ABSTRACT
Germline activating mutations in STAT3 cause a multi-systemic autoimmune and autoinflammatory condition. By studying a mouse model, Toth et al. (https://doi.org/10.1084/jem.20232091) propose a role for dysregulated IL-22 production by Th17 cells in causing some aspects of immune-mediated skin inflammation in human STAT3 GOF syndrome.
Subject(s)
Interleukin-22 , STAT3 Transcription Factor , Skin , Th17 Cells , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , Animals , Humans , Th17 Cells/immunology , Th17 Cells/metabolism , Skin/metabolism , Skin/pathology , Interleukins/genetics , Interleukins/metabolism , Gain of Function Mutation , Mice , Inflammation/metabolismABSTRACT
Hypertension affects one billion people worldwide and is the most common risk factor for cardiovascular disease, yet a comprehensive picture of its underlying genetic factors is incomplete. Amongst regulators of blood pressure is the renal outer medullary potassium (ROMK) channel. While select ROMK mutants are prone to premature degradation and lead to disease, heterozygous carriers of some of these same alleles are protected from hypertension. Therefore, we hypothesized that gain-of-function (GoF) ROMK variants which increase potassium flux may predispose people to hypertension. To begin to test this hypothesis, we employed genetic screens and a candidate-based approach to identify six GoF variants in yeast. Subsequent functional assays in higher cells revealed two variant classes. The first group exhibited greater stability in the endoplasmic reticulum, enhanced channel assembly, and/or increased protein at the cell surface. The second group of variants resided in the PIP2-binding pocket, and computational modeling coupled with patch-clamp studies demonstrated lower free energy for channel opening and slowed current rundown, consistent with an acquired PIP2-activated state. Together, these findings advance our understanding of ROMK structure-function, suggest the existence of hyperactive ROMK alleles in humans, and establish a system to facilitate the development of ROMK-targeted antihypertensives.
Subject(s)
Potassium Channels, Inwardly Rectifying , Humans , Potassium Channels, Inwardly Rectifying/metabolism , Potassium Channels, Inwardly Rectifying/genetics , Gain of Function Mutation , Potassium/metabolism , Hypertension/genetics , Hypertension/metabolism , Kidney/metabolism , Mutation/genetics , HEK293 Cells , Endoplasmic Reticulum/metabolism , Ion Transport , AllelesABSTRACT
Gasdermin E (GSDME), a member of the gasdermin protein family, is associated with post-lingual hearing loss. All GSDME pathogenic mutations lead to skipping exon 8; however, the molecular mechanisms underlying hearing loss caused by GSDME mutants remain unclear. GSDME was recently identified as one of the mediators of programmed cell death, including apoptosis and pyroptosis. Therefore, in this study, we injected mice with GSDME mutant (MT) and examined the expression levels to assess its effect on hearing impairment. We observed loss of hair cells in the organ of Corti and spiral ganglion neurons. Further, the N-terminal release from the GSDME mutant in HEI-OC1 cells caused pyroptosis, characterized by cell swelling and rupture of the plasma membrane, releasing lactate dehydrogenase and cytokines such as interleukin-1ß. We also observed that the N-terminal release from GSDME mutants could permeabilize the mitochondrial membrane, releasing cytochromes and activating the mitochondrial apoptotic pathway, thereby generating possible positive feedback on the cleavage of GSDME. Furthermore, we found that treatment with disulfiram or dimethyl fumarate might inhibit pyroptosis and apoptosis by inhibiting the release of GSDME-N from GSDME mutants. In conclusion, this study elucidated the molecular mechanism associated with hearing loss caused by GSDME gene mutations, offering novel insights for potential treatment strategies.
Subject(s)
Apoptosis , Pyroptosis , Pyroptosis/genetics , Animals , Mice , Gain of Function Mutation , Hearing Loss/genetics , Hearing Loss/pathology , Humans , Spiral Ganglion/metabolism , Spiral Ganglion/pathology , Organ of Corti/metabolism , Organ of Corti/pathology , Hair Cells, Auditory/metabolism , Hair Cells, Auditory/pathology , GasderminsABSTRACT
BACKGROUND: We clinically and genetically evaluated a Taiwanese boy presenting with developmental delay, organomegaly, hypogammaglobulinemia and hypopigmentation without osteopetrosis. Whole-exome sequencing revealed a de novo gain-of-function variant, p.Tyr715Cys, in the C-terminal domain of ClC-7 encoded by CLCN7. METHODS: Nicoli et al. (2019) assessed the functional impact of p.Tyr715Cys by heterologous expression in Xenopus oocytes and evaluating resulting currents. RESULTS: The variant led to increased outward currents, indicating it underlies the patient's phenotype of lysosomal hyperacidity, storage defects and vacuolization. This demonstrates the crucial physiological role of ClC-7 antiporter activity in maintaining appropriate lysosomal pH. CONCLUSION: Elucidating mechanisms by which CLCN7 variants lead to lysosomal dysfunction will advance understanding of genotype-phenotype correlations. Identifying modifier genes and compensatory pathways may reveal therapeutic targets. Ongoing functional characterization of variants along with longitudinal clinical evaluations will continue advancing knowledge of ClC-7's critical roles and disease mechanisms resulting from its dysfunction. Expanded cohort studies are warranted to delineate the full spectrum of associated phenotypes.
Subject(s)
Chloride Channels , Humans , Male , Chloride Channels/genetics , Chloride Channels/metabolism , Gain of Function Mutation , Osteopetrosis/genetics , Osteopetrosis/pathology , Phenotype , Child, PreschoolABSTRACT
Gain-of-function mutations in STING cause STING-associated vasculopathy with onset in infancy (SAVI) characterized by early-onset systemic inflammation, skin vasculopathy, and interstitial lung disease. Here, we report and characterize a novel STING variant (F269S) identified in a SAVI patient. Single-cell transcriptomics of patient bone marrow revealed spontaneous activation of interferon (IFN) and inflammatory pathways across cell types and a striking prevalence of circulating naïve T cells was observed. Inducible STING F269S expression conferred enhanced signaling through ligand-independent translocation of the protein to the Golgi, protecting cells from viral infections but preventing their efficient immune priming. Additionally, endothelial cell activation was promoted and further exacerbated by cytokine secretion by SAVI immune cells, resulting in inflammation and endothelial damage. Our findings identify STING F269S mutation as a novel pathogenic variant causing SAVI, highlight the importance of the crosstalk between endothelial and immune cells in the context of lung disease, and contribute to a better understanding of how aberrant STING activation can cause pathology.
Subject(s)
Endothelial Cells , Membrane Proteins , Humans , Infant , Endothelial Cells/metabolism , Endothelial Cells/pathology , Gain of Function Mutation , Golgi Apparatus/metabolism , Interferons/metabolism , Interferons/genetics , Lung Diseases, Interstitial/genetics , Lung Diseases, Interstitial/pathology , Lung Diseases, Interstitial/immunology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutation , Signal Transduction , Vascular Diseases/genetics , Vascular Diseases/pathology , Infant, Newborn , Child, Preschool , FemaleSubject(s)
Atrial Fibrillation , Gain of Function Mutation , Homeobox Protein PITX2 , Homeodomain Proteins , Transcription Factors , Atrial Fibrillation/genetics , Atrial Fibrillation/physiopathology , Atrial Fibrillation/diagnosis , Humans , Transcription Factors/genetics , Transcription Factors/metabolism , Homeodomain Proteins/genetics , Mitochondria, Heart/genetics , Mitochondria, Heart/metabolism , Mitochondria, Heart/pathology , Animals , Membrane Potential, Mitochondrial/genetics , Genetic Predisposition to Disease , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , PhenotypeABSTRACT
KRAS gain-of-function mutations are frequently observed in sporadic arteriovenous malformations. The mechanisms underlying the progression of such KRAS-driven malformations are still incompletely understood, and no treatments for the condition are approved. Here, we show the effectiveness of sotorasib, a specific KRAS G12C inhibitor, in reducing the volume of vascular malformations and improving survival in two mouse models carrying a mosaic Kras G12C mutation. We then administered sotorasib to two adult patients with severe KRAS G12C-related arteriovenous malformations. Both patients had rapid reductions in symptoms and arteriovenous malformation size. Targeting KRAS G12C appears to be a promising therapeutic approach for patients with KRAS G12C-related vascular malformations. (Funded by the European Research Council and others.).
Subject(s)
Arteriovenous Malformations , Proto-Oncogene Proteins p21(ras) , Animals , Female , Humans , Male , Mice , Middle Aged , Arteriovenous Malformations/diagnosis , Arteriovenous Malformations/diagnostic imaging , Arteriovenous Malformations/drug therapy , Arteriovenous Malformations/genetics , Disease Models, Animal , Gain of Function Mutation , Mutation , Piperazines/therapeutic use , Proto-Oncogene Proteins p21(ras)/genetics , Pyridines/therapeutic use , Pyrimidines , Cardiovascular Agents/therapeutic use , Young AdultABSTRACT
FOXP3+ regulatory T cells (Treg) are required for maintaining immune tolerance and preventing systemic autoimmunity. PI3Kδ is required for normal Treg development and function. However, the impacts of dysregulated PI3Kδ signaling on Treg function remain incompletely understood. In this study, we used a conditional mouse model of activated PI3Kδ syndrome to investigate the role of altered PI3Kδ signaling specifically within the Treg compartment. Activated mice expressing a PIK3CD gain-of-function mutation (aPIK3CD) specifically within the Treg compartment exhibited weight loss and evidence for chronic inflammation, as demonstrated by increased memory/effector CD4+ and CD8+ T cells with enhanced IFN-γ secretion, spontaneous germinal center responses, and production of broad-spectrum autoantibodies. Intriguingly, aPIK3CD facilitated Treg precursor development within the thymus and an increase in peripheral Treg numbers. Peripheral Treg, however, exhibited an altered phenotype, including increased PD-1 expression and reduced competitive fitness. Consistent with these findings, Treg-specific aPIK3CD mice mounted an elevated humoral response following immunization with a T cell-dependent Ag, which correlated with a decrease in follicular Treg. Taken together, these findings demonstrate that an optimal threshold of PI3Kδ activity is critical for Treg homeostasis and function, suggesting that PI3Kδ signaling in Treg might be therapeutically targeted to either augment or inhibit immune responses.
Subject(s)
Class I Phosphatidylinositol 3-Kinases , Homeostasis , T-Lymphocytes, Regulatory , Animals , T-Lymphocytes, Regulatory/immunology , Mice , Class I Phosphatidylinositol 3-Kinases/genetics , Class I Phosphatidylinositol 3-Kinases/immunology , Homeostasis/immunology , Signal Transduction/immunology , Mice, Inbred C57BL , Germinal Center/immunology , Gain of Function Mutation , Primary Immunodeficiency DiseasesABSTRACT
Germline gain-of-function (GOF) variants in STAT3 cause an inborn error of immunity associated with early-onset poly-autoimmunity and immune dysregulation. To study tissue-specific immune dysregulation, we used a mouse model carrying a missense variant (p.G421R) that causes human disease. We observed spontaneous and imiquimod (IMQ)-induced skin inflammation associated with cell-intrinsic local Th17 responses in STAT3 GOF mice. CD4+ T cells were sufficient to drive skin inflammation and showed increased Il22 expression in expanded clones. Certain aspects of disease, including increased epidermal thickness, also required the presence of STAT3 GOF in epithelial cells. Treatment with a JAK inhibitor improved skin disease without affecting local Th17 recruitment and cytokine production. These findings collectively support the involvement of Th17 responses in the development of organ-specific immune dysregulation in STAT3 GOF and suggest that the presence of STAT3 GOF in tissues is important for disease and can be targeted with JAK inhibition.
Subject(s)
Gain of Function Mutation , Imiquimod , STAT3 Transcription Factor , Th17 Cells , Animals , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , Th17 Cells/immunology , Mice , Humans , Imiquimod/pharmacology , Skin/pathology , Skin/metabolism , Skin/immunology , Interleukin-22 , Dermatitis/immunology , Dermatitis/genetics , Dermatitis/pathology , Dermatitis/metabolism , Mice, Inbred C57BL , Interleukins/genetics , Interleukins/metabolism , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Inflammation/genetics , Inflammation/metabolism , Inflammation/immunology , Inflammation/pathologyABSTRACT
UNC93B1 is a transmembrane domain protein mediating the signaling of endosomal Toll-like receptors (TLRs). We report five families harboring rare missense substitutions (I317M, G325C, L330R, R466S, and R525P) in UNC93B1 causing systemic lupus erythematosus (SLE) or chilblain lupus (CBL) as either autosomal dominant or autosomal recessive traits. As for a D34A mutation causing murine lupus, we recorded a gain of TLR7 and, to a lesser extent, TLR8 activity with the I317M (in vitro) and G325C (in vitro and ex vivo) variants in the context of SLE. Contrastingly, in three families segregating CBL, the L330R, R466S, and R525P variants were isomorphic with respect to TLR7 activity in vitro and, for R525P, ex vivo. Rather, these variants demonstrated a gain of TLR8 activity. We observed enhanced interaction of the G325C, L330R, and R466S variants with TLR8, but not the R525P substitution, indicating different disease mechanisms. Overall, these observations suggest that UNC93B1 mutations cause monogenic SLE or CBL due to differentially enhanced TLR7 and TLR8 signaling.
Subject(s)
Chilblains , Lupus Erythematosus, Systemic , Toll-Like Receptor 7 , Female , Humans , Male , Chilblains/genetics , Gain of Function Mutation , HEK293 Cells , Lupus Erythematosus, Cutaneous/genetics , Lupus Erythematosus, Cutaneous/pathology , Lupus Erythematosus, Systemic/genetics , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mutation, Missense , Pedigree , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/genetics , Toll-Like Receptor 8/metabolism , Child, Preschool , Child , Young Adult , AdultABSTRACT
Together with its ß-subunit OSTM1, ClC-7 performs 2Cl-/H+ exchange across lysosomal membranes. Pathogenic variants in either gene cause lysosome-related pathologies, including osteopetrosis and lysosomal storage. CLCN7 variants can cause recessive or dominant disease. Different variants entail different sets of symptoms. Loss of ClC-7 causes osteopetrosis and mostly neuronal lysosomal storage. A recently reported de novo CLCN7 mutation (p.Tyr715Cys) causes widespread severe lysosome pathology (hypopigmentation, organomegaly, and delayed myelination and development, "HOD syndrome"), but no osteopetrosis. We now describe two additional HOD individuals with the previously described p.Tyr715Cys and a novel p.Lys285Thr mutation, respectively. Both mutations decreased ClC-7 inhibition by PI(3,5)P2 and affected residues lining its binding pocket, and shifted voltage-dependent gating to less positive potentials, an effect partially conferred to WT subunits in WT/mutant heteromers. This shift predicts augmented pH gradient-driven Cl- uptake into vesicles. Overexpressing either mutant induced large lysosome-related vacuoles. This effect depended on Cl-/H+-exchange, as shown using mutants carrying uncoupling mutations. Fibroblasts from the p.Y715C patient also displayed giant vacuoles. This was not observed with p.K285T fibroblasts probably due to residual PI(3,5)P2 sensitivity. The gain of function caused by the shifted voltage-dependence of either mutant likely is the main pathogenic factor. Loss of PI(3,5)P2 inhibition will further increase current amplitudes, but may not be a general feature of HOD. Overactivity of ClC-7 induces pathologically enlarged vacuoles in many tissues, which is distinct from lysosomal storage observed with the loss of ClC-7 function. Osteopetrosis results from a loss of ClC-7, but osteoclasts remain resilient to increased ClC-7 activity.
Subject(s)
Chloride Channels , Lysosomal Storage Diseases , Lysosomes , Humans , Male , Chloride Channels/genetics , Chloride Channels/metabolism , Gain of Function Mutation , HEK293 Cells , Lysosomal Storage Diseases/genetics , Lysosomal Storage Diseases/metabolism , Lysosomal Storage Diseases/pathology , Lysosomes/metabolism , Lysosomes/genetics , Membrane Proteins , Mutation, Missense , Phosphatidylinositol Phosphates/metabolism , Ubiquitin-Protein Ligases , Vacuoles/metabolism , Vacuoles/genetics , Vacuoles/pathologyABSTRACT
Hypertrophic cardiomyopathy (HCM) is the major contributor to morbidity and mortality in Noonan syndrome (NS). Gain-of-function variants in RAF1 are associated with high prevalence of HCM. Among these, NM_002880.4:c.770C > T, NP_002871.1:p.(Ser257Leu) accounts for approximately half of cases and has been reported as associated with a particularly severe outcome. Nevertheless, comprehensive studies on cases harboring this variant are missing. To precisely define the phenotype associated to the RAF1:c.770C > T, variant, an observational retrospective analysis on patients carrying the c.770C > T variant was conducted merging 17 unpublished patients and literature-derived ones. Data regarding prenatal findings, clinical features and cardiac phenotypes were collected to provide an exhaustive description of the associated phenotype. Clinical information was collected in 107 patients. Among them, 92% had HCM, mostly diagnosed within the first year of life. Thirty percent of patients were preterm and 47% of the newborns was admitted in a neonatal intensive care unit, mainly due to respiratory complications of HCM and/or pulmonary arterial hypertension. Mortality rate was 13%, mainly secondary to HCM-related complications (62%) at the average age of 7.5 months. Short stature had a prevalence of 91%, while seizures and ID of 6% and 12%, respectively. Two cases out of 75 (3%) developed neoplasms. In conclusion, patients with the RAF1:c.770C > T pathogenic variant show a particularly severe phenotype characterized by rapidly progressive neonatal HCM and high mortality rate suggesting the necessity of careful monitoring and early intervention to prevent or slow down the progression of HCM.
Subject(s)
Cardiomyopathy, Hypertrophic , Noonan Syndrome , Phenotype , Proto-Oncogene Proteins c-raf , Humans , Noonan Syndrome/genetics , Noonan Syndrome/pathology , Proto-Oncogene Proteins c-raf/genetics , Female , Male , Cardiomyopathy, Hypertrophic/genetics , Cardiomyopathy, Hypertrophic/pathology , Infant , Infant, Newborn , Child, Preschool , Child , Adolescent , Adult , Gain of Function MutationABSTRACT
Gain of function mutations in the pore forming Kir6 subunits of the ATP sensitive K+ channels (K(ATP) channels) of pancreatic ß-cells are the major cause of neonatal diabetes in humans. In this study, we show that in insulin secreting mouse ß-cell lines, gain of function mutations in Kir6.1 result in a significant connexin36 (Cx36) overexpression, which form gap junctional connections and mediate electrical coupling between ß-cells within pancreatic islets. Using computational modeling, we show that upregulation in Cx36 might play a functional role in the impairment of glucose stimulated Ca2+ oscillations in a cluster of ß-cells with Kir6.1 gain of function mutations in their K(ATP) channels (GoF-K(ATP) channels). Our results show that without an increase in Cx36 expression, a gain of function mutation in Kir6.1 might not be sufficient to diminish glucose stimulated Ca2+ oscillations in a ß-cell cluster. We also show that a reduced Cx36 expression, which leads to loss of coordination in a wild-type ß-cell cluster, restores coordinated Ca2+ oscillations in a ß-cell cluster with GoF-K(ATP) channels. Our results indicate that in a heterogenous ß-cell cluster with GoF-K(ATP) channels, there is an inverted u-shaped nonmonotonic relation between the cluster activity and Cx36 expression. These results show that in a neonatal diabetic ß-cell model, gain of function mutations in the Kir6.1 cause Cx36 overexpression, which aggravates the impairment of glucose stimulated Ca2+ oscillations.
Subject(s)
Insulin-Secreting Cells , KATP Channels , Up-Regulation , Insulin-Secreting Cells/metabolism , Animals , Mice , KATP Channels/genetics , KATP Channels/metabolism , Connexins/genetics , Connexins/metabolism , Gain of Function Mutation , Gap Junction delta-2 Protein , Calcium Signaling , Models, Biological , Calcium/metabolism , HumansABSTRACT
Signal Transducer and Activator of Transcription (STAT) proteins play pivotal roles in immune regulation. The dysregulation of these proteins, attributed to both gain-of-function (GOF) and loss-of-function (LOF) variants, has emerged as a substantial and intricate area of research. This comprehensive review delves into the intricate details of the diverse clinical spectrum associated with STAT variants and the immunological findings linked to these genetic alterations. Although this review does not encompass the treatment of each individual disease, we discuss investigative approaches ranging from immunophenotyping assessment to evaluation of STAT protein activity. These investigations play a crucial role in identifying affected patients and understanding the complexities of STAT.
Subject(s)
Gain of Function Mutation , STAT Transcription Factors , Humans , STAT Transcription Factors/genetics , STAT Transcription Factors/metabolism , STAT Transcription Factors/immunology , Loss of Function Mutation , Immunogenetics/methods , Genetic Predisposition to Disease , AnimalsABSTRACT
PURPOSES: STAT1 is a transduction and transcriptional regulator that functions within the classical JAK/STAT pathway. In addition to chronic mucocutaneous candidiasis, bacterial infections are a common occurrence in patients with STAT1 gain-of-function (GOF) mutations. These patients often exhibit skewing of B cell subsets; however, the impact of STAT1-GOF mutations on B cell-mediated humoral immunity remains largely unexplored. It is also unclear whether these patients with IgG within normal range require regular intravenous immunoglobulin (IVIG) therapy. METHODS: Eleven patients (harboring nine different STAT1-GOF mutations) were enrolled. Reporter assays and immunoblot analyses were performed to confirm STAT1 mutations. Flow cytometry, deep sequencing, ELISA, and ELISpot were conducted to assess the impact of STAT1-GOF on humoral immunity. RESULTS: All patients exhibited increased levels of phospho-STAT1 and total STAT1 protein, with two patients carrying novel mutations. In vitro assays showed that these two novel mutations were GOF mutations. Three patients with normal total IgG levels received regular IVIG infusions, resulting in effective control of bacterial infections. Four cases showed impaired affinity and specificity of pertussis toxin-specific antibodies, accompanied by reduced generation of class-switched memory B cells. Patients also had a disrupted immunoglobulin heavy chain (IGH) repertoire, coupled with a marked reduction in the somatic hypermutation frequency of switched Ig transcripts. CONCLUSION: STAT1-GOF mutations disrupt B cell compartments and skew IGH characteristics, resulting in impaired affinity and antigen-specificity of antibodies and recurrent bacterial infections. Regular IVIG therapy can control these infections in patients, even those with normal total IgG levels.
Subject(s)
B-Lymphocytes , Bacterial Infections , Gain of Function Mutation , Immunoglobulins, Intravenous , STAT1 Transcription Factor , Humans , STAT1 Transcription Factor/genetics , Bacterial Infections/immunology , Bacterial Infections/genetics , Female , Male , Child , Immunoglobulins, Intravenous/therapeutic use , B-Lymphocytes/immunology , Adult , Immunoglobulin G/immunology , Immunoglobulin G/blood , Child, Preschool , Adolescent , Young Adult , Immunity, HumoralABSTRACT
Transient receptor potential vanilloid (TRPV) ion channels play a crucial role in various cellular functions by regulating intracellular Ca2+ levels and have been extensively studied in the context of several metabolic diseases. However, the regulatory effects of TRPV3 in obesity and lipolysis are not well understood. In this study, utilizing a TRPV3 gain-of-function mouse model (TRPV3G568V/G568V), we assessed the metabolic phenotype of both TRPV3G568V/G568V mice and their control littermates, which were randomly assigned to either a 12-week high-fat diet or a control diet. We investigated the potential mechanisms underlying the role of TRPV3 in restraining obesity and promoting lipolysis both in vivo and in vitro. Our findings indicate that a high-fat diet led to significant obesity, characterized by increased epididymal and inguinal white adipose tissue weight and higher fat mass. However, the gain-of-function mutation in TRPV3 appeared to counteract these adverse effects by enhancing lipolysis in visceral fat through the upregulation of the major lipolytic enzyme, adipocyte triglyceride lipase (ATGL). In vitro experiments using carvacrol, a TRPV3 agonist, demonstrated the promotion of lipolysis and antioxidation in 3T3-L1 adipocytes after TRPV3 activation. Notably, carvacrol failed to stimulate Ca2+ influx, lipolysis, and antioxidation in 3T3-L1 adipocytes treated with BAPTA-AM, a cell-permeable calcium chelator. Our results revealed that TRPV3 activation induced the action of transcriptional factor nuclear factor erythroid 2-related factor 2 (NRF2), resulting in increased expression of ferroptosis suppressor protein 1 (FSP1) and superoxide dismutase2 (SOD2). Moreover, the inhibition of NRF2 impeded carvacrol-induced lipolysis and antioxidation in 3T3-L1 adipocytes, with downregulation of ATGL, FSP1, and SOD2. In summary, our study suggests that TRPV3 promotes visceral fat lipolysis and inhibits diet-induced obesity through the activation of the NRF2/FSP1 signaling axis. We propose that TRPV3 may be a potential therapeutic target in the treatment of obesity.
Subject(s)
Diet, High-Fat , Lipolysis , NF-E2-Related Factor 2 , Obesity , Signal Transduction , TRPV Cation Channels , Animals , Male , Mice , 3T3-L1 Cells , Acyltransferases , Adipocytes/metabolism , Adipocytes/pathology , Diet, High-Fat/adverse effects , Gain of Function Mutation , Lipase/metabolism , Lipase/genetics , Mice, Inbred C57BL , NF-E2-Related Factor 2/metabolism , NF-E2-Related Factor 2/genetics , Obesity/metabolism , Obesity/genetics , Obesity/pathology , Obesity/etiology , TRPV Cation Channels/metabolism , TRPV Cation Channels/geneticsABSTRACT
Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 posttranslational regulation. Here, we characterize a phosphorylation site at Ser325 situated C terminal to the catalytic domain of PTPN22 and its roles in altering protein function. In human T cells, Ser325 is phosphorylated by glycogen synthase kinase-3 (GSK3) following TCR stimulation, which promotes its TCR-inhibitory activity. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9-mediated suppression of Ser325 phosphorylation and inhibited by mimicking it via glutamic acid substitution. Global phospho-mass spectrometry showed Ser325 phosphorylation state alters downstream transcriptional activity through enrichment of Swi3p, Rsc8p, and Moira domain binding proteins, and next-generation sequencing revealed it differentially regulates the expression of chemokines and T cell activation pathways. Moreover, in vitro kinetic data suggest the modulation of activity depends on a cellular context. Finally, we begin to address the structural and mechanistic basis for the influence of Ser325 phosphorylation on the protein's properties by deuterium exchange mass spectrometry and NMR spectroscopy. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22.