Role Of SNAREs Research Articles

Role of SNAREs in Unconventional Secretion-Focus on the VAMP7-Dependent Secretion.

Intracellular membrane protein trafficking is crucial for both normal cellular physiology and cell-cell communication. The conventional secretory route follows transport from the Endoplasmic reticulum (ER) to the plasma membrane via the Golgi apparatus. Alternative modes of secretion which can bypass the need for passage through the Golgi apparatus have been collectively termed as Unconventional protein secretion (UPS). UPS can comprise of cargo without a signal peptide or proteins which escape the Golgi in spite of entering the ER. UPS has been classified further depending on the mode of transport. Type I and Type II unconventional secretion are non-vesicular and non-SNARE protein dependent whereas Type III and Type IV dependent on vesicles and on SNARE proteins. In this review, we focus on the Type III UPS which involves the import of cytoplasmic proteins in membrane carriers of autophagosomal/endosomal origin and release in the extracellular space following SNARE-dependent intracellular membrane fusion. We discuss the role of vesicular SNAREs with a strong focus on VAMP7, a vesicular SNARE involved in exosome, lysosome and autophagy mediated secretion. We further extend our discussion to the role of unconventional secretion in health and disease with emphasis on cancer and neurodegeneration.

Role of SNAREs in Atopic Dermatitis–Related Cytokine Secretion and Skin-Nerve Communication

The role of soluble N-ethylmaleimide-sensitive factor attachment protein receptors in atopic dermatitis (AD) is unknown. This study identifies the function of soluble N-ethylmaleimide sensitive factor attachment protein receptor in AD-related cytokine secretion and epidermis-nerve communication. Herein, we report that various cytokines were simultaneously upregulated and coreleased in innate immunity-activated primary human keratinocytes. AD-related cytokines thymic stromal lymphopoietin, endothelin-1, and inflammatory tumor necrosis factor-α activated distinct but overlapping sensory neurons. Tumor necrosis factor-α potentiated thymic stromal lymphopoietin-induced Ca2+-influx, whereas endothelin-1 caused itch-selective B-type natriuretic peptide release. In primary human keratinocytes, B-type natriuretic peptide upregulated genes promoting dermatological and neuroinflammatory diseases and conditions. VAMP3, SNAP-29, and syntaxin 4 proved important in driving cytokine release from primary human keratinocytes. Depletion of VAMP3 inhibited nearly all the cytokine release including thymic stromal lymphopoietin and endothelin-1. Accordingly, VAMP3 co-occurred with endothelin-1 in the skins of patients with AD. Our study pinpoints the pivotal role of soluble N-ethylmaleimide sensitive factor attachment protein receptors in mediating cytokine secretion related to AD. VAMP3 is identified as a suitable target for developing broad-spectrum anticytokine therapeutics for controlling itch and atopic skin inflammation.

Recent new insights into the role of SNARE and associated proteins in insulin granule exocytosis.

Initial work on the exocytotic machinery of predocked insulin secretory granules (SGs) in pancreatic β-cells mimicked the SNARE hypothesis work in neurons, which includes SM/SNARE complex and associated priming proteins, fusion clamps and Ca2+ sensors. However, β-cell SGs, unlike neuronal synaptic vesicles, exhibit a biphasic secretory response that requires additional distinct features in exocytosis including newcomer SGs that undergo minimal docking time at the plasma membrane (PM) before fusion and multi-SG (compound) fusion. These exocytotic events are mediated by Munc18/SNARE complexes distinct from that which mediates predocked SG fusion. We review some recent insights in SNARE complex assembly and the promiscuity in SM/SNARE complex formation, whereby both contribute to conferring different insulin SG fusion kinetics. Some SNARE and associated proteins play non-fusion roles, including tethering SGs to Ca2+ channels, SG recruitment from cell interior to PM, and inhibitory SNAREs that block the action of profusion SNAREs. We discuss new insights into how sub-PM cytoskeletal mesh gates SG access to the PM and the targeting of SG exocytosis to PM domains in functionally polarized β-cells within intact islets. These recent developments have major implications on devising clever SNARE replacement therapies that could restore the deficient insulin secretion in diabetic islet β-cells.

Membrane traffic during axon development.

Brain formation requires the establishment of complex neural circuits between a diverse array of neuronal subtypes in an intricate and ever changing microenvironment and yet with a large degree of specificity and reproducibility. In the last three decades, mounting evidence has established that neuronal development relies on the coordinated regulation of gene expression, cytoskeletal dynamics, and membrane trafficking. Membrane trafficking has been considered important in that it brings new membrane and proteins to the plasma membrane of developing neurons and because it also generates and maintains the polarized distribution of proteins into neuronal subdomains. More recently, accumulating evidence suggests that membrane trafficking may have an even more active role during development by regulating the distribution and degree of activation of a wide variety of proteins located in plasma membrane subdomains and endosomes. In this article the evidence supporting the different roles of membrane trafficking during axonal development, particularly focusing on the role of SNAREs and Rabs was reviewed. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1185-1200, 2016.

SNARE Protein in Cellular Membrane Trafficking, Its Regulation and as a Potential Target for Cancer Treatment

The role of SNARE [soluble NSF (N-ethylmaleimide-sensitive factor) accessory protein receptor] protein in cellular trafficking, membrane fusion and vesicle release in synaptic nerve terminals is described. The purpose of this review is to highlight the role of SNAREs in vital inflammatory conditions in maturing dendritic cells in order to retain the capacity to present new antigens together with altered cytokine secretory functions. This role of SNAREs can be used as novel targets for therapy in inflammatory diseases. The essential mechanism of SNAREs proteins for regulation of tumour formation through multiple signals and transportation pathways is also discussed. Finally, this review summarizes the current knowledge of SNARE proteins in regulating endocytosis in cancer cells and the possible therapeutic applications related to the pathogenesis of tumor formation.

The Gos28 SNARE Protein Mediates Intra-Golgi Transport of Rhodopsin and Is Required for Photoreceptor Survival

SNARE proteins play indispensable roles in membrane fusion events in many cellular processes, including synaptic transmission and protein trafficking. Here, we characterize the Golgi SNARE protein, Gos28, and its role in rhodopsin (Rh1) transport through Drosophila photoreceptors. Mutations in gos28 lead to defective Rh1 trafficking and retinal degeneration. We have pinpointed a role for Gos28 in the intra-Golgi transport of Rh1, downstream from α-mannosidase-II in the medial- Golgi. We have confirmed the necessity of key residues in Gos28's SNARE motif and demonstrate that its transmembrane domain is not required for vesicle fusion, consistent with Gos28 functioning as a t-SNARE for Rh1 transport. Finally, we show that human Gos28 rescues both the Rh1 trafficking defects and retinal degeneration in Drosophila gos28 mutants, demonstrating the functional conservation of these proteins. Our results identify Gos28 as an essential SNARE protein in Drosophila photoreceptors and provide mechanistic insights into the role of SNAREs in neurodegenerative disease.

Plant vacuole morphology and vacuolar trafficking.

Plant vacuoles are essential organelles for plant growth and development, and have multiple functions. Vacuoles are highly dynamic and pleiomorphic, and their size varies depending on the cell type and growth conditions. Vacuoles compartmentalize different cellular components such as proteins, sugars, ions and other secondary metabolites and play critical roles in plants response to different biotic/abiotic signaling pathways. In this review, we will summarize the patterns of changes in vacuole morphology in certain cell types, our understanding of the mechanisms of plant vacuole biogenesis, and the role of SNAREs and Rab GTPases in vacuolar trafficking.

Intracellular Electroporation as Part of the Mechanism of Fusion Pore Formation in Exocytosis

The molecular physics of fusion pore formation has remained an open question for the past half century following the confirmation of exocytosis as the pathway of cellular secretion. While a number of proteins have been found to be required for docking and exocytotic secretion in a variety of biological systems, the physical mechanism of fusion of secretory vesicle and plasma membrane remains a mystery. It is possible that the role of SNARE and “fusion proteins” together with a final cytosolic Ca2+ spike may simply be to create close apposition between secretory vesicle and plasma membranes as a prelude to electroporation and fusion pore formation. In the early 1990's the similarities between electropores in secretory granule membranes generated by high electric fields and fusion pores between secretory granules and plasma membranes were pointed out by Oberhauser and Fernandez (1993, Biophys. J. 65: 1844-1852). Then in 1998, Rosenheck, (Biophys. J. 75: 1237-1243) in a theoretical analysis concluded that the electrostatic field strength generated from the inner face of the chromaffin cell membrane by negatively charged phosphatidylserine is sufficient to electroporate the chromaffin granule membrane when the latter is in close proximity (< 30 A). Further confirmation of this conclusion was recently put forward by Lutiel and coworkers ( 2007, J. Biomol. Struct & Dynamics 24: 495-503). In this poster these and related results will be presented for consideration and their strengths and limitations discussed. An essential feature of the proposed model is the idea that fusion pore formation starts as an electrostatic field-induced perturbation in the structure of the lipid bilayer of the secretory vesicle membrane. The proposed model offers new insights into the physics underlying exocytotic membrane fusion.

Is lipid flippase activity of SNARE transmembrane domains required for membrane fusion?

It has been suggested that lipids translocate between the outer and inner leaflets of fusing membranes, or flip–flop, to facilitate changes in bilayer leaflet areas at various stages of fusion. Here, we investigated the lipid flip activity of synthetic peptides that mimic SNARE transmembrane domains (TMDs). These peptides indeed induce flip of marker lipids. However, mutations that reduce flip activity do not diminish fusogenicity and cholesterol blocks flip much more efficiently than fusion. Therefore, our data do not support a role for flip in membrane fusion. On the other hand, the ability of SNARE TMDs to catalyze flip is consistent with a role of SNAREs in biogenic lipid flip.

A targeted siRNA screen to identify SNAREs required for constitutive secretion in mammalian cells.

The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry-based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein-specific siRNA screen (38 SNAREs, 4 SNARE-like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post-Golgi SNAREs SNAP-29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP-29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP-29-depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19-interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP-23, SNAP-25, SNAP-29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post-Golgi-specific R-SNARE in this process.

Membrane Fusion: Role of SNAREs and Calcium

Life processes are governed at the chemical level, and therefore knowledge of how single molecules interact, provides a fundamental understanding of nature. The molecular mechanism of membrane fusion essential to vital cellular activities such as intracellular transport, hormone secretion, enzyme release, or neurotransmission, involve the assembly and disassembly of a specialized set of proteins present in opposing bilayers. Target membrane proteins at the cell plasma membrane SNAP-25 and syntaxin termed t-SNAREs, and secretory vesicle-associated protein VAMP or v-SNARE, are part of the conserved protein complex involved in fusion of opposing membranes. It has been demonstrated that in the presence of Ca2+, t-SNAREs and v-SNARE in opposing bilayers interact and self-assemble in a circular pattern, to form conducting channels. Such self-assembly of t-/v-SNAREs in a ring conformation occurs only when the respective SNAREs are in association with membrane. X-ray diffraction measurements further demonstrate that t-SNAREs in the target membrane and v-SNARE in the vesicle membrane overcome repulsive forces to bring opposing membranes close to within a distance of 2.8 A. Studies suggest that calcium bridging of the opposing bilayers, lead to release of water from hydrated Ca2+ ions as well as the loosely coordinated water at PO-lipid head groups, leading to membrane destabilization and fusion. The t-/v-SNARE is a tight complex, who's disassembly requires an ATPase called NSF, which functions as a right-handed molecular motor.

Functions of SNAREs in intracellular membrane fusion and lipid bilayer mixing

Intracellular membrane fusion occurs with exquisite coordination and specificity. Each fusion event requires three basic components: Rab-GTPases organize the fusion site; SNARE proteins act during fusion; and N-ethylmaleimide-sensitive factor (NSF) plus its cofactor alpha-SNAP are required for recycling or activation of the fusion machinery. Whereas Rab-GTPases seem to mediate the initial membrane contact, SNAREs appear to lie at the center of the fusion process. It is known that formation of complexes between SNAREs from apposed membranes is a prerequisite for lipid bilayer mixing; however, the biophysics and many details of SNARE function are still vague. Nevertheless, recent observations are shedding light on the role of SNAREs in membrane fusion. Structural studies are revealing the mechanisms by which SNARES form complexes and interact with other proteins. Furthermore, it is now apparent that the SNARE transmembrane segment not only anchors the protein but engages in SNARE-SNARE interactions and plays an active role in fusion. Recent work indicates that the fusion process itself may comprise two stages and proceed via a hemifusion intermediate.