Sensitive Factor Attachment Receptor Research Articles

Assessment of Stx-1A gene polymorphism (rs1569061) in relation to the development of multiple sclerosis in Egyptian patients

Multiple sclerosis (MS) is a multifactorial polygenic disease; results from autoimmune and neurodegenerative processes which lead to multifocal lesions of the central nervous system. Axonal degeneration was found to be prominent in the inflammation period of MS and contribute to the progression of disability. Soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE) complex plays a vital role in the release of neurotransmitter by synaptic vesicle fusion. Stx-1A protein (Stx-1A), a major component of the SNARE complex, is widely expressed in brain tissue. This study intended to evaluate the prevalence of the Stx-1A gene polymorphism (rs1569061) in the Egyptian population with MS and to investigate its association with various clinical factors. This study included 65 adult Egyptian MS patients and 35 age- and sex-matched normal control subjects. Diagnosis of MS was made by an experienced neurologist according to revised McDonald criteria. All Patients underwent full history taking, included Age of onset of MS, disease duration, disease course and degree of disability according to the Expanded Disability Status Scale (EDSS) and family history of neurological diseases. Stx-1A gene polymorphism (rs1569061) genotyping was performed by TaqMan assay based quantitative real time (qPCR) and verified by sanger sequencer. Genotype and allele frequencies of (rs1569061) did not differ significantly between case and control groups. No difference was detected when comparing the genotype frequency and the allele frequency to different disease parameters. Discrepancy of the minor allele frequency (MAF) of Stx-1A gene (rs1569061) between different populations was noted. In conclusion, our study in Stx-1A gene polymorphism (rs1569061) and MS showed that no difference between the patient and control as regards gene frequency and allele frequency. Predicting no association between the studied polymorphism and MS in the Egyptian population. However, discrepancy between different population was noted as regards the MAF for Stx-1A gene (rs1569061).

Stx4 is required to regulate cardiomyocyte Ca2+ handling during vertebrate cardiac development.

SummaryRequirements for vesicle fusion within the heart remain poorly understood, despite the multitude of processes that necessitate proper intracellular trafficking within cardiomyocytes. Here, we show that Syntaxin 4 (STX4), a target-Soluble N-ethylmaleimide sensitive factor attachment receptor (t-SNARE) protein, is required for normal vertebrate cardiac conduction and vesicular transport. Two patients were identified with damaging variants in STX4. A patient with a homozygous R240W missense variant displayed biventricular dilated cardiomyopathy, ectopy, and runs of non-sustained ventricular tachycardia, sensorineural hearing loss, global developmental delay, and hypotonia, while a second patient displayed severe pleiotropic abnormalities and perinatal lethality. CRISPR/Cas9-generated stx4 mutant zebrafish exhibited defects reminiscent of these patients’ clinical presentations, including linearized hearts, bradycardia, otic vesicle dysgenesis, neuronal atrophy, and touch insensitivity by 3 days post fertilization. Imaging of Vamp2+ vesicles within stx4 mutant zebrafish hearts showed reduced docking to the cardiomyocyte sarcolemma. Optical mapping of the embryonic hearts coupled with pharmacological modulation of Ca2+ handling together support that zebrafish stx4 mutants have a reduction in L-type Ca2+ channel modulation. Transgenic overexpression of zebrafish Stx4R241W, analogous to the first patient’s STX4R240W variant, indicated that the variant is hypomorphic. Thus, these data show an in vivo requirement for SNAREs in regulating normal embryonic cardiac function and that variants in STX4 are associated with pleiotropic human disease, including cardiomyopathy.

A Functional Core of IncA Is Required for Chlamydia trachomatis Inclusion Fusion.

Chlamydia trachomatis is an obligate intracellular pathogen that is the etiological agent of a variety of human diseases, including blinding trachoma and sexually transmitted infections. Chlamydiae replicate within a membrane-bound compartment, termed an inclusion, which they extensively modify by the insertion of type III secreted proteins called Inc proteins. IncA is an inclusion membrane protein that encodes two coiled-coil domains that are homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) motifs. Recent biochemical evidence suggests that a functional core, composed of SNARE-like domain 1 (SLD-1) and part of SNARE-like domain 2 (SLD-2), is required for the characteristic homotypic fusion of C. trachomatis inclusions in multiply infected cells. To verify the importance of IncA in homotypic fusion in Chlamydia, we generated an incA::bla mutant. Insertional inactivation of incA resulted in the formation of nonfusogenic inclusions, a phenotype that was completely rescued by complementation with full-length IncA. Rescue of homotypic inclusion fusion was dependent on the presence of the functional core consisting of SLD-1 and part of SLD-2. Collectively, these results confirm in vitro membrane fusion assays identifying functional domains of IncA and expand the genetic tools available for identification of chlamydia with a method for complementation of site-specific mutants. Chlamydia trachomatis replicates within a parasitophorous vacuole termed an inclusion. The chlamydial inclusions are nonfusogenic with vesicles in the endocytic pathway but, in multiply infected cells, fuse with each other to form a single large inclusion. This homotypic fusion is dependent upon the presence of a chlamydial inclusion membrane-localized protein, IncA. Specificity of membrane fusion in eukaryotic cells is regulated by SNARE (soluble N-ethylmaleimide sensitive factor attachment receptor) proteins on the cytosolic face of vesicles and target membranes. IncA contains two SNARE-like domains. Newly developed genetic tools for the complementation of targeted mutants in C. trachomatis are used to confirm the minimal requirement of SNARE-like motifs necessary to promote the homotypic fusion of inclusions.

Syntaxin 3 is necessary for cAMP- and cGMP-regulated exocytosis of CFTR: implications for enterotoxigenic diarrhea

Enterotoxins elaborated by Vibrio cholerae and Escherichia coli cannot elicit fluid secretion in the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. After enterotoxin exposure, CFTR channels are rapidly recruited from endosomes and undergo exocytic insertion into the apical plasma membrane of enterocytes to increase the number of channels on the cell surface by at least fourfold. However, the molecular machinery that orchestrates exocytic insertion of CFTR into the plasma membrane is largely unknown. The present study used immunofluorescence, immunoblotting, surface biotinylation, glutathione S-transferase (GST) pulldown assays, and immunoprecipitation to identify components of the exocytic soluble N-ethylmaleimide (NEM)-sensitive factor attachment receptor (SNARE) vesicle fusion machinery in cyclic nucleotide-activated exocytosis of CFTR in rat jejunum and polarized intestinal Caco-2(BB)e cells. Syntaxin 3, an intestine-specific SNARE, colocalized with CFTR on the apical domain of enterocytes in rat jejunum and polarized Caco-2(BB)e cells. Coimmunoprecipitation and GST binding studies confirmed that syntaxin 3 interacts with CFTR in vivo. Moreover, heat-stable enterotoxin (STa) activated exocytosis of both CFTR and syntaxin 3 to the surface of rat jejunum. Silencing of syntaxin 3 by short hairpin RNA (shRNA) interference abrogated cyclic nucleotide-stimulated exocytosis of CFTR in cells. These observations reveal a new and important role for syntaxin 3 in the pathophysiology of enterotoxin-elicited diarrhea.

Increased formation of VAMP-3-containing SNARE complexes in mast cells from VAMP-8 deficient cells

The regulated secretion of inflammatory mediators by mast cells plays an important role in the orchestration of inflammatory responses [1]. However, little is known about the late steps that control secretion although research in the last decade has revealed the importance of soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE) fusion proteins in the mechanisms leading to fusion of secretory granules with the plasma membrane (PM). It was shown that PM-localized target SNAREs SNAP-23 and Syntaxin-4 join together with the vesicular SNARE VAMP-8 in a complex to promote the merger of lipid bilayers [2, 3]. During IgE-dependent stimulation of bone marrow derived mast cells (BMMCs) one can detect increased complex formation between Syntaxin-4 and VAMP-8 or SNAP-23 and VAMP-8 [2]. Complex formation was transient, declining after longer stimulation times. As degranulation was not completely inhibited in VAMP-8-deficient BMMCs, we explored other v-SNARE proteins for their ability to engage in SNARE complex formation. Initial data showed some compensatory effects by VAMP-2, as increased complex formation was seen in VAMP-8 deficient cells [2]. Another potential candidatewasVAMP-3, as the introduction of recombinant protein into streptolysine O permeabilized mast cells revealed some blocking of exocytosis [4].We therefore analyzed the ability of VAMP-3 to form complexes with SNAP-23 upon IgE-dependent stimulation. Material and Methods

A search for synthetic peptides that inhibit soluble N‐ethylmaleimide sensitive‐factor attachment receptor‐mediated membrane fusion

Soluble N-ethylmaleimide sensitive-factor attachment receptor (SNARE) proteins have crucial roles in driving exocytic membrane fusion. Molecular recognition between vesicle-associated (v)-SNARE and target membrane (t)-SNARE leads to the formation of a four-helix bundle, which facilitates the merging of two apposing membranes. Synthetic peptides patterned after the SNARE motifs are predicted to block SNARE complex formation by competing with the parental SNAREs, inhibiting neuronal exocytosis. As an initial attempt to identify the peptide sequences that block SNARE assembly and membrane fusion, we created thirteen 17-residue synthetic peptides derived from the SNARE motifs of v- and t-SNAREs. The effects of these peptides on SNARE-mediated membrane fusion were investigated using an in vitro lipid-mixing assay, in vivo neurotransmitter release and SNARE complex formation assays in PC12 cells. Peptides derived from the N-terminal region of SNARE motifs had significant inhibitory effects on neuroexocytosis, whereas middle- and C-terminal-mimicking peptides did not exhibit much inhibitory function. N-terminal mimicking peptides blocked N-terminal zippering of SNAREs, a rate-limiting step in SNARE-driven membrane fusion. Therefore, the results suggest that the N-terminal regions of SNARE motifs are excellent targets for the development of drugs to block SNARE-mediated membrane fusion and neurotransmitter release.

Tag team action at the synapse

Communication between neurons relies on chemical synapses and the release of neurotransmitters into the synaptic cleft. Neurotransmitter release is an exquisitely regulated membrane fusion event that requires the linking of an electrical nerve stimulus to Ca(2+) influx, which leads to the fusion of neurotransmitter-filled vesicles with the cell membrane. The timing of neurotransmitter release is controlled through the regulation of the soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE) proteins-the core of the membrane fusion machinery. Assembly of the fusion-competent SNARE complex is regulated by several neuronal proteins, including complexin and the Ca(2+)-sensor synaptotagmin. Both complexin and synaptotagmin bind directly to SNAREs, but their mechanism of action has so far remained unclear. Recent studies revealed that synaptotagmin-Ca(2+) and complexin collaborate to regulate membrane fusion. These compelling new results provide a molecular mechanistic insight into the functions of both proteins: complexin 'clamps' the SNARE complex in a pre-fusion intermediate, which is then released by the action of Ca(2+)-bound synaptotagmin to trigger rapid fusion.

Syntaxin, but not soluble NSF attachment protein (SNAP), biosynthesis by rat pancreatic islets is regulated by glucose in parallel with proinsulin biosynthesis.

Recent studies have revealed that soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE)-related proteins, originally identified in neural tissues, are also expressed in pancreatic beta cells. In this study, we investigated the effect of glucose on syntaxin 1 and alpha/beta SNAP biosynthesis in pancreatic beta cells and we demonstrated that syntaxin 1, but not alpha/beta SNAP biosynthesis by rat isolated pancreatic islets was stimulated specifically by glucose nearly in parallel with proinsulin biosynthesis. Stimulation of syntaxin 1 and proinsulin biosynthesis by glucose was dose-dependent (Km = approximately 8 mmol/l) and reached the maximum (about 8-12 fold) at concentrations over 11 mmol/l. In contrast, 22 mmol/l glucose increased alpha/beta SNAP biosynthesis about 2-fold only, similar to the increase in total protein synthesis. Stimulation of syntaxin 1 biosynthesis by glucose was also time-dependent, taking around 3 h to reach the maximum, and was not affected by actinomycin-D, suggesting regulation at the translational level. On the other hand, glucose had a similar stimulating effect on both syntaxin 1 and alpha/beta SNAP biosynthesis by mouse insulinoma betaTC3 cells as it did on proinsulin biosynthesis. The evidence showing coordinated stimulation of syntaxin 1 and proinsulin biosynthesis by glucose in rat islets suggested the critical functional role of syntaxin 1 in the insulin exocytotic mechanism.