Project description:Toll-like receptor 4 (TLR4), a pattern-recognition receptor located on the plasma membrane, senses extracellular danger signals to initiate inflammatory immune responses. It is initially synthesized in the endoplasmic reticulum (ER), undergoes N-linked glycosylation, and is subsequently transported to the Golgi before ultimately reaching the plasma membrane. However, the mechanisms underlying the processing and maturation of TLR4 in the ER remain elusive. Through whole genome-wide CRISPR screening, CCDC134 was identified as a critical and essential factor for TLR4-dependent inflammatory response. Localization of CCDC134 in the ER lumen rather than its exosome-mediated secretion is required for its role in TLR4 signaling. Loss of CCDC134 results in the retention of TLR4 in the ER for subsequent ER-associated degradation, and thus blockade of TLR4 maturation and plasma membrane trafficking. Defects in TLR4 processing and maturation in the ER in CCDC134-depleted cells are caused by aberrant hyperglycosylation and destabilization of glycoprotein 96 (gp96), a key chaperone of TLR4. These results suggest that CCDC134 controls gp96 glycosylation to facilitate TLR4 maturation in the ER.
Project description:This dataset consists of 1 raw MS file and associated peak list and result file, acquired on an Orbitrap Elite mass spectrometer operated in Data Dependent Acquisition mode. The files are associated with a manuscript submitted for publication. Publication title: "JAK2-CHK2 signaling safeguards the integrity of the mitotic spindle assembly checkpoint and genome stability"
Project description:The toll-like receptor 4 (TLR4) is a central regulator of innate immune signaling that primarily recognizes bacterial lipopolysaccharide (LPS) cell wall constituents to trigger cytokine secretion. We identify the intramembrane protease RHBDL4 as a negative regulator of TLR4 signaling. We show that RHBDL4-triggers the degradation of TLR4’s trafficking factor TMED7, a member of the p24 family of COPII adaptor proteins, which counteracts the transport of TLR4 to the cell surface. Besides TMED7, RHBDL4 cleaves a subset of related p24 cargo receptors, suggesting that this is a general protein abundance control mechanism to regulate the loading of specific secretory proteins into COPII vesicles. Notably, TLR4 activation by LPS mediates transcriptional upregulation of RHBDL4. Hence, TLR4 activation triggers an RHBDL4-dependent negative feedback loop to reduce the export of newly synthesized TLR4 molecules from the endoplasmic reticulum into COPII-coated vesicles. This secretory cargo tuning mechanism prevents the over-activation of TLR4-dependent signaling and consequently alleviates septic shock in a mouse model.
Project description:The aetiology of bowel inflammatory disease (IBD) is a multifactorial interplay between heredity and environment1-3. Here we report SETDB1, a histone methyltransferase (HMTs) for histone H3 lysine 9 trimethylation (H3K9me3) whose deficiency participates in the pathogenesis of IBD. We found that SETDB1 level decreased in IBD patients and that mice with reduced SETDB1 in intestinal stem cells (ISCs) developed spontaneous terminal ileitis and colitis. SETDB1 safeguards genome stability4, of which the loss in ISCs released repression of endogenous retrovirus (ERVs), the retrovirus-like elements with long repeats that comprise ~8% of genome5. Excessive viral mimicry generated by motivated ERVs triggered Z-DNA-binding protein 1 (ZBP1) dependent necroptosis, which irreversibly disrupted the homeostasis of epithelial barrier and promoted bowel inflammation. Of note, genome instability, reactive ERVs, upregulation of ZBP1 and necroptosis were seen in IBD patients. Pharmaceutic inhibition of RIP3 showed curative effect in SETDB1 deficient mice, suggesting therapy targeting ISC necroptosis may represent a new approach for severe IBD treatment.
Project description:Common missense mutations (D299G, T399I) have been recently identified in the human TLR4 gene. The aim of this study was to determine how TLR4 and associated mutants affect gene expression in Caco-2 cells. We used microarrays to asses gene expression profiles in Caco-2 stably overexpressing TLR4-WT, TLR4-D299G, TLR4-T399I or untransfected. Caco-2 clones stably overexpressing HA-tagged wildtype TLR4-WT, mutant TLR4-D299G or TLR4-T399I were generated. Prior to analysis, cell clones were cultured for 8 days in all experiments. RNA (triplicate) was extracted and hybridized on Affymetrix microarrays.
Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. Here we define the role of TLR4 and MD-2 in LPS signaling by using genome wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin, and are both absolutely required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation. Bone marrow-derived macrophages were pooled from four individual WT or TLR4-deficient mice and stimulated with either 10 ng LPS /mL, 100 ng lipid A/mL or 10 nM Pam2 for 2 hours and compared to PBS-stimulated control cells. We also compared PBS-stimulated WT cells directly to PBS-stimulated TLR4-deficient cells to compare the basal expression of genes in the two genotypes. This experiment was repeated once in its entirety.
Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. Here we define the role of TLR4 and MD-2 in LPS signaling by using genome wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin, and are both absolutely required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation.
Project description:Analysis of hepatic stellate cells isoltaed from wild-type, TLR4-/-, and CD44-/- mice. TLR4 and CD44 are major hyaluronic acid receptors. Results provide insight into the effects of TLR4 and CD44 loss in hepatic stellate cells.
Project description:Common missense mutations (D299G, T399I) have been recently identified in the human TLR4 gene. The aim of this study was to determine how TLR4 and associated mutants affect gene expression in Caco-2 cells. We used microarrays to asses gene expression profiles in Caco-2 stably overexpressing TLR4-WT, TLR4-D299G, TLR4-T399I or untransfected.