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.

Vesicle-associated membrane protein 8 knockdown exerts anti-proliferative, pro-apoptotic, anti-autophagic, and pro-ferroptotic effects on colorectal cancer cells by inhibition of the JAK/STAT3 pathway.

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.

Upregulation of vesicle-associated membrane protein 7 in breast cancer tissues.

BACKGROUND: Vesicle-associated membrane protein 7 (VAMP7) plays oncogenic roles in cancers. However, its clinical significance in breast cancer (BC) tissues remains unknown. OBJECTIVE: To elucidate the clinical implications of VAMP7, as well as its involvement in the tumor microenvironment and molecular pathways of breast cancer. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

CENPN suppresses autophagy and increases paclitaxel resistance in nasopharyngeal carcinoma cells by inhibiting the CREB-VAMP8 signaling axis.

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.

VAMP7j: A Splice Variant of Human VAMP7 That Modulates Neurite Outgrowth by Regulating L1CAM Transport to 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.

Sec22b and Stx4 Depletion Has No Major Effect on Cross-Presentation of PLGA Microsphere-Encapsulated Antigen and a Synthetic Long Peptide In Vitro.

The induction of CTL responses by vaccines is important to combat infectious diseases and cancer. Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres and synthetic long peptides are efficiently internalized by professional APCs and prime CTL responses after cross-presentation of Ags on MHC class I molecules. Specifically, they mainly use the cytosolic pathway of cross-presentation that requires endosomal escape, proteasomal processing, and subsequent MHC class I loading of Ags in the endoplasmic reticulum (ER) and/or the endosome. The vesicle SNARE protein Sec22b has been described as important for this pathway by mediating vesical trafficking for the delivery of ER-derived proteins to the endosome. As this function has also been challenged, we investigated the role of Sec22b in cross-presentation of the PLGA microsphere-encapsulated model Ag OVA and a related synthetic long peptide. Using CRISPR/Cas9-mediated genome editing, we generated Sec22b knockouts in two murine C57BL/6-derived APC lines and found no evidence for an essential role of Sec22b. Although pending experimental evidence, the target SNARE protein syntaxin 4 (Stx4) has been suggested to promote cross-presentation by interacting with Sec22b for the fusion of ER-derived vesicles with the endosome. In the current study, we show that, similar to Sec22b, Stx4 knockout in murine APCs had very limited effects on cross-presentation under the conditions tested. This study contributes to characterizing cross-presentation of two promising Ag delivery systems and adds to the discussion about the role of Sec22b/Stx4 in related pathways. Our data point toward SNARE protein redundancy in the cytosolic pathway of cross-presentation.

VAMP8 suppresses the metastasis via DDX5/ß-catenin signal pathway in osteosarcoma.

Osteosarcoma is a highly metastatic malignant bone tumor, necessitating the development of new treatments to target its metastasis. Recent studies have revealed the significance of VAMP8 in regulating various signaling pathways in various types of cancer. However, the specific functional role of VAMP8 in osteosarcoma progression remains unclear. In this study, we observed a significant downregulation of VAMP8 in osteosarcoma cells and tissues. Low levels of VAMP8 in osteosarcoma tissues were associated with patients' poor prognosis. VAMP8 inhibited the migration and invasion capability of osteosarcoma cells. Mechanically, we identified DDX5 as a novel interacting partner of VAMP8, and the conjunction of VAMP8 and DDX5 promoted the degradation of DDX5 via the ubiquitin-proteasome system. Moreover, reduced levels of DDX5 led to the downregulation of ß-catenin, thereby suppressing the epithelial-mesenchymal transition (EMT). Additionally, VAMP8 promoted autophagy flux, which may contribute to the suppression of osteosarcoma metastasis. In conclusion, our study anticipated that VAMP8 inhibits osteosarcoma metastasis by promoting the proteasomal degradation of DDX5, consequently inhibiting WNT/ß-catenin signaling and EMT. Dysregulation of autophagy by VAMP8 is also implicated as a potential mechanism. These findings provide new insights into the biological nature driving osteosarcoma metastasis and highlight the modulation of VAMP8 as a potential therapeutic strategy for targeting osteosarcoma metastasis.

A bacterial type III effector targets plant vesicle-associated membrane proteins.

The soilborne bacterial pathogen Ralstonia solanacearum is one of the most destructive plant pathogens worldwide, and its infection process involves the manipulation of numerous plant cellular functions. In this work, we found that the R. solanacearum effector protein RipD partially suppressed different levels of plant immunity triggered by R. solanacearum elicitors, including specific responses triggered by pathogen-associated molecular patterns and secreted effectors. RipD localized in different subcellular compartments in plant cells, including vesicles, and its vesicular localization was enriched in cells undergoing R. solanacearum infection, suggesting that this specific localization may be particularly relevant during infection. Among RipD-interacting proteins, we identified plant vesicle-associated membrane proteins (VAMPs). We also found that overexpression of Arabidopsis thaliana VAMP721 and VAMP722 in Nicotiana benthamiana leaves promoted resistance to R. solanacearum, and this was abolished by the simultaneous expression of RipD, suggesting that RipD targets VAMPs to contribute to R. solanacearum virulence. Among proteins secreted in VAMP721/722-containing vesicles, CCOAOMT1 is an enzyme required for lignin biosynthesis, and mutation of CCOAOMT1 enhanced plant susceptibility to R. solanacearum. Altogether our results reveal the contribution of VAMPs to plant resistance against R. solanacearum and their targeting by a bacterial effector as a pathogen virulence strategy.

SEC22B inhibition attenuates colorectal cancer aggressiveness and autophagic flux under unfavorable environment.

Autophagy has bidirectional functions in cancer by facilitating cell survival and death in a context-dependent manner. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a large family of proteins essential for numerous biological processes, including autophagy; nevertheless, their potential function in cancer malignancy remains unclear. Here, we explored the gene expression patterns of SNAREs in tissues of patients with colorectal cancer (CRC) and discovered that SEC22B expression, a vesicle SNARE, was higher in tumor tissues than in normal tissues, with a more significant increase in metastatic tissues. Interestingly, SEC22B knockdown dramatically decreased CRC cell survival and growth, especially under stressful conditions, such as hypoxia and serum starvation, and decreased the number of stress-induced autophagic vacuoles. Moreover, SEC22B knockdown successfully attenuated liver metastasis in a CRC cell xenograft mouse model, with histological signs of decreased autophagic flux and proliferation within cancer cells. Together, this study posits that SEC22B plays a crucial role in enhancing the aggressiveness of CRC cells, suggesting that SEC22B might be an attractive therapeutic target for CRC.

Inhibition of STX17-SNAP29-VAMP8 complex formation by costunolide sensitizes ovarian cancer cells to cisplatin via the AMPK/mTOR signaling pathway.

Ovarian cancer (OC) is the most common gynecological malignancy. Chemotherapy failure is a major challenge in OC treatment. Targeting autophagy is a promising strategy to enhance the cytotoxicity of chemotherapeutic agents. In this study, we found that costunolide (CTD) inhibits autophagic flux and exhibits high therapeutic efficacy for OC treatment in an in vitro model. Mechanistically, CTD inactivates AMPK/mTOR signaling to inhibit autophagy initiation at the early stage and blocks mTORC1-dependent autophagosome-lysosome fusion at the late stage during autophagy by disrupting SNARE complex (STX17-SNAP29-VAMP8) formation, resulting in lethal autophagy arrest in OC cells. Furthermore, CTD sensitizes OC cells to cisplatin (CDDP) by blocking CDDP-induced autophagy both in vitro and in vivo. Together, our data provide novel mechanistic insights into CTD-induced autophagy arrest and suggest a new autophagy inhibitor for effective treatment of OC.

NRF2 controls iron homeostasis and ferroptosis through HERC2 and VAMP8.

Enhancing the intracellular labile iron pool (LIP) represents a powerful, yet untapped strategy for driving ferroptotic death of cancer cells. Here, we show that NRF2 maintains iron homeostasis by controlling HERC2 (E3 ubiquitin ligase for NCOA4 and FBXL5) and VAMP8 (mediates autophagosome-lysosome fusion). NFE2L2/NRF2 knockout cells have low HERC2 expression, leading to a simultaneous increase in ferritin and NCOA4 and recruitment of apoferritin into the autophagosome. NFE2L2/NRF2 knockout cells also have low VAMP8 expression, which leads to ferritinophagy blockage. Therefore, deletion of NFE2L2/NRF2 results in apoferritin accumulation in the autophagosome, an elevated LIP, and enhanced sensitivity to ferroptosis. Concordantly, NRF2 levels correlate with HERC2 and VAMP8 in human ovarian cancer tissues, as well as ferroptosis resistance in a panel of ovarian cancer cell lines. Last, the feasibility of inhibiting NRF2 to increase the LIP and kill cancer cells via ferroptosis was demonstrated in preclinical models, signifying the impact of NRF2 inhibition in cancer treatment.

Oxidative Stress Impairs Autophagy via Inhibition of Lysosomal Transport of VAMP8.

Autophagy is a highly conserved intracellular degrading system and its dysfunction is considered related to the cause of neurodegenerative disorders. A previous study showed that the inhibition of endocytosis transport attenuates soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein transport to lysosomes and block autophagy. The other studies demonstrated oxidative stress, one of the inducers of neurodegenerative diseases inhibits endocytosis transport. Thus, we hypothesized that oxidative stress-induced endocytosis inhibition causes alteration of SNARE protein transport to lysosomes and impairs autophagy. Here, we demonstrated that oxidative stress inhibits endocytosis and decreased the lysosomal localization of VAMP8, one of the autophagy-related SNARE proteins in a human neuroblastoma cell line. Moreover, this oxidative stress induction blocked the autophagosome-lysosome fusion step. Since we also observed decreased lysosomal localization of VAMP8 and inhibition of autophagosome-lysosome fusion in endocytosis inhibitor-treated cells, oxidative stress may inhibit VAMP8 trafficking by suppressing endocytosis and impair autophagy. Our findings suggest that oxidative stress-induced inhibition of VAMP8 trafficking to lysosomes is associated with the development of neurodegenerative diseases due to the blocked autophagosome-lysosome fusion, and may provide a new therapeutic target for restoring the autophagic activity.

Loss of Activity-Induced Mitochondrial ATP Production Underlies the Synaptic Defects in a Drosophila Model of ALS.

Mutations in the gene encoding vesicle-associated membrane protein B (VAPB) cause a familial form of amyotrophic lateral sclerosis (ALS). Expression of an ALS-related variant of vapb (vapbP58S ) in Drosophila motor neurons results in morphologic changes at the larval neuromuscular junction (NMJ) characterized by the appearance of fewer, but larger, presynaptic boutons. Although diminished microtubule stability is known to underlie these morphologic changes, a mechanism for the loss of presynaptic microtubules has been lacking. By studying flies of both sexes, we demonstrate the suppression of vapbP58S -induced changes in NMJ morphology by either a loss of endoplasmic reticulum (ER) Ca2+ release channels or the inhibition Ca2+/calmodulin (CaM)-activated kinase II (CaMKII). These data suggest that decreased stability of presynaptic microtubules at vapbP58S NMJs results from hyperactivation of CaMKII because of elevated cytosolic [Ca2+]. We attribute the Ca2+ dyshomeostasis to delayed extrusion of cytosolic Ca2+ Suggesting that this defect in Ca2+ extrusion arose from an insufficient response to the bioenergetic demand of neural activity, depolarization-induced mitochondrial ATP production was diminished in vapbP58S neurons. These findings point to bioenergetic dysfunction as a potential cause for the synaptic defects in vapbP58S -expressing motor neurons.SIGNIFICANCE STATEMENT Whether the synchrony between the rates of ATP production and demand is lost in degenerating neurons remains poorly understood. We report that expression of a gene equivalent to an amyotrophic lateral sclerosis (ALS)-causing variant of vesicle-associated membrane protein B (VAPB) in fly neurons decouples mitochondrial ATP production from neuronal activity. Consequently, levels of ATP in mutant neurons are unable to keep up with the bioenergetic burden of neuronal activity. Reduced rate of Ca2+ extrusion, which could result from insufficient energy to power Ca2+ ATPases, results in the accumulation of residual Ca2+ in mutant neurons and leads to alterations in synaptic vesicle (SV) release and synapse development. These findings suggest that synaptic defects in a model of ALS arise from the loss of activity-induced ATP production.

Recycling of cell surface membrane proteins from yeast endosomes is regulated by ubiquitinated Ist1.

Upon internalization, many surface membrane proteins are recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labor between interconnected pathways are poorly defined. In yeast, we show recycling back to the surface occurs through distinct pathways. In addition to retrograde recycling pathways via the late Golgi, used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalize to, and recycle from, endosomes marked by the ESCRT-III associated factor Ist1. This compartment serves as both "early" and "recycling" endosome. We show Ist1 is ubiquitinated and that this is required for proper endosomal recruitment and cargo recycling to the surface. Additionally, the essential ATPase Cdc48 and its adaptor Npl4 are required for recycling, potentially through regulation of ubiquitinated Ist1. This collectively suggests mechanistic features of recycling from endosomes to the plasma membrane are conserved.

Regulation of the SNARE protein Ykt6 function by diprenylation and phosphorylation.

For proper intracellular vesicle transport, it is essential for transport vesicle membranes to fuse with the appropriate target membranes. Ykt6 is a SNARE protein with functions in diverse vesicle transport pathways, including secretory, endocytotic and autophagic pathways. To exert these functions, the association of Ykt6 with vesicle membranes and the change of its conformation from closed to open play key roles. Recent studies have revealed regulatory mechanisms involved in Ykt6 membrane association and conformation change. When in the cytosol, the vicinal cysteine residues within the C-terminal CCAIM sequence of Ykt6 undergo diprenylation (farnesylation of the distal cysteine residues by farnesyltransferase; this is followed by geranylgeranylation of the proximal cysteine residue by geranylgeranyltransferase-III). Phosphorylation of a serine residue within the SNARE domain triggers the conversion of the Ykt6 conformation from closed to open, allowing Ykt6 membrane association. In this commentary, I briefly summarize and discuss the recently revealed regulatory mechanisms of Ykt6 function by diprenylation and phosphorylation.

Extended-synaptotagmin 1 engages in unconventional protein secretion mediated via SEC22B+ vesicle pathway in liver cancer.

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER-plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B+ vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER-PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.

Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody.

Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP substrate sensitive enough. The immune detection of the cleaved substrate is much more sensitive, as we have previously shown for botulinum neurotoxin type A. Here, we describe the production in rabbit of a polyclonal antibody raised versus a peptide encompassing the 13 residues C-terminal with respect to the neurotoxin cleavage site. The antibody was affinity purified and found to recognize, with high specificity and selectivity, the novel N-terminus of VAMP that becomes exposed after cleavage by tetanus toxin and botulinum toxin type B. This antibody recognizes the neoepitope not only in native and denatured VAMP but also in cultured neurons and in neurons in vivo in neurotoxin-treated mice or rats, suggesting the great potential of this novel tool to elucidate tetanus and botulinum B toxin activity in vivo.

lncRNA DANCR Promotes Proliferation and Metastasis of Breast Cancer Cells Through Sponging miR-4319 and Upregulating VAPB.

Background: Breast cancer is one of the most prevalent cancers that often occur in females. Long noncoding RNA differentiation antagonizing nonprotein coding RNA (DANCR) has been involved in the pathogenesis of various tumors, including breast cancer. This study aimed to investigate the role and underlying mechanism of DANCR in breast cancer. Materials and Methods: The level of DANCR was detected in breast cancer tissues and cells by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Cell apoptosis was assessed using flow cytometry. Cell migration and invasion were estimated by the Transwell assay. The relationship between DANCR, miR-4319, and vesicle-associated membrane protein-associated protein B (VAPB) was confirmed by bioinformatic analysis and dual-luciferase reporter assay. The level of microRNA-4319 (miR-4319) was tested by qRT-PCR. The expression of VAPB was measured by qRT-PCR or Western blot assay. Results: DANCR and VAPB were upregulated, while miR-4319 was downregulated in breast cancer tissues and cells. Knockdown of DANCR hindered proliferation, migration, and invasion and promoted apoptosis of breast cancer cells. DANCR knockdown inhibited breast cancer development through regulating miR-4319. Inhibition of miR-4319 restrained breast cancer cell progression by targeting VAPB. Moreover, DANCR regulated VAPB expression by sponging miR-4319 in breast cancer cells. Conclusion: DANCR facilitated breast cancer cell progression through regulating the miR-4319/VAPB axis, indicating that DANCR might be a potential biomarker and therapeutic target for breast cancer treatment.

Prefused lysosomes cluster on autophagosomes regulated by VAMP8.

Lysosome-autophagosome fusion is critical to autophagosome maturation. Although several proteins that regulate this fusion process have been identified, the prefusion architecture and its regulation remain unclear. Herein, we show that upon stimulation, multiple lysosomes form clusters around individual autophagosomes, setting the stage for membrane fusion. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein on lysosomes-vesicle-associated membrane protein 8 (VAMP8)-plays an important role in forming this prefusion state of lysosomal clusters. To study the potential role of phosphorylation on spontaneous fusion, we investigated the effect of phosphorylation of C-terminal residues of VAMP8. Using a phosphorylation mimic, we observed a decrease of fusion in an ensemble lipid mixing assay and an increase of unfused lysosomes associated with autophagosomes. These results suggest that phosphorylation not only reduces spontaneous fusion for minimizing autophagic flux under normal conditions, but also preassembles multiple lysosomes to increase the fusion probability for resuming autophagy upon stimulation. VAMP8 phosphorylation may thus play an important role in chemotherapy drug resistance by influencing autophagosome maturation.

Tumor-derived exosomes drive immunosuppressive macrophages in a pre-metastatic niche through glycolytic dominant metabolic reprogramming.

One of the defining characteristics of a pre-metastatic niche, a fundamental requirement for primary tumor metastasis, is infiltration of immunosuppressive macrophages. How these macrophages acquire their phenotype remains largely unexplored. Here, we demonstrate that tumor-derived exosomes (TDEs) polarize macrophages toward an immunosuppressive phenotype characterized by increased PD-L1 expression through NF-kB-dependent, glycolytic-dominant metabolic reprogramming. TDE signaling through TLR2 and NF-κB leads to increased glucose uptake. TDEs also stimulate elevated NOS2, which inhibits mitochondrial oxidative phosphorylation resulting in increased conversion of pyruvate to lactate. Lactate feeds back on NF-κB, further increasing PD-L1. Analysis of metastasis-negative lymph nodes of non-small-cell lung cancer patients revealed that macrophage PD-L1 positively correlates with levels of GLUT-1 and vesicle release gene YKT6 from primary tumors. Collectively, our study provides a novel mechanism by which macrophages within a pre-metastatic niche acquire their immunosuppressive phenotype and identifies an important link among exosomes, metabolism, and metastasis.