SNARE Complex Assembly Research Articles

Atractylenolide I inhibits angiogenesis and reverses sunitinib resistance in clear cell renal cell carcinoma through ATP6V0D2-mediated autophagic degradation of EPAS1/HIF2α

ABSTRACT Clear cell renal cell carcinoma (ccRCC) is tightly associated with VHL (von Hippel-Lindau tumor suppressor) mutation and dysregulated angiogenesis. Accumulating evidence indicates that antiangiogenic treatment abolishing tumor angiogenesis can achieve longer disease-free survival in patients with ccRCC. Atractylenolide I (ATL-I) is one of the main active compounds in Atractylodes macrocephala root extract and exhibits various pharmacological effects, including anti-inflammatory and antitumor effects. In this study, we revealed the potent antitumor activity of ATL-I in ccRCC. ATL-I exhibited robust antiangiogenic capacity by inhibiting EPAS1/HIF2α-mediated VEGFA production in VHL-deficient ccRCC, and it promoted autophagic degradation of EPAS1 by upregulating the ATPase subunit ATP6V0D2 (ATPase H+ transporting V0 subunit d2) to increase lysosomal function and facilitated fusion between autophagosomes and lysosomes. Mechanistically, ATP6V0D2 directly bound to RAB7 and VPS41 and promoted the RAB7-HOPS interaction, facilitating SNARE complex assembly and autophagosome-lysosome fusion. Moreover, ATP6V0D2 promoted autolysosome degradation by increasing the acidification and activity of lysosomes during the later stages of macroautophagy/autophagy. Additionally, we found that ATL-I could decrease the level of EPAS1, which was upregulated in sunitinib-resistant cells, thus reversing sunitinib resistance. Collectively, our findings demonstrate that ATL-I is a robust antiangiogenic and antitumor lead compound with potential clinical application for ccRCC therapy.

Migfilin promotes autophagic flux through direct interaction with SNAP29 and Vamp8.

Autophagy plays a crucial role in cancer cell survival by facilitating the elimination of detrimental cellular components and the recycling of nutrients. Understanding the molecular regulation of autophagy is critical for developing interventional approaches for cancer therapy. In this study, we report that migfilin, a focal adhesion protein, plays a novel role in promoting autophagy by increasing autophagosome-lysosome fusion. We found that migfilin is associated with SNAP29 and Vamp8, thereby facilitating Stx17-SNAP29-Vamp8 SNARE complex assembly. Depletion of migfilin disrupted the formation of the SNAP29-mediated SNARE complex, which consequently blocked the autophagosome-lysosome fusion, ultimately suppressing cancer cell growth. Restoration of the SNARE complex formation rescued migfilin-deficiency-induced autophagic flux defects. Finally, we found depletion of migfilin inhibited cancer cell proliferation. SNARE complex reassembly successfully reversed migfilin-deficiency-induced inhibition of cancer cell growth. Taken together, our study uncovers a new function of migfilin as an autophagy-regulatory protein and suggests that targeting the migfilin-SNARE assembly could provide a promising therapeutic approach to alleviate cancer progression.

Tomosyns attenuate SNARE assembly and synaptic depression by binding to VAMP2-containing template complexes

Tomosyns are widely thought to attenuate membrane fusion by competing with synaptobrevin-2/VAMP2 for SNARE-complex assembly. Here, we present evidence against this scenario. In a novel mouse model, tomosyn-1/2 deficiency lowered the fusion barrier and enhanced the probability that synaptic vesicles fuse, resulting in stronger synapses with faster depression and slower recovery. While wild-type tomosyn-1m rescued these phenotypes, substitution of its SNARE motif with that of synaptobrevin-2/VAMP2 did not. Single-molecule force measurements indeed revealed that tomosyn’s SNARE motif cannot substitute synaptobrevin-2/VAMP2 to form template complexes with Munc18-1 and syntaxin-1, an essential intermediate for SNARE assembly. Instead, tomosyns extensively bind synaptobrevin-2/VAMP2-containing template complexes and prevent SNAP-25 association. Structure-function analyses indicate that the C-terminal polybasic region contributes to tomosyn’s inhibitory function. These results reveal that tomosyns regulate synaptic transmission by cooperating with synaptobrevin-2/VAMP2 to prevent SNAP-25 binding during SNARE assembly, thereby limiting initial synaptic strength and equalizing it during repetitive stimulation.

The role of Munc18 in regulating the spatial arrangement of Syntaxin-1A and SNARE complex assembly

The role of Munc18 in regulating the spatial arrangement of Syntaxin-1A and SNARE complex assembly

Domain 3a mutation of VPS33A suppresses larval arrest phenotype in the loss of VPS45 in Caenorhabditis elegans.

The Sec1/Munc18 (SM) protein VPS45 is a key regulator of SNARE-mediated membrane fusion in endosomal trafficking, but its precise role remains unknown. To understand the function of VPS45 in vivo , we performed a genetic suppressor screen in Caenorhabditis elegans . We found that the temperature-sensitive lethality caused by the loss of VPS-45 can be suppressed by a mutation in another SM protein, VPS33A. The VPS33A M376I mutation is located in domain 3a, which is predicted to be essential for SNARE complex assembly. These results highlight the functional importance of domain 3a in endosomal SM proteins and its role in specific membrane fusion.

α-Synuclein is the major platelet isoform but is dispensable for activation, secretion, and thrombosis

Platelets play many roles in the vasculature ensuring proper hemostasis and maintaining integrity. These roles are facilitated, in part, by cargo molecules released from platelet granules via Soluble NSF Attachment Protein Receptor (SNARE) mediated membrane fusion, which is controlled by several protein-protein interactions. Chaperones have been characterized for t-SNAREs (i.e. Munc18b for Syntaxin-11), but none have been clearly identified for v-SNAREs. α-Synuclein has been proposed as a v-SNARE chaperone which may affect SNARE-complex assembly, fusion pore opening, and thus secretion. Despite its abundance and that it is the only isoform present, α-synuclein’s role in platelet secretion is uncharacterized. In this study, immunofluorescence showed that α-synuclein was present on punctate structures that co-stained with markers for α-granules and lysosomes and in a cytoplasmic pool. We analyzed the phenotype of α-synuclein−/− mice and their platelets. Platelets from knockout mice had a mild, agonist-dependent secretion defect but aggregation and spreading in vitro were unaffected. Consistently, thrombosis/hemostasis were unaffected in the tail-bleeding, FeCl3 carotid injury and jugular vein puncture models. None of the platelet secretory machinery examined, e.g. the v-SNAREs, were affected by α-synuclein’s loss. The results indicate that, despite its abundance, α-synuclein has only a limited role in platelet function and thrombosis.

The release of inhibition model reproduces kinetics and plasticity of neurotransmitter release in central synapses

Calcium-evoked release of neurotransmitters from synaptic vesicles (SVs) is catalysed by SNARE proteins. The predominant view is that, at rest, complete assembly of SNARE complexes is inhibited (‘clamped’) by synaptotagmin and complexin molecules. Calcium binding by synaptotagmins releases this fusion clamp and triggers fast SV exocytosis. However, this model has not been quantitatively tested over physiological timescales. Here we describe an experimentally constrained computational modelling framework to quantitatively assess how the molecular architecture of the fusion clamp affects SV exocytosis. Our results argue that the ‘release-of-inhibition’ model can indeed account for fast calcium-activated SV fusion, and that dual binding of synaptotagmin-1 and synaptotagmin-7 to the same SNARE complex enables synergistic regulation of the kinetics and plasticity of neurotransmitter release. The developed framework provides a powerful and adaptable tool to link the molecular biochemistry of presynaptic proteins to physiological data and efficiently test the plausibility of calcium-activated neurotransmitter release models.

Expression and function of Caenorhabditis elegans UNCP-18, a paralog of the SM protein UNC-18.

Sec1/Munc18 (SM) proteins are important regulators of SNARE complex assembly during exocytosis throughout all major animal tissue types. However, expression of a founding member of the SM family, UNC-18, is mostly restricted to the nervous system of the nematode Caenorhabditis elegans, where it is important for synaptic transmission. Moreover, unc-18 null mutants do not display the lethality phenotype associated with (a) loss of all Drosophila and mouse orthologs of unc-18 and (b) with complete elimination of synaptic transmission in C. elegans. We investigated whether a previously uncharacterized unc-18 paralog, which we named uncp-18, may be able to explain the restricted expression and limited phenotypes of unc-18 null mutants. A reporter allele shows ubiquitous expression of uncp-18. Analysis of uncp-18 null mutants, unc-18 and uncp-18 double null mutants, as well as overexpression of uncp-18 in an unc-18 null mutant background, shows that these 2 genes can functionally compensate for one another and are redundantly required for embryonic viability. Our results indicate that the synaptic transmission defects of unc-18 null mutants cannot necessarily be interpreted as constituting a null phenotype for SM protein function at the synapse.

Docosahexaenoic acid promotes vesicle clustering mediated by alpha-Synuclein via electrostatic interaction.

α-Synuclein (α-Syn) is an intrinsically disordered protein whose aggregation is associated with Parkinson's disease, dementia, and other neurodegenerative diseases known as synucleinopathies. However, the functional role of α-Syn is still unclear, although it has been shown to be involved in the regulation of neurotransmitter release via the interaction with synaptic vesicles (SVs), vesicle clustering, and SNARE complex assembly. Fatty acids have significant occupancy in synaptic vesicles; and recent studies suggest the interaction of fatty acids with α-Syn affect the formation of (pathological) aggregates, but it is less clear how fatty acids affects the functional role of α-Syn including α-Syn-membrane interactions, in particular with (SV-like) vesicles. Here, we report the concentration dependent effect of docosahexaenoic acid (DHA) in synaptic-like vesicle clustering via α-Syn interaction. Through molecular dynamics simulation, we revealed that DHA promoted vesicle clustering is due to the electrostatic interaction between DHA in the membrane and the N-terminal region of α-Syn. Moreover, this increased electrostatic interaction arises from a change in the macroscopic properties of the protein-membrane interface induced by (preferential solvation of) DHA. Our results provide insight as to how DHA regulates vesicle clustering mediated by α-Syn and may further be useful to understand its physiological as well as pathological role. Description: In physiological environments, α-Synuclein (α-Syn) localizes at nerve termini and synaptic vesicles and interacts with anionic phospholipid membranes to promote vesicle clustering. Docosahexaenoic acid (DHA) increases binding affinity between α-Syn and lipid membranes by increasing electrostatic interaction energy through modulating the local and global membrane environment and conformational properties of α-Syn.

Identification of residues critical for the extension of Munc18-1 domain 3a

BackgroundNeurotransmitter release depends on the fusion of synaptic vesicles with the presynaptic membrane and is mainly mediated by SNARE complex assembly. During the transition of Munc18-1/Syntaxin-1 to the SNARE complex, the opening of the Syntaxin-1 linker region catalyzed by Munc13-1 leads to the extension of the domain 3a hinge loop, which enables domain 3a to bind SNARE motifs in Synaptobrevin-2 and Syntaxin-1 and template the SNARE complex assembly. However, the exact mechanism of domain 3a extension remains elusive.ResultsHere, we characterized residues on the domain 3a hinge loop that are crucial for the extension of domain 3a by using biophysical and biochemical approaches and electrophysiological recordings. We showed that the mutation of residues T323/M324/R325 disrupted Munc13-1-mediated SNARE complex assembly and membrane fusion starting from Munc18-1/Syntaxin-1 in vitro and caused severe defects in the synaptic exocytosis of mouse cortex neurons in vivo. Moreover, the mutation had no effect on the binding of Synaptobrevin-2 to isolated Munc18-1 or the conformational change of the Syntaxin-1 linker region catalyzed by the Munc13-1 MUN domain. However, the extension of the domain 3a hinge loop in Munc18-1/Syntaxin-1 was completely disrupted by the mutation, leading to the failure of Synaptobrevin-2 binding to Munc18-1/Syntaxin-1.ConclusionsTogether with previous results, our data further support the model that the template function of Munc18-1 in SNARE complex assembly requires the extension of domain 3a, and particular residues in the domain 3a hinge loop are crucial for the autoinhibitory release of domain 3a after the MUN domain opens the Syntaxin-1 linker region.

The exocyst complex in neurological disorders.

Exocytosis is the process by which secretory vesicles fuse with the plasma membrane to deliver materials to the cell surface or to release cargoes to the extracellular space. The exocyst-an evolutionarily conserved octameric protein complex-mediates spatiotemporal control of SNARE complex assembly for vesicle fusion and tethering the secretory vesicles to the plasma membrane. The exocyst participates in diverse cellular functions, including protein trafficking to the plasma membrane, membrane extension, cell polarity, neurite outgrowth, ciliogenesis, cytokinesis, cell migration, autophagy, host defense, and tumorigenesis. Exocyst subunits are essential for cell viability; and mutations or variants in several exocyst subunits have been implicated in human diseases, mostly neurodevelopmental disorders and ciliopathies. These conditions often share common features such as developmental delay, intellectual disability, and brain abnormalities. In this review, we summarize the mutations and variants in exocyst subunits that have been linked to disease and discuss the implications of exocyst dysfunction in other disorders.

The complement inhibitor CD59 is required for GABAergic synaptic transmission in the dentate gyrus

Complement-dependent microglia pruning of excitatory synapses has been widely reported in physiological and pathological conditions, with few reports concerning pruning of inhibitory synapses or direct regulation of synaptic transmission by complement components. Here, we report that loss of CD59, an important endogenous inhibitor of the complement system, leads to compromised spatial memory performance. Furthermore, CD59 deficiency impairs GABAergic synaptic transmission in the hippocampal dentate gyrus (DG). This depends on regulation of GABA release triggered by Ca2+ influx through voltage-gated calcium channels (VGCCs) rather than inhibitory synaptic pruning by microglia. Notably, CD59 colocalizes with inhibitory pre-synaptic terminals and regulates SNARE complex assembly. Together, these results demonstrate that the complement regulator CD59 plays an important role in normal hippocampal function.

STING activation in platelets aggravates septic thrombosis by enhancing platelet activation and granule secretion

Sepsis is a dysregulated inflammatory consequence of systemic infection. As a result, excessive platelet activation leads to thrombosis and coagulopathy, but we currently lack sufficient understanding of these processes. Here, using the cecal ligation and puncture (CLP) model of sepsis, we observed septic thrombosis and neutrophil extracellular trap formation (NETosis) within the mouse vasculature by intravital microscopy. STING activation in platelets was a critical driver of sepsis-induced pathology. Platelet-specific STING deficiency suppressed platelet activation and granule secretion, which alleviated sepsis-induced intravascular thrombosis and NETosis in mice. Mechanistically, sepsis-derived cGAMP promoted the binding of STING to STXBP2, the assembly of SNARE complex, granule secretion, and subsequent septic thrombosis, which probably depended on the palmitoylation of STING. We generated a peptide, C-ST5, to block STING binding to STXBP2. Septic mice treated with C-ST5 showed reduced thrombosis. Overall, platelet activation via STING reveals a potential strategy for limiting life-threatening sepsis-mediated coagulopathy.

SMN linked to SNARE complex assembly.

SMN linked to SNARE complex assembly.

A spinal muscular atrophy modifier implicates the SMN protein in SNARE complex assembly at neuromuscular synapses

Reduced survival motor neuron (SMN) protein triggers the motor neuron disease, spinal muscular atrophy (SMA). Restoring SMN prevents disease, but it is not known how neuromuscular function is preserved. We used model mice to map and identify an Hspa8G470R synaptic chaperone variant, which suppressed SMA. Expression of the variant in the severely affected mutant mice increased lifespan >10-fold, improved motor performance, and mitigated neuromuscular pathology. Mechanistically, Hspa8G470R altered SMN2 splicing and simultaneously stimulated formation of a tripartite chaperone complex, critical for synaptic homeostasis, by augmenting its interaction with other complex members. Concomitantly, synaptic vesicular SNARE complex formation, which relies on chaperone activity for sustained neuromuscular synaptic transmission, was found perturbed in SMA mice and patient-derived motor neurons and was restored in modified mutants. Identification of the Hspa8G470R SMA modifier implicates SMN in SNARE complex assembly and casts new light on how deficiency of the ubiquitous protein causes motor neuron disease.

Mass spectrometry uncovers intermediates and off-pathway complexes for SNARE complex assembly

The SNARE complex assembles from vesicular Synaptobrevin-2 as well as Syntaxin-1 and SNAP25 both anchored to the presynaptic membrane. It mediates fusion of synaptic vesicles with the presynaptic plasma membrane resulting in exocytosis of neurotransmitters. While the general sequence of SNARE complex formation is well-established, our knowledge on possible intermediates and stable off-pathway complexes is incomplete. We, therefore, follow the stepwise assembly of the SNARE complex and target individual SNAREs, binary sub-complexes, the ternary SNARE complex as well as interactions with Complexin-1. Using native mass spectrometry, we identify the stoichiometry of sub-complexes and monitor oligomerisation of various assemblies. Importantly, we find that interactions with Complexin-1 reduce multimerisation of the ternary SNARE complex. Chemical cross-linking provides detailed insights into these interactions suggesting a role for membrane fusion. In summary, we unravel the stoichiometry of intermediates and off-pathway complexes and compile a road map of SNARE complex assembly including regulation by Complexin-1.

MitoSNARE Assembly and Disassembly Factors Regulate Basal Autophagy and Aging in C. elegans

SNARE proteins reside between opposing membranes and facilitate vesicle fusion, a physiological process ubiquitously required for secretion, endocytosis and autophagy. With age, neurosecretory SNARE activity drops and is pertinent to age-associated neurological disorders. Despite the importance of SNARE complex assembly and disassembly in membrane fusion, their diverse localization hinders the complete understanding of their function. Here, we revealed a subset of SNARE proteins, the syntaxin SYX-17, the synaptobrevins VAMP-7, SNB-6 and the tethering factor USO-1, to be either localized or in close proximity to mitochondria, in vivo. We term them mitoSNAREs and show that animals deficient in mitoSNAREs exhibit increased mitochondria mass and accumulation of autophagosomes. The SNARE disassembly factor NSF-1 seems to be required for the effects of mitoSNARE depletion. Moreover, we find mitoSNAREs to be indispensable for normal aging in both neuronal and non-neuronal tissues. Overall, we uncover a previously unrecognized subset of SNAREs that localize to mitochondria and propose a role of mitoSNARE assembly and disassembly factors in basal autophagy regulation and aging.

Elucidating the role of the Hsc70 chaperone in SNARE complex assembly.

The complex mechanism of synaptic vesicle fusion with the plasma membrane for neurotransmitter release is initiated by the formation of the SNARE complex at the presynaptic terminal. SNARE is a proteinaceous complex composed of four helices contributed by three proteins: one helix from syntaxin, two from SNAP-25 (both at the plasma membrane) and, one helix from synaptobrevin (at the vesicle). SNAP-25 is an intrinsically disordered protein that is prone to aggregation. The fusion process is tightly regulated and requires the constitutively expressed Hsp70 chaperone (Hsc70) and its co-chaperones CSPα (J protein) and Hsp110 (nucleotide exchange factor). However, the precise molecular details by which the Hsc70 system participates in the SNARE complex formation remain unknown. SNAP-25 must neither aggregate nor fold prematurely before SNARE formation. We hypothesize that Hsc70 and CSPα cooperate to chaperone SNAP-25 to keep it in folding competent state for SNARE complex formation and facilitates the bringing of the membranes to proximity for exocytosis. To test this hypothesis, we employed a bottom-up approach and studied the interaction between Hsc70 and CSPα with SNAP-25 in vitro. Using a peptide array that spans the sequence of SNAP-25, we identified three potential Hsc70-interacting sequences and designed peptides containing these sequences to test binding in solution. We characterized the interaction of SNAP-25 peptides with Hsc70 and CSPα using a combination of biochemical and biophysical techniques, such as native-PAGE, binding affinity by fluorescence anisotropy, ATP-hydrolysis activity of Hsc70, and NMR. Our peptide binding data show different affinities of SNAP-25 peptides with Hsc70 in solution. We have tentatively identified an Hsc70 binding site within SNAP-25 that is likely to represent the site used in the cell. Importantly, we showed that the aggregation of SNAP-25 is delayed in the presence of Hsc70 and CSPα.

Complexins: Ubiquitously Expressed Presynaptic Regulators of SNARE-Mediated Synaptic Vesicle Fusion.

Neurotransmitter release is a spatially and temporally tightly regulated process, which requires assembly and disassembly of SNARE complexes to enable the exocytosis of transmitter-loaded synaptic vesicles (SVs) at presynaptic active zones (AZs). While the requirement for the core SNARE machinery is shared by most membrane fusion processes, SNARE-mediated fusion at AZs is uniquely regulated to allow very rapid Ca2+-triggered SV exocytosis following action potential (AP) arrival. To enable a sub-millisecond time course of AP-triggered SV fusion, synapse-specific accessory SNARE-binding proteins are required in addition to the core fusion machinery. Among the known SNARE regulators specific for Ca2+-triggered SV fusion are complexins, which are almost ubiquitously expressed in neurons. This chapter summarizes the structural features of complexins, models for their molecular interactions with SNAREs, and their roles in SV fusion.

On the difficulties of characterizing weak protein interactions that are critical for neurotransmitter release.

The mechanism of neurotransmitter release has been extensively characterized, showing that vesicle fusion is mediated by the SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin. This complex is disassembled by N-ethylmaleimide sensitive factor (NSF) and SNAPs to recycle the SNAREs, whereas Munc18-1 and Munc13s organize SNARE complex assembly in an NSF-SNAP-resistant manner. Synaptotagmin-1 acts as the Ca2+ sensor that triggers exocytosis in a tight interplay with the SNAREs and complexins. Here, we review technical aspects associated with investigation of protein interactions underlying these steps, which is hindered because the release machinery is assembled between two membranes and is highly dynamic. Moreover, weak interactions, which are difficult to characterize, play key roles in neurotransmitter release, for instance by lowering energy barriers that need to be overcome in this highly regulated process. We illustrate the crucial role that structural biology has played in uncovering mechanisms underlying neurotransmitter release, but also discuss the importance of considering the limitations of the techniques used, including lessons learned from research in our lab and others. In particular, we emphasize: (a) the promiscuity of some protein sequences, including membrane-binding regions that can mediate irrelevant interactions with proteins in the absence of their native targets; (b) the need to ensure that weak interactions observed in crystal structures are biologically relevant; and (c) the limitations of isothermal titration calorimetry to analyze weak interactions. Finally, we stress that even studies that required re-interpretation often helped to move the field forward by improving our understanding of the system and providing testable hypotheses.