RESUMO
The control over the extent and timing of G protein signaling is provided by the regulator of G protein signaling (RGS) proteins that deactivate G protein α subunits (Gα). Mammalian genomes encode 20 canonical RGS and 16 Gα genes with key roles in physiology and disease. To understand the principles governing the selectivity of Gα regulation by RGS, we examine the catalytic activity of all canonical human RGS proteins and their selectivity for a complete set of Gα substrates using real-time kinetic measurements in living cells. The data reveal rules governing RGS-Gα recognition, the structural basis of its selectivity, and provide principles for engineering RGS proteins with defined selectivity. The study also explores the evolution of RGS-Gα selectivity through ancestral reconstruction and demonstrates how naturally occurring non-synonymous variants in RGS alter signaling. These results provide a blueprint for decoding signaling selectivity and advance our understanding of molecular recognition principles.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Proteínas RGS/genética , Animais , Feminino , Reguladores de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Cultura Primária de Células , Ligação Proteica , Proteínas RGS/metabolismo , Proteínas RGS/fisiologia , Transdução de Sinais/genéticaRESUMO
Early prenatal inflammatory conditions are thought to be a risk factor for different neurodevelopmental disorders. Maternal interleukin-6 (IL-6) elevation during pregnancy causes abnormal behavior in offspring, but whether these defects result from altered synaptic developmental trajectories remains unclear. Here we showed that transient IL-6 elevation via injection into pregnant mice or developing embryos enhanced glutamatergic synapses and led to overall brain hyperconnectivity in offspring into adulthood. IL-6 activated synaptogenesis gene programs in glutamatergic neurons and required the transcription factor STAT3 and expression of the RGS4 gene. The STAT3-RGS4 pathway was also activated in neonatal brains during poly(I:C)-induced maternal immune activation, which mimics viral infection during pregnancy. These findings indicate that IL-6 elevation at early developmental stages is sufficient to exert a long-lasting effect on glutamatergic synaptogenesis and brain connectivity, providing a mechanistic framework for the association between prenatal inflammatory events and brain neurodevelopmental disorders.
Assuntos
Hipocampo/metabolismo , Interleucina-6/biossíntese , Exposição Materna , Neurônios/metabolismo , Efeitos Tardios da Exposição Pré-Natal , Sinapses/metabolismo , Animais , Citocinas/biossíntese , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Hipocampo/fisiopatologia , Mediadores da Inflamação/metabolismo , Camundongos , Gravidez , Transdução de Sinais , Transmissão SinápticaRESUMO
Genetic variation influences how the genome is interpreted in individuals and in mouse strains used to model immune responses. We developed approaches to utilize next-generation sequencing datasets to identify sequence variation in genes and enhancer elements in congenic and backcross mouse models. We defined genetic variation in the widely used B6-CD45.2 and B6.SJL-CD45.1 congenic model, identifying substantial differences in SJL genetic content retained in B6.SJL-CD45.1 strains on the basis of the vendor source of the mice. Genes encoding PD-1, CD62L, Bcl-2, cathepsin E, and Cxcr4 were within SJL genetic content in at least one vendor source of B6.SJL-CD45.1 mice. SJL genetic content affected enhancer elements, gene regulation, protein expression, and amino acid content in CD4+ T helper 1 cells, and mice infected with influenza showed reduced expression of Cxcr4 on B6.SJL-CD45.1 T follicular helper cells. These findings provide information on experimental variables and aid in creating approaches that account for genetic variables.
Assuntos
Catepsina E/metabolismo , Elementos Facilitadores Genéticos/genética , Imunidade/genética , Receptores CXCR4/metabolismo , Células Th1/imunologia , Animais , Catepsina E/genética , Comércio , Regulação da Expressão Gênica , Patrimônio Genético , Variação Genética , Centro Germinativo/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Endogamia , Antígenos Comuns de Leucócito/genética , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Modelos Animais , Receptores CXCR4/genéticaRESUMO
Disease-associated variants identified from genome-wide association studies (GWASs) frequently map to non-coding areas of the genome such as introns and intergenic regions. An exclusive reliance on gene-agnostic methods of genomic investigation could limit the identification of relevant genes associated with polygenic diseases such as Alzheimer disease (AD). To overcome such potential restriction, we developed a gene-constrained analytical method that considers only moderate- and high-risk variants that affect gene coding sequences. We report here the application of this approach to publicly available datasets containing 181,388 individuals without and with AD and the resulting identification of 660 genes potentially linked to the higher AD prevalence among Africans/African Americans. By integration with transcriptome analysis of 23 brain regions from 2,728 AD case-control samples, we concentrated on nine genes that potentially enhance the risk of AD: AACS, GNB5, GNS, HIPK3, MED13, SHC2, SLC22A5, VPS35, and ZNF398. GNB5, the fifth member of the heterotrimeric G protein beta family encoding Gß5, is primarily expressed in neurons and is essential for normal neuronal development in mouse brain. Homozygous or compound heterozygous loss of function of GNB5 in humans has previously been associated with a syndrome of developmental delay, cognitive impairment, and cardiac arrhythmia. In validation experiments, we confirmed that Gnb5 heterozygosity enhanced the formation of both amyloid plaques and neurofibrillary tangles in the brains of AD model mice. These results suggest that gene-constrained analysis can complement the power of GWASs in the identification of AD-associated genes and may be more broadly applicable to other polygenic diseases.
Assuntos
Doença de Alzheimer , Subunidades beta da Proteína de Ligação ao GTP , Camundongos , Humanos , Animais , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Estudo de Associação Genômica Ampla , Emaranhados Neurofibrilares/metabolismo , Fenótipo , Genômica , Peptídeos beta-Amiloides/genética , Encéfalo/metabolismo , Membro 5 da Família 22 de Carreadores de Soluto/genética , Membro 5 da Família 22 de Carreadores de Soluto/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismoRESUMO
Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate an optogenetic tool that disrupts Gαq signaling through membrane recruitment of a minimal regulator of G protein signaling (RGS) domain. This approach, Photo-induced Gα Modulator-Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. Using PiGM-Iq we alter the behavior of Caenorhabditis elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq changes axon guidance in cultured dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. Furthermore, by altering the minimal RGS domain, we show that this approach is amenable to Gαi signaling. Our unique and robust optogenetic Gα inhibiting approaches complement existing neurobiological tools and can be used to investigate the functional effects neuromodulators that signal through GPCR and trimeric G proteins.
Assuntos
Caenorhabditis elegans , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Optogenética , Proteínas RGS , Transdução de Sinais , Peixe-Zebra , Animais , Optogenética/métodos , Caenorhabditis elegans/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Proteínas RGS/metabolismo , Proteínas RGS/genética , Peixe-Zebra/embriologia , Neurônios/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Domínios Proteicos , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Drosophila/metabolismoRESUMO
Lung adenocarcinoma (LUAD) is the leading cause of cancer-related death worldwide, but the underlying molecular mechanisms remain largely unclear. The transcription factor (TF) specificity protein 1 (SP1) plays a crucial role in the development of various cancers, including LUAD. Recent studies have indicated that master TFs may form phase-separated macromolecular condensates to promote super-enhancer (SE) assembly and oncogene expression. In this study, we demonstrated that SP1 undergoes phase separation and that its zinc finger 3 in the DNA-binding domain is essential for this process. Through Cleavage Under Targets & Release Using Nuclease (CUT&RUN) using antibodies against SP1 and H3K27ac, we found a significant correlation between SP1 enrichment and SE elements, identified the regulator of the G protein signaling 20 (RGS20) gene as the most likely target regulated by SP1 through SE mechanisms, and verified this finding using different approaches. The oncogenic activity of SP1 relies on its phase separation ability and RGS20 gene activation, which can be abolished by glycogen synthase kinase J4 (GSK-J4), a demethylase inhibitor. Together, our findings provide evidence that SP1 regulates its target oncogene expression through phase separation and SE mechanisms, thereby promoting LUAD cell progression. This study also revealed an innovative target for LUAD therapies through intervening in SP1-mediated SE formation.
Assuntos
Adenocarcinoma de Pulmão , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares , Proteínas RGS , Fator de Transcrição Sp1 , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp1/genética , Humanos , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Proteínas RGS/metabolismo , Proteínas RGS/genética , Linhagem Celular Tumoral , Animais , Elementos Facilitadores Genéticos , Progressão da Doença , Camundongos , Separação de FasesRESUMO
Dose-limiting cardiotoxicity remains a major limitation in the clinical use of cancer chemotherapeutics. Here, we describe a role for Regulator of G protein Signaling 7 (RGS7) in chemotherapy-dependent heart damage, the demonstration for a functional role of RGS7 outside of the nervous system and retina. Though expressed at low levels basally, we observed robust up-regulation of RGS7 in the human and murine myocardium following chemotherapy exposure. In ventricular cardiomyocytes (VCM), RGS7 forms a complex with Ca2+/calmodulin-dependent protein kinase (CaMKII) supported by key residues (K412 and P391) in the RGS domain of RGS7. In VCM treated with chemotherapeutic drugs, RGS7 facilitates CaMKII oxidation and phosphorylation and CaMKII-dependent oxidative stress, mitochondrial dysfunction, and apoptosis. Cardiac-specific RGS7 knockdown protected the heart against chemotherapy-dependent oxidative stress, fibrosis, and myocyte loss and improved left ventricular function in mice treated with doxorubicin. Conversely, RGS7 overexpression induced fibrosis, reactive oxygen species generation, and cell death in the murine myocardium that were mitigated following CaMKII inhibition. RGS7 also drives production and release of the cardiokine neuregulin-1, which facilitates paracrine communication between VCM and neighboring vascular endothelial cells (EC), a maladaptive mechanism contributing to VCM dysfunction in the failing heart. Importantly, while RGS7 was both necessary and sufficient to facilitate chemotherapy-dependent cytotoxicity in VCM, RGS7 is dispensable for the cancer-killing actions of these same drugs. These selective myocyte-intrinsic and myocyte-extrinsic actions of RGS7 in heart identify RGS7 as an attractive therapeutic target in the mitigation of chemotherapy-driven cardiotoxicity.
Assuntos
Antineoplásicos , Cardiotoxicidade , Proteínas RGS , Animais , Humanos , Camundongos , Antineoplásicos/efeitos adversos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiotoxicidade/metabolismo , Células Endoteliais/metabolismo , Fibrose , Miócitos Cardíacos/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismoRESUMO
Regulators of G protein signaling (RGS) proteins constrain G protein-coupled receptor (GPCR)-mediated and other responses throughout the body primarily, but not exclusively, through their GTPase-activating protein activity. Asthma is a highly prevalent condition characterized by airway hyper-responsiveness (AHR) to environmental stimuli resulting in part from amplified GPCR-mediated airway smooth muscle contraction. Rgs2 or Rgs5 gene deletion in mice enhances AHR and airway smooth muscle contraction, whereas RGS4 KO mice unexpectedly have decreased AHR because of increased production of the bronchodilator prostaglandin E2 (PGE2) by lung epithelial cells. Here, we found that knockin mice harboring Rgs4 alleles encoding a point mutation (N128A) that sharply curtails RGS4 GTPase-activating protein activity had increased AHR, reduced airway PGE2 levels, and augmented GPCR-induced bronchoconstriction compared with either RGS4 KO mice or WT controls. RGS4 interacted with the p85α subunit of PI3K and inhibited PI3K-dependent PGE2 secretion elicited by transforming growth factor beta in airway epithelial cells. Together, these findings suggest that RGS4 affects asthma severity in part by regulating the airway inflammatory milieu in a G protein-independent manner.
Assuntos
Asma , Proteínas RGS , Animais , Humanos , Camundongos , Asma/metabolismo , Asma/genética , Asma/patologia , Broncoconstrição/genética , Dinoprostona/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/genética , Hipersensibilidade Respiratória/patologia , Proteínas RGS/metabolismo , Proteínas RGS/genética , Linhagem CelularRESUMO
Microsomal glutathione transferase 3 (MGST3) regulates eicosanoid and glutathione metabolism. These processes are associated with oxidative stress and apoptosis, suggesting that MGST3 might play a role in the pathophysiology of Alzheimer's disease. Here, we report that knockdown (KD) of MGST3 in cell lines reduced the protein level of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and the resulting amyloidogenesis. Interestingly, MGST3 KD did not alter intracellular reactive oxygen species level but selectively reduced the expression of apoptosis indicators which could be associated with the receptor of cysteinyl leukotrienes, the downstream metabolites of MGST3 in arachidonic acid pathway. We then showed that the effect of MGST3 on BACE1 was independent of cysteinyl leukotrienes but involved a translational mechanism. Further RNA-seq analysis identified that regulator of G-protein signaling 4 (RGS4) was a target gene of MGST3. Silencing of RGS4 inhibited BACE1 translation and prevented MGST3 KD-mediated reduction of BACE1. The potential mechanism was related to AKT activity, as the protein level of phosphorylated AKT was significantly reduced by silencing of MGST3 and RGS4, and the AKT inhibitor abolished the effect of MGST3/RGS4 on phosphorylated AKT and BACE1. Together, MGST3 regulated amyloidogenesis by controlling BACE1 protein expression, which was mediated by RGS4 and downstream AKT signaling pathway.
Assuntos
Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Glutationa Transferase , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-akt , Proteínas RGS , Transdução de Sinais , Proteínas RGS/metabolismo , Proteínas RGS/genética , Humanos , Ácido Aspártico Endopeptidases/metabolismo , Ácido Aspártico Endopeptidases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Glutationa Transferase/metabolismo , Glutationa Transferase/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Apoptose , Peptídeos beta-Amiloides/metabolismo , AnimaisRESUMO
Heterotrimeric GTP-binding protein alpha subunit (Gα) and its cognate regulator of G-protein signaling (RGS) protein transduce signals in eukaryotes spanning protists, amoeba, animals, fungi, and plants. The core catalytic mechanisms of the GTPase activity of Gα and the interaction interface with RGS for the acceleration of GTP hydrolysis seem to be conserved across these groups; however, the RGS gene is under low selective pressure in plants, resulting in its frequent loss. Our current understanding of the structural basis of Gα:RGS regulation in plants has been shaped by Arabidopsis Gα, (AtGPA1), which has a cognate RGS protein. To gain a comprehensive understanding of this regulation beyond Arabidopsis, we obtained the x-ray crystal structures of Oryza sativa Gα, which has no RGS, and Selaginella moellendorffi (a lycophyte) Gα that has low sequence similarity with AtGPA1 but has an RGS. We show that the three-dimensional structure, protein-protein interaction with RGS, and the dynamic features of these Gα are similar to AtGPA1 and metazoan Gα. Molecular dynamic simulation of the Gα-RGS interaction identifies the contacts established by specific residues of the switch regions of GTP-bound Gα, crucial for this interaction, but finds no significant difference due to specific amino acid substitutions. Together, our data provide valuable insights into the regulatory mechanisms of plant G-proteins but do not support the hypothesis of adaptive co-evolution of Gα:RGS proteins in plants.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP , Modelos Moleculares , Proteínas de Plantas , Proteínas RGS , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cristalografia por Raios X , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Oryza/metabolismo , Oryza/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ligação Proteica , Proteínas RGS/metabolismo , Proteínas RGS/química , Proteínas RGS/genética , Relação Estrutura-Atividade , Selaginellaceae/genética , Selaginellaceae/metabolismo , Estrutura Quaternária de ProteínaRESUMO
Anaplastic thyroid cancer (ATC), an aggressive malignancy with virtually 100% disease-specific mortality, has long posed a formidable challenge in oncology due to its resistance to conventional treatments and the severe side effects associated with current regimens such as doxorubicin chemotherapy. Consequently, there was urgent need to identify novel candidate compounds that could provide innovative therapeutic strategies for ATC. Ophiopogonin D' (OPD'), a triterpenoid saponin extracted, yet its roles in ATC has not been reported. Our data demonstrated that OPD' potently inhibited proliferation and metastasis of ATC cells, promoting cell cycle arrest and apoptosis. Remarkably, OPD' impeded growth and metastasis of ATC in vitro and in vivo, displaying an encouraging safety profile. Regulator of G-protein signalling 4 (RGS4) expression was significantly up-regulated in ATC compared to normal tissues, and this upregulation was suppressed by OPD' treatment. Mechanistically, we elucidated that the transcription factor JUN bound to the RGS4 promoter, driving its transactivation. However, OPD' interacted with JUN, attenuating its transcriptional activity and thereby disrupting RGS4 overexpression. In summary, our research revealed that OPD' bound with JUN, which in turn resulted in the suppression of transcriptional activation of RGS4, thereby eliciting cell cycle arrest and apoptosis in ATC cells. These findings could offer promise in the development of high-quality candidate compounds for treatment in ATC.
Assuntos
Apoptose , Proliferação de Células , Proteínas RGS , Saponinas , Transdução de Sinais , Espirostanos , Carcinoma Anaplásico da Tireoide , Humanos , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Carcinoma Anaplásico da Tireoide/metabolismo , Carcinoma Anaplásico da Tireoide/patologia , Saponinas/farmacologia , Proteínas RGS/metabolismo , Proteínas RGS/genética , Proliferação de Células/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Espirostanos/farmacologia , Camundongos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Camundongos Nus , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Metástase NeoplásicaRESUMO
Regulator of G protein signaling 1 (RGS1) is closely associated with the tumor immune microenvironment and is highly expressed in various tumors and immune cells. The specific effects of RGS1 in the dynamic progression from chronic gastritis to gastric cancer have not been reported, and the role of tumor-associated macrophages (TAMs) is also unclear. In the present study, RGS1 was identified as an upregulated gene in different pathological stages ranging from chronic gastritis to gastric cancer by using Gene Expression Omnibus (GEO) screening together with pancancer analysis of The Cancer Genome Atlas and clinical prognostic analysis. The results indicated that RGS1 is highly expressed in gastric cancer and has potential prognostic value. We confirmed through in vivo experiments that RGS1 inhibited the proliferation of gastric cancer cells and promoted apoptosis, which was further corroborated by in vitro experiments. Additionally, RGS1 influenced cell migration and invasion. In our subsequent investigation of RGS1, we discovered its role in the immune response. Through analyses of single-cell and GEO database data, we confirmed its involvement in immune cell regulation, specifically TAM activation. Subsequently, we conducted in vivo and in vitro experiments to confirm the involvement of RGS1 in polarizing M1 macrophages while indirectly regulating M2 macrophages through tumor cells. In conclusion, RGS1 could be a potential target for the transformation of chronic gastritis into gastric cancer and has a measurable impact on TAMs, which warrants further in-depth research.
Assuntos
Gastrite , Neoplasias Gástricas , Humanos , Macrófagos Associados a Tumor/metabolismo , Neoplasias Gástricas/patologia , Transdução de Sinais , Proteínas de Ligação ao GTP/metabolismo , Microambiente TumoralRESUMO
The incidence and mortality rates of gastric cancer (GC) remain alarmingly high worldwide, imposing a substantial healthcare burden. In this study, we utilized data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. A 4-gene prognostic model was developed to predict patient prognosis, and its accuracy was validated across multiple datasets. Patients with a low-risk score exhibited improved prognosis, elevated tumor mutation burden, heightened sensitivity to both immunotherapy and conventional chemotherapy. Notably, our investigation revealed that the key gene RGS5 positively modulates the expression of mismatch repair proteins via c-Myc. Furthermore, co-immunoprecipitation (COIP) assays demonstrated the interaction between RGS5 and c-Myc. Additionally, we confirmed that RGS5 regulates c-Myc through the ubiquitin-proteasome pathway. Moreover, RGS5 was identified as a positive regulator of PD-L1 expression and exhibited a negative correlation with the majority of immune cells. These findings underscore the potential of RGS5 as a novel biomarker and therapeutic target in the context of GC.
Assuntos
Biomarcadores Tumorais , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-myc , Proteínas RGS , Neoplasias Gástricas , Humanos , Proteínas RGS/genética , Proteínas RGS/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Reparo de Erro de Pareamento de DNA , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genéticaRESUMO
BACKGROUND: Prior evidence demonstrated that Regulator of G protein Signaling 6 (RGS6) translocates to the nucleolus in response to cytotoxic stress though the functional significance of this phenomenon remains unknown. METHODS: Utilizing in vivo gene manipulations in mice, primary murine cardiac cells, human cell lines and human patient samples we dissect the participation of a RGS6-nucleolin complex in chemotherapy-dependent cardiotoxicity. RESULTS: Here we demonstrate that RGS6 binds to a key nucleolar protein, Nucleolin, and controls its expression and activity in cardiomyocytes. In the human myocyte AC-16 cell line, induced pluripotent stem cell derived cardiomyocytes, primary murine cardiomyocytes, and the intact murine myocardium tuning RGS6 levels via overexpression or knockdown resulted in diametrically opposed impacts on Nucleolin mRNA, protein, and phosphorylation.RGS6 depletion provided marked protection against nucleolar stress-mediated cell death in vitro, and, conversely, RGS6 overexpression suppressed ribosomal RNA production, a key output of the nucleolus, and triggered death of myocytes. Importantly, overexpression of either Nucleolin or Nucleolin effector miRNA-21 counteracted the pro-apoptotic effects of RGS6. In both human and murine heart tissue, exposure to the genotoxic stressor doxorubicin was associated with an increase in the ratio of RGS6/Nucleolin. Preventing RGS6 induction via introduction of RGS6-directed shRNA via intracardiac injection proved cardioprotective in mice and was accompanied by restored Nucleolin/miRNA-21 expression, decreased nucleolar stress, and decreased expression of pro-apoptotic, hypertrophy, and oxidative stress markers in heart. CONCLUSION: Together, these data implicate RGS6 as a driver of nucleolar stress-dependent cell death in cardiomyocytes via its ability to modulate Nucleolin. This work represents the first demonstration of a functional role for an RGS protein in the nucleolus and identifies the RGS6/Nucleolin interaction as a possible new therapeutic target in the prevention of cardiotoxicity.
Assuntos
MicroRNAs , Proteínas RGS , Animais , Humanos , Camundongos , Cardiotoxicidade , MicroRNAs/genética , Miócitos Cardíacos , Nucleolina , Proteínas RGS/genética , Transdução de Sinais/fisiologiaRESUMO
Regulator of G protein signaling 5 (RGS5) is a GTPase activator for heterotrimeric G-protein α-subunits, shown to be a marker of pericytes. Bone marrow stromal cell population (BMSCs) is heterogeneous. Populations of mesenchymal progenitors, cells supportive of hematopoiesis, and stromal cells regulating bone remodeling have been recently identified. Periosteal and bone marrow mesenchymal stem cells (MSCs) are participating in fracture healing, but it is difficult to distinguish the source of cells within the callus. Considering that perivascular cells exert osteoprogenitor potential, we generated an RGS5 transgenic mouse model (Rgs5-CreER) which when crossed with Ai9 reporter animals (Rgs5/Tomato), is suitable for lineage tracing during growth and post-injury. Flow cytometry analysis and histology confirmed the presence of Rgs5/Tomato+ cells within CD31+ endothelial, CD45+ hematopoietic, and CD31-CD45- mesenchymal/perivascular cells. A tamoxifen chase showed expansion of Rgs5/Tomato+ cells expressing osterix within the trabeculae positioned between mineralized matrix and vasculature. Long-term chase showed proportion of Rgs5/Tomato+ cells contributes to mature osteoblasts expressing osteocalcin. Following femoral fracture, Rgs5/Tomato+ cells are observed around newly formed bone within the BM cavity and expressed osterix and osteocalcin, while contribution within periosteum was low and limited to fibroblastic callus with very few positive chondrocytes. In addition, BM injury model confirmed that RGS5-Cre labels population of BMSCs expands during injury and participates in osteogenesis. Under homeostatic conditions, lineage-traced RGS5 cells within the trabecular area demonstrate osteoprogenitor capacity that in an injury model contributes to new bone formation primarily within the BM niche.
Assuntos
Calo Ósseo , Proteínas RGS , Camundongos , Animais , Osteocalcina/metabolismo , Calo Ósseo/metabolismo , Calo Ósseo/patologia , Osteogênese , Consolidação da Fratura/fisiologia , Condrócitos/metabolismo , Camundongos Transgênicos , Osteoblastos/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismoRESUMO
RGS (Regulator of G protein signaling) proteins have long captured the fascination of researchers due to their intricate involvement across a wide array of signaling pathways within cellular systems. Their diverse and nuanced functions have positioned them as continual subjects of scientific inquiry, especially given the implications of certain family members in various cancer types. Of particular note in this context is RGS20, whose clinical relevance and molecular significance in hepatocellular carcinoma we have recently investigated. These investigations have prompted questions into the prevalence of pathogenic mutations within the RGS20 gene and the intricate network of interacting proteins that could contribute to the complex landscape of cancer biology. In our study, we aim to unravel the mutations within the RGS20 gene and the multifaceted interplay between RGS20 and other proteins within the context of cancer. Expanding on this line of inquiry, our research is dedicated to uncovering the intricate mechanisms of RGS20 in various cancers. In particular, we have redirected our attention to examining the role of RGS20 within hematological malignancies, with a specific focus on multiple myeloma and follicular lymphoma. These hematological cancers hold significant promise for further investigation, as understanding the involvement of RGS20 in their pathogenesis could unveil novel therapeutic strategies and treatment avenues. Furthermore, our exploration has extended to encompass the latest discoveries concerning the potential involvement of RGS20 in diseases affecting the central nervous system, thereby broadening the scope of its implications beyond oncology to encompass neurobiology and related fields.
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BACKGROUND: Novel therapeutic targets are urgently needed for treating drug-resistant non-small cell lung cancer (NSCLC) and overcoming drug resistance to molecular-targeted therapies. Regulator of G protein signaling 20 (RGS20) is identified as an upregulated factor in many cancers, yet its specific role and the mechanism through which RGS20 functions in NSCLC remain unclear. Our study aimed to identify the role of RGS20 in NSCLC prognosis and delineate associated cellular and molecular pathways. METHODS: Immunohistochemistry and lung cancer tissue microarray were used to verify the expression of RGS20 between NSCLC patients. CCK8 and cell cloning were conducted to determine the proliferation ability of H1299 and Anip973 cells in vitro. Furthermore, Transcriptome sequencing was performed to show enrichment genes and pathways. Immunofluorescence was used to detect the translocation changes of YAP to nucleus. Western blotting demonstrated different expressions of autophagy and the Hippo-PKA signal pathway. In vitro and in vivo experiments verified whether overexpression of RGS20 affect the proliferation and autophagy of NSCLC through regulating the Hippo pathway. RESULTS: The higher RGS20 expression was found to be significantly correlated with a poorer five-year survival rate. Further, RGS20 accelerated cell proliferation by increasing autophagy. Transcriptomic sequencing suggested the involvement of the Hippo signaling pathway in the action of RGS20 in NSCLC. RGS20 activation reduced YAP phosphorylation and facilitated its nuclear translocation. Remarkably, inhibiting Hippo signaling with GA-017 promoted cell proliferation and activated autophagy in RGS20 knock-down cells. However, forskolin, a GPCR activator, increased YAP phosphorylation and reversed the promoting effect of RGS20 in RGS20-overexpressing cells. Lastly, in vivo experiments further confirmed role of RGS20 in aggravating tumorigenicity, as its overexpression increased NSCLC cell proliferation. CONCLUSION: Our findings indicate that RGS20 drives NSCLC cell proliferation by triggering autophagy via the inhibition of PKA-Hippo signaling. These insights support the role of RGS20 as a promising novel molecular marker and a target for future targeted therapies in lung cancer treatment.
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Off target damage to vital organ systems is an unfortunate side effect of cancer chemotherapy and remains a major limitation to the use of these essential drugs in the clinic. Despite decades of research, the mechanisms conferring susceptibility to chemotherapy driven cardiotoxicity and hepatotoxicity remain unclear. In the livers of patients with a history of chemotherapy, we observed a twofold increase in expression of G protein regulator RGS7 and a corresponding decrease in fellow R7 family member RGS11. Knockdown of RGS7 via introduction of RGS7 shRNA via tail vein injection decreased doxorubicin-induced hepatic collagen and lipid deposition, glycogen accumulation, and elevations in ALT, AST, and triglycerides by approximately 50%. Surprisingly, a similar result could be achieved via introduction of RGS7 shRNA directly to the myocardium without impacting RGS7 levels in the liver directly. Indeed, doxorubicin-treated cardiomyocytes secrete the endocrine factors transforming growth factor ß1 (TGFß1) and TGFß superfamily binding protein follistatin-related protein 1 (FSTL1). Importantly, RGS7 overexpression in the heart was sufficient to recapitulate the impacts of doxorubicin on the liver and inhibition of TGFß1 signaling with the receptor blocker GW788388 ameliorated the effect of cardiac RGS7 overexpression on hepatic fibrosis, steatosis, oxidative stress, and cell death as well as the resultant elevation in liver enzymes. Together these data demonstrate that RGS7 controls both the release of TGFß1 from the heart and the profibrotic and pro-oxidant actions of TGFß1 in the liver and emphasize the functional significance of endocrine cardiokine signaling in the pathogenesis of chemotherapy drive multiorgan damage.
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Proteínas Relacionadas à Folistatina , Proteínas RGS , Humanos , Proteínas RGS/genética , Proteínas RGS/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Transporte/metabolismo , Fígado/metabolismo , Doxorrubicina/efeitos adversos , Proteínas Relacionadas à Folistatina/metabolismoRESUMO
G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors in eukaryotes that sense and transduce extracellular signals into cells. In Aspergillus oryzae, 16 canonical GPCR genes are identified and classified into nine classes based on the sequence similarity and proposed functions. Class VI GPCRs (AoGprK-1, AoGprK-2, and AoGprR in A. oryzae), unlike other GPCRs, feature a unique hybrid structure containing both the seven transmembrane (7-TM) and regulator of G-protein signaling (RGS) domains, which is not found in animal GPCRs. We report here that the mutants with double or triple deletion of class VI GPCR genes produced significantly increased number of sclerotia compared to the control strain when grown on agar plates. Interestingly, complementation analysis demonstrated that the expression of the RGS domain without the 7-TM domain is sufficient to restore the phenotype. In line with this, among the three Gα subunits in A. oryzae, AoGpaA, AoGpaB, and AoGanA, forced expression of GTPase-deficient mutants of either AoGpaA or AoGpaB caused an increase in the number of sclerotia formed, suggesting that RGS domains of class VI GPCRs are the negative regulators of these two GTPases. Finally, we measured the expression of velvet complex genes and sclerotia formation-related genes and found that the expression of velB was significantly increased in the multiple gene deletion mutants. Taken together, these results demonstrate that class VI GPCRs negatively regulate sclerotia formation through their GTPase-activating activity in the RGS domains. KEY POINTS: ⢠Class VI GPCRs in A. oryzae regulate sclerotia formation in A. oryzae ⢠RGS function of class VI GPCRs is responsible for regulation of sclerotia formation ⢠Loss of class VI GPCRs resulted in increased expression of sclerotia-related genes.
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Aspergillus oryzae , Animais , Aspergillus oryzae/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Ágar , GTP Fosfo-HidrolasesRESUMO
Cardiotoxicity remains a major limitation in the clinical utility of anthracycline chemotherapeutics. Regulator of G-protein Signaling 7 (RGS7) and inflammatory markers are up-regulated in the hearts of patients with a history of chemotherapy particularly those with reduced left-ventricular function. RGS7 knockdown in either the murine myocardium or isolated murine ventricular cardiac myocytes (VCM) or cultured human VCM provided marked protection against doxorubicin-dependent oxidative stress, NF-κB activation, inflammatory cytokine production, and cell death. In exploring possible mechanisms causally linking RGS7 to pro-inflammatory signaling cascades, we found that RGS7 forms a complex with acetylase Tip60 and deacetylase sirtuin 1 (SIRT1) and controls the acetylation status of the p65 subunit of NF-κB. In VCM, the detrimental impact of RGS7 could be mitigated by inhibiting Tip60 or activating SIRT1, indicating that the ability of RGS7 to modulate cellular acetylation capacity is critical for its pro-inflammatory actions. Further, RGS7-driven, Tip60/SIRT1-dependent cytokines released from ventricular cardiac myocytes and transplanted onto cardiac fibroblasts increased oxidative stress, markers of transdifferentiation, and activity of extracellular matrix remodelers emphasizing the importance of the RGS7-Tip60-SIRT1 complex in paracrine signaling in the myocardium. Importantly, while RGS7 overexpression in heart resulted in sterile inflammation, fibrotic remodeling, and compromised left-ventricular function, activation of SIRT1 counteracted the detrimental impact of RGS7 in heart confirming that RGS7 increases acetylation of SIRT1 substrates and thereby drives cardiac dysfunction. Together, our data identify RGS7 as an amplifier of inflammatory signaling in heart and possible therapeutic target in chemotherapeutic drug-induced cardiotoxicity.