Soluble N-ethylmaleimide-sensitive Factor Activating Protein Receptor Research Articles

Analysis of Hyperosmotic Tolerance Mechanisms in Gracilariopsis lemaneiformis Based on Weighted Co-Expression Network Analysis.

We conducted transcriptome sequencing on salt-tolerant mutants X5 and X3, and a control (Ctr) strain of Gracilariopsis lemaneiformis after treatment with artificial seawater at varying salinities (30‱, 45‱, and 60‱) for 3 weeks. Differentially expressed genes were identified and a weighted co-expression network analysis was conducted. The blue, red, and tan modules were most closely associated with salinity, while the black, cyan, light cyan, and yellow modules showed a close correlation with strain attributes. KEGG enrichment of genes from the aforementioned modules revealed that the key enrichment pathways for salinity attributes included the proteasome and carbon fixation in photosynthesis, whereas the key pathways for strain attributes consisted of lipid metabolism, oxidative phosphorylation, soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE) interactions in vesicular transport, and porphyrin and chlorophyll metabolism. Gene expression for the proteasome and carbon fixation in photosynthesis was higher in all strains at 60‱. In addition, gene expression in the proteasome pathway was higher in the X5-60 than Ctr-60 and X3-60. Based on the above data and relevant literature, we speculated that mutant X5 likely copes with high salt stress by upregulating genes related to lysosome and carbon fixation in photosynthesis. The proteasome may be reset to adjust the organism's proteome composition to adapt to high-salt environments, while carbon fixation may aid in maintaining material and energy metabolism for normal life activities by enhancing carbon dioxide uptake via photosynthesis. The differences between the X5-30 and Ctr-30 expression of genes involved in the synthesis of secondary metabolites, oxidative phosphorylation, and SNARE interactions in vesicular transport suggested that the X5-30 may differ from Ctr-30 in lipid metabolism, energy metabolism, and vesicular transport. Finally, among the key pathways with good correlation with salinity and strain traits, the key genes with significant correlation with salinity and strain traits were identified by correlation analysis.

Abstract 17384: Unraveling the Impact of VAMP-8 Deficiency on Atherogenesis: A Fresh Perspective on Platelet Involvement

Introduction and Objective: Recognized for their intricate role in atherogenesis, platelets interact with oxidized low-density lipoprotein (oxLDL), immune cells, and endothelial cells and release granule proteins and microparticles, contributing to the onset and advancement of atherosclerosis. Despite the challenges of antiplatelet treatment in primary prevention, exploring alternative platelet targets could provide key insights into their role in atherogenesis. This study pivots on the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins, notably vesicle associated membrane protein 8 (VAMP-8) - the primary vesicle membrane (v-SNARE) and vital facilitator of platelet secretion. Approach and Results: The profound impairment in α-granule, dense granule, and lysosome release due to VAMP-8 deficiency directed our investigation towards its potential role in atherogenesis. Using RNA-seq data from washed platelets of VAMP-8-/- mice versus wildtypes, we first analyzed the molecular signature of VAMP-8 deficiency. Gene ontology analysis revealed that the major differences in enriched genes between the two groups involved triglyceride biosynthetic process and platelet calcium homeostasis. We initiated atherosclerosis studies using ApoE -/- mice crossed with VAMP-8 -/- mice (n=11) alongside littermate controls (n=9), all fed a high cholesterol diet for 15 weeks. Remarkably, the deletion of VAMP-8 led to a substantial reduction in atherosclerotic lesion development when compared to the control group. Notably, this phenotype manifested irrespective of total cholesterol levels, as plasma cholesterol remained consistent across all groups. Conclusion: Our findings reveal that VAMP8 deficiency markedly mitigates atherogenesis, thereby proposing a novel perspective to explore the influence of platelet cargo secretion on the inception of atherosclerosis. We are currently conducting additional studies to uncover the underlying mechanisms responsible for these observations

Syntaxin4-Munc18c Interaction Promotes Breast Tumor Invasion and Metastasis by Regulating MT1-MMP Trafficking.

Disrupting the interaction of Syntaxin4 and Munc18c may be a useful approach to perturb trafficking of MT1-MMP and reduce metastatic potential of breast cancers.

Function and molecular mechanism of N-terminal acetylation in autophagy.

Acetyl ligation to the amino acids in a protein is an important posttranslational modification. However, in contrast to lysine acetylation, N-terminal acetylation is elusive in terms of its cellular functions. Here, we identify Nat3 as an N-terminal acetyltransferase essential for autophagy, a catabolic pathway for bulk transport and degradation of cytoplasmic components. We identify the actin cytoskeleton constituent Act1 and dynamin-like GTPase Vps1 (vacuolar protein sorting 1) as substrates for Nat3-mediated N-terminal acetylation of the first methionine. Acetylated Act1 forms actin filaments and therefore promotes the transport of Atg9 vesicles for autophagosome formation; acetylated Vps1 recruits and facilitates bundling of the SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor) complex for autophagosome fusion with vacuoles. Abolishment of the N-terminal acetylation of Act1 and Vps1 is associated with blockage of upstream and downstream steps of the autophagy process. Therefore, our work shows that protein N-terminal acetylation plays a critical role in controlling autophagy by fine-tuning multiple steps in the process.

Novel Native and Engineered Botulinum Neurotoxins.

Botulinum neurotoxins (BoNTs), produced by Clostridia and other bacteria, are the most potent toxins known. Their cleavage of the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins in neurons prevents the release of neurotransmitters, thus resulting in the muscle paralysis that is characteristic of botulism. This mechanism of action has been exploited for a variety of therapeutic and cosmetic applications of BoNTs. This chapter provides an overview of the native BoNTs, including the classical serotypes and their clinical use, mosaic BoNTs, and novel BoNTs that have been recently identified in clostridial and non-clostridial strains. In addition, the modular structure of native BoNTs, which are composed of a light chain and a heavy chain, is amenable to a multitude of novel fusions and mutations using molecular biology techniques. These novel recombinant BoNTs have been used or are being developed to further characterize the biology of toxins, to assist in vaccine production, to serve as delivery vehicles to neurons, and to be utilized as novel therapeutics for both neuronal and non-neuronal cells.

Hyperoxidized Albumin Modulates Platelets and Promotes Inflammation Through CD36 Receptor in Severe Alcoholic Hepatitis.

Hyperoxidized albumin promotes inflammation and modulates several immune cells in severe alcoholic hepatitis (SAH). Platelets mediate inflammation by interacting with immune cells, endothelium, and other cells. The role of hyperoxidized albumin in platelet activation and alteration of platelet phenotype/functions is not known. Quantitative platelet proteomics performed in 10 patients with SAH was compared with 10 patients with alcoholic cirrhosis and 10 healthy controls, respectively. Dysregulated pathways were identified and validated in a separate cohort (n = 40). Healthy platelets were exposed to patient plasma or purified albumin or ex vivo modified albumin (human‐mercaptalbumin, humannonmercaptalbumin‐1, and human nonmercaptalbumin 2) in the presence or absence of CD36 blockade, and platelet secretome was analyzed. Two hundred and two up‐regulated proteins linked to platelet activation, complement regulation, lipid transportation, and 321 down‐regulated proteins related to platelet hemostasis and coagulation (fold change ± 1.5, P < 0.01) were identified. Blood transcription module enrichment showed an inflammatory phenotype of SAH platelet. Increased level of platelet factor‐4, P‐selectin, and soluble cluster of differentiation‐40 ligand correlated with severity (Model for End‐Stage Liver Disease score, r > 0.3, P < 0.05) in SAH. Transcripts linked to platelet activation (increased) and granular secretions (decreased in SAH) correlated with disease severity. SNARE (soluble‐N‐ethylmaleimide‐sensitive‐factor‐activating‐protein‐receptor) complex proteins (SNAP‐23 [synaptosomal‐associated protein 23] and VAMP‐8 [vesicle‐associated membrane protein 3]) were down‐regulated in SAH platelets (P < 0.05). In vitro stimulation of healthy platelets showed enhanced activation with patient plasma, or purified albumin‐treatment blocking of CD36 blunted this effect (P < 0.05). Ex vivo modified albumin (primarily nonmercaptalbumin–human nonmercaptalbumin 2 [HNA2; 1 mg/mL]) showed high activation and aggregation and intracellular reactive oxygen species production in healthy platelets (P < 0.05), which significantly reduced after CD36 neutralization. Platelet secretome showed reduced inflammatory mediators and increased repair proteins. Conclusion: Hyperoxidized albumin triggers platelet activation (possibly through the CD36 receptor), promotes inflammation and oxidative stress, and contributes to disease severity in patients with SAH.

A Hyperactive Form of unc-13 Enhances Ca2+ Sensitivity and Synaptic Vesicle Release Probability in C. elegans

SUMMARYMunc13 proteins play several roles in regulating shortterm synaptic plasticity. However, the underlying molecular mechanisms remain largely unclear. Here we report that C. elegans UNC-13L, a Munc13-1 ortholog, has three domains that inhibit synaptic vesicle (SV) exocytosis. These include the X (sequence between C2A and C1), C1, and C2B domains. Deleting all three inhibitory domains produces a hyperactive UNC-13 (sUNC-13) that exhibits dramatically increased neurotransmitter release, Ca2+ sensitivity of release, and release probability. The vesicular pool in unc-13 mutants rescued by sUNC-13 exhibits a faster synaptic recovery and replenishment rate, demonstrating an important role of sUNC-13 in regulating synaptic plasticity. Analysis of double mutants suggests that sUNC-13 enhances tonic release by increasing the open probability of UNC-64/syntaxin-1A, whereas its effects on evoked release appear to be mediated by additional functions, presumably by further regulating the activity of the assembled soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) complex.

A Bilayer-Based In Vitro Assay for Botulinum Holotoxin Potency Assessments

The standard procedure for botulinum toxin potency assessments is the mouse lethality assay by intraperitoneal injection. However, the mouse assay is laborious, expensive and ethically controversial. We have developed and are refining an in vitro, synthetic bilayer-based assay (BBA) for characterizing the potency of botulinum neurotoxin (BoNT) containing therapeutics, as a replacement for the mouse lethality assay. The BBA employs interfacial planar lipid bilayer, preconfigured to mimic a neuronal cell membrane, along with a nanopore-based endopeptidase assay for assessing the peptidase activity of the toxin. When the BoNT-containing sample is introduced into the assay, the BoNT heavy chain (HC) binds to its target receptors before forming a transmembrane channel that then transports the light chain (LC) across the lipid bilayer. Once the LC has traversed the membrane and is released from the HC, it then cleaves and inactivates its respective target protein, i.e., its soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) protein target. In the BBA, both the rate of HC insertions into the planar lipid bilayer and LC peptidase activity can be independently, but simultaneously, monitored, with the rate of SNARE protein cleavage being directly proportional to the potency of the sample introduced. While the assay is being specifically developed for BoNT testing, the platform will be generally applicable to a large number of other biomolecules with transmembrane activity for which there is currently no standard in vitro characterization technique.

Botulinum Toxin Induced Atrophy: An Uncharted Territory.

Botulinum neurotoxins (BoNTs) produce local chemo-denervation by cleaving soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins. Botulinum neurotoxins are therapeutically indicated in several neurological disorders and have been in use for three decades. The long-term efficacy, safety, and side effects of BoNTs have been well documented in the literature. However, the development of muscle atrophy following chronic exposure to BoNTs has not received sufficient attention. Muscle atrophy is not only cosmetically distressing, but also has an impact on future injections. An extensive literature search was conducted on atrophy and mechanisms of atrophy. Five hundred and four relevant articles in the English language were reviewed. This review revealed the surprising lack of documentation of atrophy within the literature. In addition, as demonstrated in this review, the mechanisms and the clinical factors that may lead to atrophy have also been poorly studied. However, even with this limited information it is possible to indicate factors that could modify the clinical approach to botulinum toxin injections. This review highlights the need for further study of atrophy following BoNT injections.

The cytotoxicity of coxsackievirus B3 is associated with a blockage of autophagic flux mediated by reduced syntaxin 17 expression

Coxsackievirus B3 (CVB3) is an important human pathogen linked to cardiac arrhythmias and acute heart failure. CVB3 infection has been reported to induce the formation of autophagosomes that support the viral replication in host cells. Interestingly, our study shows that the accumulation of autophagosomes during CVB3 infection is caused by a blockage of autophagosome–lysosome fusion rather than the induction of autophagosome biogenesis. Moreover, CVB3 decreases the transcription and translation of syntaxin 17 (STX17), a SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor) protein involved in autophagosome–lysosome fusion. Overexpression of STX17 restored the autophagic flux, alleviated the virus-induced lysosomal dysfunction, and decreased the apoptosis induced by CVB3 infection in HeLa cells. Taken together, our results suggest that CVB3 infection impairs the autophagic flux by blocking autophagosome–lysosome fusion. These findings thus point to potential new therapeutic strategies targeting STX17 or autophagosome–lysosome fusion for treating CVB3-associated diseases.

Targeting a Potassium Channel/Syntaxin Interaction Ameliorates Cell Death in Ischemic Stroke.

The voltage-gated K+ channel Kv2.1 has been intimately linked with neuronal apoptosis. After ischemic, oxidative, or inflammatory insults, Kv2.1 mediates a pronounced, delayed enhancement of K+ efflux, generating an optimal intracellular environment for caspase and nuclease activity, key components of programmed cell death. This apoptosis-enabling mechanism is initiated via Zn2+-dependent dual phosphorylation of Kv2.1, increasing the interaction between the channel's intracellular C-terminus domain and the SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor) protein syntaxin 1A. Subsequently, an upregulation of de novo channel insertion into the plasma membrane leads to the critical enhancement of K+ efflux in damaged neurons. Here, we investigated whether a strategy designed to interfere with the cell death-facilitating properties of Kv2.1, specifically its interaction with syntaxin 1A, could lead to neuroprotection following ischemic injury in vivo The minimal syntaxin 1A-binding sequence of Kv2.1 C terminus (C1aB) was first identified via a far-Western peptide screen and used to create a protherapeutic product by conjugating C1aB to a cell-penetrating domain. The resulting peptide (TAT-C1aB) suppressed enhanced whole-cell K+ currents produced by a mutated form of Kv2.1 mimicking apoptosis in a mammalian expression system, and protected cortical neurons from slow excitotoxic injury in vitro, without influencing NMDA-induced intracellular calcium responses. Importantly, intraperitoneal administration of TAT-C1aB in mice following transient middle cerebral artery occlusion significantly reduced ischemic stroke damage and improved neurological outcome. These results provide strong evidence that targeting the proapoptotic function of Kv2.1 is an effective and highly promising neuroprotective strategy.SIGNIFICANCE STATEMENT Kv2.1 is a critical regulator of apoptosis in central neurons. It has not been determined, however, whether the cell death-enabling function of this K+ channel can be selectively targeted to improve neuronal survival following injury in vivo The experiments presented here demonstrate that the cell death-specific role of Kv2.1 can be uniquely modulated to provide neuroprotection in an animal model of acute ischemic stroke. We thus reveal a novel therapeutic strategy for neurological disorders that are accompanied by Kv2.1-facilitated forms of cell death.

Rapid SNARE-Mediated Fusion of Liposomes and Chromaffin Granules with Giant Unilamellar Vesicles

Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins are the main catalysts for membrane fusion in the secretory pathway of eukaryotic cells. In vitro, SNAREs are sufficient to mediate effective fusion of both native and artificial membranes. Here we have established, to our knowledge, a new platform for monitoring SNARE-mediated docking and fusion between giant unilamellar vesicles (GUVs) and smaller liposomes or purified secretory granules with high temporal and spatial resolution. Analysis of fusion is restricted to the free-standing part of the GUV-membrane exhibiting low curvature and a lack of surface contact, thus avoiding adhesion-mediated interference with the fusion reaction as in fusion with supported bilayers or surface-immobilized small vesicles. Our results show that liposomes and chromaffin granules fuse with GUVs containing activated SNAREs with only few milliseconds delay between docking and fusion. We conclude that after initial contact in trans, SNAREs alone can complete fusion at a rate close to fast neuronal exocytosis.

An Immunohistochemical Survey of SNARE Proteins Shows Distinct Patterns of Expression in Hematolymphoid Neoplasia.

Five proteins from the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) complex family were studied in normal hematopoietic cells in bone marrow; normal lymphocytes at different stages of maturation and differentiation in bone marrow, thymus, tonsil, and lymph node; malignant lymphomas; and leukemias. Sixty-eight reactive and 380 hematopoietic and lymphoid neoplasms were immunohistochemically stained for syntaxin 7 (STX7), vesicle-associated membrane proteins (VAMP2, VAMP7, VAMP8), and synaptosomal-associated protein 23 (SNAP23). STX7 has potential for being a useful marker for distinguishing between normal B precursors (hematogones) vs B lymphoblasts, as well as between the "popcorn" cells of nodular lymphocyte-predominant Hodgkin lymphoma vs the Reed-Sternberg cells of classic Hodgkin lymphoma or the B cells of T-cell, histiocyte-rich B-cell lymphoma. VAMP2 is uniquely expressed by both reactive and malignant plasma cells, in contrast to B-cell non-Hodgkin lymphoma. There is differential expression of SNARE proteins in normal and neoplastic lymphoid tissue depending on lymphocyte maturation stage. Differential SNARE protein expression in the lymphoid system may have potential use in diagnosis and may offer clues to lymphoma biology. VAMP2 is a promising new plasma cell marker.

Morphine Antidependence of Erythroxylum cuneatum (Miq.) Kurz in Neurotransmission Processes In Vitro.

Opiate abuse has been studied to cause adaptive changes observed in the presynaptic release and the mediated-synaptic plasticity proteins. The involvement of neuronal SNARE proteins reveals the role of the neurotransmitter release in expressing the opioid actions. The present study was designed to determine the effect of the alkaloid extract of Erythroxylum cuneatum (E. cuneatum) against chronic morphine and the influences of E. cuneatum on neurotransmission processes observed in vitro. The human neuroblastoma cell line, SK-N-SH, was treated with the morphine, methadone, or E. cuneatum. The cell lysates were collected and tested for α-synuclein, calmodulin, vesicle-associated membrane protein 2 (VAMP 2), and synaptotagmin 1. The extract of E. cuneatum was observed to upregulate the decreased expression of dependence proteins, namely, α-synuclein and calmodulin. The effects were comparable to methadone and control. The expressions of VAMP 2 and synaptotagmin 1 were normalised by the plant and methadone. The extract of E. cuneatum was postulated to treat dependence symptoms after chronic morphine and improve the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) protein involved in synaptic vesicle after.

Calcium sensitive ring-like oligomers formed by synaptotagmin.

The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear. Here we report an unexpected circular arrangement (ring) of SYT's cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18-43 nm, corresponding to 11-26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded.

Characterization of SNARE Proteins in Human Pituitary Adenomas: Targeted Secretion Inhibitors as a New Strategy for the Treatment of Acromegaly?

Targeted secretion inhibitors (TSIs), a new class of recombinant biotherapeutic proteins engineered from botulinum toxin, represent a novel approach for treating diseases with excess secretion. They inhibit hormone secretion from targeted cell types through cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor-activating protein receptor) proteins. qGHRH-LH(N)/D is a TSI targeting pituitary somatotroph through binding to the GHRH-receptor and cleavage of the vesicle-associated membrane protein (VAMP) family of SNARE proteins. Our objective was to study SNARE protein expression in pituitary adenomas and to inhibit GH secretion from somatotropinomas using qGHRH-LH(N)/D. We analyzed human pituitary adenoma analysis for SNARE expression and response to qGHRH-LH(N)/D treatment. The study was conducted in University Hospitals. We used pituitary adenoma samples from 25 acromegaly and 47 nonfunctioning pituitary adenoma patients. Vesicle-SNARE (VAMP1-3), target-SNARE (syntaxin1, SNAP-23, and SNAP-25), and GHRH-receptor detection with RT-qPCR, immunocytochemistry, and immunoblotting. Assessment of TSI catalytic activity on VAMPs and release of GH from adenoma cells. SNARE proteins were variably expressed in pituitary samples. In vitro evidence using recombinant GFP-VAMP2&3 or pituitary adenoma lysates suggested sufficient catalytic activity of qGHRH-LH(N)/D to degrade VAMPs, but was unable to inhibit GH secretion in somatotropinoma cell cultures. SNARE proteins are present in human pituitary somatotroph adenomas that can be targeted by TSIs to inhibit GH secretion. qGHRH-LH(N)/D was unable to inhibit GH secretion from human somatotroph adenoma cells. Further studies are required to understand how the SNARE proteins drive GH secretion in human somatotrophs to allow the development of novel TSIs with a potential therapeutic benefit.

What is the role of SNARE proteins in membrane fusion?

Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins have been at the fore-front of research on biological membrane fusion for some time. The subcellular localization of SNAREs and their ability to form the so-called SNARE complex may be integral to determining the specificity of intracellular fusion (the SNARE hypothesis) and/or serving as the minimal fusion machinery. Both the SNARE hypothesis and the idea of the minimal fusion machinery have been challenged by a number of experimental observations in various model systems, suggesting that SNAREs may have other functions. Considering recent advances in the SNARE literature, it appears that SNAREs may actually function as part of a complex fusion "machine." Their role in the machinery could be any one or a combination of roles, including establishing tight membrane contact, formation of a scaffolding on which to build the machine, binding of lipid surfaces, and many others. It is also possible that complexations other than the classic SNARE complex participate in membrane fusion.