Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNAREs.

Mammalian homologs of yeast Atg8 protein (mAtg8s) are important in autophagy, but their exact mode of action remains ill-defined. Syntaxin 17 (Stx17), a SNARE with major roles in autophagy, was recently shown to bind mAtg8s. Here, we identified LC3-interacting regions (LIRs) in several SNAREs that broaden the landscape of the mAtg8-SNARE interactions. We found that Syntaxin 16 (Stx16) and its cognate SNARE partners all have LIR motifs and bind mAtg8s. Knockout of Stx16 caused defects in lysosome biogenesis, whereas a Stx16 and Stx17 double knockout completely blocked autophagic flux and decreased mitophagy, pexophagy, xenophagy, and ribophagy. Mechanistic analyses revealed that mAtg8s and Stx16 control several properties of lysosomal compartments including their function as platforms for active mTOR. These findings reveal a broad direct interaction of mAtg8s with SNAREs with impact on membrane remodeling in eukaryotic cells and expand the roles of mAtg8s to lysosome biogenesis.

Syntaxin 6: A novel predictive and prognostic biomarker in papillary renal cell carcinoma.

Syntaxin 6 is a SNARE family protein known to play an important role in intracellular trafficking. Here, we examined the tumorogenic role of syntaxin 6 in renal cell carcinoma (RCC). The Cancer Genome Atlas (TCGA) was queried for clinicopathologic data and syntaxin 6 expression. We found a significant difference in overall survival (OS) between groups, with high syntaxin 6 expression correlating with decreased survival. When stratifying the data based on histological subtype, the papillary RCC subtype exhibited a significant correlation between syntaxin 6 expression and survival. Using ROC curve, we calculated the area under the curve (AUC) to determine the ability of syntaxin 6 to predict 3-year overall survival. The AUC for syntaxin 6 was 0.73, significantly higher compared to 0.52 for T stage. Next, syntaxin 6 expression was evaluated in clear cell (786-O and Caki-1) and papillary (Caki-2 and ACHN) RCC cells. Syntaxin 6 expression was higher in Caki-1 and ACHN RCC cells. Silencing of syntaxin 6 in ACHN cells significantly decreased the cell viability (p < 0.001). Overall, syntaxin 6 could be a prognostic biomarker for patients with papillary RCC and syntaxin 6 inhibitors hold promise as a novel therapy against RCC.

SNARE priming is essential for maturation of autophagosomes but not for their formation.

Autophagy, a unique intracellular membrane-trafficking pathway, is initiated by the formation of an isolation membrane (phagophore) that engulfs cytoplasmic constituents, leading to generation of the autophagosome, a double-membrane vesicle, which is targeted to the lysosome. The outer autophagosomal membrane consequently fuses with the lysosomal membrane. Multiple membrane-fusion events mediated by SNARE molecules have been postulated to promote autophagy. αSNAP, the adaptor molecule for the SNARE-priming enzyme N-ethylmaleimide-sensitive factor (NSF) is known to be crucial for intracellular membrane fusion processes, but its role in autophagy remains unclear. Here we demonstrated that knockdown of αSNAP leads to inhibition of autophagy, manifested by an accumulation of sealed autophagosomes located in close proximity to lysosomes but not fused with them. Under these conditions, moreover, association of both Atg9 and the autophagy-related SNARE protein syntaxin17 with the autophagosome remained unaffected. Finally, our results suggested that under starvation conditions, the levels of αSNAP, although low, are nevertheless sufficient to partially promote the SNARE priming required for autophagy. Taken together, these findings indicate that while autophagosomal-lysosomal membrane fusion is sensitive to inhibition of SNARE priming, the initial stages of autophagosome biogenesis and autophagosome expansion remain resistant to its loss.

Accumulation of autophagosomes confers cytotoxicity.

Autophagy comprises the processes of autophagosome synthesis and lysosomal degradation. In certain stress conditions, increased autophagosome synthesis may be associated with decreased lysosomal activity, which may result in reduced processing of the excessive autophagosomes by the rate-limiting lysosomal activity. Thus, the excessive autophagosomes in such situations may be largely unfused to lysosomes, and their formation/accumulation under these conditions is assumed to be futile for autophagy. The role of cytotoxicity in accumulating autophagosomes (representing synthesis of autophagosomes subsequently unfused to lysosomes) has not been investigated previously. Here, we found that accumulation of autophagosomes compromised cell viability, and this effect was alleviated by depletion of autophagosome machinery proteins. We tested whether reduction in autophagosome synthesis could affect cell viability in cell models expressing mutant huntingtin and α-synuclein, given that both of these proteins cause increased autophagosome biogenesis and compromised lysosomal activity. Importantly, partial depletion of autophagosome machinery proteins Atg16L1 and Beclin 1 significantly ameliorated cell death in these conditions. Our data suggest that production/accumulation of autophagosomes subsequently unfused to lysosomes (or accumulation of autophagosomes) directly induces cellular toxicity, and this process may be implicated in the pathogenesis of neurodegenerative diseases. Therefore, lowering the accumulation of autophagosomes may represent a therapeutic strategy for tackling such diseases.

Targeting a Potassium Channel/Syntaxin Interaction Ameliorates Cell Death in Ischemic Stroke.

The voltage-gated K+ channel Kv2.1 has been intimately linked with neuronal apoptosis. After ischemic, oxidative, or inflammatory insults, Kv2.1 mediates a pronounced, delayed enhancement of K+ efflux, generating an optimal intracellular environment for caspase and nuclease activity, key components of programmed cell death. This apoptosis-enabling mechanism is initiated via Zn2+-dependent dual phosphorylation of Kv2.1, increasing the interaction between the channel's intracellular C-terminus domain and the SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor) protein syntaxin 1A. Subsequently, an upregulation of de novo channel insertion into the plasma membrane leads to the critical enhancement of K+ efflux in damaged neurons. Here, we investigated whether a strategy designed to interfere with the cell death-facilitating properties of Kv2.1, specifically its interaction with syntaxin 1A, could lead to neuroprotection following ischemic injury in vivo The minimal syntaxin 1A-binding sequence of Kv2.1 C terminus (C1aB) was first identified via a far-Western peptide screen and used to create a protherapeutic product by conjugating C1aB to a cell-penetrating domain. The resulting peptide (TAT-C1aB) suppressed enhanced whole-cell K+ currents produced by a mutated form of Kv2.1 mimicking apoptosis in a mammalian expression system, and protected cortical neurons from slow excitotoxic injury in vitro, without influencing NMDA-induced intracellular calcium responses. Importantly, intraperitoneal administration of TAT-C1aB in mice following transient middle cerebral artery occlusion significantly reduced ischemic stroke damage and improved neurological outcome. These results provide strong evidence that targeting the proapoptotic function of Kv2.1 is an effective and highly promising neuroprotective strategy.SIGNIFICANCE STATEMENT Kv2.1 is a critical regulator of apoptosis in central neurons. It has not been determined, however, whether the cell death-enabling function of this K+ channel can be selectively targeted to improve neuronal survival following injury in vivo The experiments presented here demonstrate that the cell death-specific role of Kv2.1 can be uniquely modulated to provide neuroprotection in an animal model of acute ischemic stroke. We thus reveal a novel therapeutic strategy for neurological disorders that are accompanied by Kv2.1-facilitated forms of cell death.

Monoubiquitination of Syntaxin 5 Regulates Golgi Membrane Dynamics during the Cell Cycle.

The Golgi apparatus undergoes a ubiquitin-dependent disassembly and reassembly process during each cycle of cell division. Here we report the identification of the Golgi t-SNARE syntaxin 5 (Syn5) as the ubiquitinated substrate. Syn5 is monoubiquitinated by the ubiquitin ligase HACE1 in early mitosis and deubiquitinated by the deubiquitinase VCIP135 in late mitosis. Syn5 ubiquitination on lysine 270 (K270) in the SNARE domain impairs the interaction between Syn5 and the cognate v-SNARE Bet1 but increases its binding to p47, the adaptor protein of p97. Expression of the Syn5 K270R mutant in cells impairs post-mitotic Golgi reassembly. Therefore, monoubiquitination of Syn5 in early mitosis disrupts SNARE complex formation. Subsequently, ubiquitinated Syn5 recruits p97/p47 to the mitotic Golgi fragments and promotes post-mitotic Golgi reassembly upon ubiquitin removal by VCIP135. Overall, this study reveals both the substrate and the mechanism of ubiquitin-mediated regulation of Golgi membrane dynamics during the cell cycle.

The Autophagosomal SNARE Protein Syntaxin 17 Is an Essential Factor for the Hepatitis C Virus Life Cycle.

UNLABELLED: Syntaxin 17 is an autophagosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein required for the fusion of autophagosomes with lysosomes to form autolysosomes and thereby to deliver the enclosed contents for degradation. Hepatitis C virus (HCV) induces autophagy. In light of the observation that the number of viral particles formed by HCV-infected cells is much greater than the number of infectious viral particles finally released by HCV-infected cells, the regulation of fusion between autophagosomes and lysosomes might fulfill a key function controlling the number of released virions. HCV-replicating cells possess a decreased amount of syntaxin 17 due to impaired expression and increased turnover of syntaxin 17. Overexpression of syntaxin 17 in HCV-replicating cells diminishes the number of released infectious viral particles and decreases the amount of intracellular retained viral particles by favoring the formation of autolysosomes, in which HCV particles are degraded. Inhibition of lysosomal acidification by bafilomycin rescues the decreased release of virions from syntaxin 17-overexpressing cells, while induction of autophagy by rapamycin enforces the impairment of release under these conditions. Vice versa, inhibition of syntaxin 17 expression by specific small interfering RNAs results in an elevated amount of intracellular retained viral particles and facilitates the release of HCV virions by impairment of autophagosome-lysosome fusion. HCV genome replication, however, is not affected by modulation of syntaxin 17 expression. These data identify syntaxin 17 to be a novel factor controlling the release of HCV. This is achieved by regulation of autophagosome-lysosome fusion, which affects the equilibrium between the release of infectious viral particles and lysosomal degradation of intracellular retained viral particles. IMPORTANCE: Hepatitis C virus (HCV) induces autophagy. Syntaxin 17 is an autophagosomal SNARE protein required for the fusion of autophagosomes with lysosomes. In HCV-infected cells, a major fraction of the de novo-synthesized viral particles is not released but is intracellularly degraded. In this context, the effect of HCV on the amount and distribution of syntaxin 17 and the relevance of syntaxin 17 for the viral life cycle were investigated. This study demonstrates that the amount of syntaxin 17 decreased in HCV-replicating cells. In addition, syntaxin 17 is identified to be a novel factor controlling the release of HCV, and the relevance of autophagosome-lysosome fusion as a regulator of the amount of released viral particles is revealed. Taken together, these findings indicate that syntaxin 17 is involved in the regulation of autophagosome-lysosome fusion and thereby affects the equilibrium between the release of infectious viral particles and the lysosomal degradation of intracellularly retained viral particles.

Essential role of STX6 in esophageal squamous cell carcinoma growth and migration.

Abnormalities in endosomes, or dysregulation in their trafficking, play an important role directly in many diseases including oncogenesis. Syntaxin-6 (STX6) is involved in diverse cellular functions in a variety of cell types and has been shown to regulate many intracellular membrane trafficking events such as endocytosis, recycling and anterograde and retrograde trafficking. However, its expression pattern and biological functions in esophageal squamous cell carcinoma (ESCC) remained unknown. Here, we have found that the expression of STX6 was up-regulated in ESCC samples, its expression was significantly correlated with tumor size, histological differentiation, lymph node metastasis and depth. On one hand, STX6 silencing inhibited ESCC cells viability and proliferation in a p53-dependent manner. On the other hand, STX6 effect integrin trafficking and regulate ESCC cells migration. Taken together, our study revealed the oncogenic roles of STX6 in the progression of ESCC, and it might be a valuable target for ESCC therapy.

Annexin A6 and Late Endosomal Cholesterol Modulate Integrin Recycling and Cell Migration.

Annexins are a family of proteins that bind to phospholipids in a calcium-dependent manner. Earlier studies implicated annexin A6 (AnxA6) to inhibit secretion and participate in the organization of the extracellular matrix. We recently showed that elevated AnxA6 levels significantly reduced secretion of the extracellular matrix protein fibronectin (FN). Because FN is directly linked to the ability of cells to migrate, this prompted us to investigate the role of AnxA6 in cell migration. Up-regulation of AnxA6 in several cell models was associated with reduced cell migration in wound healing, individual cell tracking and three-dimensional migration/invasion assays. The reduced ability of AnxA6-expressing cells to migrate was associated with decreased cell surface expression of αVß3 and α5ß1 integrins, both FN receptors. Mechanistically, we found that elevated AnxA6 levels interfered with syntaxin-6 (Stx6)-dependent recycling of integrins to the cell surface. AnxA6 overexpression caused mislocalization and accumulation of Stx6 and integrins in recycling endosomes, whereas siRNA-mediated AnxA6 knockdown did not modify the trafficking of integrins. Given our recent findings that inhibition of cholesterol export from late endosomes (LEs) inhibits Stx6-dependent integrin recycling and that elevated AnxA6 levels cause LE cholesterol accumulation, we propose that AnxA6 and blockage of LE cholesterol transport are critical for endosomal function required for Stx6-mediated recycling of integrins in cell migration.

A functional circadian clock is required for proper insulin secretion by human pancreatic islet cells.

AIM: To determine the impact of a functional human islet clock on insulin secretion and gene transcription. METHODS: Efficient circadian clock disruption was achieved in human pancreatic islet cells by small interfering RNA-mediated knockdown of CLOCK. Human islet secretory function was assessed in the presence or absence of a functional circadian clock by stimulated insulin secretion assays, and by continuous around-the-clock monitoring of basal insulin secretion. Large-scale transcription analysis was accomplished by RNA sequencing, followed by quantitative RT-PCR analysis of selected targets. RESULTS: Circadian clock disruption resulted in a significant decrease in both acute and chronic glucose-stimulated insulin secretion. Moreover, basal insulin secretion by human islet cells synchronized in vitro exhibited a circadian pattern, which was perturbed upon clock disruption. RNA sequencing analysis suggested alterations in 352 transcript levels upon circadian clock disruption. Among them, key regulators of the insulin secretion pathway (GNAQ, ATP1A1, ATP5G2, KCNJ11) and transcripts required for granule maturation and release (VAMP3, STX6, SLC30A8) were affected. CONCLUSIONS: Using our newly developed experimental approach for efficient clock disruption in human pancreatic islet cells, we show for the first time that a functional ß-cell clock is required for proper basal and stimulated insulin secretion. Moreover, clock disruption has a profound impact on the human islet transcriptome, in particular, on the genes involved in insulin secretion.

The trans-Golgi SNARE syntaxin 10 is required for optimal development of Chlamydia trachomatis.

Chlamydia trachomatis, an obligate intracellular pathogen, grows inside of a vacuole, termed the inclusion. Within the inclusion, the organisms differentiate from the infectious elementary body (EB) into the reticulate body (RB). The RB communicates with the host cell through the inclusion membrane to obtain the nutrients necessary to divide, thus expanding the chlamydial population. At late time points within the developmental cycle, the RBs respond to unknown molecular signals to redifferentiate into infectious EBs to perpetuate the infection cycle. One strategy for Chlamydia to obtain necessary nutrients and metabolites from the host is to intercept host vesicular trafficking pathways. In this study we demonstrate that a trans-Golgi soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE), syntaxin 10, and/or syntaxin 10-associated Golgi elements colocalize with the chlamydial inclusion. We hypothesized that Chlamydia utilizes the molecular machinery of syntaxin 10 at the inclusion membrane to intercept specific vesicular trafficking pathways in order to create and maintain an optimal intra-inclusion environment. To test this hypothesis, we used siRNA knockdown of syntaxin 10 to examine the impact of the loss of syntaxin 10 on chlamydial growth and development. Our results demonstrate that loss of syntaxin 10 leads to defects in normal chlamydial maturation including: variable inclusion size with fewer chlamydial organisms per inclusion, fewer infectious progeny, and delayed or halted RB-EB differentiation. These defects in chlamydial development correlate with an overabundance of NBD-lipid retained by inclusions cultured in syntaxin 10 knockdown cells. Overall, loss of syntaxin 10 at the inclusion membrane negatively affects Chlamydia. Understanding host machinery involved in maintaining an optimal inclusion environment to support chlamydial growth and development is critical toward understanding the molecular signals involved in successful progression through the chlamydial developmental cycle.

Population distribution analyses reveal a hierarchy of molecular players underlying parallel endocytic pathways.

Single-cell-resolved measurements reveal heterogeneous distributions of clathrin-dependent (CD) and -independent (CLIC/GEEC: CG) endocytic activity in Drosophila cell populations. dsRNA-mediated knockdown of core versus peripheral endocytic machinery induces strong changes in the mean, or subtle changes in the shapes of these distributions, respectively. By quantifying these subtle shape changes for 27 single-cell features which report on endocytic activity and cell morphology, we organize 1072 Drosophila genes into a tree-like hierarchy. We find that tree nodes contain gene sets enriched in functional classes and protein complexes, providing a portrait of core and peripheral control of CD and CG endocytosis. For 470 genes we obtain additional features from separate assays and classify them into early- or late-acting genes of the endocytic pathways. Detailed analyses of specific genes at intermediate levels of the tree suggest that Vacuolar ATPase and lysosomal genes involved in vacuolar biogenesis play an evolutionarily conserved role in CG endocytosis.

SNAP-23 and VAMP-3 contribute to the release of IL-6 and TNFα from a human synovial sarcoma cell line.

Fibroblast-like synoviocytes are important mediators of inflammatory joint damage in arthritis through the release of cytokines, but it is unknown whether their exocytosis from these particular cells is SNARE-dependent. Here, the complement of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in human synovial sarcoma cells (SW982) was examined with respect to the secretion of interleukin-6 (IL-6) and tumour necrosis factor α (TNFα), before and after knockdown of a synaptosome-associated protein of molecular mass 23 kDa (SNAP-23) or the vesicle-associated membrane protein 3 (VAMP-3). Wild-type SW982 cells expressed SNAP-23, VAMP-3, syntaxin isoforms 2-4 and synaptic vesicle protein 2C (SV2C). These cells showed Ca²âº-dependent secretion of IL-6 and TNFα when stimulated by interleukin-1ß (IL-1ß) or in combination with K⁺ depolarization. Specific knockdown of SNAP-23 or VAMP-3 decreased the exocytosis of IL-6 and TNFα; the reduced expression of SNAP-23 caused accumulation of SV2 in the peri-nuclear area. A monoclonal antibody specific for VAMP-3 precipitated SNAP-23 and syntaxin-2 (and syntaxin-3 to a lesser extent). The formation of SDS-resistant complexes by SNAP-23 and VAMP-3 was reduced upon knockdown of SNAP-23. Although the syntaxin isoforms 2, 3 and 4 are expressed in SW982 cells, knockdown of each did not affect the release of cytokines. Collectively, these results show that SNAP-23 and VAMP-3 participate in IL-1ß-induced Ca²âº-dependent release of IL-6 and TNFα from SW982 cells.

SNARE proteins synaptobrevin, SNAP-25, and syntaxin are involved in rapid and slow endocytosis at synapses.

Rapid endocytosis, which takes only a few seconds, is widely observed in secretory cells. Although it is more efficient in recycling vesicles than in slow clathrin-mediated endocytosis, its underlying mechanism, thought to be clathrin independent, is largely unclear. Here, we report that cleavage of three SNARE proteins essential for exocytosis, including synaptobrevin, SNAP-25, and syntaxin, inhibited rapid endocytosis at the calyx of Held nerve terminal, suggesting the involvement of the three SNARE proteins in rapid endocytosis. These SNARE proteins were also involved in slow endocytosis. In addition, SNAP-25 and syntaxin facilitated vesicle mobilization to the readily releasable pool, most likely via their roles in endocytosis and/or exocytosis. We conclude that both rapid and slow endocytosis share the involvement of SNARE proteins. The dual role of three SNARE proteins in exo- and endocytosis suggests that SNARE proteins may be molecular substrates contributing to the exocytosis-endocytosis coupling, which maintains exocytosis in secretory cells.

Targeting host syntaxin-5 preferentially blocks Leishmania parasitophorous vacuole development in infected cells and limits experimental Leishmania infections.

Our previous observations established a role for syntaxin-5 in the development of Leishmania parasitophorous vacuoles (LPVs). In this study, we took advantage of the recent identification of Retro-2, a small organic molecule that can cause the redistribution of syntaxin-5; we show herein that Retro-2 blocks LPV development within 2 hours of adding it to cells infected with Leishmania amazonensis. In infected cells incubated for 48 hours with Retro-2, LPV development was significantly limited; furthermore, infected cells harbored four to five times fewer parasites than infected cells incubated in vehicle alone. In vivo studies revealed that Retro-2 curbed experimental L. amazonensis infections in a dose-dependent manner. Retro-2 did not have any appreciable effect on the host cell physiological characteristics; furthermore, it had no apparent toxicity in experimental animals. An unexpected, but welcome, finding was that Retro-2 inhibited the replication of Leishmania parasites in axenic cultures. This study is significant because it identifies an endoplasmic reticulum/Golgi SNARE as a potential target for the control of Leishmania infections; moreover, it suggests that small organic molecules can be identified that can selectively disrupt the vesicle fusion machinery that promotes the development of pathogen-containing compartments without exerting toxic effects on the host.

Interaction of α-taxilin localized on intracellular components with the microtubule cytoskeleton.

Intracellular vesicle traffic plays an essential role in the establishment and maintenance of organelle identity and biosynthetic transport. We have identified α-taxilin as a binding partner of the syntaxin family, which is involved in intracellular vesicle traffic. Recently, we have found that α-taxilin is over-expressed in malignant tissues including hepatocellular carcinoma and renal cell carcinoma. However, a precise role of α-taxilin in intracellular vesicle traffic and carcinogenesis remains unclear. Then, we first investigated here the intracellular distribution of α-taxilin in Hela cells. Immunofluorescence studies showed that α-taxilin distributes throughout the cytoplasm and exhibits a tubulo-vesicular pattern. Biochemical studies showed that α-taxilin is abundantly localized on intracellular components as a peripheral membrane protein. Moreover, we found that α-taxilin distributes in microtubule-dependent and syntaxin-independent manners, that α-taxilin directly binds to polymerized tubulin in vitro, and that N-ethylmaleimide but not brefeldin A affects the intracellular distribution of α-taxilin. These results indicate that α-taxilin is localized on intracellular components in a syntaxin-independent manner and that the α-taxilin-containing intracellular components are associated with the microtubule cytoskeleton and suggest that α-taxilin functions as a linker protein between the α-taxilin-containing intracellular components and the microtubule cytoskeleton.

Regulation of vascular endothelial growth factor receptor 2 trafficking and angiogenesis by Golgi localized t-SNARE syntaxin 6.

Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in physiologic and pathologic angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by endocytosis and secretory transport through the Golgi apparatus. To date, the mechanism whereby the VEGFR2 traffics through the Golgi apparatus remains incompletely characterized. We show in human endothelial cells that binding of VEGF to the cell surface localized VEGFR2 stimulates exit of intracellular VEGFR2 from the Golgi apparatus. Brefeldin A treatment reduced the level of surface VEGFR2, confirming that VEGFR2 traffics through the Golgi apparatus en route to the PM. Mechanistically, we show that inhibition of syntaxin 6, a Golgi-localized target membrane-soluble N-ethylmaleimide attachment protein receptor (t-SNARE) protein, interferes with VEGFR2 trafficking to the PM and facilitates lysosomal degradation of the VEGFR2. In cell culture, inhibition of syntaxin 6 also reduced VEGF-induced cell proliferation, cell migration, and vascular tube formation. Furthermore, in a mouse ear model of angiogenesis, an inhibitory form of syntaxin 6 reduced VEGF-induced neovascularization and permeability. Our data demonstrate the importance of syntaxin 6 in the maintenance of cellular VEGFR2 levels, and suggest that the inhibitory form of syntaxin 6 has good potential as an antiangiogenic agent.

Syntaxin 6, a regulator of the protein trafficking machinery and a target of the p53 family, is required for cell adhesion and survival.

The p53 family consists of p53, p63, and p73. It has been well characterized that all of the p53 family proteins are transcription factors and capable of regulating cell cycle and apoptosis. To determine whether the p53 family exerts tumor suppression by other mechanisms, we set to identify novel p53 family target genes. Here, we found that the gene encoding STX6 (syntaxin 6), a vesicle transporter protein, is directly regulated by each of the p53 family proteins. In addition, STX6 can be induced by DNA damage and Mdm2 inhibitor Nutlin-3 in a p53-dependent manner. To examine how STX6 mediates the activity of the p53 family, STX6 is inducibly overexpressed or knocked down in various cell lines. We found that overexpression of STX6 alone has limited effect on cell proliferation. In contrast, we found that knockdown of STX6 inhibits cell proliferation and survival. We also found that knockdown of STX6 leads to cell cycle arrest and apoptosis. Interestingly, we found that p53 is necessary for STX6 knockdown-induced cell cycle arrest and apoptosis. Furthermore, we found that STX6 is necessary for proper expression of focal adhesion kinase and integrin alpha5 adhesion receptor. Consistent with this observation, STX6 knockdown inhibits cell adhesion. Together, we postulate that STX6 is an effector and a modulator of the p53 family in the regulation of cell adhesion and survival.

Effective stimulation of growth in MCF-7 human breast cancer cells by inhibition of syntaxin18 by external guide sequence and ribonuclease P.

Syntaxin18 (Stx18) is an endoplasmic reticulum (ER)-membrane bound SNARE protein involved in membrane trafficking between the ER and Golgi as well as in phagocytosis. Stx18 has also been shown to physically interact with proteins involved in the cell cycle and apoptosis. These findings suggest the possible role of Stx18 in regulating cell growth. In this study, we used theoretically designed external guide sequence molecule which utilizes RNase P to cleave Stx18 mRNA and down-regulate Stx18 levels in MCF-7 human breast cancer cells. We showed that down-regulation of Stx18 leads to significant enhancement of growth in MCF-7 cells. Consistent with this finding was the observation that over-expression of Stx18 using the CMV promoter led to suppression of cell growth. Over-expressing Stx18 had no effect on c-myc mRNA expression and half-life, suggesting that the mechanism does not involve control at the transcriptional and post-transcriptional level of the c-myc gene. Finally, we showed that Stx18 is over-expressed in clinical human breast cancer. Overall, this study showed that Stx18 plays a role in the growth of human breast cancer cells and provided the basis for further investigation in determining whether it can be used as a prognostic marker and as a molecular target in the treatment of breast cancer.

The syntaxin SYP132 contributes to plant resistance against bacteria and secretion of pathogenesis-related protein 1.

In contrast to many mammalian pathogens, potential bacterial pathogens of plants remain outside the host cell. The plant must, therefore, promote an active resistance mechanism to combat the extracellular infection. How this resistance against bacteria is manifested and whether similar processes mediate basal, gene-for-gene, and salicylate-associated defense, however, are poorly understood. Here, we identify a specific plasma membrane syntaxin, NbSYP132, as a component contributing to gene-for-gene resistance in Nicotiana benthamiana. Silencing NbSYP132 but not NbSYP121, the apparent orthologue of a syntaxin required for resistance to powdery mildew fungus, compromised AvrPto-Pto resistance. Because syntaxins may play a role in secretion of proteins to the extracellular space, we performed a limited proteomic analysis of the apoplastic fluid. We found that NbSYP132-silenced plants were impaired in the accumulation of at least a subset of pathogenesis-related (PR) proteins in the cell wall. These results were confirmed by both immunoblot analysis and imunolocalization of a PR protein, PR1a. These results implicate NbSYP132 as the cognate target soluble N-ethylmaleimide-sensitive factor attachment protein receptor for exocytosis of vesicles containing antimicrobial PR proteins. NbSYP132 also contributes to basal and salicylate-associated defense, indicating that SYP132-dependent secretion is a component of multiple forms of defense against bacterial pathogens in plants.