RESUMEN
Macroautophagy, the degradation and recycling of cytosolic components in the lysosome, is an important cellular mechanism. It is a membrane-mediated process that is linked to vesicular trafficking events. The sorting nexin (SNX) protein family controls the sorting of a large array of cargoes, and various SNXs impact autophagy. To improve our understanding of their functions in vivo, we screened all Drosophila SNXs using inducible RNA interference in the fat body. Significantly, depletion of Snazarus (Snz) led to decreased autophagic flux. Interestingly, we observed altered distribution of Vamp7-positive vesicles with Snz depletion, and the roles of Snz were conserved in human cells. SNX25, the closest human ortholog to Snz, regulates both VAMP8 endocytosis and lipid metabolism. Through knockout-rescue experiments, we demonstrate that these activities are dependent on specific SNX25 domains and that the autophagic defects seen upon SNX25 loss can be rescued by ethanolamine addition. We also demonstrate the presence of differentially spliced forms of SNX14 and SNX25 in cancer cells. This work identifies a conserved role for Snz/SNX25 as a regulator of autophagic flux and reveals differential isoform expression between paralogs.
Asunto(s)
Proteínas de Drosophila , Nexinas de Clasificación , Animales , Autofagia/genética , Drosophila/metabolismo , Endocitosis , Humanos , Transporte de Proteínas , Proteínas R-SNARE , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismoRESUMEN
Vesicle-associated membrane protein 8 (VAMP8), a soluble n-ethylmaleimide-sensitive factor receptor protein, acts as an oncogenic gene in the progression of several malignancies. Nevertheless, the roles and mechanisms of VAMP8 in colorectal cancer (CRC) progression remain unknown. The expression and prognostic significance of VAMP8 in CRC samples were analyzed through bioinformatics analyses. Cell proliferation was detected using CCK-8 and EdU incorporation assays and apoptosis was evaluated via flow cytometry. Western blot analysis was conducted to examine the protein expression. Ferroptosis was evaluated by measurement of iron metabolism, lipid peroxidation, and glutathione (GSH) content. VAMP8 was increased in CRC samples relative to normal samples on the basis of GEPIA and HPA databases. CRC patients with high level of VAMP8 had a worse overall survival. VAMP8 depletion led to a suppression of proliferation and promotion of apoptosis in CRC cells. Additionally, VAMP8 knockdown suppressed beclin1 expression and LC3-II/LC3-I ratio, elevated p62 expression, increased Fe2+, labile iron pool, lipid reactive oxygen species, and malondialdehyde levels, and repressed GSH content and glutathione peroxidase activity. Moreover, VAMP8 knockdown inhibited the activation of janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway in CRC cells. Mechanistically, activation of the JAK/STAT3 pathway by JAK1 or JAK2 overexpression attenuated VAMP8 silencing-mediated anti-proliferative, pro-apoptotic, anti-autophagic, and pro-ferroptotic effects on CRC cells. In conclusion, VAMP8 knockdown affects the proliferation, apoptosis, autophagy, and ferroptosis by the JAK/STAT3 pathway in CRC cells.
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Apoptosis , Autofagia , Proliferación Celular , Neoplasias Colorrectales , Ferroptosis , Factor de Transcripción STAT3 , Humanos , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Técnicas de Silenciamiento del Gen , Quinasas Janus/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas R-SNARE/genética , Transducción de Señal , Factor de Transcripción STAT3/metabolismoRESUMEN
BACKGROUND: On activation, mast cells rapidly release preformed inflammatory mediators from large cytoplasmic granules via regulated exocytosis. This acute degranulation is followed by a late activation phase involving synthesis and secretion of cytokines, growth factors, and other inflammatory molecules via the constitutive pathway that remains ill defined. OBJECTIVE: We investigated the role for an insulin-responsive vesicle-like endosomal compartment, marked by insulin-regulated aminopeptidase (IRAP), in the secretion of TNF-α and IL-6 in mast cells and macrophages. METHODS: Murine knockout (KO) mouse models (IRAP-KO and kit-Wsh/sh) were used to study inflammatory disease models and to measure and mechanistically investigate cytokine secretion and degranulation in bone marrow-derived mast cells in vitro. RESULTS: IRAP-KO mice are protected from TNF-α-dependent kidney injury and inflammatory arthritis. In the absence of IRAP, TNF-α and IL-6 but not IL-10 fail to be efficiently secreted. Moreover, chemical targeting of IRAP endosomes reduced proinflammatory cytokine secretion. Mechanistically, impaired TNF-α export from the Golgi and reduced colocalization of vesicle-associated membrane protein (VAMP) 3-positive TNF-α transport vesicles with syntaxin 4 (aka Stx4) was observed in IRAP-KO mast cells, while VAMP8-dependent exocytosis of secretory granules was facilitated. CONCLUSION: IRAP plays a novel role in mast cell-mediated inflammation through the regulation of exocytic trafficking of cytokines.
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Aminopeptidasas , Citocinas , Ratones , Animales , Insulina , Mastocitos , Factor de Necrosis Tumoral alfa , Interleucina-6 , InflamaciónRESUMEN
BACKGROUND: Mast cells utilize SNAREs (soluble-N-ethyl-maleimide sensitive factor attachment protein receptors) and SM (Sec1/Munc18) proteins to secrete/exocytose a variety of proinflammatory mediators. However, whether a common SNARE-SM machinery is responsible remains unclear. METHODS: Four vesicle/granule-anchored SNAREs (VAMP2, VAMP3, VAMP7, and VAMP8) and two Munc18 homologs (Munc18a and Munc18b) were systematically knocked down or knocked out in RBL-2H3 mast cells and antigen-induced release of ß-hexosaminidase, histamine, serotonin, and TNF was examined. Phenotypes were validated by rescue experiments. Immunofluorescence studies were performed to determine the subcellular distribution of key players. RESULTS: The reduction of VAMP8 expression inhibited the exocytosis of ß-hexosaminidase, histamine, and serotonin but not TNF. Unexpectedly, however, confocal microscopy revealed substantial co-localization between VAMP8 and TNF, and between TNF and serotonin. Meanwhile, the depletion of other VAMPs, including knockout of VAMP3, had no impact on the release of any of the mediators examined. On the other hand, TNF exocytosis was diminished specifically in stable Munc18bknockdown cells, in a fashion that was rescued by exogenous, RNAi-resistant Munc18b. In line with this, TNF was co-localized with Munc18b (47%) to a much greater extent than with Munc18a (13%). CONCLUSION: Distinct exocytic pathways exist in mast cells for the release of different mediators.
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Alérgenos , Histamina , Proteína 3 de Membrana Asociada a Vesículas/metabolismo , Histamina/metabolismo , Serotonina/metabolismo , Proteínas SNARE/metabolismo , Proteínas Munc18/metabolismo , Mastocitos , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Macrophage migration into injured or infected tissue is a key aspect in the pathophysiology of many diseases where inflammation is a driving factor. Membrane-type-1 matrix metalloproteinase (MT1-MMP) cleaves extracellular matrix components to facilitate invasion. Here we show that, unlike the constitutive MT1-MMP surface recycling seen in cancer cells, unactivated macrophages express low levels of MT1-MMP. Upon lipopolysaccharide (LPS) activation, MT1-MMP synthesis dramatically increases 10-fold at the surface by 15 hours. MT1-MMP is trafficked from the Golgi complex to the surface via late endosomes/lysosomes in a pathway regulated by the late endosome/lysosome R-SNAREs VAMP7 and VAMP8. These form two separate complexes with the surface Q-SNARE complex Stx4/SNAP23 to regulate MT1-MMP delivery to the plasma membrane. Loss of either one of these SNAREs leads to a reduction in surface MT1-MMP, gelatinase activity and reduced invasion. Thus, inhibiting MT1-MMP transport through this pathway could reduce macrophage migration and the resulting inflammation.
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Membrana Celular/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismo , Activación de Macrófagos , Metaloproteinasa 14 de la Matriz/metabolismo , Animales , Movimiento Celular , Aparato de Golgi/metabolismo , Ratones , Transporte de Proteínas , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Células RAW 264.7RESUMEN
In Alzheimer's disease (AD), tau, a microtubule-associated protein (MAP), becomes hyperphosphorylated, aggregates, and accumulates in the somato-dendritic compartment of neurons. In parallel to its intracellular accumulation in AD, tau is also released in the extracellular space, as revealed by its increased presence in cerebrospinal fluid (CSF). Consistent with this, recent studies, including ours, have reported that neurons secrete tau, and several therapeutic strategies aim to prevent the intracellular tau accumulation. Previously, we reported that late endosomes were implicated in tau secretion. Here, we explore the possibility of preventing intracellular tau accumulation by increasing tau secretion. Using neuronal models, we investigated whether overexpression of the vesicle-associated membrane protein 8 (VAMP8), an R-SNARE found on late endosomes, could increase tau secretion. The overexpression of VAMP8 significantly increased tau secretion, decreasing its intracellular levels in the neuroblastoma (N2a) cell line. Increased tau secretion by VAMP8 was also observed in murine hippocampal slices. The intracellular reduction of tau by VAMP8 overexpression correlated to a decrease of acetylated tubulin induced by tau overexpression in N2a cells. VAMP8 staining was preferentially found on late endosomes in N2a cells. Using total internal reflection fluorescence (TIRF) microscopy, the fusion of VAMP8-positive vesicles with the plasma membrane was correlated to the depletion of tau in the cytoplasm. Finally, overexpression of VAMP8 reduced the intracellular accumulation of tau mutants linked to frontotemporal dementia with parkinsonism and α-synuclein by increasing their secretion. Collectively, the present data indicate that VAMP8 could be used to increase tau and α-synuclein clearance to prevent their intracellular accumulation.
Asunto(s)
Proteínas R-SNARE/metabolismo , Proteínas tau/metabolismo , Acetilación , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Regulación hacia Abajo , Endosomas/metabolismo , Demencia Frontotemporal/patología , Hipocampo/metabolismo , Ratones , Microscopía Fluorescente , Mutagénesis , Neuronas/citología , Neuronas/metabolismo , Fosforilación , Proteínas R-SNARE/genética , Vesículas Secretoras/metabolismo , Tubulina (Proteína)/metabolismo , alfa-Sinucleína/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Proteínas tau/genéticaRESUMEN
Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways, including the antiviral type I interferon (IFN-I) system. Previously, we demonstrated that TRIM6 is involved in IFN-I induction and signaling. In the absence of TRIM6, optimal IFN-I signaling is reduced, allowing increased replication of interferon-sensitive viruses. Despite having evolved numerous mechanisms to restrict the vertebrate host's IFN-I response, West Nile virus (WNV) replication is sensitive to pretreatment with IFN-I. However, the regulators and products of the IFN-I pathway that are important in regulating WNV replication are incompletely defined. Consistent with WNV's sensitivity to IFN-I, we found that in TRIM6 knockout (TRIM6-KO) A549 cells, WNV replication is significantly increased and IFN-I induction and signaling are impaired compared to wild-type (wt) cells. IFN-ß pretreatment was more effective in protecting against subsequent WNV infection in wt cells than TRIM6-KO, indicating that TRIM6 contributes to the establishment of an IFN-induced antiviral response against WNV. Using next-generation sequencing, we identified VAMP8 as a potential factor involved in this TRIM6-mediated antiviral response. VAMP8 knockdown resulted in reduced JAK1 and STAT1 phosphorylation and impaired induction of several interferon-stimulated genes (ISGs) following WNV infection or IFN-ß treatment. Furthermore, VAMP8-mediated STAT1 phosphorylation required the presence of TRIM6. Therefore, the VAMP8 protein is a novel regulator of IFN-I signaling, and its expression and function are dependent on TRIM6 activity. Overall, these results provide evidence that TRIM6 contributes to the antiviral response against WNV and identify VAMP8 as a novel regulator of the IFN-I system.IMPORTANCE WNV is a mosquito-borne flavivirus that poses a threat to human health across large discontinuous areas throughout the world. Infection with WNV results in febrile illness, which can progress to severe neurological disease. Currently, there are no approved treatment options to control WNV infection. Understanding the cellular immune responses that regulate viral replication is important in diversifying the resources available to control WNV. Here, we show that the elimination of TRIM6 in human cells results in an increase in WNV replication and alters the expression and function of other components of the IFN-I pathway through VAMP8. Dissecting the interactions between WNV and host defenses both informs basic molecular virology and promotes the development of host- and virus-targeted antiviral strategies.
Asunto(s)
Inmunidad Innata , Interferón Tipo I/inmunología , Proteínas R-SNARE/inmunología , Proteínas de Motivos Tripartitos/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Replicación Viral/inmunología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/fisiología , Células A549 , Eliminación de Gen , Células HEK293 , Humanos , Janus Quinasa 1/genética , Janus Quinasa 1/inmunología , Fosforilación/genética , Fosforilación/inmunología , Proteínas R-SNARE/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/inmunología , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Replicación Viral/genética , Fiebre del Nilo Occidental/genética , Fiebre del Nilo Occidental/patologíaRESUMEN
Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α-helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.
Asunto(s)
Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas R-SNARE/metabolismo , Vesículas Secretoras/metabolismo , Animales , Línea Celular , Mastocitos/fisiología , Fosforilación , Proteómica , RatasRESUMEN
BACKGROUND: Long non-coding RNAs (lncRNAs) has been extensively reported play important roles in regulating the development and progression of cancers, including Glioblastoma (GBM). LINC01426 is a novel lncRNA that has been identified as an oncogenic gene in GBM. Herein, we attempted to elucidate the detailed functions and underlying mechanisms of LINC01426 in GBM. METHODS: LINC01426 expression in GBM cell lines and tissues were detected by quantitative real-time PCR (qRT-PCR). Cell Counting Kit-8 (CCK8) assays, colony formation assays, subcutaneous tumor formation assays were utilized to investigate the biological functions of LINC01426 in GBM. Dual-luciferase reporter assays, RNA immunoprecipitation (RIP) and bioinformatic analysis were performed to determine the underlying mechanisms. RESULTS: LINC01426 is up-regulated in malignant GBM tissues and cell lines and it is capable to promote GBM cell proliferation and growth. Mechanistically, LINC01426 serves as a molecular sponge to sequester the miR345-3p and thus enhancing the level of VAMP8, an oncogenic coding gene, to promote GBM progression. CONCLUSIONS: Our results revealed the detailed mechanisms of LINC01426 facilitated cell proliferation and growth in GBM and report the clinical value of LINC01426 for GBM prognosis and treatment.
RESUMEN
Hepatitis B virus (HBV) replication and envelopment is dependent on cellular autophagy. Previously, we have provided evidence for the extensive lysosomal degradation of HBV virions and the hepatitis B surface antigen (HBsAg), which is likely controlled by autophagosome-lysosome fusion. Synaptosomal-associated protein 29 (SNAP29) has been identified as a protein specifically mediating autophagosome-lysosome fusion. Thus, in the present study, we addressed the hypothesis that SNAP29 is required for the autophagic degradation of HBV virions and HBsAg. We found that silencing SNAP29 significantly increased the number of autophagosomes and concomitantly promoted HBV replication and HBsAg production. Conversely, SNAP29 overexpression decreased HBV production. Consistent with this, SNAP29 modulated HBV production by interacting with vesicle-associated membrane protein 8 (VAMP8) and synergistically regulated HBV replication with Rab7 complexes. Moreover, the production and release of the small HBsAg is strongly regulated by SNAP29 expression, suggesting that its export occurs partly through the autophagic pathway. Our findings provide new evidence, strongly suggesting that autophagic degradation critically determines the production of HBV virions and HBsAg and that this is controlled by the SNAP29-VAMP8 interaction.-Lin, Y., Wu, C., Wang, X., Liu, S., Kemper, T., Li, F., Squire, A., Zhu, Y., Zhang, J., Chen, X., Lu, M. Synaptosomal-associated protein 29 is required for the autophagic degradation of hepatitis B virus.
Asunto(s)
Autofagia , Antígenos de Superficie de la Hepatitis B/metabolismo , Hepatitis B/metabolismo , Proteínas Qb-SNARE/fisiología , Proteínas Qc-SNARE/fisiología , Proteínas R-SNARE/metabolismo , Sinaptosomas/metabolismo , Animales , Autofagosomas/metabolismo , Bovinos , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Silenciador del Gen , Células Hep G2 , Hepatitis B/virología , Virus de la Hepatitis B , Humanos , Lisosomas/metabolismo , Fusión de Membrana , ARN Interferente Pequeño/metabolismo , Albúmina Sérica Bovina/metabolismo , Virión , Replicación ViralRESUMEN
We have previously shown that Rab17, a small GTPase associated with epithelial polarity, is specifically suppressed by ERK2 (also known as MAPK1) signalling to promote an invasive phenotype. However, the mechanisms through which Rab17 loss permits invasiveness, and the endosomal cargoes that are responsible for mediating this, are unknown. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to a highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp8 is associated with Rab17 at late endosomes. Reduced levels of Vamp8 promote transition between ductal carcinoma in situ (DCIS) and a more invasive phenotype. We developed an unbiased proteomic approach to elucidate the complement of receptors that redistributes between endosomes and the plasma membrane, and have pin-pointed neuropilin-2 (NRP2) as a key pro-invasive cargo of Rab17- and Vamp8-regulated trafficking. Indeed, reduced Rab17 or Vamp8 levels lead to increased mobilisation of NRP2-containing late endosomes and upregulated cell surface expression of NRP2. Finally, we show that NRP2 is required for the basement membrane disruption that accompanies the transition between DCIS and a more invasive phenotype.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Endosomas/metabolismo , Proteómica/métodos , Aminoácidos/metabolismo , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Membranas Intracelulares/metabolismo , Marcaje Isotópico , Modelos Biológicos , Clasificación del Tumor , Invasividad Neoplásica , Neuropilina-2/metabolismo , Unión Proteica , Transporte de Proteínas , Proteínas R-SNARE/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Estrógenos/metabolismo , Proteínas SNARE/metabolismo , Análisis de Supervivencia , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Stimulated exocytic events provide a means for physiological communication and are a hallmark of the mast cell-mediated allergic response. In mast cells these processes are triggered by antigen crosslinking of IgE bound to its high-affinity receptor, FcϵRI, on the cell surface. Here we use the endosomal v-SNARE VAMP8, and the lysosomal hydrolase ß-hexosaminidase (ß-Hex), each C-terminally fused to super-ecliptic pHluorin, to monitor stimulated exocytosis. Using these pHluorin-tagged constructs, we monitor stimulated exocytosis by fluorimetry and visualize individual exocytic events with total internal reflection (TIRF) microscopy. Similar to constitutive recycling endosome (RE) trafficking, we find that stimulated RE exocytosis, monitored by VAMP8, is attenuated by expression of dominant negative (S25N) Rab11. Stimulated ß-Hex exocytosis is also reduced in the presence of S25N Rab11, suggesting that expression of this mutant broadly impacts exocytosis. Interestingly, pretreatment with inhibitors of actin polymerization, cytochalasin D or latrunculin A, substantially restores both RE and lysosome exocytosis in cells expressing S25N Rab11. Conversely, stabilizing F-actin with jasplakinolide inhibits antigen-stimulated exocytosis but is not additive with S25N Rab11-mediated inhibition, suggesting that these reagents inhibit related processes. Together, our results suggest that Rab11 participates in the regulation necessary for depolymerization of the actin cytoskeleton during stimulated exocytosis in mast cells.
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Endosomas/metabolismo , Exocitosis/fisiología , Mastocitos/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Degranulación de la Célula , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Endosomas/ultraestructura , Exocitosis/inmunología , Fluorometría , Humanos , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Microscopía Fluorescente , Transporte de Proteínas , Ratas , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestructura , Proteínas de Unión al GTP rab/genéticaRESUMEN
Endocytosis is an essential cellular process that is often hijacked by pathogens and pathogenic products. Endocytic processes can be classified into two broad categories, those that are dependent on clathrin and those that are not. The SNARE proteins VAMP2, VAMP3 and VAMP8 are internalized in a clathrin-dependent manner. However, the full scope of their endocytic behavior has not yet been elucidated. Here, we found that VAMP2, VAMP3 and VAMP8 are localized on plasma membrane invaginations and very early uptake structures that are induced by the bacterial Shiga toxin, which enters cells by clathrin-independent endocytosis. We show that toxin trafficking into cells and cell intoxication rely on these SNARE proteins. Of note, the cellular uptake of VAMP3 is increased in the presence of Shiga toxin, even when clathrin-dependent endocytosis is blocked. We therefore conclude that VAMP2, VAMP3 and VAMP8 are removed from the plasma membrane by non-clathrin-mediated pathways, in addition to by clathrin-dependent uptake. Moreover, our study identifies these SNARE proteins as the first transmembrane trafficking factors that functionally associate at the plasma membrane with the toxin-driven clathrin-independent invaginations during the uptake process.
Asunto(s)
Endocitosis/fisiología , Transporte de Proteínas/fisiología , Proteínas R-SNARE/metabolismo , Toxina Shiga I/farmacología , Toxinas Shiga/farmacología , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Proteína 3 de Membrana Asociada a Vesículas/metabolismo , Línea Celular , Membrana Celular/fisiología , Clatrina/metabolismo , Receptores ErbB/metabolismo , Células HeLa , Humanos , Unión Proteica/genética , Proteínas R-SNARE/genética , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Toxinas Shiga/metabolismo , Transferrina/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 3 de Membrana Asociada a Vesículas/genéticaRESUMEN
Autophagy, the process for recycling cytoplasm in the lysosome, depends on membrane trafficking. We previously identified Drosophila Sbf as a Rab21 guanine nucleotide exchange factor (GEF) that acts with Rab21 in endosomal trafficking. Here, we show that Sbf/MTMR13 and Rab21 have conserved functions required for starvation-induced autophagy. Depletion of Sbf/MTMR13 or Rab21 blocked endolysosomal trafficking of VAMP8, a SNARE required for autophagosome-lysosome fusion. We show that starvation induces Sbf/MTMR13 GEF and RAB21 activity, as well as their induced binding to VAMP8 (or closest Drosophila homolog, Vamp7). MTMR13 is required for RAB21 activation, VAMP8 interaction and VAMP8 endolysosomal trafficking, defining a novel GEF-Rab-effector pathway. These results identify starvation-responsive endosomal regulators and trafficking that tunes membrane demands with changing autophagy status.
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Autofagia/fisiología , Lisosomas/metabolismo , Fagosomas/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Proteínas R-SNARE/metabolismo , Inanición/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Cruzamientos Genéticos , Drosophila , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , ARN Interferente Pequeño/genética , Estadísticas no ParamétricasRESUMEN
Mast cells orchestrate the allergic response through the release of proinflammatory mediators, which is driven by the fusion of cytoplasmic secretory granules with the plasma membrane. During this process, SNARE proteins including Syntaxin4, SNAP23 and VAMP8 play a key role. Following stimulation, the kinase IKKß interacts with and phosphorylates the t-SNARE SNAP23. Phosphorylated SNAP23 then associates with Syntaxin4 and the v-SNARE VAMP8 to form a ternary SNARE complex, which drives membrane fusion and mediator release. Interestingly, mast cell degranulation is impaired following exposure to bacteria such as Escherichia coli. However, the molecular mechanism(s) by which this occurs is unknown. Here, we show that E. coli exposure rapidly and additively inhibits degranulation in the RBL-2H3 rat mast cell line. Following co-culture with E. coli, the interaction between IKKß and SNAP23 is disrupted, resulting in the hypophosphorylation of SNAP23. Subsequent formation of the ternary SNARE complex between SNAP23, Syntaxin4 and VAMP8 is strongly reduced. Collectively, these results demonstrate that E. coli exposure inhibits the formation of VAMP8-containing exocytic SNARE complexes and thus the release of VAMP8-dependent granules by interfering with SNAP23 phosphorylation.
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Escherichia coli/metabolismo , Mastocitos/metabolismo , Mastocitos/fisiología , Fusión de Membrana/fisiología , Proteínas SNARE/metabolismo , Animales , Línea Celular , Técnicas de Cocultivo , Escherichia coli/fisiología , Quinasa I-kappa B/metabolismo , Microdominios de Membrana/metabolismo , Microdominios de Membrana/fisiología , Proteínas de la Membrana/metabolismo , Fosforilación/fisiología , Unión Proteica/fisiología , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Ratas , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Mast cells undergo degranulation in response to various stimuli and rapidly release pre-formed mediators present in secretory granules, leading to immediate-type allergic reactions. Mast cell degranulation is commonly detected and quantified in vitro by measuring histamine or ß-hexosaminidase released to culture medium. However, this type of assay cannot monitor degranulation of individual cells in real time, and it is not suitable for in vivo detection of degranulation. At the aim of real time imaging of mast cell degranulation at single cell level, we here developed a fluorescent protein-based indicator of degranulation, designated immuno-pHluorin (impH). When expressed in mast cells, impH is located in the membrane of secretory granules and non-fluorescent under homeostatic conditions while it turns fluorescent following degranulation, due to the pH change inside of granules during exocytosis. impH enabled us to detect polarized degranulation within one single cell when mast cells were stimulated via the small area of cell surface. Transplantation of impH-expressing mast cells into mast cell-deficient mice demonstrated that impH could function as a real-time indicator of degranulation in vivo. Thus, impH is a useful tool for imaging of mast cell activation and degranulation in vitro and in vivo, and may be applied for screening of reagents regulating mast cell degranulation.
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Degranulación de la Célula , Proteínas Fluorescentes Verdes/química , Liberación de Histamina , Mastocitos/citología , Animales , Células de la Médula Ósea/citología , Medios de Cultivo , Exocitosis , Colorantes Fluorescentes/química , Histamina/química , Homeostasis , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/química , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Fluorescente , Proteínas R-SNARE/química , Vesículas Secretoras/metabolismo , Factores de Tiempo , beta-N-Acetilhexosaminidasas/química , Proteína Fluorescente RojaRESUMEN
Chorea-acanthocytosis (ChAc), a lethal disease caused by defective chorein, is characterized by neurodegeneration and erythrocyte acanthocytosis. The functional significance of chorein in other cell types remained ill-defined. The present study revealed chorein expression in blood platelets. As compared to platelets from healthy volunteers, platelets from patients with ChAc displayed a 47% increased globular/filamentous actin ratio, indicating actin depolymerization. Moreover, phosphoinositide-3-kinase subunit p85 phosphorylation, p21 protein-activated kinase (PAK1) phosphorylation, as well as vesicle-associated membrane protein 8 (VAMP8) expression were significantly reduced in platelets from patients with ChAc (by 17, 22, and 39%, respectively) and in megakaryocytic (MEG-01) cells following chorein silencing (by 16, 54, and 11%, respectively). Activation-induced platelet secretion from dense granules (ATP release) and α granules (P-selectin exposure) were significantly less (by 55% after stimulation with 1 µg/ml CRP and by 33% after stimulation with 5 µM TRAP, respectively) in ChAc platelets than in control platelets. Furthermore, platelet aggregation following stimulation with different platelet agonists was significantly impaired. These observations reveal a completely novel function of chorein, i.e., regulation of secretion and aggregation of blood platelets.
Asunto(s)
Plaquetas/metabolismo , Degranulación de la Célula , Citoesqueleto/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Actinas/metabolismo , Adulto , Plaquetas/fisiología , Plaquetas/ultraestructura , Western Blotting , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Femenino , Humanos , Masculino , Microscopía Confocal , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Neuroacantocitosis/sangre , Neuroacantocitosis/genética , Neuroacantocitosis/metabolismo , Fosforilación , Agregación Plaquetaria , Proteínas R-SNARE/metabolismo , Interferencia de ARN , Proteínas de Transporte Vesicular/genética , Adulto Joven , Quinasas p21 Activadas/metabolismoRESUMEN
Background and objective: Epithelial ovarian cancer (EOC) is associated with latent onset and poor prognosis, with drug resistance being a main concern in improving the prognosis of these patients. The resistance of cancer cells to most chemotherapeutic agents can be related to autophagy mechanisms. This study aimed to assess the therapeutic effect of MK8722, a small-molecule compound that activates AMP-activated protein kinase (AMPK), on EOC cells and to propose a novel strategy for the treatment of EOC. Purpose: To explore the therapeutic effects of MK8722 on EOC cells, and to elucidate the underlying mechanism. Methods and results: It was found that MK8722 effectively inhibited the malignant biological behaviors of EOC cells. In vitro experiments showed that MK8722 targeted and decreased the lipid metabolic pathway-related fatty acid synthase (FASN) expression levels, causing the accumulation of lipid droplets. In addition, transmission electron microscopy revealed the presence of autophagosome-affected mitochondria. Western blotting confirmed that MK8722 plays a role in activating autophagy upstream (PI3K/AKT/mTOR) and inhibiting autophagy downstream via FASN-dependent reprogramming of lipid metabolism. Plasmid transient transfection demonstrated that MK8722 suppressed late-stage autophagy by blocking autophagosome-lysosome fusion. Immunofluorescence and gene silencing revealed that this effect was achieved by inhibiting the interaction of FASN with the SNARE complexes STX17-SNP29-VAMP8. Furthermore, the antitumor effect of MK8722 was verified using a subcutaneous xenograft mouse model. Conclusion: The findings suggest that using MK8722 may be a new strategy for treating EOC, as it has the potential to be a new autophagy/mitophagy inhibitor. Its target of action, FASN, is a molecular crosstalk between lipid metabolism and autophagy, and exploration of the underlying mechanism of FASN may provide a new research direction.
Asunto(s)
Metabolismo de los Lípidos , Neoplasias Ováricas , Humanos , Femenino , Ratones , Animales , Fosfatidilinositol 3-Quinasas/metabolismo , Autofagia , Ácido Graso Sintasas/metabolismo , Ácido Graso Sintasas/farmacología , Carcinoma Epitelial de Ovario , Acido Graso Sintasa Tipo I/metabolismoRESUMEN
Osteosarcoma is a highly metastatic malignant bone tumor, necessitating the development of new treatments to target its metastasis. Recent studies have revealed the significance of VAMP8 in regulating various signaling pathways in various types of cancer. However, the specific functional role of VAMP8 in osteosarcoma progression remains unclear. In this study, we observed a significant downregulation of VAMP8 in osteosarcoma cells and tissues. Low levels of VAMP8 in osteosarcoma tissues were associated with patients' poor prognosis. VAMP8 inhibited the migration and invasion capability of osteosarcoma cells. Mechanically, we identified DDX5 as a novel interacting partner of VAMP8, and the conjunction of VAMP8 and DDX5 promoted the degradation of DDX5 via the ubiquitin-proteasome system. Moreover, reduced levels of DDX5 led to the downregulation of ß-catenin, thereby suppressing the epithelial-mesenchymal transition (EMT). Additionally, VAMP8 promoted autophagy flux, which may contribute to the suppression of osteosarcoma metastasis. In conclusion, our study anticipated that VAMP8 inhibits osteosarcoma metastasis by promoting the proteasomal degradation of DDX5, consequently inhibiting WNT/ß-catenin signaling and EMT. Dysregulation of autophagy by VAMP8 is also implicated as a potential mechanism. These findings provide new insights into the biological nature driving osteosarcoma metastasis and highlight the modulation of VAMP8 as a potential therapeutic strategy for targeting osteosarcoma metastasis.
Asunto(s)
Neoplasias Óseas , Osteosarcoma , Humanos , beta Catenina/metabolismo , Línea Celular Tumoral , Vía de Señalización Wnt , Osteosarcoma/patología , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Movimiento Celular , Proliferación Celular , Proteínas R-SNARE/metabolismoRESUMEN
In Alzheimer's disease, Tau, a microtubule-associated protein, becomes hyperphosphorylated, detaches from microtubules, and accumulates in the somato-dendritic compartment where it forms insoluble aggregates. Tau also accumulates in the CSF of patients indicating that it is released by neurons. Consistent with this, several laboratories including ours have shown that Tau is secreted by neurons through unconventional secretory pathways. Recently, we reported that VAMP8, an R-SNARE found on late endosomes, increased Tau secretion and that secreted Tau was cleaved at the C-terminal. In the present study, we examined whether the increase of Tau secretion by VAMP8 affected its intra- and extracellular cleavage. Upon VAMP8 overexpression, an increase of Tau cleaved by caspase-3 in the cell lysate and medium was observed. This was correlated to an increase of active caspase-3 in the cell lysate and medium. Using a Tau mutant not cleavable by caspase-3, we demonstrated that Tau cleavage by caspase-3 was not necessary for its secretion upon VAMP8 overexpression. By adding recombinant Tau to the culture medium, we demonstrated that extracellular Tau cleavage by caspase-3 could occur because of the release of active caspase-3, which was the highest when VAMP8 was overexpressed. When cleavage of Tau by caspase-3 was prevented by using a non-cleavable mutant, secreted Tau was still cleaved at the C-terminal, the asparagine N410 contributing to it. Lastly, we demonstrated that N-terminal of Tau regulated the secretion pattern of a Tau fragment containing the microtubule-binding domain and the C-terminal of Tau upon VAMP8 overexpression. Collectively, the above observations indicate that VAMP8 overexpression affects the intra- and extracellular cleavage pattern of Tau.