Dataset Information

Syntaxin 11 regulates the stimulus-dependent transport of Toll-like receptor 4 to the plasma membrane by cooperating with SNAP-23 in macrophages.

ABSTRACT: Syntaxin 11 (stx11) is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) that is selectively expressed in immune cells; however, its precise role in macrophages is unclear. We showed that stx11 knockdown reduces the phagocytosis of Escherichia coli in interferon-?-activated macrophages. stx11 knockdown decreased Toll-like receptor 4 (TLR4) localization on the plasma membrane without affecting total expression. Plasma membrane-localized TLR4 was primarily endocytosed within 1 h by lipopolysaccharide (LPS) stimulation and gradually relocalized 4 h after removal of LPS. This relocalization was significantly impaired by stx11 knockdown. The lack of TLR4 transport to the plasma membrane is presumably related to TLR4 degradation in acidic endosomal organelles. Additionally, an immunoprecipitation experiment suggested that stx11 interacts with SNAP-23, a plasma membrane-localized SNARE protein, whose depletion also inhibits TLR4 replenishment in LPS-stimulated cells. Using an intramolecular Förster resonance energy transfer (FRET) probe for SNAP-23, we showed that the high FRET efficiency caused by LPS stimulation is reduced by stx11 knockdown. These findings suggest that stx11 regulates the stimulus-dependent transport of TLR4 to the plasma membrane by cooperating with SNAP-23 in macrophages. Our results clarify the regulatory mechanisms underlying intracellular transport of TLR4 and have implications for microbial pathogenesis and immune responses.

SUBMITTER: Kinoshita D

PROVIDER: S-EPMC6724512 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Syntaxin 11 regulates the stimulus-dependent transport of Toll-like receptor 4 to the plasma membrane by cooperating with SNAP-23 in macrophages.

Kinoshita Daiki D Sakurai Chiye C Morita Maya M Tsunematsu Masashi M Hori Naohiro N Hatsuzawa Kiyotaka K

Molecular biology of the cell 20190227 9

Syntaxin 11 (stx11) is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) that is selectively expressed in immune cells; however, its precise role in macrophages is unclear. We showed that stx11 knockdown reduces the phagocytosis of Escherichia coli in interferon-γ-activated macrophages. stx11 knockdown decreased Toll-like receptor 4 (TLR4) localization on the plasma membrane without affecting total expression. Plasma membrane-localized TLR4 was primarily endocytosed ...[more]

PMID: 30811271

Similar Datasets

Project description:The innate immune system is the body's first line of defense against pathogens and its protection against infectious diseases. On the surface of host myeloid cells, Toll-like receptor 4 (TLR4) senses lipopolysaccharide (LPS), the major outer membrane component of Gram-negative bacteria. Intracellularly, LPS is recognized by caspase 11 through the noncanonical inflammasome to induce pyroptosis-an inflammatory form of lytic cell death. While TLR4-mediated signaling perturbations result in secretion of cytokines and chemokines that help clear infection and facilitate adaptive immunity, caspase 11-mediated pyroptosis leads to the release of damage-associated molecular patterns and inflammatory mediators. Although the core signaling events and many associated proteins in the TLR4 signaling pathway are known, the complex signaling events and protein networks within the noncanonical inflammasome pathway remain obscure. Moreover, there is mounting evidence for pathogen-specific innate immune tuning. We characterized the major LPS structures from two different pathogens, modeled their binding to the surface receptors, systematically examined macrophage inflammatory responses to these LPS molecules, and surveyed the temporal differences in global protein secretion resulting from TLR4 and caspase 11 activation in macrophages using mass spectrometry (MS)-based quantitative proteomics. This integrated strategy, spanning functional activity assays, top-down structural elucidation of endotoxins, and secretome analysis of stimulated macrophages, allowed us to identify crucial differences in TLR4- and caspase 11-mediated protein secretion in response to two Gram-negative bacterial endotoxins. IMPORTANCE Macrophages and monocytes are innate immune cells playing an important role in orchestrating the initial innate immune response to bacterial infection and the tissue damage. This response is facilitated by specific receptors on the cell surface and intracellularly. One of the bacterial molecules recognized is a Gram-negative bacteria cell wall component, lipopolysaccharide (LPS). The structure of LPS differs between different species. We have characterized the innate immune responses to the LPS molecules from two bacteria, Escherichia coli and Bordetella pertussis, administered either extracellularly or intracellularly, whose structures we first determined. We observed marked differences in the temporal dynamics and amounts of proteins secreted by the innate immune cells stimulated by any of these molecules and routes. This suggests that there is specificity in the first line of response to different Gram-negative bacteria that can be explored to tailor specific therapeutic interventions.

Project description:?-SNAP has an essential role in membrane fusion that consists of bridging cis SNARE complexes to NSF. ?-SNAP stimulates NSF, which releases itself, ?-SNAP, and individual SNAREs that subsequently re-engage in the trans arrays indispensable for fusion. ?-SNAP also binds monomeric syntaxin and NSF disengages the ?-SNAP/syntaxin dimer. Here, we examine why recombinant ?-SNAP blocks secretion in permeabilized human sperm despite the fact that the endogenous protein is essential for membrane fusion. The only mammalian organism with a genetically modified ?-SNAP is the hyh mouse strain, which bears a M105I point mutation; males are subfertile due to defective sperm exocytosis. We report here that recombinant ?-SNAP-M105I has greater affinity for the cytosolic portion of immunoprecipitated syntaxin than the wild type protein and in consequence NSF is less efficient in releasing the mutant. ?-SNAP-M105I is a more potent sperm exocytosis blocker than the wild type and requires higher concentrations of NSF to rescue its effect. Unlike other fusion scenarios where SNAREs are subjected to an assembly/disassembly cycle, the fusion machinery in sperm is tuned so that SNAREs progress uni-directionally from a cis configuration in resting cells to monomeric and subsequently trans arrays in cells challenged with exocytosis inducers. By means of functional and indirect immunofluorescense assays, we show that recombinant ?-SNAPs--wild type and M105I--inhibit exocytosis because they bind monomeric syntaxin and prevent this SNARE from assembling with its cognates in trans. Sequestration of free syntaxin impedes docking of the acrosome to the plasma membrane assessed by transmission electron microscopy. The N-terminal deletion mutant ?-SNAP-(160-295), unable to bind syntaxin, affects neither docking nor secretion. The implications of this study are twofold: our findings explain the fertility defect of hyh mice and indicate that assembly of SNAREs in trans complexes is essential for docking.

Dataset Information

Syntaxin 11 regulates the stimulus-dependent transport of Toll-like receptor 4 to the plasma membrane by cooperating with SNAP-23 in macrophages.

Publications

Syntaxin 11 regulates the stimulus-dependent transport of Toll-like receptor 4 to the plasma membrane by cooperating with SNAP-23 in macrophages.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets