N-ethylmaleimide-sensitive Factor Research Articles

N-ethylmaleimide-sensitive factor elicits a neuroprotection against ischemic neuronal injury by restoring autophagic/lysosomal dysfunction

Autophagosome-lysosome fusion defects play a critical role in driving autolysosomal dysfunction, leading to autophagic/lysosomal impairment in neurons following ischemic stroke. However, the mechanisms hindering autophagosome-lysosome fusion remain unclear. Soluble N-ethylmaleimide-sensitive factor (NSF) is an essential ATPase to reactivate STX17 and VAMP8, which are the paired molecules to mediate fusion between autophagosomes and lysosomes. However, NSF is frequently inactivated to inhibit the reactivation of STX17 and VAMP8 in ischemic neurons. Herein, we investigated whether autophagosome-lysosome fusion could be facilitated to alleviate autophagic/lysosomal impairment in ischemic neurons by over-expressing NSF. Rat model of middle cerebral artery occlusion (MCAO) and HT22 neuron ischemia model of oxygen-glucose deprivation (OGD) were prepared, respectively. The results demonstrated that NSF activity was significantly suppressed, accompanied by reduced expressions of STX17 and VAMP8 in penumbral neurons 48 h post-MCAO and in HT22 neurons 2 h post-OGD. Moreover, the attenuated autolysosome formation accompanied by autophagic/lysosomal dysfunction was observed. Thereafter, NSF activity in HT22 neurons was altered by over-expression and siRNA knockdown, respectively. After transfection with recombinant NSF-overexpressing lentiviruses, both STX17 and VAMP8 expressions were concurrently elevated to boost autophagosome-lysosome fusion, as shown by enhanced immunofluorescence intensity co-staining with LC3 and LAMP-1. Consequently, the OGD-created autophagic/lysosomal dysfunction was prominently ameliorated, as reflected by augmented autolysosomal functions and decreased autophagic substrates. By contrast, NSF knockdown conversely aggravated the autophagic/lysosomal impairment, and thereby exacerbated neurological damage. Our study indicates that NSF over-expression induces neuroprotection against ischemic neuronal injury by restoring autophagic/lysosomal dysfunction via the facilitation of autophagosome-lysosome fusion.Over-expression of NSF promotes fusion by reactivating STX17 and VAMP8. Black arrows represent the pathological process after cerebral ischemia, green arrows represent the mechanism of remission after NSF over-expression, and red arrows represent the effect on the pathological process after NSF knockdown.

Mutations of Single Residues in the Complexin N-terminus Exhibit Distinct Phenotypes in Synaptic Vesicle Fusion.

The release of neurotransmitters (NTs) at central synapses is dependent on a cascade of protein interactions, specific to the presynaptic compartment. Among those dedicated molecules, the cytosolic complexins play an incompletely defined role as synaptic transmission regulators. Complexins are multidomain proteins that bind soluble N-ethylmaleimide sensitive factor attachment protein receptor complexes, conferring both inhibitory and stimulatory functions. Using systematic mutagenesis and comparing reconstituted in vitro membrane fusion assays with electrophysiology in cultured neurons from mice of either sex, we deciphered the function of the N-terminus of complexin (Cpx) II. The N-terminus (amino acid 1-27) starts with a region enriched in hydrophobic amino acids (1-12), which binds lipids. Mutants maintaining this hydrophobic character retained the stimulatory function of Cpx, whereas exchanges introducing charged residues perturbed both spontaneous and evoked exocytosis. Mutants in the more distal region of the N-terminal domain (amino acid 11-18) showed a spectrum of effects. On the one hand, mutation of residue A12 increased spontaneous release without affecting evoked release. On the other hand, replacing D15 with amino acids of different shapes or hydrophobic properties (but not charge) not only increased spontaneous release but also impaired evoked release. Most surprising, this substitution reduced the size of the readily releasable pool, a novel function for Cpx at mammalian synapses. Thus, the exact amino acid composition of the Cpx N-terminus fine-tunes the degree of spontaneous and evoked NT release.

Investigating the potential role of α-SNAP in preventing chemotherapy-induced ovarian dysfunction: Insights from cellular and animal models

BackgroundThe phosphoinositide 3-kinase/Akt/mammalian target of rapamycin complex 1 (PI3K/Akt/mTORC1) pathway plays a crucial role in the activation of primordial follicles. However, excessive activation and the loss of primordial follicles can lead to ovarian dysfunction. The alpha-soluble N-ethylmaleimide sensitive factor attachment protein (α-SNAP) protein has been implicated in PI3K/Akt/mTORCl signaling, suggesting its potential involvement in follicle activation. Thus, this study aimed to explore the role of α-SNAP in the activation of the PI3K/Akt/mTORC1 signaling pathway and its ability to mitigate the effects of cisplatin on ovarian function, using both in vitro and in vivo models. MethodsWe transfected KGN human ovarian granulosa cells (GCs) with small interfering RNA (siRNA) targeting α-SNAP to investigate the effects of α-SNAP inhibition on GC proliferation and apoptosis, as well as on the activity of the PI3K/Akt/mTORC1 pathway. In a mouse model, α-SNAP siRNA was delivered via an adeno-associated virus before treatment with cisplatin to assess its effects on follicle activation and ovarian function. Follicle counts at various growth stages, western blotting, and immunohistochemistry analyses were conducted to detect the expression of cleaved caspase-3, Ki67, α-SNAP, and p-mTOR. Additionally, the serum concentrations of anti-Müllerian hormone (AMH) were measured through an enzyme-linked immunosorbent assay. ResultsIn vitro, α-SNAP depletion prevented GC proliferation by inhibiting the PI3K/Akt/mTORC1 pathway, thereby indicating its role in the regulation of cell growth. In vivo, α-SNAP knockdown attenuated the cisplatin-induced overactivation of primordial follicles by suppressing the PI3K/Akt/mTORC1 signaling pathway and partially restoring AMH levels. In addition, the expression and distribution patterns of cleaved caspase-3, Ki67, α-SNAP, and p-mTOR varied across different follicular growth stages, suggesting a protective effect against chemotherapy-induced ovarian damage. ConclusionsInhibiting α-SNAP may attenuate GC proliferation by suppressing the PI3K/Akt/mTORC1 pathway, thereby mitigating the overactivation and loss of primordial follicles induced by cisplatin. Targeting α-SNAP may emerge as a novel strategy to prevent ovarian damage resulting from chemotherapy. However, these conclusions warrant repeated testing, and the mechanistic underpinnings of α-SNAP must be further elucidated in the future.

#718 N-Ethylmaleimide Sensitive Factor (NSF) is essential for maintaining podocyte focal adhesion

Abstract Background and Aims Cell-matrix adhesion molecules in podocytes play a critical role in attaching these cells to the glomerular basement membrane, ensuring the proper functioning of the glomerular filtration barrier. Previous studies have shown that patients with proteinuric glomerular diseases often have higher levels of podocyturia. This increase is notably linked to a reduction in focal adhesion proteins, especially in cases of focal segmental glomerulosclerosis. Our study aims to explore the regulation of these focal adhesion molecules in podocytes. Method Doxycycline (Dox) inducible podocyte-specific Tln1 knockout mice (Tln1fl/fl Nphs2-rtTA TetO-Cre (Tln1 KO)) were used as the model for podocyte injury. These mice are genetically modified to lack Talin 1, a key protein in focal adhesion. The absence of Talin 1 in these mice leads to the development of pathological changes akin to focal segmental glomerulosclerosis, characterized by extensive proteinuria, thereby providing a valuable model for understanding this kidney disease (J Clin Invest. 2019 1;129(3):1295-13). As Tln1 KO mice 10 days after Dox induction (Tln1 KO 10 d) showed proteinuria without elevated serum creatinine, these mice were considered as the early phase of podocyte injury. Single-cell RNA sequencing (scRNAseq) was performed using these mice kidney samples, followed by differentially expressed genes (DEG) analyses. Podocyte-specific Nsf KO (Nphs2-Cre Rosa-DTRflox Nsf fl/fl (Nsf cKO)) mice were generated based on the result of the DEG analyses and the effect of NSF on focal adhesion was examined using primary podocyte culture. Results Nsf was identified as one of the most significantly down-regulated DEGs in the podocytes ofTln1 KO 10d mice. Further analysis of public scRNAseq data revealed a decrease in Nsf mRNA expression levels also in lipopolysaccharide-treated mouse podocytes. Notably, proteinuria was observed in Nsf conditional knockout (cKO) mice as early as 2 weeks of age, with all Nsf cKO mice succumbing to end-stage kidney disease by 6 weeks post-birth. Examination of isolated mutant podocytes showed a marked reduction in cell spreading. Additionally, a significant decrease in Integrin β1 expression was observed in primary podocytes of Nsf cKO mice, as evidenced by immunoblot analysis. In vivo studies using transmission electron microscopy revealed endoplasmic reticulum (ER) expansion in the podocytes of Nsf cKO mice. Immunofluorescence staining indicated a substantial increase in the ER and ER-Golgi intermediate compartment (ERGIC) in isolated Nsf cKO podocytes. These findings suggest that the deletion of Nsf in podocytes leads to a disruption in ER-to-Golgi protein transport, subsequently impairing the trafficking of focal adhesion proteins. Conclusion This study provides new insights into the molecular mechanisms underlying podocyte injury and the progression of kidney diseases. NSF plays a pivotal role in maintaining podocyte focal adhesion.

The role of syntaxins in retinal function and health.

The soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) superfamily plays a pivotal role in cellular trafficking by facilitating membrane fusion events. These SNARE proteins, including syntaxins, assemble into complexes that actively facilitate specific membrane fusion events. Syntaxins, as integral components of the SNARE complex, play a crucial role in initiating and regulating these fusion activities. While specific syntaxins have been extensively studied in various cellular processes, including neurotransmitter release, autophagy and endoplasmic reticulum (ER)-to-Golgi protein transport, their roles in the retina remain less explored. This review aims to enhance our understanding of syntaxins' functions in the retina by shedding light on how syntaxins mediate membrane fusion events unique to the retina. Additionally, we seek to establish a connection between syntaxin mutations and retinal diseases. By exploring the intricate interplay of syntaxins in retinal function and health, we aim to contribute to the broader comprehension of cellular trafficking in the context of retinal physiology and pathology.

Abstract 1063: G Protein 12 Alpha Subunit Covalent Binding To Alpha-snap And Its Potential Role In Von Willebrand Factor Secretion

Rationale: α-SNAP is part of a large 20S complex composed of 3 SNARE, 4 α-SNAP, and 6 N-ethylmaleimide sensitive factor proteins that together regulate endothelial cell Weibel Palade body fusion and exocytosis of von Willebrand factor (vWF). We previously showed that Gα12 is essential for basal and thrombin-induced vWF secretion, that GST-α-SNAP pulldown of Gα12 is dependent on N-terminal domain aa 10-15 in Gα12, and that a myristoylated 6 aa α-SNAP Binding Domain Gα12 peptide (Myr-SBD6) reduces vWF secretion, thrombo-inflammation, and lethality in septic mice. Here, using direct binding assays (Surface Plasmon Resonance (SPR), AlphaLISA, and mass spectrometry (MS)), we assessed the molecular underpinnings of SBD6 and modified higher affinity peptide (SBD15) binding directly to α-SNAP. Methods: GST-α-SNAP was incubated alone or with SBD6, SBD15-PEG-biotin, or SBD6 with C to S mutation and then evaluated by Maldi MS. SPR was performed for both SBD6 and SBD15 to assess binding affinity ( K D ) and kinetics ( k a and k d ). After demonstrating the covalent linkage of α-SNAP with SBD15-PEG-biotin, cysteine mapping was performed to identify the SBD binding site on α-SNAP. Free cysteines in SBD15-biotin conjugated α-SNAP were capped with N-ethylmaleimide, disulfide bonds were reduced with DTT, reduced Cys residues were capped with iodoacetamide (IAA), and tryptic peptide digests were analyzed by LC/MS/MS. Results: Maldi MS studies showed that SBD15-PEG-biotin exhibited covalent binding to GST-α-SNAP. Further, proteomic analysis of Cys mapped fragments identified a peptide containing C103, KADPQEAINC 103 LMR, that was modified by NEM in control samples and shifted in the presence of SBD15, indicating modification by IAA in α-SNAP - SBD15 conjugates. Further, the C103S α-SNAP mutant was unable to bind to SBD6, and C11S-SBD15-PEG-biotin was unable to bind to α-SNAP. Conclusion: We propose that amino-terminal Gα12 Cys11 covalently binds to α-SNAP Cys103 and that this plays a critical role in the mechanism regulating vWF secretion. The discovery of this disulfide binding mechanism between Gα12 and α-SNAP opens up new avenues for therapeutic intervention in thrombotic disorders associated with elevated plasma vWF.

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).

Proteomics study of Mongolian medicine warm acupuncture in the treatment of p-chlorophenylalanine-induced insomnia in rats

Mongolian medical warm acupuncture (MMWA) therapy is a traditional external therapy method that has a definite clinical effect in the treatment of insomnia, but research on its underlying neural mechanisms remains limited. iTRAQ-based quantitative proteomics was used to identify proteins that are potential neural molecules involved in the treatment of insomnia by MMWA. In this study, 6383 proteins were identified, including 45 proteins with increased expression and 31 proteins with decreased expression in the insomnia model group (M) compared to the control group (C); 101 proteins showed an increase and 48 proteins showed a decrease between the warm acupuncture group (W) and C; 26 proteins showed an increase and 22 proteins showed a decrease between W and M. GO and KEGG analysis showed that MMWA treatment of insomnia was closely related to metabolism and hormone synthesis. Differentially expressed proteins included albumin (ALB), Pro-MCH (PMCH), acetoacetyl-CoA synthetase (AACS), N-ethylmaleimide sensitive factor (NSF), potassium/sodium hyperpolarization-activated cyclic-nucleotide gated channel 4 (HCN4) and Ciapin1, which are important proteins for MMWA treatment of insomnia. The iTRAQ-based proteomics data analyses presented here elucidate variations in the expression of proteins involved in the treatment of insomnia by MMWA.

Cathepsin B knockout confers significant brain protection in the mouse model of stroke

BackgroundSignificant advances have been made in our understanding of the endolysosomal cycle. Disruption of this cycle leads to cell death. The objective of this study aims to investigate the role of disrupted endolysosomal cycle in brain ischemia-reperfusion injury. MethodsA total of 57 mice were randomly assigned into four experimental groups: (i) wildtype (wt) sham control; (ii) wt middle cerebral artery occlusion (MCAO); (iii) cathepsin B (CTSB) knockout (KO) sham control; and (iv) CTSB KO MCAO. Mice were subjected either to 0 min (sham) or 40 min of MCAO, followed by reperfusion for 1 or 7 days. Physical and behavioral examinations were conducted in the 7-day reperfusion group for 7 consecutive days after MCAO. Confocal microscopy was used to assess the levels, redistributions, and co-localizations of key endolysosomal cycle-related proteins. Histopathology was examined by light microscopy. ResultsConfocal microscopy revealed a significant accumulation of CTSB in post-ischemic penumbral neurons relative to those in the sham group. In addition, N-ethylmaleimide sensitive factor ATPase (NSF) was irreversibly depleted in these neurons. Furthermore, CTSB-immunostained structures were enlarged and diffusely distributed in both the cytoplasm and extracellular space, indicating the release of CTSB from post-ischemic neurons. Compared to wt mice, CTSB KO mice showed a significant decrease in hippocampal injury area, a significant increase in the number of survival neurons in the striatal core area, and a significant improvement in physical and functional performance in post-MCAO mice. ConclusionBrain ischemia leads to a cascade of events leading to inactivation of NSF, disruption of the endolysosomal cycle, endolysosomal structural buildup and damage, and the release of CTSB, eventually resulting in brain ischemia reperfusion injury. CTSB KO in mice protected the brain from ischemia-reperfusion injury.

NSF/αSNAP2-mediated cis-SNARE complex disassembly precedes vesicle fusion in Arabidopsis cytokinesis.

Eukaryotic membrane fusion requires trans-SNARE complexes bridging the gap between adjacent membranes1. Fusion between a transport vesicle and its target membrane transforms the trans- into a cis-SNARE complex. The latter interacts with the hexameric AAA+-ATPase N-ethylmaleimide-sensitive factor (NSF) and its co-factor alpha-soluble NSF attachment protein (αSNAP), forming a 20S complex2,3. ATPase activity disassembles the SNAP receptor (SNARE) complex into Qa-SNARE, which folds back onto itself, and its partners4,5. The fusion of identical membranes has a different sequence of events6. The fusion partners each have cis-SNARE complexes to be broken up by NSF and αSNAP. The Qa-SNARE monomers are then stabilized by interaction with Sec1/Munc18-type regulators (SM proteins) to form trans-SNARE complexes, as shown for the yeast vacuole7. Membrane fusion in Arabidopsis cytokinesis is formally akin to vacuolar fusion8. Membrane vesicles fuse with one another to form the partitioning membrane known as the cell plate. Cis-SNARE complexes of cytokinesis-specific Qa-SNARE KNOLLE and its SNARE partners are assembled at the endoplasmic reticulum and delivered by traffic via the Golgi/trans-Golgi network to the cell division plane9. The SM protein KEULE is required for the formation of trans-SNARE complexes between adjacent membrane vesicles10. Here we identify NSF and its adaptor αSNAP2 as necessary for the disassembly of KNOLLE cis-SNARE complexes, which is a prerequisite for KNOLLE-KEULE interaction in cytokinesis. In addition, we show that NSF is required for other trafficking pathways and interacts with the respective Q-SNAREs. The SNARE complex disassembly machinery is conserved in plants and plays a unique essential role in cytokinesis.

Early Endosomes Act as Local Exocytosis Hubs to Repair Endothelial Membrane Damage.

The plasma membrane of a cell is subject to stresses causing ruptures that must be repaired immediately to preserve membrane integrity and ensure cell survival. Yet, the spatio-temporal membrane dynamics at the wound site and the source of the membrane required for wound repair are poorly understood. Here, it is shown that early endosomes, previously only known to function in the uptake of extracellular material and its endocytic transport, are involved in plasma membrane repair in human endothelial cells. Using live-cell imaging and correlative light and electron microscopy, it is demonstrated that membrane injury triggers a previously unknown exocytosis of early endosomes that is induced by Ca2+ entering through the wound. This exocytosis is restricted to the vicinity of the wound site and mediated by the endosomal soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) VAMP2, which is crucial for efficient membrane repair. Thus, the newly identified Ca2+ -evoked and localized exocytosis of early endosomes supplies the membrane material required for rapid resealing of a damaged plasma membrane, thereby providing the first line of defense against damage in mechanically challenged endothelial cells.

CT hyperdense cerebral artery sign reflects distinct proteomic composition in acute ischemic stroke thrombus

BackgroundHyperdense cerebral artery sign (HCAS) is an imaging biomarker in acute ischemic stroke (AIS) that has been shown to be associated with various clinical outcomes and stroke etiology. While prior...

Genetic overlap between cortical brain morphometry and frontotemporal dementia risk.

Frontotemporal dementia (FTD) has a complex genetic etiology, where the precise mechanisms underlying the selective vulnerability of brain regions remain unknown. We leveraged summary-based data from genome-wide association studies (GWAS) and performed LD score regression to estimate pairwise genetic correlations between FTD risk and cortical brain imaging. Then, we isolated specific genomic loci with a shared etiology between FTD and brain structure. We also performed functional annotation, summary-data-based Mendelian randomization for eQTL using human peripheral blood and brain tissue data, and evaluated the gene expression in mice targeted brain regions to better understand the dynamics of the FTD candidate genes. Pairwise genetic correlation estimates between FTD and brain morphology measures were high but not statistically significant. We identified 5 brain regions with a strong genetic correlation (rg > 0.45) with FTD risk. Functional annotation identified 8 protein-coding genes. Building upon these findings, we show in a mouse model of FTD that cortical N-ethylmaleimide sensitive factor (NSF) expression decreases with age. Our results highlight the molecular and genetic overlap between brain morphology and higher risk for FTD, specifically for the right inferior parietal surface area and right medial orbitofrontal cortical thickness. In addition, our findings implicate NSF gene expression in the etiology of FTD.

Role of complexin-lipid interactions in controlling snare-mediated membrane fusion.

Fast and reliable neurotransmitter release is essential for signaling fidelity at neuronal synapses. Synaptic exocytosis is a tightly controlled process accomplished through Ca2+ triggered fusion of neurotransmitter-laden vesicles with the presynaptic plasma membrane. This process is mediated by SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins that zipper into a four-helix bundle to drive membrane fusion. The neuronal SNARE complex, consisting of Syntaxin-1a, SNAP-25, and Synaptobrevin-2, is intrinsically capable of catalyzing slow and Ca2+ independent fusion in reconstituted fusion assays in vitro. A single model of how interactions with SNARE regulatory proteins impart physiological properties seen in neurons, such as Ca2+ specificity, has yet to be established. Complexin-1 (Cpx) is a small, soluble regulatory protein that binds SNAREs and lipid membranes. Cpx is proposed to facilitate fusion in the presence of Ca2+ as well as suppress fusion in the absence of Ca2+ through interactions of its central domain with SNARE complexes in their prefusion and postfusion states. Additionally, the Cpx C-terminal domain interacts with membranes, an interaction that has been proposed to facilitate function. Recent work has shown that Cpx's lipid-binding domains can differentiate between membranes of different compositions, though the functional relevance of this finding has yet to be established. Our group has previously developed a hybrid fusion assay that combines synthetic, reconstituted, and native components to reproduce key features of synaptic exocytosis. This method mimics intracellular conditions more closely than a reconstituted system with only purified components while maintaining the ability to easily alter lipid, protein, and ion concentrations. We utilize this system with wild-type and mutant Cpx, along with other protein and lipid regulators of SNAREs, to investigate how interaction with and composition of the fusing membranes controls Cpx's regulatory function.

Involvement of mTOR pathway in neurodegeneration in NSF-related developmental and epileptic encephalopathy.

Membrane fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. During neurotransmitter exocytosis, SNARE proteins on a synaptic vesicle and the target membrane form a complex, resulting in neurotransmitter release. N-ethylmaleimide-sensitive factor (NSF), a homohexameric ATPase, disassembles the complex, allowing individual SNARE proteins to be recycled. Recently, the association between pathogenic NSF variants and developmental and epileptic encephalopathy (DEE) was reported; however, the molecular pathomechanism of NSF-related DEE remains unclear. Here, three patients with de novo heterozygous NSF variants were presented, of which two were associated with DEE and one with a very mild phenotype. One of the DEE patients also had hypocalcemia from parathyroid hormone deficiency and neuromuscular junction impairment. Using PC12 cells, a neurosecretion model, we show that NSF with DEE-associated variants impaired the recycling of vesicular membrane proteins and vesicle enlargement in response to exocytotic stimulation. In addition, DEE-associated variants caused neurodegenerative change and defective autophagy through overactivation of the mammalian/mechanistic target of rapamycin (mTOR) pathway. Treatment with rapamycin, an mTOR inhibitor or overexpression of wild-type NSF ameliorated these phenotypes. Furthermore, neurons differentiated from patient-derived induced pluripotent stem cells showed neurite degeneration, which was also alleviated by rapamycin treatment or gene correction using genome editing. Protein structure analysis of NSF revealed that DEE-associated variants might disrupt the transmission of the conformational change of NSF monomers and consequently halt the rotation of ATP hydrolysis, indicating a dominant negative mechanism. In conclusion, this study elucidates the pathomechanism underlying NSF-related DEE and identifies a potential therapeutic approach.

A novel association between platelet filamin A and soluble N-ethylmaleimide sensitive factor attachment proteins regulates granule secretion

Background and ObjectiveThe molecular mechanisms that underpin platelet granule secretion remain poorly defined. Filamin A (FLNA) is an actin-crosslinking and signaling scaffold protein whose role in granule exocytosis has not been explored despite evidence that FLNA gene mutations confer platelet defects in humans. Methods and ResultsUsing platelets from platelet-specific conditional Flna-knockout mice, we showed that the loss of FLNA confers a severe defect in alpha (α)- and dense (δ)-granule exocytosis, as measured based on the release of platelet factor 4 (aka CXCL4) and adenosine triphosphate (ATP), respectively. This defect was observed following activation of both immunoreceptor tyrosine-based activation motif (ITAM) signaling by collagen-related peptide (CRP) and G protein–coupled receptor (GPCR) signaling by thrombin and the thromboxane mimetic U46619. CRP–induced spikes in intracellular calcium [Ca2+]i were impaired in FLNA-null platelets relative to controls, confirming that FLNA regulates ITAM-driven proximal signaling. In contrast, GPCR-mediated spikes in [Ca2+]i in response to thrombin and U46619 were unaffected by FLNA. Normal platelet secretion requires complexing of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins synaptosomal-associated protein 23 (SNAP23) and syntaxin-11 (STX11). We determined that FLNA coimmunoprecipitates with both SNAP23 and STX11 upon platelet stimulation. ConclusionFLNA regulates GPCR-driven platelet granule secretion and associates with SNAP23 and STX11 in an activation-dependent manner.

SNARE Modulators and SNARE Mimetic Peptides.

The soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) proteins play a central role in most forms of intracellular membrane trafficking, a key process that allows for membrane and biocargo shuffling between multiple compartments within the cell and extracellular environment. The structural organization of SNARE proteins is relatively simple, with several intrinsically disordered and folded elements (e.g., SNARE motif, N-terminal domain, transmembrane region) that interact with other SNAREs, SNARE-regulating proteins and biological membranes. In this review, we discuss recent advances in the development of functional peptides that can modify SNARE-binding interfaces and modulate SNARE function. The ability of the relatively short SNARE motif to assemble spontaneously into stable coiled coil tetrahelical bundles has inspired the development of reduced SNARE-mimetic systems that use peptides for biological membrane fusion and for making large supramolecular protein complexes. We evaluate two such systems, based on peptide-nucleic acids (PNAs) and coiled coil peptides. We also review how the self-assembly of SNARE motifs can be exploited to drive on-demand assembly of complex re-engineered polypeptides.

Differentially expressed whey proteins of donkey and bovine colostrum revealed with a label-free proteomics approach

This study aimed to analyze and compare the differentially expressed whey proteins (DEWPs) of donkey and bovine colostrum using high-performance liquid chromatography with tandem mass spectrometry-based proteomics. A total of 620 and 696 whey proteins were characterized in the donkey and bovine colostrum, respectively, including 383 common whey proteins. Among these common proteins, 80 were identified as DEWPs, including 21 upregulated and 59 downregulated DEWPs in donkey colostrum compared to bovine colostrum. Gene Ontology analysis revealed that these DEWPs were mainly related to cellular components, such as extracellular exosome, plasma membrane, and mitochondrion; biological processes, such as oxidation-reduction process, cell-cell adhesion, and small guanosine triphosphate (GTP) ase-mediated signal transduction; and molecular functions, such as GTP binding, GTPase activity, and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor activity. Metabolic pathway analysis suggested that the majority of the DEWPs were associated with soluble NSF factor attachment protein receptor interactions in vesicular transport, fatty acid biosynthesis, and estrogen signaling pathways. Our results provide a vital insight into the differences between donkey and bovine colostrum, along with important information on the significant components as nutritional and functional factors to be included in infant formula based on multiple milk sources.

Listeria monocytogenes Co-Opts the Host Exocyst Complex To Promote Internalin A-Mediated Entry.

The bacterial pathogen Listeria monocytogenes induces its internalization (entry) into intestinal epithelial cells through interaction of its surface protein, internalin A (InlA), with the human cell-cell adhesion molecule, E-cadherin. While InlA-mediated entry requires bacterial stimulation of actin polymerization, it remains unknown whether additional host processes are manipulated to promote internalization. Here, we show that interaction of InlA with E-cadherin induces the host membrane-trafficking process of polarized exocytosis, which augments uptake of Listeria. Imaging studies revealed that exocytosis is stimulated at sites of InlA-dependent internalization. Experiments inhibiting human N-ethylmaleimide-sensitive factor (NSF) demonstrated that exocytosis is needed for efficient InlA-mediated entry. Polarized exocytosis is mediated by the exocyst complex, which comprises eight proteins, including Sec6, Exo70, and Exo84. We found that Exo70 was recruited to sites of InlA-mediated entry. In addition, depletion of Exo70, Exo84, or Sec6 by RNA interference impaired entry without affecting surface levels of E-cadherin. Similar to binding of InlA to E-cadherin, homophilic interaction of E-cadherin molecules mobilized the exocyst and stimulated exocytosis. Collectively, these results demonstrate that ligation of E-cadherin induces exocytosis that promotes Listeria entry, and they raise the possibility that the exocyst might also control the normal function of E-cadherin in cell-cell adhesion.

Vesicle trafficking with snares: a perspective for autism.

Vesicle-mediated membrane traffic is the mechanism fundamental to many biological events, especially the release of neurotransmitters. The main proteins of the mechanism that mediates membrane fusion in vesicle-mediated membrane traffic are N-ethylmaleimide sensitive factor (NSF) supplemental protein (SNAP) receptor (SNAREs) proteins. SNAREs are classified into vesicle-associated SNAREs (vesicle-SNAREs/v-SNAREs) and target membrane-associated SNAREs (target-SNARE/t-SNAREs). Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by many symptoms, especially complications in social communication and stereotypical behaviours. Defects in synaptogenesis and neurotransmission, oxidative stress, and developmental defects in the early stages of development are defined in the pathogenesis of the disease. SNARE proteins are on the basis of synaptogenesis and neurotransmission. Although the formation mechanisms and underlying causes of the SNARE complex are not fully understood, expression differences, polymorphisms, abnormal expressions or dysfunctions of the proteins that make up the SNARE complex have been associated with many neurodevelopmental diseases, including autism. Further understanding of SNARE mechanisms is crucial both for understanding ASD and for developing new treatments. In this review, the formation mechanisms of the SNARE complex and the roles of various factors involved in this process are explained. In addition, a brief evaluation of clinical and basic studies on the SNARE complex in autism spectrum disorders was made.