RESUMEN
Metabolic demands fluctuate rhythmically and rely on coordination between the circadian clock and nutrient-sensing signalling pathways, yet mechanisms of their interaction remain not fully understood. Surprisingly, we find that class 3 phosphatidylinositol-3-kinase (PI3K), known best for its essential role as a lipid kinase in endocytosis and lysosomal degradation by autophagy, has an overlooked nuclear function in gene transcription as a coactivator of the heterodimeric transcription factor and circadian driver Bmal1-Clock. Canonical pro-catabolic functions of class 3 PI3K in trafficking rely on the indispensable complex between the lipid kinase Vps34 and regulatory subunit Vps15. We demonstrate that although both subunits of class 3 PI3K interact with RNA polymerase II and co-localize with active transcription sites, exclusive loss of Vps15 in cells blunts the transcriptional activity of Bmal1-Clock. Thus, we establish non-redundancy between nuclear Vps34 and Vps15, reflected by the persistent nuclear pool of Vps15 in Vps34-depleted cells and the ability of Vps15 to coactivate Bmal1-Clock independently of its complex with Vps34. In physiology we find that Vps15 is required for metabolic rhythmicity in liver and, unexpectedly, it promotes pro-anabolic de novo purine nucleotide synthesis. We show that Vps15 activates the transcription of Ppat, a key enzyme for the production of inosine monophosphate, a central metabolic intermediate for purine synthesis. Finally, we demonstrate that in fasting, which represses clock transcriptional activity, Vps15 levels are decreased on the promoters of Bmal1 targets, Nr1d1 and Ppat. Our findings open avenues for establishing the complexity for nuclear class 3 PI3K signalling for temporal regulation of energy homeostasis.
Asunto(s)
Relojes Circadianos , Relojes Circadianos/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/metabolismo , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Purinas , LípidosRESUMEN
The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.
Asunto(s)
Membrana Celular/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/química , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteínas de Unión al GTP rab1/química , Proteínas de Unión al GTP rab1/metabolismo , Proteínas de Unión al GTP rab5/química , Proteínas de Unión al GTP rab5/metabolismo , Beclina-1/química , Beclina-1/genética , Beclina-1/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/genética , Endosomas/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Estructura Secundaria de Proteína , Tomografía , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína de Clasificación Vacuolar VPS15/química , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/metabolismo , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab5/genéticaRESUMEN
Differentiation of trophoblast stem (TS) cells into various cell lineages of the placenta during mammalian development is accompanied by dynamic changes in its proteome for exerting the highly specialized functions of various cell subtypes. In the present study, we demonstrate that the autophagic machinery, which includes proteins for initiation, vesicle nucleation, and autophagosome maturation are robustly upregulated during differentiation of TS cells. Interestingly, basal levels of autophagy were detectable in the developing mouse placenta as well as TS cells. However, autophagic flux was actively triggered by induction of differentiation evident from LC3 maturation. Formation of Beclin1, Vps34, and PIK3R4 ternary complex at the phagophore assembly site that is typically known to induce autophagy was also enhanced during differentiation. Degradation of the p62/SQSTM1 cargo protein and its colocalization with LC3, a mature autophagosome marker, was most prevalent in the trophoblast giant cells (TGCs) and negligible in other trophoblast cells at day 6 of differentiation. Furthermore, disruption of autophagy by impairing lysosomal fusion in TS cells before induction of differentiation led to a decrease in the giant cell and spongiotrophoblast cell markers Prl3d1, Prl2c2, Prl4a1, and Tpbpα upon differentiation. In addition, inhibition of autophagy was associated with a decrease in nuclear size of TGCs. Taken together, these data highlight that autophagy is a necessary prelude in commitment of trophoblast differentiation from the multipotent TS cells probably by regulating protein turnover at the onset of differentiation.
Asunto(s)
Autofagia , Diferenciación Celular , Células Madre Embrionarias de Ratones/metabolismo , Trofoblastos/citología , Animales , Beclina-1/genética , Beclina-1/metabolismo , Células Cultivadas , Fosfatidilinositol 3-Quinasas Clase III/genética , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Células Madre Embrionarias de Ratones/citología , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/metabolismoRESUMEN
AIMS: Vps15 is an important regulator on the activity of class III PI3K in autophagy induction. AngII plays a positive role of autophagy in the early protection of endothelial cells. In this study, the expression of Vps15 was knocked down using the specific shRNA to investigate the effects of Vps15 on cell autophagy, senescence and apoptosis in HUVECs stimulated by AngII. The associated cell signaling pathway was also explored. MATERIALS AND METHODS: MDC staining was applied to show autophagic bodies. Cell senescence was detected using ß-galactosidase staining. Cell apoptosis was examined by flow cytometry using Annexin V-FITC/PI staining. And western blot was used to evaluate the ratio of LC3-II/I and the activation of associated cell signaling pathway. KEY FINDINGS: Cell autophagy induced by AngII was inhibited in HUVECs transfected with Vps15-shRNA, while cell senescence and apoptosis were enhanced. Rescue experiment revealed that cell autophagy was activated after Vps15 reexpression, while cell senescence and apoptosis were inhibited. Moreover, the phosphorylations of PDK1 and PKC substrates were increased after AngII treatment, which were decreased by Vps15 knockdown. Pretreatment of cells with the inhibitor for PDK1 or PKC attenuated cell autophagy after AngII stimulation, yet promoted cell senescence and apoptosis. The phosphorylations of both PDK1 and PKC were inhibited in cells pretreated with PDK1 inhibitor. Only the activation of PKC was inhibited when the inhibitor for pan-PKC was used. SIGNIFICANCE: These results suggested that Vps15 was critical to the protective autophagy in HUVECs induced by AngII, and PDK1/PKC signaling pathway was probably involved.
Asunto(s)
Angiotensina II/farmacología , Autofagia , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Clasificación Vacuolar VPS15/metabolismo , Apoptosis , Senescencia Celular , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína Quinasa C/genética , Proteínas Serina-Treonina Quinasas/genética , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , ARN Interferente Pequeño/genética , Transducción de Señal , Proteína de Clasificación Vacuolar VPS15/genéticaRESUMEN
The class 3 phosphoinositide 3-kinase (PI3K) is required for lysosomal degradation by autophagy and vesicular trafficking, assuring nutrient availability. Mitochondrial lipid catabolism is another energy source. Autophagy and mitochondrial metabolism are transcriptionally controlled by nutrient sensing nuclear receptors. However, the class 3 PI3K contribution to this regulation is unknown. We show that liver-specific inactivation of Vps15, the essential regulatory subunit of the class 3 PI3K, elicits mitochondrial depletion and failure to oxidize fatty acids. Mechanistically, transcriptional activity of Peroxisome Proliferator Activated Receptor alpha (PPARα), a nuclear receptor orchestrating lipid catabolism, is blunted in Vps15-deficient livers. We find PPARα repressors Histone Deacetylase 3 (Hdac3) and Nuclear receptor co-repressor 1 (NCoR1) accumulated in Vps15-deficient livers due to defective autophagy. Activation of PPARα or inhibition of Hdac3 restored mitochondrial biogenesis and lipid oxidation in Vps15-deficient hepatocytes. These findings reveal roles for the class 3 PI3K and autophagy in transcriptional coordination of mitochondrial metabolism.
Asunto(s)
Autofagia/fisiología , Metabolismo de los Lípidos , Mitocondrias/metabolismo , PPAR alfa/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Fenofibrato/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histona Desacetilasas/fisiología , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Co-Represor 1 de Receptor Nuclear/genética , Co-Represor 1 de Receptor Nuclear/metabolismo , Co-Represor 1 de Receptor Nuclear/fisiología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Transcripción Genética/efectos de los fármacos , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/metabolismo , Proteína de Clasificación Vacuolar VPS15/fisiologíaRESUMEN
The class III phosphatidylinositol 3-kinase (PI3K) Vps34 is an important regulator of key cellular functions, including cell growth, survival, intracellular trafficking, autophagy and nutrient sensing. In yeast, Vps34 is associated with the putative serine/threonine protein kinase Vps15, however, its role in signaling has not been deeply evaluated. Here, we have identified the Vps15 orthologue in Trypanosoma brucei, named TbVps15. Knockdown of TbVps15 expression by interference RNA resulted in inhibition of cell growth and blockage of cytokinesis. Scanning electron microcopy revealed a variety of morphological abnormalities, with enlarged parasites and dividing cells that often exhibited a detached flagellum. Transmission electron microscopy analysis of TbVps15 RNAi cells showed an increase in intracellular vacuoles of the endomembrane system and some cells displayed an enlargement of the flagellar pocket, a common feature of cells defective in endocytosis. Moreover, uptake of dextran, transferrin and Concanavalin A was impaired. Finally, TbVps15 downregulation affected the PI3K activity, supporting the hypothesis that TbVps15 and TbVps34 form a complex as occurs in other organisms. In summary, we propose that TbVps15 has a role in the maintenance of cytokinesis, endocytosis and intracellular trafficking in T. brucei.
Asunto(s)
Citocinesis , Endocitosis , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/fisiología , Proteína de Clasificación Vacuolar VPS15/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Transmisión de Enfermedad Infecciosa , Técnicas de Silenciamiento del Gen , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Fosfatidilinositol 3-Quinasa/análisis , Unión Proteica , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/genética , Proteína de Clasificación Vacuolar VPS15/genéticaRESUMEN
The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is required for the initiation of essentially all macroautophagic processes. PI3KC3-C1 consists of the lipid kinase catalytic subunit VPS34, the VPS15 scaffold, and the regulatory BECN1 and ATG14 subunits. The VPS34 catalytic domain and BECN1:ATG14 subcomplex do not touch, and it is unclear how allosteric signals are transmitted to VPS34. We used EM and crosslinking mass spectrometry to dissect five conformational substates of the complex, including one in which the VPS34 catalytic domain is dislodged from the complex but remains tethered by an intrinsically disordered linker. A "leashed" construct prevented dislodging without interfering with the other conformations, blocked enzyme activity in vitro, and blocked autophagy induction in yeast cells. This pinpoints the dislodging and tethering of the VPS34 catalytic domain, and its regulation by VPS15, as a master allosteric switch in autophagy induction.
Asunto(s)
Autofagia , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Regulación Alostérica , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Beclina-1/genética , Beclina-1/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/química , Fosfatidilinositol 3-Quinasas Clase III/genética , Células HEK293 , Humanos , Espectrometría de Masas/métodos , Mutación , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Relación Estructura-Actividad , Proteína de Clasificación Vacuolar VPS15/química , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/metabolismoRESUMEN
The autophagy-lysosome system is critical for muscle homeostasis and defects in lysosomal function result in a number of inherited muscle diseases, generally referred to as autophagic vacuolar myopathies (AVMs). Among them, Danon Disease (DD) and glycogen storage disease type II (GSDII) are due to primary lysosomal protein defects. DD is characterized by mutations in the lysosome-associated membrane protein 2 (LAMP2) gene. The DD mouse model suggests that inefficient lysosome biogenesis/maturation and impairment of autophagosome-lysosome fusion contribute to the pathogenesis of muscle wasting. To define the role of autophagy in human disease, we analyzed the muscle biopsies of DD patients and monitored autophagy and several autophagy regulators like transcription factor EB (TFEB), a master player in lysosomal biogenesis, and vacuolar protein sorting 15 (VPS15), a critical factor for autophagosome and endosome biogenesis and trafficking. Furthermore, to clarify whether the mechanisms involved are shared by other AVMs, we extended our mechanistic study to a group of adult GSDII patients. Our data show that, similar to GSDII, DD patients display an autophagy block that correlates with the severity of the disease. Both DD and GSDII show accumulation and altered localization of VPS15 in autophagy-incompetent fibers. However, TFEB displays a different pattern between these two lysosomal storage diseases. Although in DD TFEB and downstream targets are activated, in GSDII patients TFEB is inhibited. These findings suggest that these regulatory factors may have an active role in the pathogenesis of these diseases. Therapeutic approaches targeted to normalize these factors and restore the autophagic flux in these patients should therefore be considered.
Asunto(s)
Autofagia/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Enfermedad por Depósito de Glucógeno de Tipo IIb/genética , Proteína de Clasificación Vacuolar VPS15/genética , Adolescente , Adulto , Animales , Modelos Animales de Enfermedad , Femenino , Enfermedad del Almacenamiento de Glucógeno Tipo II/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/patología , Enfermedad por Depósito de Glucógeno de Tipo IIb/metabolismo , Enfermedad por Depósito de Glucógeno de Tipo IIb/patología , Humanos , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Lisosomas/genética , Lisosomas/patología , Masculino , Ratones , Músculos/metabolismo , Músculos/patología , MutaciónRESUMEN
Autophagy is a degradative process by which eukaryotic cells digest their own components to provide aminoacids that may function as energy source under nutritional stress conditions. There is experimental evidence for autophagy in parasitic protists belonging to the family Trypanosomatidae. However, few proteins implicated in this process have been characterized so far in these parasites. Moreover, it has been shown that autophagy is involved in Trypanosoma cruzi differentiation and thus might have a role in pathogenicity. Here, we report the cloning and biochemical characterization of TcVps15. In addition, we demonstrate that TcVps15 interact with the PI3K TcVps34 and that both proteins associate with cellular membranes. Under nutritional stress conditions, TcVps15 and TcVps34 modify their subcellular distribution showing a partial co-localization in autophagosomes with TcAtg8.1 and using an active site TcVps15-mutated version (TcVps15-K219D-HA) we demonstrated that this relocalization depends on the TcVps15 catalytic activity. Overexpression of TcVps15-HA and TcVps15-K219D-HA also leads to increased accumulation of monodansylcadaverine (MDC) in autophagic vacuoles under nutritional stress conditions compared to wild-type cells. In addition, the MDC-specific activity shows to be significantly higher in TcVps15-HA overexpressing cells when compared with TcVps15-K219D-HA. Our results reveal for the first time a role of TcVps15 as a key regulator of TcVps34 enzymatic activity and implicate the TcVps15-Vps34 complex in autophagy in T. cruzi, exposing a new key pathway to explore novel chemotherapeutic targets.
Asunto(s)
Autofagia , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/metabolismo , Proteína de Clasificación Vacuolar VPS15/metabolismo , Animales , Cadaverina/análogos & derivados , Cadaverina/metabolismo , Técnicas de Cultivo de Célula , Membrana Celular/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/genética , Fosfatidilinositol 3-Quinasas Clase III/fisiología , Clonación Molecular , ADN Protozoario , Pruebas de Enzimas , Regulación Enzimológica de la Expresión Génica , Estadios del Ciclo de Vida , Mutagénesis Sitio-Dirigida , Fagosomas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Análisis de Secuencia , Transfección , Trypanosoma cruzi/citología , Trypanosoma cruzi/genética , Técnicas del Sistema de Dos Híbridos , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/fisiología , Vacuolas/metabolismoRESUMEN
Ciliopathies are a group of diseases that affect kidney and retina among other organs. Here, we identify a missense mutation in PIK3R4 (phosphoinositide 3-kinase regulatory subunit 4, named VPS15) in a family with a ciliopathy phenotype. Besides being required for trafficking and autophagy, we show that VPS15 regulates primary cilium length in human fibroblasts, as well as ciliary processes in zebrafish. Furthermore, we demonstrate its interaction with the golgin GM130 and its localization to the Golgi. The VPS15-R998Q patient mutation impairs Golgi trafficking functions in humanized yeast cells. Moreover, in VPS15-R998Q patient fibroblasts, the intraflagellar transport protein IFT20 is not localized to vesicles trafficking to the cilium but is restricted to the Golgi. Our findings suggest that at the Golgi, VPS15 and GM130 form a protein complex devoid of VPS34 to ensure the IFT20-dependent sorting and transport of membrane proteins from the cis-Golgi to the primary cilium.
Asunto(s)
Proteínas Portadoras/metabolismo , Cilios/metabolismo , Ciliopatías/genética , Aparato de Golgi/metabolismo , Proteína de Clasificación Vacuolar VPS15/genética , Anomalías Múltiples/genética , Adolescente , Animales , Estudios de Casos y Controles , Células Cultivadas , Niño , Preescolar , Anomalías Craneofaciales/complicaciones , Anomalías Craneofaciales/genética , Femenino , Fibroblastos/metabolismo , Deformidades Congénitas de la Mano/complicaciones , Deformidades Congénitas de la Mano/genética , Humanos , Discapacidades para el Aprendizaje/complicaciones , Discapacidades para el Aprendizaje/genética , Masculino , Mutación , Mutación Missense , Insuficiencia Renal/complicaciones , Insuficiencia Renal/genética , Retinitis Pigmentosa/complicaciones , Retinitis Pigmentosa/genética , Saccharomyces cerevisiae , Hermanos , Piel/citología , Adulto Joven , Pez CebraRESUMEN
Defective hepatic insulin receptor (IR) signalling is a pathogenic manifestation of metabolic disorders including obesity and diabetes. The endo/lysosomal trafficking system may coordinate insulin action and nutrient homeostasis by endocytosis of IR and the autophagic control of intracellular nutrient levels. Here we show that class III PI3K--a master regulator of endocytosis, endosomal sorting and autophagy--provides negative feedback on hepatic insulin signalling. The ultraviolet radiation resistance-associated gene protein (UVRAG)-associated class III PI3K complex interacts with IR and is stimulated by insulin treatment. Acute and chronic depletion of hepatic Vps15, the regulatory subunit of class III PI3K, increases insulin sensitivity and Akt signalling, an effect that requires functional IR. This is reflected by FoxO1-dependent transcriptional defects and blunted gluconeogenesis in Vps15 mutant cells. On depletion of Vps15, the metabolic syndrome in genetic and diet-induced models of insulin resistance and diabetes is alleviated. Thus, feedback regulation of IR trafficking and function by class III PI3K may be a therapeutic target in metabolic conditions of insulin resistance.
Asunto(s)
Glucosa/metabolismo , Insulina/metabolismo , Hígado/metabolismo , Proteína de Clasificación Vacuolar VPS15/metabolismo , Animales , Diabetes Mellitus/metabolismo , Retroalimentación Fisiológica , Homeostasis , Humanos , Resistencia a la Insulina , Hígado/enzimología , Masculino , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína de Clasificación Vacuolar VPS15/genéticaRESUMEN
Papillary thyroid carcinoma (PTC) is the most frequent thyroid malignant neoplasia. Oncogene activation occurs in more than 70% of the cases. Indeed, about 40% of PTCs harbor mutations in BRAF gene, whereas RET rearrangements (RET/PTC oncogenes) are present in about 20% of cases. Finally, RAS mutations and TRK rearrangements account for about 5% each of these malignancies. We used RNA-Sequencing to identify fusion transcripts and mutations in cancer driver genes in a cohort of 18 PTC patients. Furthermore, we used targeted DNA sequencing to validate identified mutations. We extended the screening to 50 PTC patients and 30 healthy individuals. Using this approach we identified new missense mutations in CBL, NOTCH1, PIK3R4 and SMARCA4 genes. We found somatic mutations in DICER1, MET and VHL genes, previously found mutated in other tumors, but not described in PTC. We identified a new chimeric transcript generated by the fusion of WNK1 and B4GALNT3 genes, correlated with B4GALNT3 overexpression. Our data confirmed PTC genetic heterogeneity, revealing that gene expression correlates more with the mutation pattern than with tumor staging. Overall, this study provides new data about mutational landscape of this neoplasia, suggesting potential pharmacological adjuvant therapies against Notch signaling and chromatin remodeling enzymes.
Asunto(s)
Biomarcadores de Tumor/genética , Carcinoma/genética , Fusión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación Missense , N-Acetilgalactosaminiltransferasas/genética , Proteínas Serina-Treonina Quinasas/genética , Neoplasias de la Tiroides/genética , Carcinoma/enzimología , Carcinoma/patología , Carcinoma Papilar , Estudios de Casos y Controles , ADN Helicasas/genética , Análisis Mutacional de ADN , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Antígenos de Histocompatibilidad Menor , Estadificación de Neoplasias , Proteínas Nucleares/genética , Fenotipo , Valor Predictivo de las Pruebas , Proteínas Proto-Oncogénicas c-cbl/genética , Receptor Notch1/genética , Reproducibilidad de los Resultados , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides/enzimología , Neoplasias de la Tiroides/patología , Factores de Transcripción/genética , Proteína de Clasificación Vacuolar VPS15/genética , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
The yeast class III phosphoinositide 3-kinase (PI3K) that catalyses production of the lipid signalling molecule, phosphatidylinositol-3-phosphate, is primarily implicated in vesicle-mediated transport and autophagy. In this study, we identified, through a genetic screen, the Candida glabrata CgVPS15 gene, an orthologue of the Saccharomyces cerevisiaeâ PI3K regulatory subunit-encoding open reading frame (ORF) to be required for impairment of phagosomal maturation in human macrophages. We also disrupted catalytic subunit of the C. glabrataâ PI3K complex, CgVps34, and found it to be pivotal to arrest mature phagolysosome biogenesis. Further, deletion of either CgVPS15 or CgVPS34 rendered C. glabrata cells hyperadherent to epithelial cells and susceptible to the antimicrobial arsenal of primary murine and cultured human macrophages and diverse stresses. Despite no growth retardation at 37°C, Cgvps15Δ and Cgvps34Δ mutants were severely virulence attenuated in mice. We demonstrate that trafficking and/or processing of the vacuolar lumenal hydrolase, carboxypeptidase Y, and the major adhesin, Epa1, rely on PI3K regulatory mechanisms in C. glabrata. By disrupting autophagy-related PI3K complex genes, we show that C. glabrataâ PI3K-impeded phagolysosomal acidification is primarily owing to its role in cellular trafficking events. Altogether, our findings underscore the essentiality of PI3K signalling in modulation of host immune response, intracellular survival and virulence in C. glabrata.
Asunto(s)
Candida glabrata/enzimología , Candida glabrata/fisiología , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Interacciones Huésped-Patógeno , Fagosomas/metabolismo , Fagosomas/microbiología , Proteína de Clasificación Vacuolar VPS15/metabolismo , Animales , Candida glabrata/crecimiento & desarrollo , Candida glabrata/inmunología , Células Cultivadas , Fosfatidilinositol 3-Quinasas Clase III/genética , Eliminación de Gen , Humanos , Ratones , Viabilidad Microbiana , Temperatura , Proteína de Clasificación Vacuolar VPS15/genéticaRESUMEN
The Saccharomyces cerevisiae NatB N-terminal acetylase contains a catalytic subunit Naa20 and an auxiliary subunit Naa25. To elucidate the cellular functions of the NatB, we utilized the Synthetic Genetic Array to screen for genes that are essential for cell growth in the absence of NAA20. The genome-wide synthetic lethal screen of NAA20 identified genes encoding for serine/threonine protein kinase Vps15, 1,3-beta-glucanosyltransferase Gas5, and a catabolic repression regulator Mig3. The present study suggests that the catalytic activity of the NatB N-terminal aceytase is involved in vacuolar protein sorting and cell wall maintenance.
Asunto(s)
Eliminación de Gen , Genes Esenciales , Acetiltransferasa B N-Terminal/genética , Acetiltransferasa B N-Terminal/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/fisiología , Dominio Catalítico/genética , Genes Fúngicos , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/metabolismoRESUMEN
Macroautophagy is a physiological cellular response to nutrient stress, which leads to the engulfment of cytosolic contents by a double-walled membrane structure, the phagophore. Phagophores seal to become autophagosomes, which then fuse with lysosomes to deliver their contents for degradation. Macroautophagy is regulated by numerous cellular factors, including the Class III PI3K (phosphoinositide 3-kinase) Vps34 (vacuolar protein sorting 34). The autophagic functions of Vps34 require its recruitment to a complex that includes Vps15, Beclin-1 and Atg14L (autophagy-related 14-like protein) and is known as Vps34 Complex I. We have now identified NRBF2 (nuclear receptor-binding factor 2) as a new member of Vps34 Complex I. NRBF2 binds to complexes that include Vps34, Vps15, Beclin-1 and ATG-14L, but not the Vps34 Complex II component UVRAG (UV radiation resistance-associated gene). NRBF2 directly interacts with Vps15 via the Vps15 WD40 domain as well as other regions of Vps15. The formation of GFP-LC3 (light chain 3) punctae and PE (phosphatidylethanolamine)-conjugated LC3 (LC3-II) in serum-starved cells was inhibited by NRBF2 knockdown in the absence and presence of lysosomal inhibitors, and p62 levels were increased. Thus NRBF2 plays a critical role in the induction of starvation-induced autophagy as a specific member of Vps34 Complex I.
Asunto(s)
Autofagia/genética , Fosfatidilinositol 3-Quinasas Clase III/genética , Transactivadores/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Relacionadas con la Autofagia , Beclina-1 , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fagosomas/metabolismo , Transporte de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Transactivadores/antagonistas & inhibidores , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/metabolismo , Vacuolas/metabolismoRESUMEN
In Saccharomyces cerevisiae, the vacuolar protein sorting complexes Vps51/52/53/54 and Vps15/30/34/38 are essential for efficient endosome-to-Golgi complex retrograde transport. Here we investigated the function of Vps15 and Vps51, representative members of these complexes, in the stress resistance, host cell interactions, and virulence of Candida albicans. We found that C. albicans vps15Δ/Δ and vps51Δ/Δ mutants had abnormal vacuolar morphology, impaired retrograde protein trafficking, and dramatically increased susceptibility to a variety of stressors. These mutants also had reduced capacity to invade and damage oral epithelial cells in vitro and attenuated virulence in the mouse model of oropharyngeal candidiasis. Proteomic analysis of the cell wall of the vps51Δ/Δ mutant revealed increased levels of the Crh11 and Utr2 transglycosylases, which are targets of the calcineurin signaling pathway. The transcript levels of the calcineurin pathway members CHR11, UTR2, CRZ1, CNA1, and CNA2 were elevated in the vps15Δ/Δ and vps51Δ/Δ mutants. Furthermore, these strains were highly sensitive to the calcineurin-specific inhibitor FK506. Also, deletion of CHR11 and UTR2 further increased the stress susceptibility of these mutants. In contrast, overexpression of CRH11 and UTR2 partially rescued their defects in stress resistance, but not host cell interactions. Therefore, intact retrograde trafficking in C. albicans is essential for stress resistance, host cell interactions, and virulence. Aberrant retrograde trafficking stimulates the calcineurin signaling pathway, leading to the increased expression of Chr11 and Utr2, which enables C. albicans to withstand environmental stress.
Asunto(s)
Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Interacciones Huésped-Patógeno , Estrés Fisiológico , Proteína de Clasificación Vacuolar VPS15/metabolismo , Animales , Calcineurina/genética , Calcineurina/metabolismo , Inhibidores de la Calcineurina , Candida albicans/genética , Candida albicans/patogenicidad , Candidiasis Bucal/microbiología , Proteínas Fúngicas/genética , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Mutación , Transporte de Proteínas , Tacrolimus/farmacología , Proteína de Clasificación Vacuolar VPS15/genética , Virulencia/genéticaRESUMEN
Autophagy is a tightly controlled degradation process of all eukaryotes. It includes the sequestration of cytoplasmic contents and organelles within a double-membraned autophagosome. Autophagy involves core autophagy related (atg) genes as well as genes regulating vesicle trafficking. Previously, we analyzed the impact of proteins of the core autophagic machinery SmATG7, SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. While deletion of Smatg8 and Smatg4 abolished fruiting-body formation and impaired vegetative growth, Smatg7 is required for viability. In yeast, the phosphatidylinositol 3-kinase vacuolar protein sorting 34 (Vps34) and its myristoylated membrane targeting unit, the protein kinase Vps15 have been shown to be important regulators of autophagy and vacuolar protein sorting. However, their exact role in filamentous ascomycetes remains elusive. To determine the function of Smvps34 and Smvps15 we isolated genes with high sequence similarity to Saccharomyces cerevisiae VPS34 and VPS15. For both genes we were not able to generate a homokaryotic knockout mutant in S. macrospora, suggesting that Smvps34 and Smvps15 are required for viability. Furthermore, we analyzed the repertoire of vps genes encoded by S. macrospora and could identify putative homologs of nearly all of the 61 VPS genes of S. cerevisiae.
Asunto(s)
Autofagia , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Viabilidad Microbiana , Sordariales/citología , Sordariales/enzimología , Proteína de Clasificación Vacuolar VPS15/metabolismo , Secuencia de Aminoácidos , Fosfatidilinositol 3-Quinasas Clase III/química , Fosfatidilinositol 3-Quinasas Clase III/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Sordariales/química , Sordariales/genética , Proteína de Clasificación Vacuolar VPS15/química , Proteína de Clasificación Vacuolar VPS15/genéticaRESUMEN
The complex of Vacuolar Protein Sorting 34 and 15 (Vps34 and Vps15) has Class III phosphatidylinositol 3-kinase activity and putative roles in nutrient sensing, mammalian Target Of Rapamycin (mTOR) activation by amino acids, cell growth, vesicular trafficking and autophagy. Contrary to expectations, here we show that Vps15-deficient mouse tissues are competent for LC3-positive autophagosome formation and maintain mTOR activation. However, an impaired lysosomal function in mutant cells is traced by accumulation of adaptor protein p62, LC3 and Lamp2 positive vesicles, which can be reverted to normal levels after ectopic overexpression of Vps15. Mice lacking Vps15 in skeletal muscles, develop a severe myopathy. Distinct from the autophagy deficient Atg7(-/-) mutants, pathognomonic morphological hallmarks of autophagic vacuolar myopathy (AVM) are observed in Vps15(-/-) mutants, including elevated creatine kinase plasma levels, accumulation of autophagosomes, glycogen and sarcolemmal features within the fibres. Importantly, Vps34/Vps15 overexpression in myoblasts of Danon AVM disease patients alleviates the glycogen accumulation. Thus, the activity of the Vps34/Vps15 complex is critical in disease conditions such as AVMs, and possibly a variety of other lysosomal storage diseases.
Asunto(s)
Autofagia , Músculo Esquelético/metabolismo , Proteína de Clasificación Vacuolar VPS15/metabolismo , Animales , Proteína 7 Relacionada con la Autofagia , Línea Celular , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Humanos , Enfermedades por Almacenamiento Lisosomal/metabolismo , Enfermedades por Almacenamiento Lisosomal/patología , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Músculo Esquelético/fisiopatología , Músculo Esquelético/ultraestructura , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factor de Transcripción TFIIH , Factores de Transcripción/metabolismo , Proteína de Clasificación Vacuolar VPS15/genéticaRESUMEN
There is increasing evidence that certain Vacuolar protein sorting (Vps) proteins, factors that mediate vesicular protein trafficking, have additional roles in regulating transcription factors at the endosome. We found that yeast mutants lacking the phosphatidylinositol 3-phosphate [PI(3)P] kinase Vps34 or its associated protein kinase Vps15 display multiple phenotypes indicating impaired transcription elongation. These phenotypes include reduced mRNA production from long or G+C-rich coding sequences (CDS) without affecting the associated GAL1 promoter activity, and a reduced rate of RNA polymerase II (Pol II) progression through lacZ CDS in vivo. Consistent with reported genetic interactions with mutations affecting the histone acetyltransferase complex NuA4, vps15Δ and vps34Δ mutations reduce NuA4 occupancy in certain transcribed CDS. vps15Δ and vps34Δ mutants also exhibit impaired localization of the induced GAL1 gene to the nuclear periphery. We found unexpectedly that, similar to known transcription elongation factors, these and several other Vps factors can be cross-linked to the CDS of genes induced by Gcn4 or Gal4 in a manner dependent on transcriptional induction and stimulated by Cdk7/Kin28-dependent phosphorylation of the Pol II C-terminal domain (CTD). We also observed colocalization of a fraction of Vps15-GFP and Vps34-GFP with nuclear pores at nucleus-vacuole (NV) junctions in live cells. These findings suggest that Vps factors enhance the efficiency of transcription elongation in a manner involving their physical proximity to nuclear pores and transcribed chromatin.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Elongación de la Transcripción Genética , Proteína de Clasificación Vacuolar VPS15/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Núcleo Celular/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/genética , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Secuencia Rica en GC , Galactoquinasa/genética , Galactoquinasa/metabolismo , Eliminación de Gen , Histona Acetiltransferasas/metabolismo , Poro Nuclear/metabolismo , Fenotipo , Fosforilación , Regiones Promotoras Genéticas , Transporte de Proteínas , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína de Clasificación Vacuolar VPS15/genética , Vacuolas/metabolismoRESUMEN
VPS15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells. The knowledge about the function of its homologue in plants remains limited. Here we report that AtVPS15, a homologue of yeast VPS15p in Arabidopsis, plays an essential role in pollen germination. Homozygous T-DNA insertion mutants of AtVPS15 could not be obtained from the progenies of self-pollinated heterozygous mutants. Reciprocal crosses between atvps15 mutants and wild-type Arabidopsis revealed that the T-DNA insertion was not able to be transmitted by male gametophytes. DAPI staining, Alexander's stain and scanning electron microscopic analysis showed that atvps15 heterozygous plants produced pollen grains that were morphologically indistinguishable from wild-type pollen, whereas in vitro germination experiments revealed that germination of the pollen grains was defective. GUS staining analysis of transgenic plants expressing the GUS reporter gene driven by the AtVPS15 promoter showed that AtVPS15 was mainly expressed in pollen grains. Finally, DUALmembrane yeast two-hybrid analysis demonstrated that AtVPS15 might interact directly with AtVPS34. These results suggest that AtVPS15 is very important for pollen germination, possibly through modulation of the activity of PI3-kinase.