RESUMEN
Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , ADN Helicasas/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Esclerosis Tuberosa/metabolismo , Secuencia de Aminoácidos , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/química , Evolución Molecular , Femenino , Humanos , Insulina/farmacología , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fenotipo , Proteínas de Unión a Poli-ADP-Ribosa/química , ARN Helicasas/química , Proteínas con Motivos de Reconocimiento de ARN/química , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Pez Cebra/metabolismoRESUMEN
The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy1. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1)2-5 is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino acid-mediated activation of the RagC GTPase activating protein FLCN6,7. TFEB lacks the TOR signalling motif responsible for the recruitment of other mTORC1 substrates. We used cryogenic-electron microscopy to determine the structure of TFEB as presented to mTORC1 for phosphorylation, which we refer to as the 'megacomplex'. Two full Rag-Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag-Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag-Ragulator complex (non-canonical) docks onto the first through a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB with a RagCGDP-dependent aspartate clamp in the cleft between the Rag G domains. In cellulo mutation of the clamp drives TFEB constitutively into the nucleus while having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state.
Asunto(s)
Lisosomas , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas de Unión al GTP Monoméricas , Aminoácidos/metabolismo , Dominio Catalítico , Guanosina Difosfato/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosforilación , Multimerización de Proteína , Proteína Reguladora Asociada a mTOR/metabolismo , Transducción de SeñalRESUMEN
The neurodevelopmental disorders Prader-Willi syndrome (PWS) and Schaaf-Yang syndrome (SYS) both arise from genomic alterations within human chromosome 15q11-q13. A deletion of the SNORD116 cluster, encoding small nucleolar RNAs, or frameshift mutations within MAGEL2 result in closely related phenotypes in individuals with PWS or SYS, respectively. By investigation of their subcellular localization, we observed that in contrast to a predominant cytoplasmic localization of wild-type (WT) MAGEL2, a truncated MAGEL2 mutant was evenly distributed between the cytoplasm and the nucleus. To elucidate regulatory pathways that may underlie both diseases, we identified protein interaction partners for WT or mutant MAGEL2, in particular the survival motor neuron protein (SMN), involved in spinal muscular atrophy, and the fragile-X-messenger ribonucleoprotein (FMRP), involved in autism spectrum disorders. The interactome of the non-coding RNA SNORD116 was also investigated by RNA-CoIP. We show that WT and truncated MAGEL2 were both involved in RNA metabolism, while regulation of transcription was mainly observed for WT MAGEL2. Hence, we investigated the influence of MAGEL2 mutations on the expression of genes from the PWS locus, including the SNORD116 cluster. Thereby, we provide evidence for MAGEL2 mutants decreasing the expression of SNORD116, SNORD115, and SNORD109A, as well as protein-coding genes MKRN3 and SNRPN, thus bridging the gap between PWS and SYS.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Proteínas Intrínsecamente Desordenadas , Síndrome de Prader-Willi , Humanos , Cromosomas Humanos Par 15/genética , Citoplasma/metabolismo , Células HEK293 , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Síndrome de Prader-Willi/genética , Proteínas/genética , Proteínas/metabolismo , ARN Nucleolar Pequeño/genéticaRESUMEN
The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1-3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB-unlike other substrates of mTORC1, such as S6K and 4E-BP1- is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt-Hogg-Dubé syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt-Hogg-Dubé syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer.
Asunto(s)
Síndrome de Birt-Hogg-Dubé/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/patología , Línea Celular , Modelos Animales de Enfermedad , Activación Enzimática , Células HeLa , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Ratones , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteína Homóloga de Ras Enriquecida en el Cerebro/metabolismo , Especificidad por Sustrato , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
BACKGROUND: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. METHODS: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/ß receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. RESULTS: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn-/-, Tlr3-/-, and Ifnar1-/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/ß receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. CONCLUSIONS: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.
Asunto(s)
Estenosis de la Válvula Aórtica , Calcinosis , Adulto , Animales , Humanos , Ratones , Válvula Aórtica/patología , Estenosis de la Válvula Aórtica/patología , Biglicano/metabolismo , Calcinosis/metabolismo , Células Cultivadas , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Pez CebraRESUMEN
Seasonally circulating viruses, such as Influenza, as well as newly emerging viruses and variants thereof, and waning immunity urge the need for safe, easy-to-use and inexpensive drugs to protect from these challenges. To prevent transmission of these viruses and subsequent excessive inflammatory reactions on mucous membranes, we tested the efficacy of the natural essence P80 as spray and in form of lozenges against respiratory infections caused by SARS-CoV-2 variants of concern (VoCs), influenza A (H3N2) and influenza B (Victoria). P80 natural essence, a Dimocarpus longan extract, shielded highly differentiated human airway epithelia from SARS-CoV-2 wildtype and Omicron variant as well as Influenza A and B infection and dampened inflammation by down-modulating pro-inflammatory cytokine and anaphylatoxin secretion. A single application of P80 natural essence spray maintained tissue integrity long-term. This also significantly reduced the release of infectious viral particles and the secretion of IP10, MCP1, RANTES and C3a, all of which mediate the migration of immune cells to the sites of infection. Even P80 lozenges dissolved in distilled water or non-neutralizing saliva efficiently prevented SARS-CoV-2 and Influenza-induced tissue destruction. Consequently, our in vitro data suggest that P80 natural essence can act as antiviral prophylactic, both in form of nasal or oral spray and in form of lozenges, independent of circulating respiratory challenges.
Asunto(s)
COVID-19 , Gripe Humana , Humanos , Gripe Humana/prevención & control , Subtipo H3N2 del Virus de la Influenza A , SARS-CoV-2 , InflamaciónRESUMEN
New SARS-CoV-2 variants of concern (VOCs) and waning immunity illustrate that quick and easy-to-use agents are needed to prevent infection. To protect from viral transmission and subsequent inflammatory reactions, we applied GlyperA™, a novel antimicrobial formulation that can be used as mouth gargling solution or as nasal spray, to highly differentiated human airway epithelia prior infection with Omicron VOCs BA.1 and BA.2. This formulation fully protected polarized human epithelium cultured in air-liquid interphase (ALI) from SARS-CoV-2-mediated tissue destruction and infection upon single application up to two days post infection. Moreover, inflammatory reactions induced by the Omicron VOCs were significantly lowered in tissue equivalents either pre-treated with the GlyperA™ solution, or even when added simultaneously. Thus, the GlyperA™ formulation significantly shielded epithelial integrity, successfully blocked infection with Omicron and release of viral particles, and decreased intracellular complement C3 activation within human airway epithelial cell cultures. Crucially, our in vitro data imply that GlyperA™ may be a simple tool to prevent from SARS-CoV-2 infection independent on the circulating variant via both, mouth and nose.
Asunto(s)
COVID-19 , Humanos , SARS-CoV-2 , Epitelio , Nariz , InflamaciónRESUMEN
Lysosomes are key cellular catabolic centers that also perform fundamental metabolic, signaling and quality control functions. Lysosomes are not static and they respond dynamically to intra- and extracellular stimuli triggering changes in organelle numbers, size and position. Such physical changes have a strong impact on lysosomal activity ultimately influencing cellular homeostasis. In this review, we summarize the current knowledge on lysosomal size regulation, on its physiological role(s) and association to several disease conditions.
Asunto(s)
Lisosomas , Transducción de Señal , Autofagia , HomeostasisRESUMEN
OBJECTIVES: Metabolic inflammation is a hallmark of obesity and related disorders, afflicting substantial morbidity and mortality to individuals worldwide. White visceral and subcutaneous adipose tissue not only serves as energy storage but also controls metabolism. Adipose tissue inflammation, commonly observed in human obesity, is considered a critical driver of metabolic perturbation while molecular hubs are poorly explored. Metabolic stress evoked by e.g. long-chain fatty acids leads to oxidative perturbation of adipocytes and production of inflammatory cytokines, fuelling macrophage infiltration and systemic low-grade inflammation. Glutathione peroxidase 4 (GPX4) protects against lipid peroxidation, accumulation of oxygen-specific epitopes and cell death, collectively referred to as ferroptosis. Here, we explore the function of adipocyte GPX4 in mammalian metabolism. METHODS: We studied the regulation and function of GPX4 in differentiated mouse adipocytes derived from 3T3-L1 fibroblasts. We generated two conditional adipocyte-specific Gpx4 knockout mice by crossing Gpx4fl/fl mice with Adipoq-Cre+ (Gpx4-/-AT) or Fabp4-Cre+ (Gpx4+/-Fabp4) mice. Both models were metabolically characterized by a glucose tolerance test and insulin resistance test, and adipose tissue lipid peroxidation, inflammation and cell death were assessed by quantifying oxygen-specific epitopes, transcriptional analysis of chemokines, quantification of F4/80+ macrophages and TUNEL labelling. RESULTS: GPX4 expression was induced during and required for adipocyte differentiation. In mature adipocytes, impaired GPX4 activity spontaneously evoked lipid peroxidation and expression of inflammatory cytokines such as TNF-α, interleukin 1ß (IL-1ß), IL-6 and the IL-8 homologue CXCL1. Gpx4-/-AT mice spontaneously displayed adipocyte hypertrophy on a chow diet, which was paralleled by the accumulation of oxygen-specific epitopes and macrophage infiltration in adipose tissue. Furthermore, Gpx4-/-AT mice spontaneously developed glucose intolerance, hepatic insulin resistance and systemic low-grade inflammation, when compared to wildtype littermates, which was similarly recapitulated in Gpx4+/-Fabp4 mice. Gpx4-/-AT mice exhibited no signs of adipocyte death. CONCLUSION: Adipocyte GPX4 protects against spontaneous metabolic dysregulation and systemic low-grade inflammation independent from ferroptosis, which could be therapeutically exploited in the future.
Asunto(s)
Resistencia a la Insulina , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Animales , Citocinas/metabolismo , Dieta Alta en Grasa , Epítopos/metabolismo , Inflamación/metabolismo , Mamíferos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/metabolismo , Oxígeno/metabolismo , Fosfolípido Hidroperóxido Glutatión PeroxidasaRESUMEN
BACKGROUND: Excessive inflammation triggered by a hitherto undescribed mechanism is a hallmark of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and is associated with enhanced pathogenicity and mortality. OBJECTIVE: Complement hyperactivation promotes lung injury and was observed in patients suffering from Middle East respiratory syndrome-related coronavirus, SARS-CoV-1, and SARS-CoV-2 infections. Therefore, we investigated the very first interactions of primary human airway epithelial cells on exposure to SARS-CoV-2 in terms of complement component 3 (C3)-mediated effects. METHODS: For this, we used highly differentiated primary human 3-dimensional tissue models infected with SARS-CoV-2 patient isolates. On infection, viral load, viral infectivity, intracellular complement activation, inflammatory mechanisms, and tissue destruction were analyzed by real-time RT-PCR, high content screening, plaque assays, luminex analyses, and transepithelial electrical resistance measurements. RESULTS: Here, we show that primary normal human bronchial and small airway epithelial cells respond to SARS-CoV-2 infection by an inflated local C3 mobilization. SARS-CoV-2 infection resulted in exaggerated intracellular complement activation and destruction of the epithelial integrity in monolayer cultures of primary human airway cells and highly differentiated, pseudostratified, mucus-producing, ciliated respiratory tissue models. SARS-CoV-2-infected 3-dimensional cultures secreted significantly higher levels of C3a and the proinflammatory cytokines IL-6, monocyte chemoattractant protein 1, IL-1α, and RANTES. CONCLUSIONS: Crucially, we illustrate here for the first time that targeting the anaphylotoxin receptors C3a receptor and C5a receptor in nonimmune respiratory cells can prevent intrinsic lung inflammation and tissue damage. This opens up the exciting possibility in the treatment of COVID-19.
Asunto(s)
Bronquios/inmunología , COVID-19/inmunología , Activación de Complemento , Células Epiteliales/inmunología , Receptor de Anafilatoxina C5a/inmunología , Mucosa Respiratoria/inmunología , SARS-CoV-2/inmunología , Bronquios/patología , Bronquios/virología , COVID-19/patología , COVID-19/virología , Línea Celular , Complemento C3/inmunología , Citocinas/inmunología , Células Epiteliales/patología , Células Epiteliales/virología , Humanos , Inflamación/inmunología , Inflamación/patología , Mucosa Respiratoria/patología , Mucosa Respiratoria/virologíaRESUMEN
Mechanisms that control lysosomal function are essential for cellular homeostasis. Lysosomes adapt in size and number to cellular needs but little is known about the underlying molecular mechanism. We demonstrate that the late endosomal/lysosomal multimeric BLOC-1-related complex (BORC) regulates the size of these organelles via PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2 ] production. Deletion of the core BORC component Diaskedin led to increased levels of PI(3,5)P2 , suggesting activation of PIKfyve, and resulted in enhanced lysosomal reformation and subsequent reduction in lysosomal size. This process required AMP-activated protein kinase (AMPK), a known PIKfyve activator, and was additionally dependent on the late endosomal/lysosomal adaptor, mitogen-activated protein kinases and mechanistic target of rapamycin activator (LAMTOR/Ragulator) complex. Consistently, in response to glucose limitation, AMPK activated PIKfyve, which induced lysosomal reformation with increased baseline autophagy and was coupled to a decrease in lysosomal size. These adaptations of the late endosomal/lysosomal system reversed under glucose replete growth conditions. In summary, our results demonstrate that BORC regulates lysosomal reformation and size in response to glucose availability.
Asunto(s)
Endosomas/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Autofagia , Células HEK293 , Células HeLa , Humanos , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Proteínas de Membrana de los Lisosomas/genética , Sistema de Señalización de MAP Quinasas , Ratones , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas/genética , Proteínas/metabolismoRESUMEN
Biallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic-intestinal and retinal-disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.
Asunto(s)
Enfermedades Hereditarias del Ojo/genética , Mucosa Intestinal/metabolismo , Síndromes de Malabsorción/genética , Microvellosidades/patología , Mucolipidosis/genética , Polimorfismo de Nucleótido Simple , Proteínas Qa-SNARE/genética , Células Fotorreceptoras Retinianas Conos/metabolismo , Distrofias Retinianas/genética , Anciano , Anciano de 80 o más Años , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Animales , Autopsia , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Enfermedades Hereditarias del Ojo/metabolismo , Enfermedades Hereditarias del Ojo/patología , Femenino , Regulación de la Expresión Génica , Homocigoto , Humanos , Mucosa Intestinal/patología , Síndromes de Malabsorción/metabolismo , Síndromes de Malabsorción/patología , Ratones , Ratones Noqueados , Microvellosidades/genética , Microvellosidades/metabolismo , Mucolipidosis/metabolismo , Mucolipidosis/patología , Fenotipo , Proteínas Qa-SNARE/deficiencia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Distrofias Retinianas/metabolismo , Distrofias Retinianas/patología , Rodopsinas Sensoriales/genética , Rodopsinas Sensoriales/metabolismo , Secuenciación del ExomaRESUMEN
Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.
Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Lisosomas/metabolismo , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Proteínas de Unión al GTP Monoméricas/metabolismo , Nucleótidos/metabolismoRESUMEN
Immunogold labeling of permeabilized whole-mount cells or thin-sectioned material is widely used for the subcellular localization of biomolecules at the high spatial resolution of electron microscopy (EM). Those approaches are well compatible with either 3-dimensional (3D) reconstruction of organelle morphology and antigen distribution or with rapid cryofixation-but not easily with both at once. We describe here a specimen preparation and labeling protocol for animal cell cultures, which represents a novel blend of specifically adapted versions of established techniques. It combines the virtues of reliably preserved organelle ultrastructure, as trapped by rapid freezing within milliseconds followed by freeze-substitution and specimen rehydration, with the advantages of robust labeling of intracellular constituents in 3D through means of pre-embedding NANOGOLD-silver immunocytochemistry. So obtained thin and semi-thick epoxy resin sections are suitable for transmission EM imaging, as well as tomographic reconstruction and modeling of labeling patterns in the 3D cellular context.
Asunto(s)
Microscopía Electrónica de Transmisión/métodos , Microscopía Inmunoelectrónica/métodos , Tomografía/métodos , Animales , Antígenos/química , Células CACO-2 , Criopreservación/métodos , Compuestos Epoxi/química , Congelación , Oro/química , Células HeLa , Humanos , Inmunohistoquímica , Nanopartículas/química , Presión , Plata/químicaRESUMEN
Congenital diarrheal disorders (CDD) comprise > 50 monogenic entities featuring chronic diarrhea of early-onset, including defects in nutrient and electrolyte absorption, enterocyte polarization, enteroendocrine cell differentiation, and epithelial integrity. Diarrhea is also a predominant symptom in many immunodeficiencies, congenital disorders of glycosylation, and in some defects of the vesicular sorting and transporting machinery. We set out to identify the etiology of an intractable diarrhea in 2 consanguineous families by whole-exome sequencing, and identified two novel AP1S1 mutations, c.269T>C (p.Leu90Pro) and c.346G>A (p.Glu116Lys). AP1S1 encodes the small subunit of the adaptor protein 1 complex (AP-1), which plays roles in clathrin coat-assembly and trafficking between trans-Golgi network, endosomes and the plasma membrane. An AP1S1 knock-out (KO) of a CaCo2 intestinal cell line was generated to characterize intestinal AP1S1 deficiency as well as identified mutations by stable expression in KO background. Morphology and prototype transporter protein distribution were comparable between parental and KO cells. We observed altered localization of tight-junction proteins ZO-1 and claudin 3, decreased transepithelial electrical resistance and an increased dextran permeability of the CaCo2-AP1S1-KO monolayer. In addition, lumen formation in 3D cultures of these cells was abnormal. Re-expression of wild-type AP1S1 in CaCo2-AP1S1-KO cells reverted these abnormalities, while expression of AP1S1 containing either missense mutation did not. Our data indicate that loss of AP1S1 function causes an intestinal epithelial barrier defect, and that AP1S1 mutations can cause a non-syndromic form of congenital diarrhea, whereas 2 reported truncating AP1S1 mutations caused MEDNIK syndrome, characterized by mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratodermia.
Asunto(s)
Complejo 1 de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Sordera/genética , Diarrea/genética , Ictiosis/genética , Discapacidad Intelectual/genética , Queratodermia Palmoplantar/genética , Mutación Missense , Complejo 1 de Proteína Adaptadora/deficiencia , Subunidades sigma de Complejo de Proteína Adaptadora/deficiencia , Secuencia de Bases , Células CACO-2 , Claudina-3/genética , Claudina-3/metabolismo , Consanguinidad , Sordera/diagnóstico , Sordera/metabolismo , Sordera/patología , Diarrea/diagnóstico , Diarrea/metabolismo , Diarrea/patología , Femenino , Expresión Génica , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Humanos , Ictiosis/diagnóstico , Ictiosis/metabolismo , Ictiosis/patología , Lactante , Recién Nacido , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Queratodermia Palmoplantar/diagnóstico , Queratodermia Palmoplantar/metabolismo , Queratodermia Palmoplantar/patología , Linaje , Permeabilidad , Secuenciación del Exoma , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Biallelic loss-of-function mutations in the centrosomal pericentrin gene (PCNT) cause microcephalic osteodysplastic primordial dwarfism type II (MOPDII), which is characterized by extreme growth retardation, microcephaly, skeletal dysplasia, and dental anomalies. Life expectancy is reduced due to a high risk of cerebral vascular anomalies. Here, we report two siblings with MOPDII and attenuated growth restriction, and pachygyria. Compound heterozygosity for two novel truncated PCNT variants was identified. Both truncated PCNT proteins were expressed in patient's fibroblasts, with a reduced total protein amount compared to control. Patient's fibroblasts showed impaired cell cycle progression. As a novel finding, 20% of patient's fibroblasts were shown to express PCNT comparable to control. This was associated with normal mitotic morphology and normal co-localization of mutated PCNT with centrosome-associated proteins γ-tubulin and centrin 3, suggesting some residual function of truncated PCNT proteins. These data expand the clinical and molecular spectrum of MOPDII and indicate that residual PCNT function might be associated with attenuated growth restriction in MOPDII.
Asunto(s)
Antígenos/genética , Enanismo/genética , Retardo del Crecimiento Fetal/genética , Predisposición Genética a la Enfermedad , Lisencefalia/genética , Microcefalia/genética , Osteocondrodisplasias/genética , Adolescente , Adulto , Alelos , Centrosoma/metabolismo , Niño , Preescolar , Enanismo/patología , Femenino , Retardo del Crecimiento Fetal/patología , Fibroblastos/metabolismo , Humanos , Lisencefalia/patología , Mutación con Pérdida de Función/genética , Masculino , Microcefalia/patología , Osteocondrodisplasias/patología , Hermanos , Tubulina (Proteína)/genética , Adulto JovenRESUMEN
Erythropoietin (EPO) is the principal cytokine regulating erythropoiesis through its receptor, EPOR. Interestingly, EPORs are also found on immune cells with incompletely understood functions. Here, we show that EPO inhibits the induction of proinflammatory genes including tumor necrosis factor (TNF)-α and inducible nitric oxide (NO) synthase in activated macrophages, which is mechanistically attributable to blockage of nuclear factor (NF)-κB p65 activation by EPO. Accordingly, in systemic Salmonella infection, treatment of mice with EPO results in reduced survival and impaired pathogen clearance because of diminished formation of anti-microbial effector molecules such as TNF-α and NO. However, neutralization of endogenous EPO or genetic ablation of Epor promotes Salmonella elimination. In contrast, in chemically induced colitis, EPO-EPOR interaction decreases the production of NF-κB-inducible immune mediators, thus limiting tissue damage and ameliorating disease severity. These immune-modulatory effects of EPO may be of therapeutic relevance in infectious and inflammatory diseases.
Asunto(s)
Colitis/inmunología , Eritropoyetina/administración & dosificación , Macrófagos Peritoneales/efectos de los fármacos , FN-kappa B/metabolismo , Receptores de Eritropoyetina/metabolismo , Infecciones por Salmonella/inmunología , Salmonella/inmunología , Animales , Línea Celular , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Sulfato de Dextran/administración & dosificación , Humanos , Mediadores de Inflamación/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico/inmunología , Óxido Nítrico/metabolismo , Receptores de Eritropoyetina/genética , Salmonella/patogenicidad , Infecciones por Salmonella/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Ácido Trinitrobencenosulfónico/administración & dosificación , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by accumulation of vesiculo-tubular endomembranes in the subapical cytoplasm of enterocytes, historically termed "secretory granules." However, neither their identity nor pathophysiological significance is well defined. Using immunoelectron microscopy and tomography, we studied biopsies from MVID patients (3× Myosin 5b mutations and 1× Syntaxin3 mutation) and compared them to controls and genome-edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from 2 patients with novel Myosin 5b mutations and typical clinical symptoms showed unusual ultrastructural phenotypes: aberrant subapical vesicles and tubules were prominent in the enterocytes, though other histological hallmarks of MVID were almost absent (ectopic intra-/intercellular microvilli, brush border atrophy). We identified these enigmatic vesiculo-tubular organelles as Rab11-Rab8-positive recycling compartments of altered size, shape and location harboring the apical SNARE Syntaxin3, apical transporters sodium-hydrogen exchanger 3 (NHE3) and cystic fibrosis transmembrane conductance regulator. Our data strongly indicate that in MVID disrupted trafficking between cargo vesicles and the apical plasma membrane is the primary cause of a defect of epithelial polarity and subsequent facultative loss of brush border integrity, leading to malabsorption. Furthermore, they support the notion that mislocalization of transporters, such as NHE3 substantially contributes to the reported sodium loss diarrhea.
Asunto(s)
Enterocitos/metabolismo , Síndromes de Malabsorción/metabolismo , Microvellosidades/patología , Mucolipidosis/metabolismo , Vesículas Secretoras/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Células CACO-2 , Membrana Celular/metabolismo , Enterocitos/ultraestructura , Humanos , Síndromes de Malabsorción/genética , Masculino , Microvellosidades/genética , Microvellosidades/metabolismo , Mucolipidosis/genética , Mutación , Miosina Tipo V/genética , Transporte de Proteínas , Proteínas Qa-SNARE/genética , Vesículas Secretoras/ultraestructuraRESUMEN
BACKGROUND & AIMS: Inactivating mutations in MYO5B cause microvillus inclusion disease (MVID), but the physiological cause of the diarrhea associated with this disease is unclear. We investigated whether loss of MYO5B results in aberrant expression of apical enterocyte transporters. METHODS: We studied alterations in apical membrane transporters in MYO5B-knockout mice, as well as mice with tamoxifen-inducible, intestine-specific disruption of Myo5b (VilCreERT2;Myo5bflox/flox mice) or those not given tamoxifen (controls). Intestinal tissues were collected from mice and analyzed by immunostaining, immunoelectron microscopy, or cultured enteroids were derived. Functions of brush border transporters in intestinal mucosa were measured in Ussing chambers. We obtained duodenal biopsy specimens from individuals with MVID and individuals without MVID (controls) and compared transporter distribution by immunocytochemistry. RESULTS: Compared to intestinal tissues from littermate controls, intestinal tissues from MYO5B-knockout mice had decreased apical localization of SLC9A3 (also called NHE3), SLC5A1 (also called SGLT1), aquaporin (AQP) 7, and sucrase isomaltase, and subapical localization of intestinal alkaline phosphatase and CDC42. However, CFTR was present on apical membranes of enterocytes from MYO5B knockout and control mice. Intestinal biopsies from patients with MVID had subapical localization of NHE3, SGLT1, and AQP7, but maintained apical CFTR. After tamoxifen administration, VilCreERT2;Myo5bflox/flox mice lost apical NHE3, SGLT1, DRA, and AQP7, similar to germline MYO5B knockout mice. Intestinal tissues from VilCreERT2;Myo5bflox/flox mice had increased CFTR in crypts and CFTR localized to the apical membranes of enterocytes. Intestinal mucosa from VilCreERT2;Myo5bflox/flox mice given tamoxifen did not have an intestinal barrier defect, based on Ussing chamber analysis, but did have decreased SGLT1 activity and increased CFTR activity. CONCLUSIONS: Although trafficking of many apical transporters is regulated by MYO5B, trafficking of CFTR is largely independent of MYO5B. Decreased apical localization of NHE3, SGLT1, DRA, and AQP7 might be responsible for dysfunctional water absorption in enterocytes of patients with MVID. Maintenance of apical CFTR might exacerbate water loss by active secretion of chloride into the intestinal lumen.
Asunto(s)
Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Enterocitos/metabolismo , Síndromes de Malabsorción/genética , Microvellosidades/patología , Mucolipidosis/genética , Miosina Tipo V/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Acuaporinas/metabolismo , Duodeno/metabolismo , Duodeno/patología , Silenciador del Gen , Humanos , Mucosa Intestinal , Intestinos/citología , Intestinos/patología , Síndromes de Malabsorción/patología , Ratones , Ratones Noqueados , Microvellosidades/genética , Mucolipidosis/patología , Transporte de Proteínas , Transportador 1 de Sodio-Glucosa/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Complejo Sacarasa-Isomaltasa/metabolismo , Tamoxifeno/administración & dosificaciónRESUMEN
Nuclear factor kappa B (NF-κB) is an inducible transcription factor that plays critical roles in immune and stress responses and is often implicated in pathologies, including chronic inflammation and cancer. Although much has been learned about NF-κB-activating pathways, the specific repression of NF-κB is far less well understood. Here we identified the type I protein arginine methyltransferase 1 (PRMT1) as a restrictive factor controlling TNFα-induced activation of NF-κB. PRMT1 forms a cellular complex with NF-κB through direct interaction with the Rel homology domain of RelA. We demonstrate that PRMT1 methylates RelA at evolutionary conserved R30, located in the DNA-binding L1 loop, which is a critical residue required for DNA binding. Asymmetric R30 dimethylation inhibits the binding of RelA to DNA and represses NF-κB target genes in response to TNFα. Molecular dynamics simulations of the DNA-bound RelA:p50 predicted structural changes in RelA caused by R30 methylation or a mutation that interferes with the stability of the DNA-NF-κB complex. Our findings provide evidence for the asymmetric arginine dimethylation of RelA and unveil a unique mechanism controlling TNFα/NF-κB signaling.