Mass spectrometry analysis of proteins interacting with cGAS
Ontology highlight
ABSTRACT: o identify the potential associated proteins of cGAS, we performed co-IP followed by mass spectrometry analysis in HEK293A cells stably expressing SFB-tagged cGAS.
Project description:Cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor that initiates a STING-dependent innate immune response, binds tightly to chromatin, where its catalytic activity is inhibited. However, the mechanisms underlying cGAS recruitment to chromatin and the functions of chromatin-bound cGAS (ccGAS) remain unclear. Here, we demonstrate that mTORC2-mediated serine 37 phosphorylation promotes human cGAS chromatin localization, regulating colorectal cancer cell growth and drug resistance independently of STING. We discovered that ccGAS recruits the SWI/SNF complex at specific chromatin regions to regulate expression of genes involved in glutaminolysis and DNA replication. Knockdown of ccGAS inhibits colorectal cancer cell growth but induces chemoresistance under fluorouracil exposure both in vitro and in vivo. Moreover, inhibition of kidney-type glutaminase (KGA), a downstream target of ccGAS, overcomes chemoresistance induced by ccGAS knockdown in human and murine colorectal cancer. Thus, our study demonstrates that ccGAS coordinates colorectal cancer plasticity and acquired chemoresistance through epigenetic patterning, and illustrates that simultaneously targeting mTORC2-ccGAS and KGA provides a promising theraputic strategy to eliminate quiescent resistant cancer cells.
Project description:DNA derived from the genetic material of pathogens or from cellular DNA damage provides a molecular pattern that can be sensed by pattern-recognition receptors of the mammalian innate immune system. In recent years, the cyclic GMP-AMP synthase (cGAS) protein has been characterized as a primary cytosolic DNA sensor during infection with bacteria, DNA viruses, or retroviruses. While the role of cGAS in downstream immune signaling through STING-TBK1-IRF3 proteins is well-defined, regulatory mechanisms of cGAS activity, such as through post-translational modifications (PTMs), are still an active area of research. Here, we report a comprehensive characterization of cGAS phosphorylations and acetylations in three different cell types. Data-dependent proteomic analyses of immunoaffinity purified cGAS was performed in HEK293T cells under control and DNA-challenged conditions (N = 3 each) and human fibroblasts (HFF) cells under control and HSV-1 infected conditions (N = 4 each) to generate candidate cGAS PTM sites. Using parallel reaction monitoring, a total of 11 PTMs (4 phosphorylations and 7 acetylations) were validated in HEK293T, HFF, and THP-1 cells. Of these, 3 phosphorylations and 5 acetylations have not been previously identified. The functions of these modifications were by generating a series of mutants and measuring cGAS-dependent apoptotic and immune signaling activities.
Project description:The cGAS-STING pathway, a central component of the innate immune system, senses cytosolic DNA and induces interferon-stimulated genes (ISGs) to mediate inflammation. Here we report the unexpected discovery that cGAS senses dysfunctional protein production. Purified ribosomes interact with and stimulate the catalytic activity of recombinant cGAS in vitro. Disruption of the ribosome-associated protein quality control pathway, which detects and resolves ribosome collisions, results in cGAS- and STING-dependent ISG expression, and causes the re-localization of cGAS from the nucleus to the cytosol. Indeed, cGAS preferentially binds collided ribosomes in vitro, and other orthogonal perturbations that lead to elevated levels of collided ribosomes cause re-localization of cGAS as well. Thus, the cGAS-STING pathway senses and responds to translation stress. These findings have implications for the inflammatory responses to viral infection and tumorigenesis, both of which substantially reprogram cellular protein synthesis.
Project description:Sample 1: Control Sample 2: Interaction proteins of Flag-cGAS Sample 3: Interaction proteins of Flag-cGAS with lactate treatment Sample 4: Interaction proteins of Flag-PSMA4 Sample 5: Interaction proteins of Flag-PSMA4 with lactate treatment
Project description:Mutation analysis suggested that phosphorylation of cGAS at serine 120 is responsible for nuclear retention of cGAS by combining nucleoplasmic separation assay with immunofluorescence assay. Then, we sought to identify the kinase that phosphorylates cGAS.
Project description:The gastrointestinal tract of mammals is inhabited by hundreds of distinct species of commensal microorganisms that exist in a mutualistic relationship with the host. The process by which the commensal microbiota influence the host immune system is poorly understood. We show here that colonization of the small intestine of mice with a single commensal microbe, segmented filamentous bacterium (SFB), is sufficient to induce the appearance of CD4+ T helper cells that produce IL-17 and IL-22 (Th17 cells) in the lamina propria. SFB adhere tightly to the surface of epithelial cells in the terminal ileum of mice with Th17 cells but are absent from mice that have few Th17 cells. Colonization with SFB was correlated with increased expression of genes associated with inflammation, anti-microbial defenses, and tissue repair, and resulted in enhanced resistance to the intestinal pathogen Citrobacter rodentium. Control of Th17 cell differentiation by SFB may thus establish a balance between optimal host defense preparedness and potentially damaging T cell responses. Manipulation of this commensal-regulated pathway may provide new opportunities for enhancing mucosal immunity and treating autoimmune disease. Experiment Overall Design: We compared the gene expression profiles in the terminal ileum of Swiss-Webster GF mice before and after colonization with SFB, which induced robust Th17 cell differentiation. To sieve out host effects, the role of other microbiota, as well as other factors, we also evaluated the transcriptional program induced in Jackson C57BL/6 mice after co-housing with Taconic B6 animals, which also induces Th17 cell differentiation. 0.5 cm of the most distal part of the small intestine was dissected. Total RNA was extracted with TRIzol. RNA was labeled and hybridized to GeneChip Mouse Genome 430 2.0 arrays following the Affymetrix protocols. Data were analyzed in GeneSpring GX10.
Project description:To elucidate the role of RNF4 in DNA replication and fork reversal, we performed tandem affinity purification (TAP) using a HEK293T cell line that stably expresses SFB-tagged(S-protein tag, Flag epitope tag, and streptavidin-binding peptide tag) wild-type and CS mutant to isolate proteins that associate with RNF4.
Project description:We have identified ZNF618 as a novel binding partner of UHRF2. We have found that UHRF2 and ZNF618 co-localize at many genomic loci. Examination of genome-wide distribution of SFB-tagged UHRF2 and ZNF618 in 293T cells using ChIP-seq.
Project description:The hypothesis tested was that lentiviral driven cGAS expression activates antiviral gene expression. Cells were transduced with lentiviruses expressing cGAS or a control (firefly luciferase). Total RNA was harvested 48 h post-transduction and processed for Illumina BeadArray.