Search Results (15)

Syntaxin 3 is a member of a large protein family of syntaxin proteins that mediate fusion between vesicles and their target membranes. Mutations in the ubiquitously expressed syntaxin 3A splice form give rise to a serious gastrointestinal disorder in humans called microvillus inclusion disorder, while mutations that additionally involve syntaxin 3B, a splice form that is expressed primarily in retinal photoreceptors and bipolar cells, additionally give rise to an early onset severe retinal dystrophy. In this review, we discuss recent studies elucidating the roles of syntaxin 3B and the regulation of syntaxin 3B functionality in membrane fusion and neurotransmitter release in the vertebrate retina. Full article

Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine that plays a critical role in insulin secretion, energy metabolism, and mitochondrial biogenesis. However, the action of serotonin in insulin production and secretion by pancreatic β cells has not yet been elucidated. Here, we investigated how exogenous nanomolar serotonin concentrations regulate insulin synthesis and secretion in rat insulinoma INS-1E cells. Nanomolar serotonin concentrations (10 and 50 nM) significantly increased insulin protein expression above the constant levels in untreated control cells and decreased insulin protein levels in the media. The reductions in insulin protein levels in the media may be associated with ubiquitin-mediated protein degradation. The levels of membrane vesicle trafficking-related proteins including Rab5, Rab3A, syntaxin6, clathrin, and EEA1 proteins were significantly decreased by serotonin treatment compared to the untreated control cells, whereas the expressions of Rab27A, GOPC, and p-caveolin-1 proteins were significantly reduced by serotonin treatment. In this condition, serotonin receptors, Gαq-coupled 5-HT2b receptor (Htr2b), and ligand-gated ion channel receptor Htr3a were significantly decreased by serotonin treatment. To confirm the serotonylation of Rab3A and Rab27A during insulin secretion, we investigated the protein levels of Rab3A and Rab27A, in which transglutaminase 2 (TGase2) serotonylated Rab3A but not Rab27A. The increases in ERK phosphorylation levels were consistent with increases in the expression of p-Akt. Also, the expression level of the Bcl-2 protein was significantly increased by 50 and 100 nM serotonin treatment compared to the untreated control cells, whereas the levels of Cu/Zn-SOD and Mn-SOD proteins decreased. These results indicate that nanomolar serotonin treatment regulates the insulin protein level but decreases this level in media through membrane vesicle trafficking-related proteins (Rab5, Rab3A, syntaxin6, clathrin, and EEA1), the Akt/ERK pathway, and Htr2b/Htr3a in INS-1E cells. Full article

Retinal neurons that form ribbon-style synapses operate over a wide dynamic range, continuously relaying visual information to their downstream targets. The remarkable signaling abilities of these neurons are supported by specialized presynaptic machinery, one component of which is syntaxin3B. Syntaxin3B is an essential t-SNARE protein of photoreceptors and bipolar cells that is required for neurotransmitter release. It has a light-regulated phosphorylation site in its N-terminal domain at T14 that has been proposed to modulate membrane fusion. However, a direct test of the latter has been lacking. Using a well-controlled in vitro fusion assay, we found that a phosphomimetic T14 syntaxin3B mutation leads to a small but significant enhancement of SNARE-mediated membrane fusion following the formation of the t-SNARE complex. While the addition of Munc18a had only a minimal effect on membrane fusion mediated by SNARE complexes containing wild-type syntaxin3B, a more significant enhancement was observed in the presence of Munc18a when the SNARE complexes contained a syntaxin3B T14 phosphomimetic mutant. Finally, we showed that the retinal-specific complexins (Cpx III and Cpx IV) inhibited membrane fusion mediated by syntaxin3B-containing SNARE complexes in a dose-dependent manner. Collectively, our results establish that membrane fusion mediated by syntaxin3B-containing SNARE complexes is regulated by the T14 residue of syntaxin3B, Munc18a, and Cpxs III and IV. Full article

Pancreatic beta cell function is an important component of glucose homeostasis. Here, we investigated the function of PIMT (PRIP-interacting protein with methyl transferase domain), a transcriptional co-activator binding protein, in the pancreatic beta cells. We observed that the protein levels of PIMT, along with key beta cell markers such as PDX1 (pancreatic and duodenal homeobox 1) and MafA (MAF bZIP transcription factor A), were reduced in the beta cells exposed to hyperglycemic and hyperlipidemic conditions. Consistently, PIMT levels were reduced in the pancreatic islets isolated from high fat diet (HFD)-fed mice. The RNA sequencing analysis of PIMT knockdown beta cells identified that the expression of key genes involved in insulin secretory pathway, Ins1 (insulin 1), Ins2 (insulin 2), Kcnj11 (potassium inwardly-rectifying channel, subfamily J, member 11), Kcnn1 (potassium calcium-activated channel subfamily N member 1), Rab3a (member RAS oncogene family), Gnas (GNAS complex locus), Syt13 (synaptotagmin 13), Pax6 (paired box 6), Klf11 (Kruppel-Like Factor 11), and Nr4a1 (nuclear receptor subfamily 4, group A, member 1) was attenuated due to PIMT depletion. PIMT ablation in the pancreatic beta cells and in the rat pancreatic islets led to decreased protein levels of PDX1 and MafA, resulting in the reduction in glucose-stimulated insulin secretion (GSIS). The results from the immunoprecipitation and ChIP experiments revealed the interaction of PIMT with PDX1 and MafA, and its recruitment to the insulin promoter, respectively. Importantly, PIMT ablation in beta cells resulted in the nuclear translocation of insulin. Surprisingly, forced expression of PIMT in beta cells abrogated GSIS, while Ins1 and Ins2 transcript levels were subtly enhanced. On the other hand, the expression of genes, PRIP/Asc2/Ncoa6 (nuclear receptor coactivator 6), Pax6, Kcnj11, Syt13, Stxbp1 (syntaxin binding protein 1), and Snap25 (synaptosome associated protein 25) associated with insulin secretion, was significantly reduced, providing an explanation for the decreased GSIS upon PIMT overexpression. Our findings highlight the importance of PIMT in the regulation of insulin synthesis and secretion in beta cells. Full article

Quantitative surface plasmon resonance (SPR) was utilized to determine binding strength and calcium dependence of direct interactions between dysferlin and proteins likely to mediate skeletal muscle repair, interrupted in limb girdle muscular dystrophy type 2B/R2. Dysferlin canonical C2A (cC2A) and C2F/G domains directly interacted with annexin A1, calpain-3, caveolin-3, affixin, AHNAK1, syntaxin-4, and mitsugumin-53, with cC2A the primary target and C2F lesser involved, overall demonstrating positive calcium dependence. Dysferlin C2 pairings alone showed negative calcium dependence in almost all cases. Like otoferlin, dysferlin directly interacted via its carboxy terminus with FKBP8, an anti-apoptotic outer mitochondrial membrane protein, and via its C2DE domain with apoptosis-linked gene (ALG-2/PDCD6), linking anti-apoptosis with apoptosis. Confocal Z-stack immunofluorescence confirmed co-compartmentalization of PDCD6 and FKBP8 at the sarcolemmal membrane. Our evidence supports the hypothesis that prior to injury, dysferlin C2 domains self-interact and give rise to a folded, compact structure as indicated for otoferlin. With elevation of intracellular Ca²⁺ in injury, dysferlin would unfold and expose the cC2A domain for interaction with annexin A1, calpain-3, mitsugumin 53, affixin, and caveolin-3, and dysferlin would realign from its interactions with PDCD6 at basal calcium levels to interact strongly with FKBP8, an intramolecular rearrangement facilitating membrane repair. Full article

Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, cleaves proteins involved in neurotransmitter release, thereby triggering flaccid paralyses, which are responsible for botulism. BoNT is classified into seven serotypes (BoNT/A-G); BoNT/A and BoNT/B are used as medical therapeutics and anti-wrinkle reagents. In this study, we investigated the efficacy of BoNT/CD, a mosaic toxin of BoNT/C and BoNT/D, to assess its potential as a therapeutic alternative for BoNT/A. In a cultured neuron assay, BoNT/CD cleaved syntaxin and SNAP-25 with higher efficacy than BoNT/C and BoNT/A. Intramuscularly administrated BoNT/CD induced dose-dependent muscle paralysis, and the paralysis lasted ~21 days in a mouse digit abduction score assay (BoNT/A-induced paralysis lasted ~30 days). BoNT/C failed to induce local paralysis without systemic toxicity. Multiple alignment analyses of the amino acid sequences of the receptor binding domain (H_C) of eight BoNT/CDs and two BoNT/Ds showed sequence clustering in five groups. Comparing BoNT/CD strain 003-9 (BoNT/CD_003-9) and strain 6813 (BoNT/CD₆₈₁₃) showed that both BoNT/CDs displayed similar efficacies in cultured neurons, but BoNT/CD_003-9 displayed higher efficacy in a mouse model than BoNT/CD₆₈₁₃. These findings suggest that BoNT/CD may be a potential alternative for patients who do not respond to existing BoNT-based therapeutics. Full article

Endocytosis and autophagy are evolutionarily conserved degradative processes in all eukaryotes. Both pathways converge to the lysosome where cargo is degraded. Improper lysosomal degradation is observed in many human pathologies, so its regulatory mechanisms are important to understand. Sec20/BNIP1 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 1) is a BH3 (Bcl-2 homology 3) domain-containing SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors) protein that has been suggested to promote Golgi-ER retrograde transport, mitochondrial fission, apoptosis and mitophagy in yeast and vertebrates. Here, we show that loss of Sec20 in Drosophila fat cells causes the accumulation of autophagic vesicles and prevents proper lysosomal acidification and degradation during bulk, starvation-induced autophagy. Furthermore, Sec20 knockdown leads to the enlargement of late endosomes and accumulation of defective endolysosomes in larval Drosophila nephrocytes. Importantly, the loss of Syx18 (Syntaxin 18), one of the known partners of Sec20, led to similar changes in nephrocytes and fat cells. Interestingly. Sec20 appears to function independent of its role in Golgi-ER retrograde transport in regulating lysosomal degradation, as the loss of its other partner SNAREs Use1 (Unconventional SNARE In The ER 1) and Sec22 or tethering factor Zw10 (Zeste white 10), which function together in the Golgi-ER pathway, does not cause defects in autophagy or endocytosis. Thus, our data identify a potential new transport route specific to lysosome biogenesis and function. Full article

Ulcers due to infections with Mycobacterium ulcerans are characterized by complete lack of wound healing processes, painless, an underlying bed of host dead cells and undermined edges due to necrosis. Mycolactone, a macrolide produced by the mycobacterium, is believed to be the toxin responsible. Of interest and relevance is the knowledge that Buruli ulcer (BU) patients remember experiencing trauma previously at the site of the ulcers, suggesting an impairment of wound healing processes, the plausible effect due to the toxin. Wound healing processes involve activation of the blood platelets to release the contents of the dense granules mainly serotonin, calcium ions, and ADP/ATP by exocytosis into the bloodstream. The serotonin release results in attracting more platelets and mast cells to the wound site, with the mast cells also undergoing degranulation, releasing compounds into the bloodstream by exocytosis. Recent work has identified interference in the co-translational translocation of many secreted proteins via the endoplasmic reticulum and cell death involving Wiskott-Aldrich syndrome protein (WASP), Sec61, and angiotensin II receptors (AT2R). We hypothesized that mycolactone by being lipophilic, passively crosses cell membranes and binds to key proteins that are involved in exocytosis by platelets and mast cells, thus inhibiting the initiation of wound healing processes. Based on this, molecular docking studies were performed with mycolactone against key soluble n-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and regulators, namely Vesicle-associated membrane protein (VAMP8), Synaptosomal-associated protein (SNAP23, syntaxin 11, Munc13-4 (its isoform Munc13-1 was used), and Munc18b; and also against known mycolactone targets (Sec61, AT2R, and WASP). Munc18b was shown to be a plausible mycolactone target after the molecular docking studies with binding affinity of −8.5 kcal/mol. Structural studies and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding energy calculations of the mycolactone and Munc18b complex was done with 100 ns molecular dynamics simulations using GROMACS. Mycolactone binds strongly to Munc18b with an average binding energy of −247.571 ± 37.471 kJ/mol, and its presence elicits changes in the structural conformation of the protein. Analysis of the binding interactions also shows that mycolactone interacts with Arg405, which is an important residue of Munc18b, whose mutation could result in impaired granule exocytosis. These findings consolidate the possibility that Munc18b could be a target of mycolactone. The implication of the interaction can be experimentally evaluated to further understand its role in granule exocytosis impairment in Buruli ulcer. Full article

Glial activation and neuroinflammation play significant roles in apoptosis as well as in the development of cognitive and memory deficits. Neuroinflammation is also a critical feature in the pathogenesis of neurodegenerative disorders such as Alzheimer and Parkinson’s diseases. Previously, hesperetin has been shown to be an effective antioxidant and anti-inflammatory agent. In the present study, in vivo and in vitro analyses were performed to evaluate the neuroprotective effects of hesperetin in lipopolysaccharide (LPS)-induced neuroinflammation, oxidative stress, neuronal apoptosis and memory impairments. Based on our findings, LPS treatment resulted in microglial activation and astrocytosis and elevated the expression of inflammatory mediators such as phosphorylated-Nuclear factor-κB (p-NF-κB), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in the cortical and hippocampal regions and in BV2 cells. However, hesperetin cotreatment markedly reduced the expression of inflammatory cytokines by ameliorating Toll-like receptor-4 (TLR4)-mediated ionized calcium-binding adapter molecule 1/glial fibrillary acidic protein (Iba-1/GFAP) expression. Similarly, hesperetin attenuated LPS-induced generation of reactive oxygen species/lipid per oxidation (ROS/LPO) and improved the antioxidant protein level such as nuclear factor erythroid 2-related factor 2 (Nrf2) and Haem-oxygenase (HO-1) in the mouse brain. Additionally, hesperetin ameliorated cytotoxicity and ROS/LPO induced by LPS in HT-22 cells. Moreover, hesperetin rescued LPS-induced neuronal apoptosis by reducing the expression of phosphorylated-c-Jun N-terminal kinases (p-JNK), B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), and Caspase-3 protein and promoting the Bcl-2 protein level. Furthermore, hesperetin enhanced synaptic integrity, cognition, and memory processes by enhancing the phosphorylated-cAMP response element binding protein (p-CREB), postsynaptic density protein-95 (PSD-95), and Syntaxin. Overall, our preclinical study suggests that hesperetin conferred neuroprotection by regulating the TLR4/NF-κB signaling pathway against the detrimental effects of LPS. Full article

A set of herpes simplex virus type 1 (HSV-1) amplicon vectors expressing the light chains (LC) of botulinum neurotoxins (BoNT) A, B, C, D, E and F was constructed. Their properties have been assessed in primary cultures of rat embryonic dorsal root ganglia (DRG) neurons, and in organotypic cultures of explanted DRG from adult rats. Following infection of primary cultures of rat embryonic DRG neurons, the different BoNT LC induced efficient cleavage of their corresponding target Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) protein (VAMP, SNAP25, syntaxin). A similar effect was observed following infection by BoNT-A LC of organotypic cultures of adult rat DRG. To quantify and compare the functional activities of the different BoNT LC, the inhibition of calcitonin gene-related protein (CGRP) secretion was assessed in DRG neurons following infection by the different vectors. All BoNT-LC were able to inhibit CGRP secretion although to different levels. Vectors expressing BoNT-F LC displayed the highest inhibitory activity, while those expressing BoNT-D and -E LC induced a significantly lower CGRP release inhibition. Cleavage of SNARE proteins and inhibition of CGRP release could be detected in neuron cultures infected at less than one transducing unit (TU) per neuron, showing the extreme efficacy of these vectors. To our knowledge this is the first study investigating the impact of vector-expressed transgenic BoNT LC in sensory neurons. Full article

The Genome Aggregation Database presently contains >120,000 human genomes. We searched in this database for the presence of mutations at the sites of tetanus (TeNT) and botulinum neurotoxins (BoNTs) cleavages of the three SNARE proteins: VAMP, SNAP-25 and Syntaxin. These mutations could account for some of the BoNT/A resistant patients. At the same time, this approach was aimed at testing the possibility that TeNT and BoNT may have acted as selective agents in the development of resistance to tetanus or botulism. We found that mutations of the SNARE proteins are very rare and concentrated outside the SNARE motif required for the formation of the SNARE complex involved in neuroexocytosis. No changes were found at the BoNT cleavage sites of VAMP and syntaxins and only one very rare mutation was found in the essential C-terminus region of SNAP-25, where Arg198 was replaced with a Cys residue. This is the P1’ cleavage site for BoNT/A and the P1 cleavage site for BoNT/C. We found that the Arg198Cys mutation renders SNAP-25 resistant to BoNT/A. Nonetheless, its low frequency (1.8 × 10⁻⁵) indicates that mutations of SNAP-25 at the BoNT/A cleavage site are unlikely to account for the existence of BoNT/A resistant patients. More in general, the present findings indicate that tetanus and botulinum neurotoxins have not acted as selective agents during human evolution as it appears to have been the case for tetanus in rats and chicken. Full article

Parallel zippering of the SNARE domains of syntaxin 1A/B, SNAP-25, and VAMP/synaptobrevin 2 is widely regarded as supplying the driving force for exocytotic events at nerve terminals and elsewhere. However, in spite of intensive research, no consensus has been reached concerning the molecular mechanism by which these SNARE proteins catalyze membrane fusion. As an alternative to SNARE-based models, a scenario was developed in which synaptotagmin 1 (or, 2) can serve as a template to guide lipid movements that underlie fast, synchronous exocytosis at nerve terminals. This “dyad model” advanced a novel proposal concerning the membrane disposition of the palmitoylated, cysteine-rich region of these synaptotagmins. Unexpectedly, it now emerges that a similar principle can be exploited to reveal how the hydrophobic, carboxyl-terminal domains of syntaxin 1A and synaptobrevin 2 can perturb membrane structure at the interface between a docked synaptic vesicle and the plasma membrane. These “β-to-α transition” models will be compared and contrasted with other proposals for how macromolecules are thought to intervene to drive membrane fusion. Full article

Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection. Full article

In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of pituitary hormone synthesis on the rough endoplasmic reticulum. A combined ISH and immunohistochemistry (IHC) under EM (EM-ISH&IHC) approach has sufficient ultrastructural resolution, and provides two-dimensional images of the subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (quantum dots, Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH&IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationships between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin has revealed that both rab3B and SNARE system proteins play important roles and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. We have developed an experimental pituitary cell line, GH3 cell, which has growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP). This stable GH3 cell secretes GH linked to EYFP upon stimulation by Ca²⁺ influx or Ca²⁺ release from storage. This GH3 cell line is useful for the real-time visualization of the intracellular transport and secretion of GH. These three methods from conventional immunohistochemistry and fluorescein imaging allow us to consecutively visualize the process of transcription, translation, transport and secretion of anterior pituitary hormone. Full article

Botulinum neurotoxins (BoNTs) are highly potent poisons produced by seven serotypes of Clostridium botulinum. The mechanism of neurotoxin action is a multistep process which leads to the cleavage of one of three different SNARE proteins essential for synaptic vesicle fusion and transmission of the nerve signals to muscles: synaptobrevin, syntaxin, or SNAP-25. In order to understand the precise mechanism of neurotoxin in a host, the domain structure of the neurotoxin was analyzed among different serotypes of C. botulinum. The results indicate that neurotoxins type A, C, D, E and F contain a coiled-coil domain while types B and type G neurotoxin do not. Interestingly, phylogenetic analysis based on neurotoxin sequences has further confirmed that serotypes B and G are closely related. These results suggest that neurotoxin has multi-domain structure, and coiled-coil domain plays an important role in oligomerisation of the neurotoxin. Domain analysis may help to identify effective antibodies to treat Botulinum toxin intoxication. Full article