Human antigen R transfers miRNA to Syntaxin 5 to synergize miRNA export from activated macrophages.

Intercellular miRNA exchange acts as a key mechanism to control gene expression post-transcriptionally in mammalian cells. Regulated export of repressive miRNAs allows the expression of inflammatory cytokines in activated macrophages. Intracellular trafficking of miRNAs from the endoplasmic reticulum to endosomes is a rate-determining step in the miRNA export process and plays an important role in controlling cellular miRNA levels and inflammatory processes in macrophages. We have identified the SNARE protein Syntaxin 5 (STX5) to show a synchronized expression pattern with miRNA activity loss in activated mammalian macrophage cells. STX5 is both necessary and sufficient for macrophage activation and clearance of the intracellular pathogen Leishmania donovani from infected macrophages. Exploring the mechanism of how STX5 acts as an immunostimulant, we have identified the de novo RNA-binding property of this SNARE protein that binds specific miRNAs and facilitates their accumulation in endosomes in a cooperative manner with human ELAVL1 protein, Human antigen R. This activity ensures the export of miRNAs and allows the expression of miRNA-repressed cytokines. Conversely, in its dual role in miRNA export, this SNARE protein prevents lysosomal targeting of endosomes by enhancing the fusion of miRNA-loaded endosomes with the plasma membrane to ensure accelerated release of extracellular vesicles and associated miRNAs.

Syntaxin-5's flexibility in SNARE pairing supports Golgi functions.

Deficiency in the conserved oligomeric Golgi (COG) complex that orchestrates SNARE-mediated tethering/fusion of vesicles that recycle the Golgi's glycosylation machinery results in severe glycosylation defects. Although two major Golgi v-SNAREs, GS28/GOSR1, and GS15/BET1L, are depleted in COG-deficient cells, the complete knockout of GS28 and GS15 only modestly affects Golgi glycosylation, indicating the existence of an adaptation mechanism in Golgi SNARE. Indeed, quantitative mass-spectrometry analysis of STX5-interacting proteins revealed two novel Golgi SNARE complexes-STX5/SNAP29/VAMP7 and STX5/VTI1B/STX8/YKT6. These complexes are present in wild-type cells, but their usage is significantly increased in both GS28- and COG-deficient cells. Upon GS28 deletion, SNAP29 increased its Golgi residency in a STX5-dependent manner. While STX5 depletion and Retro2-induced diversion from the Golgi severely affect protein glycosylation, GS28/SNAP29 and GS28/VTI1B double knockouts alter glycosylation similarly to GS28 KO, indicating that a single STX5-based SNARE complex is sufficient to support Golgi glycosylation. Importantly, co-depletion of three Golgi SNARE complexes in GS28/SNAP29/VTI1B TKO cells resulted in severe glycosylation defects and a reduced capacity for glycosylation enzyme retention at the Golgi. This study demonstrates the remarkable plasticity in SXT5-mediated membrane trafficking, uncovering a novel adaptive response to the failure of canonical intra-Golgi vesicle tethering/fusion machinery.

Dispatch and delivery at the ER-Golgi interface: how endothelial cells tune their hemostatic response.

Von Willebrand factor (VWF) is a glycoprotein that is secreted into the circulation and controls bleeding by promoting adhesion and aggregation of blood platelets at sites of vascular injury. Substantial inter-individual variation in VWF plasma levels exists among the healthy population. Prior to secretion, VWF polymers are assembled and condensed into helical tubules, which are packaged into Weibel-Palade bodies (WPBs), a highly specialized post-Golgi storage compartment in vascular endothelial cells. In the inherited bleeding disorder Von Willebrand disease (VWD), mutations in the VWF gene can cause qualitative or quantitative defects, limiting protein function, secretion, or plasma survival. However, pathogenic VWF mutations cannot be found in all VWD cases. Although an increasing number of genetic modifiers have been identified, even more rare genetic variants that impact VWF plasma levels likely remain to be discovered. Here, we summarize recent evidence that modulation of the early secretory pathway has great impact on the biogenesis and release of WPBs. Based on these findings, we propose that rare, as yet unidentified quantitative trait loci influencing intracellular VWF transport contribute to highly variable VWF levels in the population. These may underlie the thrombotic complications linked to high VWF levels, as well as the bleeding tendency in individuals with low VWF levels.

BECN2 (beclin 2) Negatively Regulates Inflammasome Sensors Through ATG9A-Dependent but ATG16L1- and LC3-Independent Non-Canonical Autophagy.

Macroautophagy/autophagy-related proteins regulate infectious and inflammatory diseases in autophagy-dependent or -independent manner. However, the role of a newly identified mammalian-specific autophagy protein-BECN2 (beclin 2) in innate immune regulation is largely unknown. Here we showed that loss of BECN2 enhanced the activities of NLRP3, AIM2, NLRP1, and NLRC4 inflammasomes upon ligand stimulations. Mechanistically, BECN2 interacted with inflammasome sensors and mediated their degradation through a ULK1- and ATG9A-dependent, but BECN1-WIPI2-ATG16L1-LC3-independent, non-canonical autophagic pathway. BECN2 recruited inflammasome sensors on ATG9A+ vesicles to form a complex (BECN2-ATG9A-sensors) upon ULK1 activation. Three soluble NSF attachment protein receptor (SNARE) proteins (SEC22A, STX5, and STX6) were further shown to mediate the BECN2-ATG9A-dependent inflammasome sensor degradation. Loss of BECN2 promoted alum-induced peritonitis, which could be rescued by the ablation of CASP1 in Becn2-deficient mice. Hence, BECN2 negatively regulated inflammasome activation to control inflammation, serving as a potential therapeutic target for the treatment of infectious and inflammatory diseases.Abbreviations: AIM2: absent in melanoma 2; ATG: autophagy related; BECN1: beclin 1; BMDC: bone marrow-derived dendritic cells; BMDM: bone marrow-derived macrophages; CASP1: caspase 1; CQ: chloroquine; gMDSC: granulocytic myeloid-derived suppressor cells; IL: interleukin; LPS: lipopolysaccharide; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; mMDSC: monocytic myeloid-derived suppressor cells; NLRC4: NLR family CARD domain containing 4; NLRP1: NLR family pyrin domain containing 1; NLRP3: NLR family pyrin domain containing 3; PECs: peritoneal exudate cells; PYCARD/ASC: apoptosis-associated speck-like protein containing a caspase activation and recruitment domain; SNAREs: soluble NSF attachment protein receptors; STX5: syntaxin 5; STX6: syntaxin 6; ULK1: unc-51 like autophagy activating kinase 1; WIPI: WD repeat domain, phosphoinositide interacting.

Stx5 is a novel interactor of VLDL-R to affect its intracellular trafficking and processing.

We identified syntaxin 5 (Stx5), a protein involved in intracellular vesicle trafficking, as a novel interaction partner of the very low density lipoprotein (VLDL)-receptor (VLDL-R), a member of the LDL-receptor family. In addition, we investigated the effect of Stx5 on VLDL-R maturation, trafficking and processing. Here, we demonstrated mutual association of both proteins using several in vitro approaches. Furthermore, we detected a special maturation phenotype of VLDL-R resulting from Stx5 overexpression. We found that Stx5 prevented advanced Golgi-maturation of VLDL-R, but did not cause accumulation of the immature protein in ER, ER to Golgi compartments, or cis-Golgi ribbon, the main expression sites of Stx5. Rather more, abundantly present Stx5 was capable of translocating ER-/N-glycosylated VLDL-R to the plasma membrane, and thus was insensitive to BFA treatment and low temperature. Furthermore, abundant presence of Stx5 significantly interfered with VLDL-R reaching the trans-Golgi network. Based on our findings, we postulate that Stx5 can directly bind to the C-terminal domain of VLDL-R, thereby influencing the receptor's glycosylation, trafficking and processing characteristics. Resulting from that, we further suggest that Stx5 might play a role in modulating VLDL-R physiology by participating in an abrasively described or completely novel Golgi-bypass pathway.