scholarly journals Syntaxin-11, but not syntaxin-2 or syntaxin-4, is required for platelet secretion

Blood ◽  
2012 ◽  
Vol 120 (12) ◽  
pp. 2484-2492 ◽  
Author(s):  
Shaojing Ye ◽  
Zubair A. Karim ◽  
Rania Al Hawas ◽  
Jeffery E. Pessin ◽  
Alexandra H. Filipovich ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Critical Role ◽  
Snare Proteins ◽  
Mouse Strains ◽  
Double Knockout ◽  
Protein Receptor ◽  
Syntaxin 4 ◽  
Syntaxin 11 ◽  
Platelet Exocytosis ◽  
Platelet Secretion

Abstract The platelet release reaction plays a critical role in thrombosis and contributes to the events that follow hemostasis. Previous studies have shown that platelet secretion is mediated by Soluble NSF Attachment Protein Receptor (SNARE) proteins from granule and plasma membranes. The SNAREs form transmembrane complexes that mediate membrane fusion and granule cargo release. Although VAMP-8 (v-SNARE) and SNAP-23 (a t-SNARE class) are important for platelet secretion, the identity of the functional syntaxin (another t-SNARE class) has been controversial. Previous studies using anti-syntaxin Abs in permeabilized platelets have suggested roles for both syntaxin-2 and syntaxin-4. In the present study, we tested these conclusions using platelets from syntaxin-knockout mouse strains and from a Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL4) patient. Platelets from syntaxin-2 and syntaxin-4 single- or double-knockout mice had no secretion defect. Platelets from a FHL4 patient deficient in syntaxin-11 had a robust defect in agonist-induced secretion although their morphology, activation, and cargo levels appeared normal. Semiquantitative Western blotting showed that syntaxin-11 is the more abundant syntaxin in both human and murine platelets. Coimmunoprecipitation experiments showed that syntaxin-11 can form SNARE complexes with both VAMP-8 and SNAP-23. The results of the present study indicate that syntaxin-11, but not syntaxin-2 or syntaxin-4, is required for platelet exocytosis.

C2C12 myocytes lack an insulin-responsive vesicular compartment despite dexamethasone-induced GLUT4 expression

2002 ◽  
Vol 283 (3) ◽  
pp. E514-E524 ◽  
Author(s):  
Lori L. Tortorella ◽  
Paul F. Pilch
Keyword(s):  
Glucose Transporter ◽  
Fold Increase ◽  
Snare Proteins ◽  
Vesicular Traffic ◽  
Glut4 Expression ◽  
Protein Receptor ◽  
Syntaxin 4 ◽  
Insulin Dependent ◽  
Glucose Transporter Glut4 ◽  
Vesicular Compartment

Insulin regulates the uptake of glucose into skeletal muscle and adipocytes by redistributing the tissue-specific glucose transporter GLUT4 from intracellular vesicles to the cell surface. To date, GLUT4 is the only protein involved in insulin-regulated vesicular traffic that has this tissue distribution, thus raising the possibility that its expression alone may allow formation of an insulin-responsive vesicular compartment. We show here that treatment of differentiating C2C12myoblasts with dexamethasone, acting via the glucocorticoid receptor, causes a ≥10-fold increase in GLUT4 expression but results in no significant change in insulin-stimulated glucose transport. Signaling from the insulin receptor to its target, Akt2, and expression of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor, or SNARE, proteins syntaxin 4 and vesicle-associated membrane protein are normal in dexamethasone-treated C2C12 cells. However, these cells show no insulin-dependent trafficking of the insulin-responsive aminopeptidase or the transferrin receptor, respective markers for intracellular GLUT4-rich compartments and endosomes that are insulin responsive in mature muscle and adipose cells. Therefore, these data support the hypothesis that GLUT4 expression by itself is insufficient to establish an insulin-sensitive vesicular compartment.

scholarly journals Snapin associates with late endocytic compartments and interacts with late endosomal SNAREs

2009 ◽  
Vol 29 (4) ◽  
pp. 261-269 ◽  
Author(s):  
Li Lu ◽  
Qian Cai ◽  
Jin-Hua Tian ◽  
Zu-Hang Sheng
Keyword(s):  
Membrane Trafficking ◽  
Critical Role ◽  
Degradation Process ◽  
Late Endosome ◽  
Snare Proteins ◽  
Genetic Mouse Model ◽  
Protein Receptor ◽  
Important Regulator ◽  
Endocytic Compartments ◽  
Synaptic Vesicle Fusion

Late endocytic membrane trafficking delivers target materials and newly synthesized hydrolases into lysosomes and is critical for maintaining an efficient degradation process and cellular homoeostasis. Although some features of late endosome–lysosome trafficking have been described, the mechanisms underlying regulation of this event remain to be elucidated. Our previous studies showed that Snapin, as a SNAP25 (25 kDa synaptosome-associated protein)-binding protein, plays a critical role in priming synaptic vesicles for synchronized fusion in neurons. In the present study, we report that Snapin also associates with late endocytic membranous organelles and interacts with the late endosome-targeted SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex. Using a genetic mouse model, we further discovered that Snapin is required to maintain a proper balance of the late endocytic protein LAMP-1 (lysosome-associated membrane protein-1) and late endosomal SNARE proteins syntaxin 8 and Vti1b (vesicle transport through interaction with target SNAREs homologue 1b). Deleting the snapin gene in mice selectively led to the accumulation of these proteins in late endocytic organelles. Thus our present study suggests that Snapin serves as an important regulator of the late endocytic fusion machinery, in addition to its established role in regulating synaptic vesicle fusion.

Temporal Secretion of α-Granular Products: Insights into the Mechanisms of Release Reaction

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-36-SCI-36
Author(s):  
Sidney W. Whiteheart
Keyword(s):  
Membrane Proteins ◽  
Membrane Fusion ◽  
Wound Repair ◽  
Conflicts Of Interest ◽  
Genetically Engineered ◽  
Snare Complex ◽  
Iκb Kinase ◽  
Syntaxin 11 ◽  
Platelet Exocytosis ◽  
Platelet Secretion

Abstract Abstract SCI-36 Upon activation, platelets secrete numerous molecules that affect the local vascular microenvironment. Many of these components are important for hemostasis; however, others play key roles in the sequelae of thrombosis, such as angiogenesis, inflammation, and wound repair. These molecules are stored in three general types of platelet granules: dense, α, and lysosome. Analysis of platelet exocytosis shows that there is a direct correlation between agonist potency (thrombin > convulxin > PAR1 agonist > PAR4 agonist) and the extent and rate of content release. Release from dense granules is most rapid, while lysosome content release is the slowest; release of α-granule contents is heterogeneous. Detailed analysis of α-granule cargo release identified three kinetic classes of release events that differ in their rate. The content released in each of these three classes appeared to be random. Mechanistically, secretion from each granule requires integral membrane proteins called soluble NSF attachment protein receptors (SNAREs). v-SNAREs (vesicle-associated membrane proteins [VAMPs] from the granules) and heterodimeric t-SNAREs (syntaxins and SNAP-23 from the plasma membrane) form a trans-membrane, trimeric complex that mediates fusion and content release. How and where the SNAREs interact is controlled by regulatory proteins that in turn are affected during platelet activation. By analyzing platelet secretion from knockout mice and patients with familial hemophagocytic lymphohistiocytosis (FHL), it has been possible to identify the machinery required for each granule secretion event. Platelets contain four v-SNAREs (VAMP-2, −3, −7, and −8), but only VAMP-8 is required for content release. Platelets contain six t-SNAREs (syntaxin 2, 4, 7, 11, 13, and SNAP-23). Deletion of only syntaxin 11 in patients with FHL4 is sufficient to diminish platelet secretion. SNAP-23 is regulated by phosphorylation on Ser 95 by IκB kinase (IKK). This is important for SNARE complex formation, membrane fusion, and secretion. Intriguingly, mice treated with IKK inhibitors or genetically engineered with a platelet-specific deletion of Ikkb show increased tail-bleeding times. A number of syntaxin regulators have been identified in platelets. MUNC18b is a syntaxin 11 chaperone that is essential for platelet secretion in FHL5 patients. MUNC13 proteins, which facilitate t-SNARE/v-SNARE binding, are also important for platelet granule release. Deletion of Munc13-4 results in a significant platelet secretion defect and a robust bleeding diathesis in mice. Biochemical analysis shows that MUNC13-4 is a docking/tethering factor required to increase the efficacy of membrane fusion. Both MUNC18b and MUNC13-4 appear to be limiting in platelets, suggesting that they might be useful therapeutic targets. A greater understanding of platelet exocytosis is unfolding. This knowledge will undoubtedly lead to the development of better antithrombotic drugs. This work is supported by HL56652 and HL091893 from the National Institutes of Health. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Munc18b/STXBP2 is required for platelet secretion

Blood ◽  
2012 ◽  
Vol 120 (12) ◽  
pp. 2493-2500 ◽  
Author(s):  
Rania Al Hawas ◽  
Qiansheng Ren ◽  
Shaojing Ye ◽  
Zubair A. Karim ◽  
Alexandra H. Filipovich ◽  
...  
Keyword(s):  
Human Platelets ◽  
Limiting Factor ◽  
Attachment Protein ◽  
Platelet Factor ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Heterozygous Patient ◽  
Syntaxin 11 ◽  
Platelet Exocytosis ◽  
Platelet Secretion

Abstract Platelets are vital for hemostasis because they release their granule contents in response to vascular damage. Platelet exocytosis is mediated by soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs), whose interactions are governed by regulators, eg, Sec/Munc18 proteins. These proteins chaperone syntaxin t-SNAREs and are required for exocytosis. Platelets contain 3 Munc18 isoforms: Munc18a, Munc18b, and Munc18c. We report that Munc18b is the major isoform and is required for platelet secretion. Familial hemophagocytic lymphohistiocytosis type 5 (FHL5) is caused by defects in the Munc18b/STXBP2 gene. We confirm a previous report showing that platelets from FHL5 patients have defective secretion. Serotonin, ADP/ATP, and platelet factor 4 release was profoundly affected in the 2 biallelic patients and partially in a heterozygous patient. Release of lysosomal contents was only affected in the biallelic platelets. Platelets from the FHL5 biallelic patients showed decreased Munc18b and syntaxin-11 levels were significantly reduced; other syntaxins were unaffected. Munc18b formed complexes with syntaxin-11, SNAP-23, and vesicle-associated membrane protein-8 in human platelets. Other potential secretion regulators, Munc13-4 and Rab27, were also found associated. These data demonstrate a key role for Munc18b, perhaps as a limiting factor, in platelet exocytosis and suggest that it regulates syntaxin-11.

scholarly journals SNAP-23 participates in SNARE complex assembly in rat adipose cells

1999 ◽  
Vol 338 (3) ◽  
pp. 709-715 ◽  
Author(s):  
Jean-François ST-DENIS ◽  
Jean-Pierre CABANIOLS ◽  
Samuel W. CUSHMAN ◽  
Paul A. ROCHE
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Snare Complex ◽  
Snare Proteins ◽  
Insulin Stimulation ◽  
Adipose Cell ◽  
Vesicle Exocytosis ◽  
Syntaxin 4 ◽  
Intracellular Vesicles ◽  
Adipose Cells

SNARE proteins are required for vesicle docking and fusion in eukaryotic cells in processes as diverse as homotypic membrane fusion and synaptic vesicle exocytosis [SNARE stands for SNAP receptor, where SNAP is soluble NSF attachment protein]. The SNARE proteins syntaxin 4 and vesicle-associated membrane protein (VAMP) 2/3 also participate in the insulin-stimulated translocation of GLUT4 from intracellular vesicles to the plasma membrane in adipose cells. We now report the molecular cloning and characterization of rat SNAP-23, a ubiquitously expressed homologue of the essential neuronal SNARE protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Rat SNAP-23 is 86% and 98% identical respectively to human and mouse SNAP-23. Southern blot analysis reveals that the rat, mouse and human SNAP-23 genes encode species-specific isoforms of the same protein. Co-immunoprecipitation of syntaxin 4 and SNAP-23 shows association of these two proteins in rat adipose cell plasma membranes, and insulin stimulation does not alter the SNAP-23/syntaxin 4 complex. In addition, we demonstrate for the first time the participation of SNAP-23, along with syntaxin 4 and VAMP2/3, in the formation of 20 S SNARE complexes prepared using rat adipose cell membranes and recombinant α-SNAP and NSF proteins. The stoichiometry of the SNARE complexes formed is essentially identical using membranes from either unstimulated or insulin-stimulated adipose cells. These data demonstrate that rat SNAP-23 associates with syntaxin 4 before insulin stimulation and is present in the SNARE complexes known to mediate the translocation of GLUT4 from intracellular vesicles to the plasma membrane of rat adipose cells.

scholarly journals Qualitative and Quantitative Comparison of Plasma Exosomes from Neonates and Adults

2021 ◽  
Vol 22 (4) ◽  
pp. 1926
Author(s):  
Julia Peñas-Martínez ◽  
María N. Barrachina ◽  
Ernesto José Cuenca-Zamora ◽  
Ginés Luengo-Gil ◽  
Susana Belén Bravo ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Critical Role ◽  
Snare Proteins ◽  
Term Neonates ◽  
Content Change ◽  
Protein Receptor ◽  
Transmission Electron ◽  
Health And Disease ◽  
Adult Plasma ◽  
Granule Secretion

Exosomes are extracellular vesicles that contain nucleic acids, lipids and metabolites, and play a critical role in health and disease as mediators of intercellular communication. The majority of extracellular vesicles in the blood are platelet-derived. Compared to adults, neonatal platelets are hyporeactive and show impaired granule release, associated with defects in Soluble N-ethylmaleimide-sensitive fusion Attachment protein REceptor (SNARE) proteins. Since these proteins participate in biogenesis of exosomes, we investigated the potential differences between newborn and adult plasma-derived exosomes. Plasma-derived exosomes were isolated by ultracentrifugation of umbilical cord blood from full-term neonates or peripheral blood from adults. Exosome characterization included size determination by transmission electron microscopy and quantitative proteomic analysis. Plasma-derived exosomes from neonates were significantly smaller and contained 65% less protein than those from adults. Remarkably, 131 proteins were found to be differentially expressed, 83 overexpressed and 48 underexpressed in neonatal (vs. adult) exosomes. Whereas the upregulated proteins in plasma exosomes from neonates are associated with platelet activation, coagulation and granule secretion, most of the underexpressed proteins are immunoglobulins. This is the first study showing that exosome size and content change with age. Our findings may contribute to elucidating the potential “developmental hemostatic mismatch risk” associated with transfusions containing plasma exosomes from adults.

scholarly journals Dosage-Dependent Effects of Akt1/Protein Kinase Bα (PKBα) and Akt3/PKBγ on Thymus, Skin, and Cardiovascular and Nervous System Development in Mice

2005 ◽  
Vol 25 (23) ◽  
pp. 10407-10418 ◽  
Author(s):  
Zhong-Zhou Yang ◽  
Oliver Tschopp ◽  
Nicolas Di-Poï ◽  
Elisabeth Bruder ◽  
Anne Baudry ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Cycle Progression ◽  
System Development ◽  
Critical Role ◽  
Nervous System Development ◽  
Mouse Development ◽  
Double Knockout ◽  
Developmental Defects ◽  
Regulation Of Metabolism ◽  
Survival And Development

ABSTRACT Akt/protein kinase B (PKB) plays a critical role in the regulation of metabolism, transcription, cell migration, cell cycle progression, and cell survival. The existence of viable knockout mice for each of the three isoforms suggests functional redundancy. We generated mice with combined mutant alleles of Akt1 and Akt3 to study their effects on mouse development. Here we show that Akt1 − / − Akt3 +/ − mice display multiple defects in the thymus, heart, and skin and die within several days after birth, while Akt1 +/ − Akt3 − / − mice survive normally. Double knockout (Akt1 − / − Akt3 − / −) causes embryonic lethality at around embryonic days 11 and 12, with more severe developmental defects in the cardiovascular and nervous systems. Increased apoptosis was found in the developing brain of double mutant embryos. These data indicate that the Akt1 gene is more essential than Akt3 for embryonic development and survival but that both are required for embryo development. Our results indicate isoform-specific and dosage-dependent effects of Akt on animal survival and development.

scholarly journals Influence of genetic background on ex vivo and in vivo cardiac function in several commonly used inbred mouse strains

2010 ◽  
Vol 42A (2) ◽  
pp. 103-113 ◽  
Author(s):  
Matthew S. Barnabei ◽  
Nathan J. Palpant ◽  
Joseph M. Metzger
Keyword(s):  
Cardiac Function ◽  
Genetic Divergence ◽  
Ex Vivo ◽  
Inbred Mouse ◽  
Critical Role ◽  
Inbred Strains ◽  
Mouse Strains ◽  
Inbred Mouse Strains ◽  
Cardiac Decompensation

Inbred mouse strains play a critical role in biomedical research. Genetic homogeneity within inbred strains and their general amenability to genetic manipulation have made them an ideal resource for dissecting the physiological function(s) of individual genes. However, the inbreeding that makes inbred mice so useful also results in genetic divergence between them. This genetic divergence is often unaccounted for but may be a confounding factor when comparing studies that have utilized distinct inbred strains. Here, we compared the cardiac function of C57BL/6J mice to seven other commonly used inbred mouse strains: FVB/NJ, DBA/2J, C3H/HeJ, BALB/cJ, 129X1/SvJ, C57BL/10SnJ, and 129S1/SvImJ. The assays used to compare cardiac function were the ex vivo isolated Langendorff heart preparation and in vivo real-time hemodynamic analysis using conductance micromanometry. We report significant strain-dependent differences in cardiac function between C57BL/6J and other commonly used inbred strains. C57BL/6J maintained better cardiac function than most inbred strains after ex vivo ischemia, particularly compared with 129S1/SvImJ, 129X1/SvJ, and C57BL/10SnJ strains. However, during in vivo acute hypoxia 129X1/SvJ and 129S1/SvImJ maintained relatively normal cardiac function, whereas C57BL/6J animals showed dramatic cardiac decompensation. Additionally, C3H/HeJ showed rapid and marked cardiac decompensation in response to esmolol infusion compared with effects of other strains. These findings demonstrate the complex effects of genetic divergence between inbred strains on cardiac function. These results may help inform analysis of gene ablation or transgenic studies and further demonstrate specific quantitative traits that could be useful in discovery of genetic modifiers relevant to cardiac health and disease.

Syntaxin 11 is associated with SNAP-23 on late endosomes and the trans-Golgi network

1999 ◽  
Vol 112 (6) ◽  
pp. 845-854 ◽  
Author(s):  
A.C. Valdez ◽  
J.P. Cabaniols ◽  
M.J. Brown ◽  
P.A. Roche
Keyword(s):  
Mammalian Cells ◽  
Protein Transport ◽  
Transmembrane Domain ◽  
Family Members ◽  
Snare Proteins ◽  
Trans Golgi Network ◽  
Late Endosomes ◽  
Syntaxin 11 ◽  
Golgi Network

SNARE proteins are known to play a role in regulating intracellular protein transport between donor and target membranes. This docking and fusion process involves the interaction of specific vesicle-SNAREs (e.g. VAMP) with specific cognate target-SNAREs (e.g. syntaxin and SNAP-23). Using human SNAP-23 as the bait in a yeast two-hybrid screen of a human B-lymphocyte cDNA library, we have identified the 287-amino-acid SNARE protein syntaxin 11. Like other syntaxin family members, syntaxin 11 binds to the SNARE proteins VAMP and SNAP-23 in vitro and also exists in a complex with SNAP-23 in transfected HeLa cells and in native human B lymphocytes. Unlike other syntaxin family members, no obvious transmembrane domain is present in syntaxin 11. Nevertheless, syntaxin 11 is predominantly membrane-associated and colocalizes with the mannose 6-phosphate receptor on late endosomes and the trans-Golgi network. These data suggest that syntaxin 11 is a SNARE that acts to regulate protein transport between late endosomes and the trans-Golgi network in mammalian cells.

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