RESUMEN

Hepatic stellate cells (HSC) are critical effector cells of liver fibrosis. In the injured liver, HSC differentiate into a myofibrobastic phenotype. A critical feature distinguishing myofibroblastic from quiescent HSC is cytoskeletal reorganization. Soluble NSF attachment receptor (SNARE) proteins are important in trafficking of newly synthesized proteins to the plasma membrane for release into the extracellular environment. The goals of this project were to determine the expression of specific SNARE proteins in myofibroblastic HSC and to test whether their alteration changed the HSC phenotype in vitro and progression of liver fibrosis in vivo. We found that HSC lack the t-SNARE protein, SNAP-25, but express a homologous protein, SNAP-23. Downregulation of SNAP-23 in HSC induced reduction in polymerization and disorganization of the actin cytoskeleton associated with loss of cell movement. In contrast, reduction in SNAP-23 in mice by monogenic deletion delayed but did not prevent progression of liver fibrosis to cirrhosis. Taken together, these findings suggest that SNAP-23 is an important regular of actin dynamics in myofibroblastic HSC, but that the role of SNAP-23 in the progression of liver fibrosis in vivo is unclear.

Asunto(s)

Citoesqueleto de Actina/ultraestructura , Células Estrelladas Hepáticas/ultraestructura , Miofibroblastos/ultraestructura , Proteínas Qb-SNARE/deficiencia , Proteínas Qc-SNARE/deficiencia , Citoesqueleto de Actina/química , Factores Despolimerizantes de la Actina/biosíntesis , Actinas/análisis , Animales , Tetracloruro de Carbono/toxicidad , Línea Celular , Movimiento Celular , Separación Celular , Técnicas de Silenciamiento del Gen , Células Estrelladas Hepáticas/metabolismo , Humanos , Hígado/citología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/patología , Ratones , Proteínas Qb-SNARE/antagonistas & inhibidores , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/fisiología , Proteínas Qc-SNARE/antagonistas & inhibidores , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Transducción de Señal , Fibras de Estrés/química , Fibras de Estrés/ultraestructura , Cicatrización de Heridas , Quinasas Asociadas a rho/fisiología

RESUMEN

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion.

Asunto(s)

Adenocarcinoma/metabolismo , Matriz Extracelular/metabolismo , Fibrosarcoma/metabolismo , Proteínas Munc18/metabolismo , Proteínas de Neoplasias/metabolismo , Podosomas/metabolismo , Proteínas Qa-SNARE/metabolismo , Adenocarcinoma/patología , Unión Competitiva , Línea Celular Tumoral , Receptores ErbB/metabolismo , Matriz Extracelular/patología , Fibrosarcoma/patología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas Munc18/antagonistas & inhibidores , Proteínas Munc18/química , Proteínas Munc18/genética , Invasividad Neoplásica , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Podosomas/patología , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Transporte de Proteínas , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/genética , Proteínas Qb-SNARE/antagonistas & inhibidores , Proteínas Qb-SNARE/química , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/antagonistas & inhibidores , Proteínas Qc-SNARE/química , Proteínas Qc-SNARE/metabolismo , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/antagonistas & inhibidores , Proteína 2 de Membrana Asociada a Vesículas/química , Proteína 2 de Membrana Asociada a Vesículas/metabolismo

RESUMEN

Tumour cells secrete exosomes that are involved in the remodelling of the tumour-stromal environment and promoting malignancy. The mechanisms governing tumour exosome release, however, remain incompletely understood. Here we show that tumour cell exosomes secretion is controlled by pyruvate kinase type M2 (PKM2), which is upregulated and phosphorylated in tumours. During exosome secretion, phosphorylated PKM2 serves as a protein kinase to phosphorylate synaptosome-associated protein 23 (SNAP-23), which in turn enables the formation of the SNARE complex to allow exosomes release. Direct phosphorylation assay and mass spectrometry confirm that PKM2 phosphorylates SNAP-23 at Ser95. Ectopic expression of non-phosphorylated SNAP-23 mutant (Ser95→Ala95) significantly reduces PKM2-mediated exosomes release whereas expression of selective phosphomimetic SNAP-23 mutants (Ser95→Glu95 but not Ser20→Glu20) rescues the impaired exosomes release induced by PKM2 knockdown. Our findings reveal a non-metabolic function of PKM2, an enzyme associated with tumour cell reliance on aerobic glycolysis, in promoting tumour cell exosome release.

Asunto(s)

Proteínas Portadoras/genética , Exosomas/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Hormonas Tiroideas/genética , Células A549 , Animales , Secuencia de Bases , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proliferación Celular , Células HeLa , Células Hep G2 , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Fosforilación , Proteínas Qb-SNARE/antagonistas & inhibidores , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/antagonistas & inhibidores , Proteínas Qc-SNARE/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Hormonas Tiroideas/metabolismo , Proteínas de Unión a Hormona Tiroide

RESUMEN

Fibroblast-like synoviocytes are important mediators of inflammatory joint damage in arthritis through the release of cytokines, but it is unknown whether their exocytosis from these particular cells is SNARE-dependent. Here, the complement of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in human synovial sarcoma cells (SW982) was examined with respect to the secretion of interleukin-6 (IL-6) and tumour necrosis factor α (TNFα), before and after knockdown of a synaptosome-associated protein of molecular mass 23 kDa (SNAP-23) or the vesicle-associated membrane protein 3 (VAMP-3). Wild-type SW982 cells expressed SNAP-23, VAMP-3, syntaxin isoforms 2-4 and synaptic vesicle protein 2C (SV2C). These cells showed Ca²âº-dependent secretion of IL-6 and TNFα when stimulated by interleukin-1ß (IL-1ß) or in combination with K⁺ depolarization. Specific knockdown of SNAP-23 or VAMP-3 decreased the exocytosis of IL-6 and TNFα; the reduced expression of SNAP-23 caused accumulation of SV2 in the peri-nuclear area. A monoclonal antibody specific for VAMP-3 precipitated SNAP-23 and syntaxin-2 (and syntaxin-3 to a lesser extent). The formation of SDS-resistant complexes by SNAP-23 and VAMP-3 was reduced upon knockdown of SNAP-23. Although the syntaxin isoforms 2, 3 and 4 are expressed in SW982 cells, knockdown of each did not affect the release of cytokines. Collectively, these results show that SNAP-23 and VAMP-3 participate in IL-1ß-induced Ca²âº-dependent release of IL-6 and TNFα from SW982 cells.

Asunto(s)

Exocitosis , Interleucina-6/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Membrana Sinovial/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteína 3 de Membrana Asociada a Vesículas/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Artritis/tratamiento farmacológico , Artritis/inmunología , Artritis/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , Exocitosis/efectos de los fármacos , Humanos , Interleucina-1beta/metabolismo , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Terapia Molecular Dirigida , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Qa-SNARE/antagonistas & inhibidores , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/antagonistas & inhibidores , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/antagonistas & inhibidores , Proteínas Qc-SNARE/genética , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Interleucina-1/metabolismo , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/inmunología , Proteína 3 de Membrana Asociada a Vesículas/antagonistas & inhibidores , Proteína 3 de Membrana Asociada a Vesículas/genética

RESUMEN

The role of exocytosis in the human neutrophil respiratory burst was determined using a fusion protein (TAT-SNAP-23) containing the HIV transactivator of transcription (TAT) cell-penetrating sequence and the N-terminal SNARE domain of synaptosome-associated protein-23 (SNAP-23). This agent inhibited stimulated exocytosis of secretory vesicles and gelatinase and specific granules but not azurophil granules. GST pulldown showed that TAT-SNAP-23 bound to the combination of vesicle-associated membrane protein-2 and syntaxin-4 but not to either individually. TAT-SNAP-23 reduced phagocytosis-stimulated hydrogen peroxide production by 60% without affecting phagocytosis or generation of HOCl within phagosomes. TAT-SNAP-23 had no effect on fMLF-stimulated superoxide release but significantly inhibited priming of this response by TNF-α and platelet-activating factor. Pretreatment with TAT-SNAP-23 inhibited the increase in plasma membrane expression of gp91(phox) in TNF-α-primed neutrophils, whereas TNF-α activation of ERK1/2 and p38 MAPK was not affected. The data demonstrate that neutrophil granule exocytosis contributes to phagocytosis-induced respiratory burst activity and plays a critical role in priming of the respiratory burst by increasing expression of membrane components of the NADPH oxidase.

Asunto(s)

Gránulos Citoplasmáticos/inmunología , Exocitosis/inmunología , Activación Neutrófila/inmunología , Estallido Respiratorio/inmunología , Apoptosis/genética , Apoptosis/inmunología , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/metabolismo , Exocitosis/genética , Productos del Gen tat/antagonistas & inhibidores , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , VIH-1/inmunología , Humanos , Activación Neutrófila/genética , Fagocitosis/genética , Fagocitosis/inmunología , Factor de Activación Plaquetaria/fisiología , Estructura Terciaria de Proteína/genética , Proteínas Qb-SNARE/antagonistas & inhibidores , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/antagonistas & inhibidores , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Estallido Respiratorio/genética , Proteínas SNARE/antagonistas & inhibidores , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/fisiología

RESUMEN

Clostridium botulinum neurotoxins (BoNTs) are effective therapeutics for a variety of neurological disorders, such as strabismus, blepharospam, hemificial spasm, and cervical dystonia, because of the toxin's tropism for neurons and specific cleavage of neuronal soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors (SNARE) proteins. Modifying BoNT to bind nonneuronal cells has been attempted to extend therapeutic applications. However, prerequisite to develop nonneuronal therapies requires the retargeting the catalytic activity of BoNTs to nonneuronal SNARE isoforms. Here, we reported the engineering of a BoNT derivative that cleaves SNAP23, a nonneuronal SNARE protein. SNAP23 mediates vesicle-plasma membrane fusion processes, including secretion of airway mucus, antibody, insulin, gastric acids, and ions. This mutated BoNT/E light chain LC/E(K(224)D) showed extended substrate specificity to cleave SNAP23, and the natural substrate, SNAP25, but not SNAP29 or SNAP47. Upon direct protein delivery into cultured human epithelial cells, LC/E(K(224)D) cleaved endogenous SNAP23, which inhibited secretion of mucin and IL-8. These studies show the feasibility of genetically modifying LCs to target a nonneuronal SNARE protein that extends therapeutic potential for treatment of human hypersecretion diseases.

Asunto(s)

Toxinas Botulínicas/química , Toxinas Botulínicas/farmacología , Moco/metabolismo , Secuencia de Aminoácidos , Secreciones Corporales , Línea Celular , Membrana Celular/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Qb-SNARE/antagonistas & inhibidores , Proteínas Qc-SNARE/antagonistas & inhibidores , Proteínas SNARE/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , Proteína 25 Asociada a Sinaptosomas/química

RESUMEN

Homotypic yeast vacuole fusion occurs in three stages: (i) priming reactions, which are independent of vacuole clustering, (ii) docking, in which vacuoles cluster and accumulate fusion proteins and fusion regulatory lipids at a ring-shaped microdomain surrounding the apposed membranes of each docked vacuole, where fusion will occur, and (iii) bilayer fusion/compartment mixing. These stages require vacuolar SNAREs, SNARE-chaperones, GTPases, effector complexes, and chemically minor but functionally important lipids. For each, we have developed specific ligands that block fusion and conditions that reverse each block. Using them, we test whether docking entails a linearly ordered series of catalytic events, marked by sequential acquisition of resistance to inhibitors, or whether docking subreactions are cooperative and/or reversible. We find that each fusion protein and regulatory lipid is needed throughout docking, indicative of a reversible or highly cooperative assembly of the fusion-competent vertex ring. In accord with this cooperativity, vertices enriched in one fusion catalyst are enriched in others. Docked vacuoles finally assemble SNARE complexes, yet still require physiological temperature and lipid rearrangements to complete fusion.

Asunto(s)

Proteínas Q-SNARE/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Vacuolas/fisiología , Catálisis , Membranas Intracelulares/fisiología , Ligandos , Fusión de Membrana , Proteínas Q-SNARE/antagonistas & inhibidores , Proteínas Q-SNARE/genética , Proteínas Qa-SNARE/antagonistas & inhibidores , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/fisiología , Proteínas Qb-SNARE/antagonistas & inhibidores , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/fisiología , Proteínas Qc-SNARE/antagonistas & inhibidores , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/fisiología , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteína 25 Asociada a Sinaptosomas