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The Ras GTPase-activating-like protein IQGAP1 bridges Gasdermin D to the ESCRT system to promote IL-1beta release via exosomes

ABSTRACT: IL-1β can exit the cytosol as an exosomal cargo following inflammasome activation in intestinal epithelial cells (IECs) in a Gasdermin D (GSDMD)-dependent manner. The mechanistic connection linking inflammasome activation and the biogenesis of exosomes has so far remained largely elusive. Here, we report the Ras GTPase-activating-like protein IQGAP1 functions as an adaptor, bridging GSDMD to the endosomal sorting complexes required for transport (ESCRT) machinery to promote the biogenesis of pro-IL-1β-containing exosomes in response to NLPR3 inflammasome activation. We identified IQGAP1 as a GSDMD-interacting protein through a non-biased proteomic analysis. Functional investigation indicated the IQGAP1-GSDMD interaction is required for LPS and ATP-induced exosome release. Further analysis revealed that IQGAP1 serves as an adaptor which bridges GSDMD and associated IL-1β complex to Tsg101, a component of the ESCRT complex, and enables the packaging of GSDMD and IL-1β into exosomes. Importantly, this process is dependent on an LPS-induced increase in GTP-bound CDC42, a small GTPase known to activate IQGAP1. Taken together, this study reveals IQGAP1 as a link between inflammasome activation and GSDMD-dependent, ESCRT-mediated exosomal release of IL-1β.

SUBMITTER: Dr. Yun Liao

PROVIDER: S-SCDT-10_15252-EMBJ_2022110780 | biostudies-other |

REPOSITORIES: biostudies-other

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The Ras GTPase-activating-like protein IQGAP1 bridges Gasdermin D to the ESCRT system to promote IL-1β release via exosomes.

Project description:IL-1β can exit the cytosol as an exosomal cargo following inflammasome activation in intestinal epithelial cells (IECs) in a Gasdermin D (GSDMD)-dependent manner. The mechanistic connection linking inflammasome activation and the biogenesis of exosomes has so far remained largely elusive. Here, we report the Ras GTPase-activating-like protein IQGAP1 functions as an adaptor, bridging GSDMD to the endosomal sorting complexes required for transport (ESCRT) machinery to promote the biogenesis of pro-IL-1β-containing exosomes in response to NLPR3 inflammasome activation. We identified IQGAP1 as a GSDMD-interacting protein through a non-biased proteomic analysis. Functional investigation indicated the IQGAP1-GSDMD interaction is required for LPS and ATP-induced exosome release. Further analysis revealed that IQGAP1 serves as an adaptor which bridges GSDMD and associated IL-1β complex to Tsg101, a component of the ESCRT complex, and enables the packaging of GSDMD and IL-1β into exosomes. Importantly, this process is dependent on an LPS-induced increase in GTP-bound CDC42, a small GTPase known to activate IQGAP1. Taken together, this study reveals IQGAP1 as a link between inflammasome activation and GSDMD-dependent, ESCRT-mediated exosomal release of IL-1β.

| S-EPMC9811620 | biostudies-literature

Nonredundant functions for Ras GTPase-activating proteins in tissue homeostasis.

Project description:Inactivation of the small guanosine triphosphate-binding protein Ras during receptor signal transduction is mediated by Ras guanosine triphosphatase (GTPase)-activating proteins (RasGAPs). Ten different RasGAPs have been identified and have overlapping patterns of tissue distribution. However, genetic analyses are revealing critical nonredundant functions for each RasGAP in tissue homeostasis and as regulators of disease processes in mouse and man. Here, we discuss advances in understanding the role of RasGAPs in the maintenance of tissue integrity.

| S-EPMC5483993 | biostudies-literature

Tumor Suppression of Ras GTPase-Activating Protein RASA5 through Antagonizing Ras Signaling Perturbation in Carcinomas.

Project description:Aberrant RAS signaling activation is common in cancers with even few Ras mutations, indicating alternative dysregulation other than genetic mutations. We identified a Ras GTPase-activating gene RASA5/SYNGAP1, at the common 6p21.3 deletion, methylated/downregulated in multiple carcinomas and different from other RASA family members (RASA1-RASA4), indicating its special functions in tumorigenesis. RASA5 mutations are rare, unlike other RASA members, whereas its promoter CpG methylation is frequent in multiple cancer cell lines and primary carcinomas and associated with patient's poor survival. RASA5 expression inhibited tumor cell migration/invasion and growth in mouse model, functioning as a tumor suppressor. RASA5 suppressed RAS signaling, depending on its Ras GTPase-activating protein catalytic activity, which could be counteracted by oncogenic HRas Q61L mutant. RASA5 knockdown enhanced Ras signaling to promote tumor cell growth. RASA5 also inhibited epithelial-mesenchymal transition (EMT) through regulating actin reorganization. Thus, epigenetic inactivation of RASA5 contributing to hyperactive RAS signaling is involved in Ras-driven human oncogenesis.

| S-EPMC6820368 | biostudies-literature

Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function.

Project description:KRas has recently been shown to be activated by monoubiquitination (mUb). Similar to oncogenic mutations, mUb of Ras at position 147 activates Ras by causing a defect in GTPase activating protein (GAP) function. To characterize the mechanism by which mUb impairs GAP-mediated downregulation of Ras, we made various modifications at position 147 of Ras and examined the impact on Ras sensitivity to GAP function. Whereas small modifications (iodoacetamide and glutathione) at position 147 of Ras do not affect GAP-mediated hydrolysis, ligation of Ras to Ub(G76C) (native linker), Ub(X77C) (one residue longer), and PDZ2 (with a native ubiquitin linker) was defective in GAP-mediated GTP hydrolysis. However, restoration of GAP activity was observed for Ras modified with the PDZ2 domain containing a shorter and stiffer linker region than ubiquitin. Therefore, the properties of the linker region dictate whether modification affects GAP-mediated hydrolysis, and our data indicate that the GAP defect requires a minimum linker length of 7 to 8 residues.

| S-EPMC3976977 | biostudies-literature

Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function.

Project description:Cell growth and differentiation are controlled by growth factor receptors coupled to the GTPase Ras. Oncogenic mutations disrupt GTPase activity, leading to persistent Ras signaling and cancer progression. Recent evidence indicates that monoubiquitination of Ras leads to Ras activation. Mutation of the primary site of monoubiquitination impairs the ability of activated K-Ras (one of the three mammalian isoforms of Ras) to promote tumor growth. To determine the mechanism of human Ras activation, we chemically ubiquitinated the protein and analyzed its function by NMR, computational modeling and biochemical activity measurements. We established that monoubiquitination has little effect on the binding of Ras to guanine nucleotide, GTP hydrolysis or exchange-factor activation but severely abrogates the response to GTPase-activating proteins in a site-specific manner. These findings reveal a new mechanism by which Ras can trigger persistent signaling in the absence of receptor activation or an oncogenic mutation.

| S-EPMC3537887 | biostudies-literature

IFN-gamma impairs release of IL-8 by IL-1beta-stimulated A549 lung carcinoma cells.

Project description:BackgroundProduction of interferon (IFN)-gamma is key to efficient anti-tumor immunity. The present study was set out to investigate effects of IFNgamma on the release of the potent pro-angiogenic mediator IL-8 by human A549 lung carcinoma cells.MethodsA549 cells were cultured and stimulated with interleukin (IL)-1beta alone or in combination with IFNgamma. IL-8 production by these cells was analyzed with enzyme linked immuno sorbent assay (ELISA). mRNA-expression was analyzed by real-time PCR and RNase protection assay (RPA), respectively. Expression of inhibitor-kappa Balpha, cellular IL-8, and cyclooxygenase-2 was analyzed by Western blot analysis.ResultsHere we demonstrate that IFNgamma efficiently reduced IL-8 secretion under the influence of IL-1beta. Surprisingly, real-time PCR analysis and RPA revealed that the inhibitory effect of IFNgamma on IL-8 was not associated with significant changes in mRNA levels. These observations concurred with lack of a modulatory activity of IFNgamma on IL-1beta-induced NF-kappaB activation as assessed by cellular IkappaB levels. Moreover, analysis of intracellular IL-8 suggests that IFNgamma modulated IL-8 secretion by action on the posttranslational level. In contrast to IL-8, IL-1beta-induced cyclooxygenase-2 expression and release of IL-6 were not affected by IFNgamma indicating that modulation of IL-1beta action by this cytokine displays specificity.ConclusionData presented herein agree with an angiostatic role of IFNgamma as seen in rodent models of solid tumors and suggest that increasing T helper type 1 (Th1)-like functions in lung cancer patients e.g. by local delivery of IFNgamma may mediate therapeutic benefit via mechanisms that potentially include modulation of pro-angiogenic IL-8.

| S-EPMC2556346 | biostudies-literature

Tissue-resident macrophages actively suppress IL-1beta release via a reactive prostanoid/IL-10 pathway.

Project description:The alarm cytokine interleukin-1β (IL-1β) is a potent activator of the inflammatory cascade following pathogen recognition. IL-1β production typically requires two signals: first, priming by recognition of pathogen-associated molecular patterns leads to the production of immature pro-IL-1β; subsequently, inflammasome activation by a secondary signal allows cleavage and maturation of IL-1β from its pro-form. However, despite the important role of IL-1β in controlling local and systemic inflammation, its overall regulation is still not fully understood. Here we demonstrate that peritoneal tissue-resident macrophages use an active inhibitory pathway, to suppress IL-1β processing, which can otherwise occur in the absence of a second signal. Programming by the transcription factor Gata6 controls the expression of prostacyclin synthase, which is required for prostacyclin production after lipopolysaccharide stimulation and optimal induction of IL-10. In the absence of secondary signal, IL-10 potently inhibits IL-1β processing, providing a previously unrecognized control of IL-1β in tissue-resident macrophages.

| S-EPMC7360975 | biostudies-literature

A Ras-GTPase-activating protein SH3-domain-binding protein.

Project description:We report the purification of a Ras-GTPase-activating protein (GAP)-binding protein, G3BP, a ubiquitously expressed cytosolic 68-kDa protein that coimmunoprecipitates with GAP. G3BP physically associates with the SH3 domain of GAP, which previously had been shown to be essential for Ras signaling. The G3BP cDNA revealed that G3BP is a novel 466-amino-acid protein that shares several features with heterogeneous nuclear RNA-binding proteins, including ribonucleoprotein (RNP) motifs RNP1 and RNP2, an RG-rich domain, and acidic sequences. Recombinant G3BP binds effectively to the GAP SH3 domain G3BP coimmunoprecipitates with GAP only when cells are in a proliferating state, suggesting a recruitment of a GAP-G3BP complex when Ras is in its activated conformation.

| S-EPMC231246 | biostudies-other

The Ras GTPase-activating protein Rasal3 supports survival of naive T cells.

Project description:The Ras-mitogen-activated protein kinase (MAPK) pathway is crucial for T cell receptor (TCR) signaling in the development and function of T cells. The significance of various modulators of the Ras-MAPK pathway in T cells, however, remains to be fully understood. Ras-activating protein-like 3 (Rasal3) is an uncharacterized member of the SynGAP family that contains a conserved Ras GTPase-activating protein (GAP) domain, and is predominantly expressed in the T cell lineage. In the current study, we investigated the function and physiological roles of Rasal3. Our results showed that Rasal3 possesses RasGAP activity, but not Rap1GAP activity, and represses TCR-stimulated ERK phosphorylation in a T cell line. In systemic Rasal3-deficient mice, T cell development in the thymus including positive selection, negative selection, and β-selection was unaffected. However, the number of naive, but not effector memory CD4 and CD8 T cell in the periphery was significantly reduced in Rasal3-deficient mice, and associated with a marked increase in apoptosis of these cells. Indeed, survival of Rasal3 deficient naive CD4 T cells in vivo by adoptive transfer was significantly impaired, whereas IL-7-dependent survival of naive CD4 T cells in vitro was unaltered. Collectively, Rasal3 is required for in vivo survival of peripheral naive T cells, contributing to the maintenance of optimal T cell numbers.

| S-EPMC4368693 | biostudies-literature

Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer.

Project description:Background & aimsAberrant activation of the RAS pathway is ubiquitous in human hepatocarcinogenesis, but the molecular mechanisms leading to RAS induction in the absence of RAS mutations remain under-investigated. We defined the role of Ras GTPase activating proteins (GAPs) in the constitutive activity of Ras signaling during human hepatocarcinogenesis.MethodsThe mutation status of RAS genes and RAS effectors was assessed in a collection of human hepatocellular carcinomas (HCC). Levels of RAS GAPs (RASA1-4, RASAL1, nGAP, SYNGAP1, DAB2IP, and NF1) and the RASAL1 upstream inducer PITX1 were determined by real-time RT-PCR and immunoblotting. The promoter and genomic status of RASAL1, DAB2IP, NF1, and PITX1 were assessed by methylation assays and microsatellite analysis. Effects of RASAL1, DAB2IP, and PITX1 on HCC growth were evaluated by transfection and siRNA analyses of HCC cell lines.ResultsIn the absence of Ras mutations, downregulation of at least one RAS GAP (RASAL1, DAB2IP, or NF1) was found in all HCC samples. Low levels of DAB2IP and PITX1 were detected mostly in a HCC subclass from patients with poor survival, indicating that these proteins control tumor aggressiveness. In HCC cells, reactivation of RASAL1, DAB2IP, and PITX1 inhibited proliferation and induced apoptosis, whereas their silencing increased proliferation and resistance to apoptosis.ConclusionsSelective suppression of RASAL1, DAB2IP, or NF1 RAS GAPs results in unrestrained activation of Ras signaling in the presence of wild-type RAS in HCC.

| S-EPMC3031080 | biostudies-literature

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