Synaptobrevin Research Articles

Transcriptome profiles of corticosterone-induced cytotoxicity reveals the involvement of neurite growth-related genes in depression

Corticosterone (CORT), the main HPA-axis glucocorticoid hormone in rodents, is involved in the regulation of animal stress responses. However, the neural mechanisms underlying the effects of corticosteroids on depression are yet to be elucidated. We found that fluoxetine reversed neurite growth inhibition induced by CORT in PC12 cells, a widely used model system for neurobiological and neurotoxicological studies. Transcriptome profiling showed that 1,609 genes were up-regulated, whereas 1,764 genes were down-regulated significantly in the CORT group in comparison with the Control group. Of them, the expression of 589 DEGs was reversed after fluoxetine treatment, and genes related to cell morphogenesis, neurite growth, and immune function were involved in the neuroprotective effect of fluoxetine against CORT. Furthermore, expression of neurite growth-related genes, such as such as Calpain 2 (Capn2), vesicle-associated membrane protein 7 (Vamp7) and C-type natriuretic peptide (Cnp), altered in a brain region- or treatment-specific manner in the animal models of depression. Therefore, the interaction between stress, glucocorticoids, and neurite growth inhibition may be a candidate pathophysiology underlying major depressive disorder (MDD), and the identification of Capn2, Vamp7 and Cnp might provide insight into treatment of MDD.

Association of VAMP8 rs1010 Polymorphism with Host Susceptibility to Pulmonary Tuberculosis in a Chinese Han Population.

Aims: Autophagic eradication of pathogenic microbes, including Mycobacterium tuberculosis (Mtb), is an effective host immune process that protects hosts from developing diseases associated with intracellular pathogens. This study was designed to investigate the association between the single nucleotide polymorphisms (SNPs) of the autophagy-related genes VAMP8 and VTI1B, and the susceptibility to pulmonary tuberculosis (PTB) in a Chinese Han population. Materials and Methods: Two SNPs, rs1010 from the VAMP8 gene and rs15493 from the VTI1B gene, were examined in 202 PTB patients and 216 healthy controls using high-resolution melt-polymerase chain reaction. Results: The rs1010 SNP genotypes AG (p = 0.028) and GG (p = 0.016) were associated with increased susceptibility to PTB. However, the VTI1B rs15493 SNP had no impact on the susceptibility to PTB (p > 0.05). Conclusions: Our study demonstrated that the rs1010 SNP of VAMP8 gene was significantly associated with the susceptibility to PTB. This result suggests that rs1010 genotyping could be used as prognostic biomarker to predict the risk of Mtb infection and/or PTB disease development after Mtb infection in the Chinese Han population.

Long non-coding RNA HOTAIR promotes exosome secretion by regulating RAB35 and SNAP23 in hepatocellular carcinoma

BackgroundEmerging evidence indicates that tumor cells release a large amount of exosomes loaded with cargos during tumorigenesis. Exosome secretion is a multi-step process regulated by certain related molecules. Long non-coding RNAs (lncRNAs) play an important role in hepatocellular carcinoma (HCC) progression. However, the role of lncRNA HOTAIR in regulating exosome secretion in HCC cells remains unclear.MethodsWe analyzed the relationship between HOTAIR expression and exosome secretion-related genes using gene set enrichment analysis (GSEA). Nanoparticle tracking analysis was performed to validate the effect of HOTAIR on exosome secretion. The transport of multivesicular bodies (MVBs) after overexpression of HOTAIR was detected by transmission electron microscopy and confocal microscopy analysis of cluster determinant 63 (CD63) with synaptosome associated protein 23 (SNAP23). The mechanism of HOTAIR’s regulation of Ras-related protein Rab-35 (RAB35), vesicle associated membrane protein 3 (VAMP3), and SNAP23 was assessed using confocal co-localization analysis, phosphorylation assays, and rescue experiments.ResultsWe found an enrichment of exosome secretion-related genes in the HOTAIR high expression group. HOTAIR promoted the release of exosomes by inducing MVB transport to the plasma membrane. HOTAIR regulated RAB35 expression and localization, which controlled the docking process. Moreover, HOTAIR facilitated the final step of fusion by influencing VAMP3 and SNAP23 colocalization. In addition, we validated that HOTAIR induced the phosphorylation of SNAP23 via mammalian target of rapamycin (mTOR) signaling.ConclusionOur study demonstrated a novel function of lncRNA HOTAIR in promoting exosome secretion from HCC cells and provided a new understanding of lncRNAs in tumor cell biology.

Interactions Between SNARE and Exocyst Complex Proteins with KCa3.1 in the Tethering and Fusion of KCa3.1‐containing Vesicles to the Basolateral Membrane of Epithelial Cells

Trafficking of cargo‐containing vesicles to a specific destination membrane is an intricate process that requires coordinated interactions between vesicle, tethering and docking proteins for the proper delivery of the cargo (e.g., ion channel) to a target membrane. KCa3.1 is a calcium‐activated, intermediate‐conductance K+ channel which is targeted to the basolateral membrane (BLM) of epithelial cells, and it plays various roles in epithelial cell physiology. Previously, we reported that trafficking of KCa3.1 to the BLM is a Rab1‐ and Rab8‐dependent process (Bertuccio et al. PlosONE 9:e92013, 2014); which requires Myosin‐Vc for membrane delivery of KCa3.1 (Farquhar et al. Front. Physiol. 7:639, 2017). At recent EB meetings, we reported that SNARE (Soluble NSF Attachment Protein REceptor) proteins including the target‐SNAREs, SNAP‐23 and syntaxin‐4 (STX4), and the vesicle‐SNARE, VAMP3 (vesicle‐associated membrane protein 3), are all crucial in the delivery of KCa3.1 to the BLM (Farquhar et al. FASEB J. 31:1007.25, 2017). Also, we reported that Sec 6 and Sec 8, which are tethering proteins of the Exocyst complex, are critical for the trafficking of KCa3.1 to the BLM (Farquhar et al. FASEB J 32: Issue 1, #750.27, 2018). Currently, there is a gap in our knowledge as to the protein‐protein interactions that occur between the SNAREs, Exocyst complex proteins and KCa3.1 resulting in the incorporation of KCa3.1 in the BLM. In this study, we hypothesized that there would be protein‐protein interactions between the SNAREs and Exocyst proteins and KCa3.1. In order to test this hypothesis, we used our well‐established Fischer rat thyroid (FRT) epithelial cell line stably expressing KCa3.1‐BLAP‐Bir‐A‐KDEL. Cells were grown on TranswellTM filters, and channels were biotinylated in the endoplasmic reticulum and trafficked to the BLM where channels were labeled with streptavidin. Co‐immunoprecipitation (co‐IP) and Western immunoblot (IB) experiments were performed to identify protein‐protein interactions between the various proteins. A typical experiment was to select one protein (e.g., KCa3.1) as the ‘bait’ with a specific antibody (e.g., streptavidin antibody), then immunoblot for a ‘target’ second protein (e.g., VAMP3). After which, the IB membrane was then stripped and blotted for another ‘target’ protein (e.g., STX4). We determined that KCa3.1 co‐IPed with VAMP3 (n=6) and STX4 (n=6), but not SNAP23 (n=5). Additionally, using SNAP‐23 as ‘bait’, SNAP‐23 co‐IPed with STX4 (n=3) and VAMP3 (n=2), but did not co‐IP with KCa3.1 (n=3). When Sec 8 was used as ‘bait’, Sec 8 co‐IPed with SNAP23 (n=2), STX‐4 (n=2), VAMP3 (n=1), but did not interact with KCa3.1. Finally, when Sec 6 was used as ‘bait’, Sec 6 co‐IPed with VAMP3 (n=2), SNAP‐23 (n=3), KCa3.1 (n=1) and no interaction was found with STX4. Currently, these results suggest that there are multiple interactions between these proteins for the incorporation of KCa3.1 into the BLM.Support or Funding InformationThis work was supported by a Dean's Fund grant from the School of Biomedical Sciences and an AIM grant from the Department of Physiology of the University of Otago.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular Docking and Dynamics Simulation Studies Predict Munc18b as a Target of Mycolactone: A Plausible Mechanism for Granule Exocytosis Impairment in Buruli Ulcer Pathogenesis.

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.

A VPS33A-binding motif on syntaxin 17 controls autophagy completion in mammalian cells

Autophagy is an intracellular degradation pathway that transports cytoplasmic material to the lysosome for hydrolysis. It is completed by SNARE-mediated fusion of the autophagosome and endolysosome membranes. This process must be carefully regulated to maintain the organization of the membrane system and prevent mistargeted degradation. As yet, models of autophagosomal fusion have not been verified within a cellular context because of difficulties with assessing protein interactions in situ. Here, we used high-resolution fluorescence lifetime imaging (FLIM)-FRET of HeLa cells to identify protein interactions within the spatiotemporal framework of the cell. We show that autophagosomal syntaxin 17 (Stx17) heterotrimerizes with synaptosome-associated protein 29 (SNAP29) and vesicle-associated membrane protein 7 (VAMP7) in situ, highlighting a functional role for VAMP7 in autophagosome clearance that has previously been sidelined in favor of a role for VAMP8. Additionally, we identified multimodal regulation of SNARE assembly by the Sec1/Munc18 (SM) protein VPS33A, mirroring other syntaxin–SM interactions and therefore suggesting a unified model of SM regulation. Contrary to current theoretical models, we found that the Stx17 N-peptide appears to interact in a positionally conserved, but mechanistically divergent manner with VPS33A, providing a late “go, no-go” step for autophagic fusion via a phosphoserine master-switch. Our findings suggest that Stx17 fusion competency is regulated by a phosphosite in its N-peptide, representing a previously unknown regulatory step in mammalian autophagy.

Abundance of Synaptic Vesicle-Related Proteins in Alpha-Synuclein-Containing Protein Inclusions Suggests a Targeted Formation Mechanism.

Proteinaceous α-synuclein-containing inclusions are found in affected brain regions in patients with Parkinson's disease (PD), Dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). These appear in neurons as Lewy bodies in both PD and DLB and as glial cytoplasmic inclusions (GCIs) in oligodendrocytes in MSA. The role they play in the pathology of the diseases is unknown, and relatively little is still known about their composition. By purifying the inclusions from the surrounding tissue and comprehensively analysing their protein composition, vital clues to the formation mechanism and role in the disease process may be found. In this study, Lewy bodies were purified from postmortem brain tissue from DLB cases (n = 2) and GCIs were purified from MSA cases (n = 5) using a recently improved purification method, and the purified inclusions were analysed by mass spectrometry. Twenty-one percent of the proteins found consistently in the GCIs and LBs were synaptic-vesicle related. Identified proteins included those associated with exosomes (CD9), clathrin-mediated endocytosis (clathrin, AP-2 complex, dynamin), retrograde transport (dynein, dynactin, spectrin) and synaptic vesicle fusion (synaptosomal-associated protein 25, vesicle-associated membrane protein 2, syntaxin-1). This suggests that the misfolded or excess α-synuclein may be targeted to inclusions via vesicle-mediated transport, which also explains the presence of the neuronal protein α-synuclein within GCIs.

Synaptosomal‐associated protein 29 is required for the autophagic degradation of hepatitis B virus

Hepatitis B virus (HBV) replication and envelopment is dependent on cellular autophagy. Previously, we have provided evidence for the extensive lysosomal degradation of HBV virions and the hepatitis B surface antigen (HBsAg), which is likely controlled by autophagosome-lysosome fusion. Synaptosomal-associated protein 29 (SNAP29) has been identified as a protein specifically mediating autophagosome-lysosome fusion. Thus, in the present study, we addressed the hypothesis that SNAP29 is required for the autophagic degradation of HBV virions and HBsAg. We found that silencing SNAP29 significantly increased the number of autophagosomes and concomitantly promoted HBV replication and HBsAg production. Conversely, SNAP29 overexpression decreased HBV production. Consistent with this, SNAP29 modulated HBV production by interacting with vesicle-associated membrane protein 8 (VAMP8) and synergistically regulated HBV replication with Rab7 complexes. Moreover, the production and release of the small HBsAg is strongly regulated by SNAP29 expression, suggesting that its export occurs partly through the autophagic pathway. Our findings provide new evidence, strongly suggesting that autophagic degradation critically determines the production of HBV virions and HBsAg and that this is controlled by the SNAP29-VAMP8 interaction.-Lin, Y., Wu, C., Wang, X., Liu, S., Kemper, T., Li, F., Squire, A., Zhu, Y., Zhang, J., Chen, X., Lu, M. Synaptosomal-associated protein 29 is required for the autophagic degradation of hepatitis B virus.

Comparative study of commercially available and homemade anti-VAMP7 antibodies using CRISPR/Cas9-depleted HeLa cells and VAMP7 knockout mice.

VAMP7 (vesicle-associated membrane protein) belongs to the intracellular membrane fusion SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein family. In this study, we used CRISPR/Cas9 genome editing technology to generate VAMP7 knockout (KO) human HeLa cells and mouse KO brain extracts in order to test the specificity and the background of a set of commercially available and homemade anti-VAMP7 antibodies. We propose a simple profiling method to analyze western blotting and use visual scoring for immunocytochemistry staining to determine the extent of the antibodies' specificity. Thus, we were able to rank the performance of a set of available antibodies and further showed an optimized procedure for VAMP7 immunoprecipitation, which we validated using wild-type and KO mouse brain extracts.

Regulation of plasma membrane H+-ATPase activity by the members of the V-SNARE VAMP7C family in arabidopsis thaliana

ABSTRACTThe plasma membrane (PM) H+-ATPase plays an important role in response to environmental stress, such as salt-alkaline stress. We have reported that the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein Vesicle-associated membrane protein 711 (VAMP711) is involved in drought stress by regulating PM H+-ATPase activity directly. In this study, we report that VAMP7C (VAMP711-14) represses PM H+-ATPase activity in response to high-pH stress. The Arabidopsis PM H+-ATPase AHA2 interacts with VAMP7C. The longin domain of VAMP711 interacts with AHA2 to inhibit PM H+-ATPase activity on the plasma membrane. The alkaline phenotype of vamp711 and vamp711 vamp712 vamp713 triple mutants suggested that VAMP7C was functionally redundant in regulating PM H+-ATPase activity in response to high-pH stress.

MicroRNA-185 inhibits proliferation, migration and invasion in human osteosarcoma MG63 cells by targeting vesicle-associated membrane protein 2

MicroRNAs (miRNAs) play an essential role in cancer development. Several studies have indicated that miRNAs mediate tumorigenesis processes, such as inflammation, proliferation, apoptosis and invasion. In the present study, we aimed to investigate the role of the microRNA-185 (miR-185) on the proliferation, migration and invasion of osteosarcoma (OS). MiR-185 expression was reduced in OS tissues and OS cell lines. MiR-185 inhibited OS cell proliferation, migration and invasion. Furthermore, a dual-luciferase assay validated that vesicle-associated membrane protein 2 (VAMP2) was a direct target of miR-185. Overexpression of miR-185 in OC cells reduced VAMP2 expression in protein and mRNA levels, whereas suppression of miR-185 led to an increase in VAMP2 protein and mRNA levels. In addition, we found that VAMP2 silencing inhibited the OS cell proliferation, migration and invasion. Further studies verified that introducing VAMP2 mRNA into cells over-expressing miR-185 abrogated the effects of miR-185 on OS cell proliferation, migration and invasion. Therefore, these results confirm that decreased expression of miR-185 might be regarded as a tumor marker for the early diagnosis of OS, by manipulating of its interactive factors with VAMP2, to provide an effective novel therapeutic target for treatment of the OS tumor.

LncSNHG14 promotes the development and progression of bladder cancer by targeting miRNA-150-5p.

To elucidate whether lncSNHG14 could influence the proliferative potential and cell cycle progression of bladder cancer cells via binding to microRNA-150-5p (miRNA-150-5p). We aim to investigate the potential mechanism of miRNA-150-5p in the occurrence and progression of bladder cancer (BCa). Expression levels of SNHG14 and miRNA-150-5p in BCa tissues and normal bladder tissues were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Their expressions in BCa cell lines were detected as well. Regulatory effects of NHG14 and miRNA-150-5p on proliferative potential and cell cycle progression were evaluated by cell counting kit-8 (CCK-8) and flow cytometry, respectively. Through the dual-luciferase reporter gene assay, binding conditions between SNHG14 and miRNA-150-5p, as well as between miRNA-150-5p and synaptic vesicle-associated membrane protein 2 (VAMP2), were verified. Finally, rescue experiments were performed to clarify whether SNHG14 regulated behaviors of BCa cells by absorbing miRNA-150-5p to degrade VAMP2. SNHG14 was highly expressed in BCa tissues and cell lines. The overexpression of SNHG14 accelerated the proliferative potential and cell cycle progression of BCa cells. SNHG14 was confirmed to bind to miRNA-150-5p. MiRNA-150-5p remained a low expression in BCa tissues. Moreover, miRNA-150-5p overexpression suppressed proliferative potential and cell cycle progression of BCa cells, which could reverse the promotive role of SNHG14 on behaviors of BCa cells. Furthermore, VAMP2 was the target gene of miRNA-150-5p. VAMP2 overexpression reversed the biological function of miRNA-150-5p in inhibiting proliferative potential and cell cycle progression of T24 and UC9 cells. LncSNHG14 overexpression accelerates proliferative potential and cell cycle progression of BCa cells through absorbing miRNA-150-5p to degrade VAMP2 expression.

Ammonia-N exposure alters neurohormone levels in the hemolymph and mRNA abundance of neurohormone receptors and associated downstream factors in the gills of Litopenaeus vannamei.

Effects of ammonia-N (0.05, 2, 10 and 20 mg l-1) on the neuroendocrine regulation of ammonia transport were investigated in Litopenaeus vannamei The results showed that corticotrophin-releasing hormone, adrenocorticotropic hormone, dopamine, noradrenaline and 5-hydroxytryptamine concentrations in all ammonia-N groups increased significantly between 3 and 12 h. Cortisol increased significantly between 3 and 24 h. All hormones except crustacean hyperglycemic hormone were reduced to control levels. mRNA abundance of guanylyl cyclase increased significantly during the experiment. Dopamine receptor D4 and α2 adrenergic receptor mRNA abundance in treatments decreased significantly at the beginning, and eventually returned to the control level, whereas mRNA abundance of the 5-HT7 receptor increased significantly only within the first 12 h. Changes in protein kinase (PKA, PKG) mRNA abundance were similar to the patterns of biogenic amines and crustacean hyperglycemic hormone, peaking at 6 and 12 h, respectively, whereas PKC mRNA abundance decreased within 24 h. 14-3-3 protein, FXYD2 and cAMP-response element binding protein mRNA abundance increased significantly and peaked at 6 h. β-catenin and T-cell factor mRNA abundance increased significantly throughout the experiment and peaked at 12 h. The upregulation of Rh protein, K+ channel, Na+/K+-ATPase, V-type H+-ATPase and vesicle associated membrane protein (VAMP) mRNA, together with downregulation of Na+/K+/2Cl- cotransporter mRNA, indicated an adjustment of general branchial ion-/ammonia-regulatory mechanisms. Meanwhile, hemolymph ammonia concentration was significantly increased in most ammonia-N exposure groups. Histological investigation revealed the hepatopancreatic damage caused by ammonia-N. Results suggest that hormones, biogenic amines and Wnt/β-catenin play a principal role in adapting to ammonia-N exposure and facilitating ammonia transport.

Evidence that dopamine is involved in neuroendocrine regulation, gill intracellular signaling pathways and ion regulation in Litopenaeus vannamei.

The transport of ions and ammonia in gills may be regulated by neuroendocrine factors. In order to explore the mechanism of dopamine (DA) regulation, we investigated hemolymph neuroendocrine hormones, gill intracellular signaling pathways, ion and ammonia transporters, hemolymph osmolality and ammonia concentration in Litopenaeus vannamei after injection of 10-7 and 10-6mol DA per shrimp. The data showed a significant increase in crustacean hyperglycemic hormone (CHH) concentration at 1-12 h and a transient significant decrease in corticotrophin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and cortisol concentration under DA stimulation. The up-regulation of guanylyl cyclase (GC) mRNA, cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG) concentration, together with the down-regulation of DA receptor D4 mRNA and up-regulation of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), diacylglycerol (DAG) and protein kinase C (PKC) concentration suggested the activation of complicated intracellular signaling pathways. The expression of cAMP response element-binding protein (CREB), FXYD2 and 14-3-3 protein mRNA was significantly increased by PKA regulation. The increase in Na+/K+-ATPase (NKA) activity and the stabilization of V-type H+-ATPase (V-rATPase) activity were accompanied by an up-regulation of K+ channel, Na+/K+/2Cl- cotransporter (NKCC), Rh protein and vesicle associated membrane protein (VAMP) mRNA, resulting in an increase in hemolymph osmolality and a decrease in hemolymph ammonia concentration. These results suggest that DA stimulates the secretion of CHH and inhibits the release of cortisol, which activates intracellular signaling factors to facilitate ion and ammonia transport across the gills, and may not affect intracellular acidification.

Systematic prediction of FFAT motifs across eukaryote proteomes identifies nucleolar and eisosome proteins with the predicted capacity to form bridges to the endoplasmic reticulum.

The endoplasmic reticulum (ER), the most pervasive organelle, exchanges information and material with many other organelles, but the extent of its inter-organelle connections and the proteins that form bridges are not well known. The integral ER membrane protein VAMP-associated protein (VAP) is found in multiple bridges, interacting with many proteins that contain a short linear motif consisting of "two phenylalanines in an acidic tract" (FFAT). The VAP-FFAT interaction is the most common mechanism by which cytoplasmic proteins, particularly inter-organelle bridges, target the ER. Therefore, predicting new FFAT motifs may both find new individual peripheral ER proteins and identify new routes of communication involving the ER. Here we searched for FFAT motifs across whole proteomes. The excess of eukaryotic proteins with FFAT motifs over background was ≥0.8%, suggesting this is the minimum number of peripheral ER proteins. In yeast, where VAP was previously known to bind 4 proteins with FFAT motifs, a detailed analysis of a subset of proteins predicted 20 FFAT motifs. Extrapolating these findings to the whole proteome estimated the number of FFAT motifs in yeast at approximately 50-55 (0.9% of proteome). Among these previously unstudied FFAT motifs, most have known functions outside the ER, so could be involved in inter-organelle communication. Many of these can target well-characterised membrane contact sites, however some are in nucleoli and eisosomes, organelles previously unknown to have molecular bridges to the ER. We speculate that the nucleolar and eisosomal proteins with predicted motifs may function while bridging to the ER, indicating novel ER-nucleolus and ER-eisosome routes of inter-organelle communication.

LAMP-2B regulates human cardiomyocyte function by mediating autophagosome–lysosome fusion

Mutations in lysosomal-associated membrane protein 2 (LAMP-2) gene are associated with Danon disease, which often leads to cardiomyopathy/heart failure through poorly defined mechanisms. Here, we identify the LAMP-2 isoform B (LAMP-2B) as required for autophagosome-lysosome fusion in human cardiomyocytes (CMs). Remarkably, LAMP-2B functions independently of syntaxin 17 (STX17), a protein that is essential for autophagosome-lysosome fusion in non-CMs. Instead, LAMP-2B interacts with autophagy related 14 (ATG14) and vesicle-associated membrane protein 8 (VAMP8) through its C-terminal coiled coil domain (CCD) to promote autophagic fusion. CMs derived from induced pluripotent stem cells (hiPSC-CMs) from Danon patients exhibit decreased colocalization between ATG14 and VAMP8, profound defects in autophagic fusion, as well as mitochondrial and contractile abnormalities. This phenotype was recapitulated by LAMP-2B knockout in non-Danon hiPSC-CMs. Finally, gene correction of LAMP-2 mutation rescues the Danon phenotype. These findings reveal a STX17-independent autophagic fusion mechanism in human CMs, providing an explanation for cardiomyopathy in Danon patients and a foundation for targeting defective LAMP-2B-mediated autophagy to treat this patient population.

Reduced SNAP25 Protein Fragmentation Contributes to SNARE Complex Dysregulation in Schizophrenia Postmortem Brain

Recent studies associated schizophrenia with enhanced functionality of the presynaptic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. Altered degradation pathways of the three core SNARE proteins: synaptosomal-associated protein 25 (SNAP25), syntaxin-1 and vesicle-associated membrane protein (VAMP) could contribute to enhanced complex function. To investigate these pathways, we first identified a 15-kDa SNAP25 fragment (f-S25) in human and rat brains, highly enriched in synaptosomal extractions, and mainly attached to cytosolic membranes with low hydrophobicity. The presence of f-S25 is consistent with reports of calpain-mediated SNAP25 cleavage. Co-immunoprecipitation assays showed that f-S25 retains the ability to bind syntaxin-1, which might prevent VAMP and/or Munc18-1 assembly into the complex. Quantitative analyses in postmortem human orbitofrontal cortex (OFC) revealed that schizophrenia (n = 35), but not major depression (n = 15), is associated with lower amounts of f-S25 (−37%, P = 0.027), and greater SNARE protein–protein interactions (35%, P < 0.001), compared with healthy matched controls (n = 28). Enhanced SNARE complex formation was strongly correlated with lower SNAP25 fragmentation rates (R = 0.563, P < 0.001). Statistical mediation analyses supported the hypothesis that reduced f-S25 density could upregulate SNARE fusion events in schizophrenia. Cortical calpain activity in schizophrenia did not differ from controls. f-S25 levels did not correlate with total calpain activity, indicating that if present, schizophrenia-related calpain dysfunction might occur locally at the presynaptic terminals. Overall, the present findings suggest the existence of an endogenous SNARE complex inhibitor related to SNAP25 proteolysis, associated with enhanced SNARE activity in schizophrenia.

A Golgi-Released Subpopulation of the Trans-Golgi Network Mediates Protein Secretion in Arabidopsis.

Spatiotemporal coordination of protein trafficking among organelles is essential for eukaryotic cells. The post-Golgi interface, including the trans-Golgi network (TGN), is a pivotal hub for multiple trafficking pathways. The Golgi-released independent TGN (GI-TGN) is a compartment described only in plant cells, and its cellular and physiological roles remain elusive. In Arabidopsis (Arabidopsis thaliana), the SYNTAXIN OF PLANTS (SYP) 4 group Qa-SNARE (soluble N-ethylmaleimide) membrane fusion proteins are shared components of TGN and GI-TGN and regulate secretory and vacuolar transport. Here we reveal that GI-TGNs mediate the transport of the R-SNARE VESICLE-ASSOCIATED MEMBRANE PROTEIN (VAMP) 721 to the plasma membrane. In interactions with a nonadapted powdery mildew pathogen, the SYP4 group of SNAREs is required for the dynamic relocation of VAMP721 to plant-fungus contact sites via GI-TGNs, thereby facilitating complex formation with its cognate SNARE partner PENETRATION1 to restrict pathogen entry. Furthermore, quantitative proteomic analysis of leaf apoplastic fluid revealed constitutive and pathogen-inducible secretion of cell wall-modification enzymes in a SYP4- and VAMP721-dependent manner. Hence, the GI-TGN acts as a transit compartment between the Golgi apparatus and the plasma membrane. We propose a model in which the GA-TGN matures into the GI-TGN and then into secretory vesicles by increasing the abundance of VAMP721-dependent secretory pathway components.

Genomics of lipid-laden human hepatocyte cultures enables drug target screening for the treatment of non-alcoholic fatty liver disease

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a major health burden in need for new medication. To identify potential drug targets a genomic study was performed in lipid-laden primary human hepatocyte (PHH) and human hepatoma cell cultures.MethodsPHH, HuH7 and HepG2 hepatoma cell cultures were treated with lipids and/or TNFα. Intracellular lipid load was quantified with the ORO assay. The Affymetrix HG-U133+ array system was employed to perform transcriptome analysis. The lipid droplet (LD) growth and fusion was determined by fluorescence microscopy. LD associated proteins were imaged by confocal immunofluorescence microscopy and confirmed by Western immunoblotting. Bioinformatics defined perturbed metabolic pathways.ResultsWhole genome expression profiling identified 227, 1031 and 571 significant regulated genes. Likewise, the combined lipid and TNFα treatment of PHH, HuH7 and HepG2 cell cultures revealed 154, 1238 and 278 differentially expressed genes. Although genomic responses differed among in-vitro systems, commonalities were ascertained by filtering the data for LD associated gene regulations. Among others the LD-growth and fusion associated cell death inducing DFFA like effector C (CIDEC), perilipins (PLIN2, PLIN3), the synaptosome-associated-protein 23 and the vesicle associated membrane protein 3 were strongly up-regulated. Likewise, the PPAR targets pyruvate-dehydrogenase-kinase-4 and angiopoietin-like-4 were up-regulated as was hypoxia-inducible lipid droplet-associated (HILPDA), flotilin and FGF21. Their inhibition ameliorates triglyceride and cholesterol accumulation. TNFα treatment elicited strong induction of the chemokine CXCL8, the kinases MAP3K8, MAP4K4 and negative regulators of cytokine signaling, i.e. SOCS2&SOCS3. Live cell imaging of DsRED calreticulin plasmid transfected HuH7 cells permitted an assessment of LD growth and fusion and confocal immunofluorescence microscopy evidenced induced LD-associated PLIN2, CIDEC, HIF1α, HILPDA, JAK1, PDK4 and ROCK2 expression. Notwithstanding, CPT1A protein was repressed to protect mitochondria from lipid overload. Pharmacological inhibition of the GTPase-dynamin and the fatty acid transporter-2 reduced lipid uptake by 28.5 and 35%, respectively. Finally, a comparisons of in-vitro/NAFLD patient biopsy findings confirmed common gene regulations thus demonstrating clinical relevance.ConclusionThe genomics of fat-laden hepatocytes revealed LD-associated gene regulations and perturbed metabolic pathways. Immunofluorescence microscopy confirmed expression of coded proteins to provide a rationale for therapeutic intervention strategies. Collectively, the in-vitro system permits testing of drug candidates.

Localizing Proton-Mediated Inhibitory Feedback at the Retinal Horizontal Cell-Cone Synapse with Genetically-Encoded pH Probes.

Lateral inhibition in the vertebrate retina depends on a negative feedback synapse between horizontal cells (HCs) and rod and cone photoreceptors. A change in pH is thought to be the signal for negative feedback, but its spatial profile in the synaptic cleft is unknown. Here we use three different membrane proteins, each fused to the same genetically-encoded pH-sensitive Green Fluorescent Protein (GFP) (pHluorin), to probe synaptic pH in retina from transgenic zebrafish (Danio rerio) of either sex. We used the cone transducin promoter to express SynaptopHluorin (pHluorin on vesicle-associated membrane protein (VAMP2)) or CalipHluorin (pHluorin on an L-type Ca2+ channel) and the HC-specific connexin-55.5 promoter to express AMPApHluorin (pHluorin on an AMPA receptor). Stimulus light led to increased fluorescence of all three probes, consistent with alkalinization of the synaptic cleft. The receptive field size, sensitivity to surround illumination, and response to activation of an alien receptor expressed exclusively in HCs, are consistent with lateral inhibition as the trigger for alkalinization. However, SynaptopHluorin and AMPApHluorin, which are displaced farther from cone synaptic ribbons than CalipHluorin, reported a smaller pH change. Hence, unlike feedforward glutamatergic transmission, which spills over to allow cross talk between terminals in the cone network, the pH change underlying HC feedback is compartmentalized to individual synaptic invaginations within a cone terminal, consistent with private line communication.SIGNIFICANCE STATEMENT Lateral inhibition (LI) is a fundamental feature of information processing in sensory systems, enhancing contrast sensitivity and enabling edge discrimination. Horizontal cells (HCs) are the first cellular substrate of LI in the vertebrate retina, but the synaptic mechanisms underlying LI are not completely understood, despite decades of study. This paper makes a significant contribution to our understanding of LI, by showing that each HC-cone synapse is a "private-line" that operates independently from other HC-cone connections. Using transgenic zebrafish expressing pHluorin, a pH-sensitive GFP variant spliced onto three different protein platforms expressed either in cones or HCs we show that the feedback pH signal is constrained to individual cone terminals, and more stringently, to individual synaptic contact sites within each terminal.