Vesicle-associated membrane protein 8 (VAMP8), a soluble n-ethylmaleimide-sensitive factor receptor protein, acts as an oncogenic gene in the progression of several malignancies. Nevertheless, the roles and mechanisms of VAMP8 in colorectal cancer (CRC) progression remain unknown. The expression and prognostic significance of VAMP8 in CRC samples were analyzed through bioinformatics analyses. Cell proliferation was detected using CCK-8 and EdU incorporation assays and apoptosis was evaluated via flow cytometry. Western blot analysis was conducted to examine the protein expression. Ferroptosis was evaluated by measurement of iron metabolism, lipid peroxidation, and glutathione (GSH) content. VAMP8 was increased in CRC samples relative to normal samples on the basis of GEPIA and HPA databases. CRC patients with high level of VAMP8 had a worse overall survival. VAMP8 depletion led to a suppression of proliferation and promotion of apoptosis in CRC cells. Additionally, VAMP8 knockdown suppressed beclin1 expression and LC3-II/LC3-I ratio, elevated p62 expression, increased Fe2+, labile iron pool, lipid reactive oxygen species, and malondialdehyde levels, and repressed GSH content and glutathione peroxidase activity. Moreover, VAMP8 knockdown inhibited the activation of janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway in CRC cells. Mechanistically, activation of the JAK/STAT3 pathway by JAK1 or JAK2 overexpression attenuated VAMP8 silencing-mediated anti-proliferative, pro-apoptotic, anti-autophagic, and pro-ferroptotic effects on CRC cells. In conclusion, VAMP8 knockdown affects the proliferation, apoptosis, autophagy, and ferroptosis by the JAK/STAT3 pathway in CRC cells.

Tuberculosis (TB) remains a significant global health concern, necessitating accurate diagnosis and treatment monitoring. Extracellular vesicles (EVs), including exosomes, play crucial roles in disease progression, with their associated genes serving as potential biomarkers and therapeutic targets. Leveraging publicly available RNA-Seq datasets of TB patients and healthy controls (HCs), to identify differentially expressed genes (DEGs) and their associated protein-protein interaction networks and immune cell profiles, the common EV-related DEGs were identified and validated in the GSE42830 and GSE40553 datasets. We have identified nine common EV-related DEGs (SERPINA1, TNFAIP6, MAPK14, STAT1, ITGA2B, VAMP5, CTSL, CEACAM1, and PLAUR) upregulated in TB patients. Immune cell infiltration analysis revealed significant differences between TB patients and HCs, highlighting increased proportions of various immune cells in TB patients. These DEGs are involved in crucial cellular processes and pathways related to exocytosis and immune response regulation. Notably, VAMP5 exhibited excellent diagnostic performance (AUC-0.993, sensitivity-93.8%, specificity-100%), with potential as a novel biomarker for TB. The EV-related genes can serve as novel potential biomarkers that can distinguish between TB and HCs. VAMP5, which functions in exosome biogenesis and showed significant upregulation in TB, can be targeted for therapeutic interventions and treatment outcomes.

The SNAP receptor (SNARE) proteins syntaxin-1, SNAP-25, and synaptobrevin mediate neurotransmitter release by forming tight SNARE complexes that fuse synaptic vesicles with the plasma membranes in microseconds. Membrane fusion is generally explained by the action of proteins on macroscopic membrane properties such as curvature, elastic modulus, and tension, and a widespread model envisions that the SNARE motifs, juxtamembrane linkers, and C-terminal transmembrane regions of synaptobrevin and syntaxin-1 form continuous helices that act mechanically as semirigid rods, squeezing the membranes together as they assemble (\"zipper\") from the N to the C termini. However, the mechanism underlying fast SNARE-induced membrane fusion remains unknown. We have used all-atom molecular dynamics simulations to investigate this mechanism. Our results need to be interpreted with caution because of the limited number and length of the simulations, but they suggest a model of membrane fusion that has a natural physicochemical basis, emphasizes local molecular events over general membrane properties, and explains extensive experimental data. In this model, the central event that initiates fast (microsecond scale) membrane fusion occurs when the SNARE helices zipper into the juxtamembrane linkers which, together with the adjacent transmembrane regions, promote encounters of acyl chains from both bilayers at the polar interface. The resulting hydrophobic nucleus rapidly expands into stalk-like structures that gradually progress to form a fusion pore, aided by the SNARE transmembrane regions and without clearly discernible intermediates. The propensity of polyunsaturated lipids to participate in encounters that initiate fusion suggests that these lipids may be important for the high speed of neurotransmitter release.

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.

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.

UNASSIGNED: Vesicle-associated membrane protein 7 (VAMP7) plays oncogenic roles in cancers. However, its clinical significance in breast cancer (BC) tissues remains unknown.
UNASSIGNED: To elucidate the clinical implications of VAMP7, as well as its involvement in the tumor microenvironment and molecular pathways of breast cancer.
UNASSIGNED: BC (n=100) and non-cancerous breast tissues (n= 100) were collected for an immunohistochemical experiment (1:200). The protein expression level of VAMP7 was determined by using a semi-quantitative scoring method. High-throughput RNA-sequencing data of BC tissues were analyzed to confirm the mRNA expression trend of VAMP7. Additionally, the largest BC prognosis cohort data were collected to mine the potential impact VAMP7 has on BC progression. The association between VAMP7 and the microenvironment of BC was evaluated by using a CIBERSORT algorithm. Moreover, we explored the co-expressed molecular mechanisms of VAMP7 in BC by calculating Pearson correlation coefficients and overexpressed genes. Finally, the biological mechanism underlying the relationship between VAMP7 and the key pathways was also explored using gene set enrichment analysis (GSEA). Potential therapeutic strategies were predicted targeting VAMP7.
UNASSIGNED: VAMP7 protein was significantly over-expressed in BC tissue than that in controls (p< 0.001). Compared with 459 normal breast tissues and 113 non-cancerous breast tissues, the expression level of VAMP7 mRNA was significantly increased in 1111 BC tissues. CD4+T cells, macrophages, and naïve B cells had a higher infiltration rate in BC tissues with high VAMP7 expression, while regulatory T cells and CD8+T cells had a lower infiltration rate. Over-expressed VAMP7 was associated with macrophages activation and transition from M1 to M2 polarization. Upregulated VAMP7 could predicted poorer OS, DMFS, PPS, and RFS outcomes. Upregulated VAMP7 co-expressed genes were significantly enriched in the cell cycle checkpoints. GSEA confirmed that over-expressed VAMP7 are markedly associated with functional enrichment in cell cycle related categories, including mitotic spindle, G2M checkpoint, and E2F targets. KU-55933 was predicted as a putative therapeutic drug for BC targeting VAMP7.
UNASSIGNED: VAMP7 was upregulated in BC tissue and correlated with poor prognosis of BC patients. VAMP7 may promote BC progression by targeting the cell cycle pathway.

Autophagy is crucial for degrading and recycling cellular components. Fusion between autophagosomes and lysosomes is pivotal, directing autophagic cargo to degradation. This process is driven by STX17-SNAP29-VAMP8 and STX7-SNAP29-YKT6 in mammalian cells. However, the interaction between STX17 and YKT6 and its significance remain to be revealed. In this study, we challenge the notion that STX17 and YKT6 function independently in autophagosome-lysosome fusion. YKT6, through its SNARE domain, forms a complex with STX17 and SNAP29 on autophagosomes, enhancing autophagy flux. VAMP8 displaces YKT6 from this complex, leading to the formation of the fusogenic complex STX17-SNAP29-VAMP8. We demonstrated that the YKT6-SNAP29-STX17 complex facilitates both lipid and content mixing driven by STX17-SNAP29-VAMP8, suggesting a priming role of YKT6 for efficient membrane fusion. Our results provide a potential regulation mechanism of autophagosome-lysosome fusion, highlighting the importance of YKT6 and its interactions with STX17 and SNAP29 in promoting autophagy flux.

In the Drosophila larval salivary gland, developmentally programmed fusions between lysosomes and secretory granules (SGs) and their subsequent acidification promote the maturation of SGs that are secreted shortly before puparium formation. Subsequently, ongoing fusions between non-secreted SGs and lysosomes give rise to degradative crinosomes, where the superfluous secretory material is degraded. Lysosomal fusions control both the quality and quantity of SGs, however, its molecular mechanism is incompletely characterized. Here we identify the R-SNARE Ykt6 as a novel regulator of crinosome formation, but not the acidification of maturing SGs. We show that Ykt6 localizes to Lamp1+ carrier vesicles, and forms a SNARE complex with Syntaxin 13 and Snap29 to mediate fusion with SGs. These Lamp1 carriers represent a distinct vesicle population that are functionally different from canonical Arl8+, Cathepsin L+ lysosomes, which also fuse with maturing SGs but are controlled by another SNARE complex composed of Syntaxin 13, Snap29 and Vamp7. Ykt6- and Vamp7-mediated vesicle fusions also determine the fate of SGs, as loss of either of these SNAREs prevents crinosomes from acquiring endosomal PI3P. Our results highlight that fusion events between SGs and different lysosome-related vesicle populations are critical for fine regulation of the maturation and crinophagic degradation of SGs.

Tethering factors play a critical role in deciphering the correct combination of vesicle and target membrane, before SNARE complex formation and membrane fusion. The exocyst plays a central role in tethering post-Golgi vesicles to the plasma membrane, although the mechanism by which this occurs is poorly understood. We recently established an assay for measuring exocyst-mediated vesicle tethering in vitro and we have adapted this assay to examine the ability of exocyst to tether vesicles in an asymmetric manner. We demonstrate that exocyst differs from another post-Golgi vesicle tethering protein, Sro7, in that it is fully capable of tethering vesicles with a functional Rab GTPase, Sec4, to vesicles lacking a functional Rab GTPase. Using this assay, we show that exocyst requires both the Rab and R-SNARE, Snc1, to be present on the same membrane surface. Using Sac1 phosphatase treatment, we demonstrate a likely role for phosphoinositides on the opposing Rab-deficient membrane. This suggests a specific model for exocyst orientation and its points of contact between membranes during heterotypic tethering of post-Golgi vesicles with the plasma membrane.

The vesicle-associated membrane protein 7 (VAMP7) is a SNARE protein of the longin family involved in a wide range of subcellular trafficking events, including neurite sprouting and elongation. The expression of the human gene SYBL1, encoding VAMP7, is finely regulated by alternative splicing. Among the minor isoforms identified so far, VAMP7j is the one most expressed and modulated in the human brain. Therefore, we focused on gaining functional evidence on VAMP7j, which lacks a functional SNARE motif but retains both the longin and transmembrane domains. In human SH-SY5Y cells, we found VAMP7j to modulate neuritogenesis by mediating transport of L1CAM toward the plasma membrane, in a fashion regulated by phosphorylation of the longin domain. VAMP7-mediated regulation of L1CAM trafficking seems at least to differentiate humans from rats, with VAMP7j CNS expression being restricted to primates, including humans. Since L1CAM is a central player in neuritogenesis and axon guidance, these findings suggest the species-specific splicing of SYBL1 is among the fine tuners of human neurodevelopmental complexity.