Intermediate steps in the formation of neuronal SNARE complexes.

Neuronal exocytosis requires the assembly of three SNARE proteins, syntaxin and SNAP25 on the plasma membrane and synaptobrevin on the vesicle membrane. However, the precise steps in this process and the points at which assembly and fusion are controlled by regulatory proteins are unclear. In the present work, we examine the kinetics and intermediate states during SNARE assembly in vitro using a combination of time resolved fluorescence and EPR spectroscopy. We show that syntaxin rapidly forms a dimer prior to forming the kinetically stable 2:1 syntaxin:SNAP25 complex, and that the 2:1 complex is not diminished by the presence of excess SNAP25. Moreover, the 2:1 complex is temperature dependent with a reduced concentration at 37°C. The two segments of SNAP25 behave differently. The N-terminal SN1 segment of SNAP25 exhibits a pronounced increase in backbone ordering from the N- to the C-terminus that is not seen in the C-terminal SNAP25 segment SN2. Both the SN1 and SN2 segments of SNAP25 will assemble with syntaxin; however, while the association of the SN1 segment with syntaxin produces a stable 2:2 (SN1:syntaxin) complex, the complex formed between SN2 and syntaxin is largely disordered. Synaptobrevin fails to bind syntaxin alone, but will associate with syntaxin in the presence of either the SN1 or SN2 segments; however, the synaptobrevin:syntaxin:SN2 complex remains disordered. Taken together, these data suggest that synaptobrevin and syntaxin do not assemble in the absence of SNAP25, and that the SN2 segment of SNAP25 is the last to enter the SNARE complex.

Legionella effectors SidC/SdcA ubiquitinate multiple small GTPases and SNARE proteins to promote phagosomal maturation.

Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and is involved in the regulation of almost all cellular signaling pathways. The intracellular bacterial pathogen Legionella pneumophila translocates at least 26 effectors to hijack host ubiquitination signaling via distinct mechanisms. Among these effectors, SidC/SdcA are novel E3 ubiquitin ligases with the adoption of a Cys-His-Asp catalytic triad. SidC/SdcA are critical for the recruitment of endoplasmic reticulum (ER)-derived vesicles to the Legionella-containing vacuole (LCV). However, the ubiquitination targets of SidC/SdcA are largely unknown, which restricts our understanding of the mechanisms used by these effectors to hijack the vesicle trafficking pathway. Here, we demonstrated that multiple Rab small GTPases and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are bona fide ubiquitination substrates of SidC/SdcA. SidC/SdcA-mediated ubiquitination of syntaxin 3 and syntaxin 4 promotes their unconventional pairing with the vesicle-SNARE protein Sec22b, thereby contributing to the membrane fusion of ER-derived vesicles with the phagosome. In addition, our data reveal that ubiquitination of Rab7 by SidC/SdcA is critical for its association with the LCV membrane. Rab7 ubiquitination could impair its binding with the downstream effector Rab-interacting lysosomal protein (RILP), which partially explains why LCVs avoid fusion with lysosomes despite the acquisition of Rab7. Taken together, our study reveals the biological mechanisms employed by SidC/SdcA to promote the maturation of the LCVs.

Ykt6 functionally overlaps with vacuolar and exocytic R-SNAREs in the yeast Saccharomyces cerevisiae.

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex forms a 4-helix coiled-coil bundle consisting of 16 layers of interacting side chains upon membrane fusion. The central layer (layer 0) is highly conserved and comprises three glutamines (Q) and one arginine (R), and thus SNAREs are classified into Qa-, Qb-, Qc-, and R-SNAREs. Homotypic vacuolar fusion in Saccharomyces cerevisiae requires the SNAREs Vam3 (Qa), Vti1 (Qb), Vam7 (Qc), and Nyv1 (R). However, the yeast strain lacking NYV1 (nyv1Δ) shows no vacuole fragmentation, whereas the vam3Δ and vam7Δ strains display fragmented vacuoles. Here, we provide genetic evidence that the R-SNAREs Ykt6 and Nyv1 are functionally redundant in vacuole homotypic fusion in vivo using a newly isolated ykt6 mutant. We observed the ykt6-104 mutant showed no defect in vacuole morphology, but the ykt6-104 nyv1Δ double mutant had highly fragmented vacuoles. Furthermore, we show the defect in homotypic vacuole fusion caused by the vam7-Q284R mutation was compensated by the nyv1-R192Q or ykt6-R165Q mutations, which maintained the 3Q:1R ratio in the layer 0 of the SNARE complex, indicating that Nyv1 is exchangeable with Ykt6 in the vacuole SNARE complex. Unexpectedly, we found Ykt6 assembled with exocytic Q-SNAREs when the intrinsic exocytic R-SNAREs Snc1 and its paralog Snc2 lose their ability to assemble into the exocytic SNARE complex. These results suggest that Ykt6 may serve as a backup when other R-SNAREs become dysfunctional and that this flexible assembly of SNARE complexes may help cells maintain the robustness of the vesicular transport network.

Apical annuli are specialised sites of post-invasion secretion of dense granules in Toxoplasma.

Apicomplexans are ubiquitous intracellular parasites of animals. These parasites use a programmed sequence of secretory events to find, invade, and then re-engineer their host cells to enable parasite growth and proliferation. The secretory organelles micronemes and rhoptries mediate the first steps of invasion. Both secrete their contents through the apical complex which provides an apical opening in the parasite's elaborate inner membrane complex (IMC) - an extensive subpellicular system of flattened membrane cisternae and proteinaceous meshwork that otherwise limits access of the cytoplasm to the plasma membrane for material exchange with the cell exterior. After invasion, a second secretion programme drives host cell remodelling and occurs from dense granules. The site(s) of dense granule exocytosis, however, has been unknown. In Toxoplasma gondii, small subapical annular structures that are embedded in the IMC have been observed, but the role or significance of these apical annuli to plasma membrane function has also been unknown. Here, we determined that integral membrane proteins of the plasma membrane occur specifically at these apical annular sites, that these proteins include SNARE proteins, and that the apical annuli are sites of vesicle fusion and exocytosis. Specifically, we show that dense granules require these structures for the secretion of their cargo proteins. When secretion is perturbed at the apical annuli, parasite growth is strongly impaired. The apical annuli, therefore, represent a second type of IMC-embedded structure to the apical complex that is specialised for protein secretion, and reveal that in Toxoplasma there is a physical separation of the processes of pre- and post-invasion secretion that mediate host-parasite interactions.

Proximity labelling reveals effects of disease-causing mutation on the DNAJC5/cysteine string protein α interactome.

Cysteine string protein α (CSPα), also known as DNAJC5, is a member of the DnaJ/Hsp40 family of co-chaperones. The name derives from a cysteine-rich domain, palmitoylation of which enables localization to intracellular membranes, notably neuronal synaptic vesicles. Mutations in the DNAJC5 gene that encodes CSPα cause autosomal dominant, adult-onset neuronal ceroid lipofuscinosis (ANCL), a rare neurodegenerative disease. As null mutations in CSP-encoding genes in flies, worms and mice similarly result in neurodegeneration, CSP is evidently an evolutionarily conserved neuroprotective protein. However, the client proteins that CSP chaperones to prevent neurodegeneration remain unclear. Traditional methods for identifying protein-protein interactions such as yeast 2-hybrid and affinity purification approaches are poorly suited to CSP, due to its requirement for membrane anchoring and its tendency to aggregate after cell lysis. Therefore, we employed proximity labelling, which enables identification of interacting proteins in situ in living cells via biotinylation. Neuroendocrine PC12 cell lines stably expressing wild type or L115R ANCL mutant CSP constructs fused to miniTurbo were generated; then the biotinylated proteomes were analysed by liquid chromatographymass spectrometry (LCMS) and validated by western blotting. This confirmed several known CSP-interacting proteins, such as Hsc70 and SNAP-25, but also revealed novel binding proteins, including STXBP1/Munc18-1. Interestingly, some protein interactions (such as Hsc70) were unaffected by the L115R mutation, whereas others (including SNAP-25 and STXBP1/Munc18-1) were inhibited. These results define the CSP interactome in a neuronal model cell line and reveal interactions that are affected by ANCL mutation and hence may contribute to the neurodegeneration seen in patients.

SNAREs: a double-edged sword for intravacuolar bacterial pathogens within host cells.

In the tug-of-war between host and pathogen, both evolve to combat each other's defence arsenals. Intracellular phagosomal bacteria have developed strategies to modify the vacuolar niche to suit their requirements best. Conversely, the host tries to target the pathogen-containing vacuoles towards the degradative pathways. The host cells use a robust system through intracellular trafficking to maintain homeostasis inside the cellular milieu. In parallel, intracellular bacterial pathogens have coevolved with the host to harbour strategies to manipulate cellular pathways, organelles, and cargoes, facilitating the conversion of the phagosome into a modified pathogen-containing vacuole (PCV). Key molecular regulators of intracellular traffic, such as changes in the organelle (phospholipid) composition, recruitment of small GTPases and associated effectors, soluble N-ethylmaleimide-sensitive factor-activating protein receptors (SNAREs), etc., are hijacked to evade lysosomal degradation. Legionella, Salmonella, Coxiella, Chlamydia, Mycobacterium, and Brucella are examples of pathogens which diverge from the endocytic pathway by using effector-mediated mechanisms to overcome the challenges and establish their intracellular niches. These pathogens extensively utilise and modulate the end processes of secretory pathways, particularly SNAREs, in repurposing the PCV into specialised compartments resembling the host organelles within the secretory network; at the same time, they avoid being degraded by the host's cellular mechanisms. Here, we discuss the recent research advances on the host-pathogen interaction/crosstalk that involves host SNAREs, conserved cellular processes, and the ongoing host-pathogen defence mechanisms in the molecular arms race against each other. The current knowledge of SNAREs, and intravacuolar bacterial pathogen interactions, enables us to understand host cellular innate immune pathways, maintenance of homeostasis, and potential therapeutic strategies to combat ever-growing antimicrobial resistance.

A SNARE protective pool antagonizes APOL1 renal toxicity in Drosophila nephrocytes.

BACKGROUND: People of Sub-Saharan African ancestry are at higher risk of developing chronic kidney disease (CKD), attributed to the Apolipoprotein L1 (APOL1) gene risk alleles (RA) G1 and G2. The underlying mechanisms by which the APOL1-RA precipitate CKD remain elusive, hindering the development of potential treatments. RESULTS: Using a Drosophila genetic modifier screen, we found that SNARE proteins (Syx7, Ykt6, and Syb) play an important role in preventing APOL1 cytotoxicity. Reducing the expression of these SNARE proteins significantly increased APOL1 cytotoxicity in fly nephrocytes, the equivalent of mammalian podocytes, whereas overexpression of Syx7, Ykt6, or Syb attenuated their toxicity in nephrocytes. These SNARE proteins bound to APOL1-G0 with higher affinity than APOL1-G1/G2, and attenuated APOL1-G0 cytotoxicity to a greater extent than either APOL1-RA. CONCLUSIONS: Using a Drosophila screen, we identified SNARE proteins (Syx7, Ykt6, and Syb) as antagonists of APOL1-induced cytotoxicity by directly binding APOL1. These data uncovered a new potential protective role for certain SNARE proteins in the pathogenesis of APOL1-CKD and provide novel therapeutic targets for APOL1-associated nephropathies.

Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis.

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

The Role of Tomosyn in the Regulation of Neurotransmitter Release.

Soluble NSF attachment protein receptor (SNARE) proteins play a central role in synaptic vesicle (SV) exocytosis. These proteins include the vesicle-associated SNARE protein (v-SNARE) synaptobrevin and the target membrane-associated SNARE proteins (t-SNAREs) syntaxin and SNAP-25. Together, these proteins drive membrane fusion between synaptic vesicles (SV) and the presynaptic plasma membrane to generate SV exocytosis. In the presynaptic active zone, various proteins may either enhance or inhibit SV exocytosis by acting on the SNAREs. Among the inhibitory proteins, tomosyn, a syntaxin-binding protein, is of particular importance because it plays a critical and evolutionarily conserved role in controlling synaptic transmission. In this chapter, we describe how tomosyn was discovered, how it interacts with SNAREs and other presynaptic regulatory proteins to regulate SV exocytosis and synaptic plasticity, and how its various domains contribute to its synaptic functions.

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

Background and Objective: The 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 Results: Using 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. Conclusion: FLNA regulates GPCR-driven platelet granule secretion and associates with SNAP23 and STX11 in an activation-dependent manner.

Lytic granule exocytosis at immune synapses: lessons from neuronal synapses.

Regulated exocytosis is a central mechanism of cellular communication. It is not only the basis for neurotransmission and hormone release, but also plays an important role in the immune system for the release of cytokines and cytotoxic molecules. In cytotoxic T lymphocytes (CTLs), the formation of the immunological synapse is required for the delivery of the cytotoxic substances such as granzymes and perforin, which are stored in lytic granules and released via exocytosis. The molecular mechanisms of their fusion with the plasma membrane are only partially understood. In this review, we discuss the molecular players involved in the regulated exocytosis of CTL, highlighting the parallels and differences to neuronal synaptic transmission. Additionally, we examine the strengths and weaknesses of both systems to study exocytosis.

SNARE Protein Snc1 Is Essential for Vesicle Trafficking, Membrane Fusion and Protein Secretion in Fungi.

Fungi are an important group of microorganisms that play crucial roles in a variety of ecological and biotechnological processes. Fungi depend on intracellular protein trafficking, which involves moving proteins from their site of synthesis to the final destination within or outside the cell. The soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are vital components of vesicle trafficking and membrane fusion, ultimately leading to the release of cargos to the target destination. The v-SNARE (vesicle-associated SNARE) Snc1 is responsible for anterograde and retrograde vesicle trafficking between the plasma membrane (PM) and Golgi. It allows for the fusion of exocytic vesicles to the PM and the subsequent recycling of Golgi-localized proteins back to the Golgi via three distinct and parallel recycling pathways. This recycling process requires several components, including a phospholipid flippase (Drs2-Cdc50), an F-box protein (Rcy1), a sorting nexin (Snx4-Atg20), a retromer submit, and the COPI coat complex. Snc1 interacts with exocytic SNAREs (Sso1/2, Sec9) and the exocytic complex to complete the process of exocytosis. It also interacts with endocytic SNAREs (Tlg1 and Tlg2) during endocytic trafficking. Snc1 has been extensively investigated in fungi and has been found to play crucial roles in various aspects of intracellular protein trafficking. When Snc1 is overexpressed alone or in combination with some key secretory components, it results in enhanced protein production. This article will cover the role of Snc1 in the anterograde and retrograde trafficking of fungi and its interactions with other proteins for efficient cellular transportation.

Biochemically Reconstituted Fusion of Phagosomes with Endosomes and Lysosomes.

Professional phagocytic cells, such as macrophages, ingest large particles into a specialized endocytic compartment, the phagosome, which eventually turns into a phagolysosome and degrades its contents. This phagosome "maturation" is governed by successive fusion of the phagosome with early sorting endosomes, late endosomes, and lysosomes. Further changes occur by fission of vesicles from the maturing phagosome and by on-and-off cycling of cytosolic proteins. We present here a detailed protocol which allows to reconstitute in a cell-free system the fusion events between phagosomes and the different endocytic compartments. This reconstitution can be used to define the identity of, and interplay between, key players of the fusion events.

Intrinsic Disorder of the Neuronal SNARE Protein SNAP25a in its Pre-fusion Conformation.

The neuronal SNARE protein SNAP25a (isoform 2) forms part of the SNARE complex eliciting synaptic vesicle fusion during neuronal exocytosis. While the post-fusion cis-SNARE complex has been studied extensively, little is known about the pre-fusion conformation of SNAP25a. Here we analyze monomeric SNAP25a by NMR spectroscopy, further supported by small-angle X-ray scattering (SAXS) experiments. SAXS data indicate that monomeric SNAP25 is more compact than a Gaussian chain but still a random coil. NMR shows that for monomeric SNAP25a, before SNAP25a interacts with its SNARE partners to drive membrane fusion, only the N-terminal part (region A5 to V36) of the first SNARE motif, SN1 (L11 - L81), is helical, comprising two α-helices (ranging from A5 to Q20 and S25 toV36). From E37 onwards, SNAP25a is mostly disordered and displays high internal flexibility, including the C-terminal part of SN1, almost the entire second SNARE motif (SN2, N144-A199), and the connecting loop region. Apart from the N-terminal helices, only the C-termini of both SN1 (E73 - K79) and SN2 (region T190 - A199), as well as two short regions in the connecting loop (D99 - K102 and E123 - M127) show a weak α-helical propensity (α-helical population < 25%). We speculate that the N-terminal helices (A5 to Q20 and S25 to V36) which constitute the N-terminus of SN1 act as a nucleation site for initiating SNARE zippering.

Forty years of the adrenal chromaffin cell through ISCCB meetings around the world.

This historical review focuses on the evolution of the knowledge accumulated during the last two centuries on the biology of the adrenal medulla gland and its chromaffin cells (CCs). The review emerged in the context of a series of meetings that started on the Spanish island of Ibiza in 1982 with the name of the International Symposium on Chromaffin Cell Biology (ISCCB). Hence, the review is divided into two periods namely, before 1982 and from this year to 2022, when the 21st ISCCB meeting was just held in Hamburg, Germany. The first historical period extends back to 1852 when Albert Kölliker first described the fine structure and function of the adrenal medulla. Subsequently, the adrenal staining with chromate salts identified the CCs; this was followed by the establishment of the embryological origin of the adrenal medulla, and the identification of adrenaline-storing vesicles. By the end of the nineteenth century, the basic morphology, histochemistry, and embryology of the adrenal gland were known. The twentieth century began with breakthrough findings namely, the experiment of Elliott suggesting that adrenaline was the sympathetic neurotransmitter, the isolation of pure adrenaline, and the deciphering of its molecular structure and chemical synthesis in the laboratory. In the 1950s, Blaschko isolated the catecholamine-storing vesicles from adrenal medullary extracts. This switched the interest in CCs as models of sympathetic neurons with an explosion of studies concerning their functions, i.e., uptake of catecholamines by chromaffin vesicles through a specific coupled transport system; the identification of several vesicle components in addition to catecholamines including chromogranins, ATP, opioids, and other neuropeptides; the calcium-dependence of the release of catecholamines; the underlying mechanism of exocytosis of this release, as indicated by the co-release of proteins; the cross-talk between the adrenal cortex and the medulla; and the emission of neurite-like processes by CCs in culture, among other numerous findings. The 1980s began with the introduction of new high-resolution techniques such as patch-clamp, calcium probes, marine toxins-targeting ion channels and receptors, confocal microscopy, or amperometry. In this frame of technological advances at the Ibiza ISCCB meeting in 1982, 11 senior researchers in the field predicted a notable increase in our knowledge in the field of CCs and the adrenal medulla; this cumulative knowledge that occurred in the last 40 years of history of the CC is succinctly described in the second part of this historical review. It deals with cell excitability, ion channel currents, the exocytotic fusion pore, the handling of calcium ions by CCs, the kinetics of exocytosis and endocytosis, the exocytotic machinery, and the life cycle of secretory vesicles. These concepts together with studies on the dynamics of membrane fusion with super-resolution imaging techniques at the single-protein level were extensively reviewed by top scientists in the field at the 21st ISCCB meeting in Hamburg in the summer of 2022; this frontier topic is also briefly reviewed here. Many of the concepts arising from those studies contributed to our present understanding of synaptic transmission. This has been studied in physiological or pathophysiological conditions, in CCs from animal disease models. In conclusion, the lessons we have learned from CC biology as a peripheral model for brain and brain disease pertain more than ever to cutting-edge research in neurobiology. In the 22nd ISCCB meeting in Israel in 2024 that Uri Asheri is organizing, we will have the opportunity of seeing the progress of the questions posed in Ibiza, and on other questions that undoubtedly will arise.

A synthetic organelle approach to probe SNARE-mediated membrane fusion in a bacterial host.

In vivo and in vitro assays, particularly reconstitution using artificial membranes, have established the role of synaptic soluble N-Ethylmaleimide-sensitive attachment protein receptors (SNAREs) VAMP2, Syntaxin-1A, and SNAP-25 in membrane fusion. However, using artificial membranes requires challenging protein purifications that could be avoided in a cell-based assay. Here, we developed a synthetic biological approach based on the generation of membrane cisternae by the integral membrane protein Caveolin in Escherichia coli and coexpression of SNAREs. Syntaxin-1A/SNAP-25/VAMP-2 complexes were formed and regulated by SNARE partner protein Munc-18a in the presence of Caveolin. Additionally, Syntaxin-1A/SNAP-25/VAMP-2 synthesis provoked increased length of E. coli only in the presence of Caveolin. We found that cell elongation required SNAP-25 and was inhibited by tetanus neurotoxin. This elongation was not a result of cell division arrest. Furthermore, electron and super-resolution microscopies showed that synaptic SNAREs and Caveolin coexpression led to the partial loss of the cisternae, suggesting their fusion with the plasma membrane. In summary, we propose that this assay reconstitutes membrane fusion in a simple organism with an easy-to-observe phenotype and is amenable to structure-function studies of SNAREs.

Beet cyst nematode HsSNARE1 interacts with both AtSNAP2 and AtPR1 and promotes disease in Arabidopsis.

INTRODUCTION: Plant parasitic cyst nematodes secrete a number of effectors into hosts to initiate formation of syncytia and infection causing huge yield losses. OBJECTIVES: The identified cyst nematode effectors are still limited, and the cyst nematode effectors-involved interaction mechanisms between cyst nematodes and plants remain largely unknown. METHODS: The t-SNARE domain-containing effector in beet cyst nematode (BCN) was identified by In situ hybridization and immunohistochemistry analyses. The mutant of effector gene was designed by protein structure modeling analysis. The functions of effector gene and its mutant were analyzed by genetic transformation in Arabidopsis and infection by BCN. The protein-protein interaction was analyzed by yeast two hybrid, BiFC and pulldown assays. Gene expression was assayed by quantitative real-time PCR. RESULTS: A t-SNARE domain-containing BCN HsSNARE1 was identified as an effector, and its mutant HsSNARE1-M1 carrying three mutations (E141D, A143T and -148S) that altered regional structure from random coils to α-helixes was designed and constructed. Transgenic analyses indicated that expression of HsSNARE1 significantly enhanced while expression of HsSNARE1-M1 and highly homologous HgSNARE1 remarkably suppressed BCN susceptibility of Arabidopsis. HsSNARE1 interacted with AtSNAP2 and AtPR1 via its t-SNARE domain and N-terminal, respectively, while HsSNARE1-M1/HgSNARE1 could not interact with AtPR1 but bound AtSNAP2. AtSNAP2, AtSHMT4 and AtPR1 interacted pairwise, but neither HsSNARE1 nor HsSNARE1-M1/HgSNARE1 could interact with AtSHMT4. Expression of HsSNARE1 significantly suppressed while expression of HsSNARE1-M1/HgSNARE1 considerably induced both AtSHMT4 and AtPR1 in transgenic Arabidopsis infected with BCN. Overexpression of AtPR1 significantly suppressed BCN susceptibility of Arabidopsis. CONCLUSIONS: This work identified a t-SNARE-domain containing cyst nematode effector HsSNARE1 and deciphered a molecular mode of action of the t-SNARE-domain containing cyst nematode effectors that HsSNARE1 promotes cyst nematode disease by interaction with both AtSNAP2 and AtPR1 and significant suppression of both AtSHMT4 and AtPR1, which is mediated by three structure change-causing amino acid residues.

Distribution of proteins for synaptic release in nerve endings associated with the trachealis muscle of rats.

The immunohistochemical localization of proteins for synaptic release was examined in smooth muscle-associated sensory nerve endings using whole-mount preparations of the rat trachea. Plant-like smooth muscle-associated nerve endings with immunoreactivity for Na+-K+-ATPase, α3-subunit were identified in the trachealis muscle. VGLUT1, synapsin1, t-SNARE proteins (SNAP25 and syntaxin1), v-SNARE proteins (VAMP1 and VAMP2), and a presynaptic active zone-related protein (piccolo) were detected in the terminal parts of these endings. These results suggest that smooth muscle-associated nerve endings secrete glutamate to modulate sensorimotor functions in the lung deflation reflex.

A GHKNN model based on the physicochemical property extraction method to identify SNARE proteins.

There is a great deal of importance to SNARE proteins, and their absence from function can lead to a variety of diseases. The SNARE protein is known as a membrane fusion protein, and it is crucial for mediating vesicle fusion. The identification of SNARE proteins must therefore be conducted with an accurate method. Through extensive experiments, we have developed a model based on graph-regularized k-local hyperplane distance nearest neighbor model (GHKNN) binary classification. In this, the model uses the physicochemical property extraction method to extract protein sequence features and the SMOTE method to upsample protein sequence features. The combination achieves the most accurate performance for identifying all protein sequences. Finally, we compare the model based on GHKNN binary classification with other classifiers and measure them using four different metrics: SN, SP, ACC, and MCC. In experiments, the model performs significantly better than other classifiers.

ß1-Integrin-A Key Player in Controlling Pancreatic Beta-Cell Insulin Secretion via Interplay With SNARE Proteins.

Shortcomings in cell-based therapies for patients with diabetes have been revealed to be, in part, a result of an improper extracellular matrix (ECM) environment. In vivo, pancreatic islets are emersed in a diverse ECM that provides physical support and is crucial for healthy function. ß1-Integrin receptors have been determined to be responsible for modulation of beneficial interactions with ECM proteins influencing beta-cell development, proliferation, maturation, and function. ß1-Integrin signaling has been demonstrated to augment insulin secretion by impacting the actin cytoskeleton via activation of focal adhesion kinase and downstream signaling pathways. In other secretory cells, evidence of a bidirectional relationship between integrins and exocytotic machinery has been demonstrated, and, thus, this relationship could be present in pancreatic beta cells. In this review, we will discuss the role of ECM-ß1-integrin interplay with exocytotic proteins in controlling pancreatic beta-cell insulin secretion through their dynamic and unique signaling pathway.