its serine phosphorylation both at the plasma membrane and within endosomes. Co-incident with glucagon receptor endocytosis, a massive internalization of both the 45- and 47-kDa Gsalpha proteins was also observed. In contrast, no change in the subcellular distribution of adenylate cyclase or beta-arrestin 1 and 2 was observed. In response to des-His(1)-[Glu(9)]glucagon amide, a glucagon receptor antagonist, the extent and rate of glucagon receptor endocytosis and Gsalpha shift were markedly reduced compared with wild-type glucagon. However, while the glucagon analog exhibited a wild-type affinity for endosomal acidic glucagonase activity and was processed at low pH with similar kinetics and rates, its proteolysis at neutral pH was 3-fold lower. In response to tetraiodoglucagon, a glucagon receptor agonist of enhanced biological potency, glucagon receptor endocytosis and Gsalpha shift were of higher magnitude and of longer duration, and a marked and prolonged activation of adenylate cyclase both at the plasma membrane and in endosomes was observed. The subsequent post-endosomal fate of internalized Gsalpha was evaluated in a cell-free rat liver endosome-lysosome fusion system following glucagon injection. A sustained endo-lysosomal transfer of the two 45- and 47-kDa Gsalpha isoforms was observed. Therefore, these results reveal that within hepatic target cells and consequent to glucagon-mediated internalization of the serine-phosphorylated glucagon receptor and the Gsalpha protein, extended signal transduction may occur in vivo at the locus of the endo-lysosomal apparatus.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Glucagón/metabolismo , Hígado/metabolismo , Fosfoserina/metabolismo , Receptores de Glucagón/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Arrestinas/metabolismo , Endocitosis , Masculino , Ratas , Ratas Sprague-Dawley , beta-Arrestina 1 , beta-ArrestinasRESUMEN
We have defined the in vivo and in vitro metabolic fate of internalized cholera toxin (CT) in the endosomal apparatus of rat liver. In vivo, CT was internalized and accumulated in endosomes where it underwent degradation in a pH-dependent manner. In vitro proteolysis of CT using an endosomal lysate required an acidic pH and was sensitive to pepstatin A, an inhibitor of aspartic acid proteases. By nondenaturating immunoprecipitation, the acidic CT-degrading activity was attributed to the luminal form of endosomal cathepsin D. The rate of toxin hydrolysis using an endosomal lysate or pure cathepsin D was found to be high for native CT and free CT-B subunit, and low for free CT-A subunit. On the basis of IC(50) values, competition studies revealed that CT-A and CT-B subunits share a common binding site on the cathepsin D enzyme, with native CT and free CT-B subunit displaying the highest affinity for the protease. By immunofluorescence, partial colocalization of internalized CT with cathepsin D was confirmed at early times of endocytosis in both hepatoma HepG2 and intestinal Caco-2 cells. Hydrolysates of CT generated at low pH by bovine cathepsin D displayed ADP-ribosyltransferase activity towards exogenous Gsalpha protein suggesting that CT cytotoxicity, at least in part, may be related to proteolytic events within endocytic vesicles. Together, these data identify the endocytic apparatus as a critical subcellular site for the accumulation and proteolytic degradation of endocytosed CT, and define endosomal cathepsin D an enzyme potentially responsible for CT cytotoxic activation.
Asunto(s)
Catepsina D/metabolismo , Toxina del Cólera/metabolismo , Endosomas/metabolismo , Hígado/metabolismo , Animales , Biodegradación Ambiental , Biotransformación , Células CACO-2 , Bovinos , Línea Celular , Toxina del Cólera/química , Endocitosis , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Masculino , Subunidades de Proteína , Ratas , Ratas Sprague-DawleyRESUMEN
Herpes simplex encephalitis (HSE) is the most common sporadic viral encephalitis of childhood. Autosomal recessive (AR) UNC-93B and TLR3 deficiencies and autosomal dominant (AD) TLR3 and TRAF3 deficiencies underlie HSE in some children. We report here unrelated HSE children with AR or AD TRIF deficiency. The AR form of the disease was found to be due to a homozygous nonsense mutation that resulted in a complete absence of the TRIF protein. Both the TLR3- and the TRIF-dependent TLR4 signaling pathways were abolished. The AD form of disease was found to be due to a heterozygous missense mutation, resulting in a dysfunctional protein. In this form of the disease, the TLR3 signaling pathway was impaired, whereas the TRIF-dependent TLR4 pathway was unaffected. Both patients, however, showed reduced capacity to respond to stimulation of the DExD/H-box helicases pathway. To date, the TRIF-deficient patients with HSE described herein have suffered from no other infections. Moreover, as observed in patients with other genetic etiologies of HSE, clinical penetrance was found to be incomplete, as some HSV-1-infected TRIF-deficient relatives have not developed HSE. Our results provide what we believe to be the first description of human TRIF deficiency and a new genetic etiology for HSE. They suggest that the TRIF-dependent TLR4 and DExD/H-box helicase pathways are largely redundant in host defense. They further demonstrate the importance of TRIF for the TLR3-dependent production of antiviral IFNs in the CNS during primary infection with HSV-1 in childhood.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Encefalitis por Herpes Simple/genética , Herpesvirus Humano 1 , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Secuencia de Aminoácidos , Preescolar , Codón sin Sentido , Consanguinidad , ARN Helicasas DEAD-box/fisiología , Femenino , Genes Dominantes , Genes Recesivos , Heterogeneidad Genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Lactante , Interferón-alfa/biosíntesis , Interferón-alfa/genética , Masculino , Datos de Secuencia Molecular , Mutación Missense , Linaje , Arabia Saudita , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Receptor Toll-Like 3/fisiología , Receptor Toll-Like 4/fisiologíaRESUMEN
Gaucher disease and Fabry disease are lysosomal storage disorders characterized by the accumulation of sphingolipids. In both cases, the goal of gene therapy is to permanently provide tissues with enzyme levels allowing to avoid storage of the undigested substrates. Different gene therapy strategies must however be designed as Gaucher disease is due to a deficiency in the membrane-associated enzyme glucocerebrosidase, whereas Fabry disease is caused by a deficiency in the soluble enzyme alpha-galactosidase. Indeed, a soluble enzyme can be provided to tissues is trans by gene-modified cells whereas gene transfer has to target the most affected cells in the case of membrane-bound enzymes. Thus, in non-neurological Gaucher disease (type 1), the hematopoietic tissue has to be targeted as the deficiency affects the monocyte/macrophage lineage. Following promising preclinical studies, clinical protocols have been initiated to explore the feasibility and safety of retroviral transfer of the glucocerebrosidase gene into CD34+ cells from patients with type 1 Gaucher disease. Although gene-marked cells were detected in vivo, the level of corrected cells was very low, a finding indicating that improved vectors along with partial myeloablation may be necessary. Here, lentiviral vectors should enable more gene transduction into the hematopoietic target cells. As concerns the diffuse neurological lesions in types 2 and 3 of Gaucher disease, they will probably be especially difficult to target by gene therapy because of the non soluble nature of glucocerebrosidase. Finally, over the last few years, Fabry disease has become a compelling target for gene therapy as an etiology-based treatment strategy. Indeed, several recent studies aiming at creating a large in vivo source of alpha-galactosidase have yielded positive long-term results in the Fabry knock-out mouse model.
Asunto(s)
Enfermedad de Fabry/terapia , Enfermedad de Gaucher/terapia , Terapia Genética , Animales , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Enfermedad de Fabry/enzimología , Enfermedad de Fabry/genética , Predicción , Enfermedad de Gaucher/clasificación , Enfermedad de Gaucher/enzimología , Enfermedad de Gaucher/genética , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Glucosilceramidasa/química , Glucosilceramidasa/deficiencia , Glucosilceramidasa/genética , Sistema Hematopoyético/enzimología , Humanos , Lentivirus/genética , Lisosomas/enzimología , Ratones , Ratones Noqueados , Sistema Nervioso/enzimología , Retroviridae/genética , Solubilidad , Trasplante de Células Madre , alfa-Galactosidasa/química , alfa-Galactosidasa/genética , alfa-Galactosidasa/uso terapéuticoRESUMEN
Gaucher disease is a lysosomal storage disorder caused by a deficiency in glucocerebrosidase which cleaves the beta-glucosidic linkage of glucosylceramide, a normal intermediate in glycolipid metabolism. Glucocerebrosidase belongs to the clan GH-A of glycoside hydrolases, a large group of enzymes which function with retention of the anomeric configuration at the hydrolysis site. Accurate three-dimensional (3D) structure data for glucocerebrosidase should help to better understand the molecular bases of Gaucher disease. As such 3D structure data were not available, we used the two-dimensional hydrophobic cluster analysis (HCA) method to make structure predictions for the catalytic domains of clan GH-A glycoside hydrolases. We found that all the enzymes of clan GH-A may share a similar catalytic domain consisting of an (alpha/beta)8 barrel with the critical acid/base and nucleophile residues located at the C-terminal ends of strands beta 4 and beta 7, respectively. In the case of glucocerebrosidase, Glu 235 was predicted to be the putative acid/base catalyst whereas the nucleophile was located at Glu 340. Next, in order to obtain experimental evidence supporting these HCA-based predictions, we used retroviral vectors to express, in murine null cells, E235A and E340A mutant proteins, in which alanine residues unable to participate in the enzymatic reaction replace the presumed critical glutamic acid residues. Both mutants were found to be catalytically inactive although they were correctly folded/processed and sorted to the lysosome. Thus, Glu 235 and Glu 340 do indeed play key roles in the active site of human glucocerebrosidase as predicted by the HCA analysis. In a broader perspective, our work points out that bioinformatics approaches may be highly useful for generating structure-function predictions based on sequence-structure interrelationships, especially in the context of a rapid increase in protein sequence information through genome sequencing.
Asunto(s)
Glucosilceramidasa/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Dominio Catalítico , Glucosilceramidasa/genética , Ácido Glutámico/química , Glicosilación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Puntual , Conformación Proteica , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase, which hydrolyzes internalized insulin and generates the major primary end product A(1--21)-B(1--24) insulin resulting from a major cleavage at residues Phe(B24)-Phe(B25). This study addresses the nature of the relevant endopeptidase activity in rat liver that is responsible for most receptor-mediated insulin degradation in vivo. The endosomal activity was shown to be aspartic acid protease cathepsin D (CD), based on biochemical similarities to purified CD in 1) the rate and site of substrate cleavage, 2) pH optimum, 3) sensitivity to pepstatin A, and 4) binding to pepstatin A-agarose. The identity of the protease was immunologically confirmed by removal of greater than 90% of the insulin-degrading activity associated with an endosomal lysate using polyclonal antibodies to CD. Moreover, the elution profile of the endosomal acidic insulinase activity on a gel-filtration TSK-GEL G3000 SW(XL) high performance liquid chromatography column corresponded exactly with the elution profile of the immunoreactive 45-kDa mature form of endosomal CD. Using nondenaturating immunoprecipitation and immunoblotting procedures, other endosomal aspartic acid proteases such as cathepsin E and beta-site amyloid precursor protein-cleaving enzyme (BACE) were ruled out as candidate enzymes for the endosomal degradation of internalized insulin. Immunofluorescence studies showed a largely vesicular staining pattern for internalized insulin in rat hepatocytes that colocalized partially with CD. In vivo pepstatin A treatment was without any observable effect on the insulin receptor content of endosomes but augmented the phosphotyrosine content of the endosomal insulin receptor after insulin injection. These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the Phe(B24)-Phe(B25) bond, generating the inactive A(1--21)-B(1--24) insulin intermediate.