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.

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.

Chemotherapeutic resistance is one of the most common reasons for poor prognosis of patients with nasopharyngeal carcinoma (NPC). We found that CENPN can promote the growth, proliferation and apoptosis resistance of NPC cells, but its relationship with chemotherapeutic resistance in NPC is unclear. Here we verified that the CENPN expression level in NPC patients was positively correlated with the degree of paclitaxel (PTX) resistance and a poor prognosis through analysis of clinical cases. VAMP8 expression was significantly increased after knockdown of CENPN by transcriptome sequencing. We found in cell experiments that CENPN inhibited macroautophagy/autophagy and VAMP8 expression and significantly increased PTX resistance. Overexpression of CENPN reduced the inhibitory effects of PTX on survival, cell proliferation, cell cycle progression and apoptosis resistance in NPC cells by inhibiting autophagy. In turn, knockdown of CENPN can affect the phenotype of NPC cells by increasing autophagy to achieve PTX sensitization. Sequential knockdown of CENPN and VAMP8 reversed the PTX-sensitizing effect of CENPN knockdown alone. Experiments in nude mice confirmed that knockdown of CENPN can increase VAMP8 expression, enhance autophagy and increase the sensitivity of NPC cells to PTX. Mechanistic studies showed that CENPN inhibited the translocation of p-CREB into the nucleus of NPC cells, resulting in the decreased binding of p-CREB to the VAMP8 promoter, thereby inhibiting the transcription of VAMP8. These results demonstrate that CENPN may be a marker for predicting chemotherapeutic efficacy and a potential target for inducing chemosensitization to agents such as PTX.Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; CENPN: centromere protein N; CQ: chloroquine; CREB: cAMP responsive element binding protein; ChIP: chromatin immunoprecipitation assay; IC50: half-maximal inhibitory concentration; LAMP2A: lysosomal associated membrane protein 2A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NPC: nasopharyngeal carcinoma; NPG: nasopharyngitis; oeCENPN: overexpressed CENPN; PTX: paclitaxel; RAPA: rapamycin; RNA-seq: transcriptome sequencing; shCENPN: small hairpin RNA expression vector targeting the human CENPN gene; shCENPN-shVAMP8: sequential knockdown targeting the human CENPN gene and VAMP8 gene; shVAMP8: small hairpin RNA expression vector targeting the human VAMP8 gene; TEM: transmission electron microscopy; TIR: tumor inhibitory rate; VAMP8: vesicle associated membrane protein 8.

Phlebopus portentosus (Berk. and Broome) Boedijn is an attractive edible mushroom and is considered the only bolete for which artificial cultivation in vitro has been achieved. Gene expression analysis has become widely used in research on edible fungi and is important for elucidating the functions of genes involved in complex biological processes. Selecting appropriate reference genes is crucial to ensuring reliable RT‒qPCR gene expression analysis results. In our study, a total of 12 candidate control genes were selected from 25 traditional housekeeping genes based on their expression stability in 9 transcriptomes of 3 developmental stages. These genes were further evaluated using geNorm, NormFinder, and RefFinder under different conditions and developmental stages. The results revealed that MSF1 domain-containing protein (MSF1), synaptobrevin (SYB), mitogen-activated protein kinase genes (MAPK), TATA-binding protein 1 (TBP1), and SPRY domain protein (SPRY) were the most stable reference genes in all sample treatments, while elongation factor 1-alpha (EF1), actin and ubiquitin-conjugating enzyme (UBCE) were the most unstably expressed. The gene SYB was selected based on the transcriptome results and was identified as a novel reference gene in P. portentosus. This is the first detailed study on the identification of reference genes in this fungus and may provide new insights into selecting genes and quantifying gene expression.

The extended tubular shape of root hairs is established by tip growth and concomitant hardening. Here, we demonstrate that a syntaxin of plants (SYP)123-vesicle-associated membrane protein (VAMP)727-dependent secretion system delivers secondary cell wall components for hardening the subapical zone and shank of Arabidopsis (Arabidopsis thaliana) root hairs. We found increased SYP123 localization at the plasma membrane (PM) of the subapical and shank zones compared with the tip region in elongating root hairs. Inhibition of phosphatidylinositol (PtdIns)(3,5)P2 production impaired SYP123 localization at the PM and SYP123-mediated root hair shank hardening. Moreover, root hair elongation in the syp123 mutant was insensitive to a PtdIns(3,5)P2 synthesis inhibitor. SYP123 interacts with both VAMP721 and VAMP727. syp123 and vamp727 mutants exhibited reduced shank cell wall stiffness due to impaired secondary cell wall component deposition. Based on these results, we conclude that SYP123 is involved in VAMP721-mediated conventional secretion for root hair elongation as well as in VAMP727-mediated secretory functions for the delivery of secondary cell wall components to maintain root hair tubular morphology.