RESUMO
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
Distant metastasis is the major cause of death in patients with breast cancer. Epithelial-mesenchymal transition (EMT) contributes to breast cancer metastasis. Regulator of G protein-signaling (RGS) proteins modulates metastasis in various cancers. This study identified a novel role for RGS10 in EMT and metastasis in breast cancer. RGS10 protein levels were significantly lower in breast cancer tissues compared to normal breast tissues, and deficiency in RGS10 protein predicted a worse prognosis in patients with breast cancer. RGS10 protein levels were lower in the highly aggressive cell line MDA-MB-231 than in the poorly aggressive, less invasive cell lines MCF7 and SKBR3. Silencing RGS10 in SKBR3 cells enhanced EMT and caused SKBR3 cell migration and invasion. The ability of RGS10 to suppress EMT and metastasis in breast cancer was dependent on lipocalin-2 and MIR539-5p. These findings identify RGS10 as a tumor suppressor, prognostic biomarker, and potential therapeutic target for breast cancer.
Assuntos
Neoplasias da Mama , Movimento Celular , Transição Epitelial-Mesenquimal , Metástase Neoplásica , Proteínas RGS , Proteínas RGS/metabolismo , Proteínas RGS/genética , Proteínas RGS/deficiência , Humanos , Transição Epitelial-Mesenquimal/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , PrognósticoRESUMO
BACKGROUND: Gut L-type enteroendocrine cells (EECs) are intestinal chemosensory cells that secrete satiety hormones GLP-1 and PYY in response to activation of G-protein coupled receptors (GPCRs) by luminal components of nutrient digestion and microbial fermentation. Regulator of G-protein Signaling (RGS) proteins are negative regulators of GPCR signaling. The expression profile of RGS in EECs, and their potential role in satiety hormone secretion and obesity is unknown. METHODS: Transcriptomic profiling of RGS was completed in native colonic EECs was completed using single-cell RNA sequencing (scRNA-Seq) in lean and obesity, and human jejunal EECs with data obtained from a publicly available RNAseq dataset (GSE114853). RGS validation studies were completed using whole mucosal intestinal tissue obtained during endoscopy in 61 patients (n = 42 OB, n = 19 Lean); a subset of patients' postprandial plasma was assayed for GLP-1 and PYY. Ex vivo human intestinal cultures and in vitro NCI-H716 cells overexpressing RGS9 were exposed to GLP-1 secretagogues in conjunction with a nonselective RGS-inhibitor and assayed for GLP-1 secretion. FINDINGS: Transcriptomic profiling of colonic and jejunal enteroendocrine cells revealed a unique RGS expression profile in EECs, and further within GLP-1+ L-type EECs. In obesity the RGS expression profile was altered in colonic EECs. Human gut RGS9 expression correlated positively with BMI and negatively with postprandial GLP-1 and PYY. RGS inhibition in human intestinal cultures increased GLP-1 release from EECs ex vivo. NCI-H716 cells overexpressing RGS9 displayed defective nutrient-stimulated GLP-1 secretion. INTERPRETATION: This study introduces the expression profile of RGS in human EECs, alterations in obesity, and suggests a role for RGS proteins as modulators of GLP-1 and PYY secretion from intestinal EECs. FUNDING: AA is supported by the NIH(C-Sig P30DK84567, K23 DK114460), a Pilot Award from the Mayo Clinic Center for Biomedical Discovery, and a Translational Product Development Fund from The Mayo Clinic Center for Clinical and Translational Science Office of Translational Practice in partnership with the University of Minnesota Clinical and Translational Science Institute.
Assuntos
Células Enteroendócrinas , Peptídeo 1 Semelhante ao Glucagon , Obesidade , Peptídeo YY , Proteínas RGS , Transdução de Sinais , Humanos , Células Enteroendócrinas/metabolismo , Obesidade/metabolismo , Proteínas RGS/metabolismo , Proteínas RGS/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Peptídeo YY/metabolismo , Masculino , Feminino , Perfilação da Expressão Gênica , Transcriptoma , Adulto , Pessoa de Meia-Idade , Regulação da Expressão Gênica , Mucosa Intestinal/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMO
BACKGROUND: The level of the regulator of G-protein signaling 4-1 (RGS4-1) isoform, the longest RGS4 isoform, is significantly reduced in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia. However, the mechanism behind this has not been clarified. The 3'untranslated regions (3'UTRs) are known to regulate the levels of their mRNA splice variants. METHODS: We constructed recombinant pmir-GLO vectors with a truncated 3' regulatory region of the RGS4 gene (3R1, 3R2, 3R3, 3R4, 3R5, and 3R6). The dual-luciferase reporter assay was conducted to find functional regions in HEK-293, SK-N-SH, and U87cells and then predicted miRNA binding to these regions. We performed a dual-luciferase reporter assay and a Western blot analysis after transiently transfecting the predicted miRNAs. RESULTS: The dual-luciferase reporter assay found that regions +401-+789, +789-+1152, and +1562-+1990 (with the last base of the termination codon being +1) might be functional regions. Hsa-miR-874-3p, associated with many psychiatric disorders, might target the +789-+1152 region in the 3'UTR of the RGS4 gene. In the dual-luciferase reporter assay, the hsa-miR-874-3p mimic, co-transfected with 3R1, down-regulated the relative fluorescence intensities. However, this was reversed when the hsa-miR-874-3p mimic was co-transfected with m3R1 (deletion of +853-+859). The hsa-miR-874-3p mimic significantly decreased the endogenous expression of the RGS4-1 isoform in HEK-293 cells. CONCLUSIONS: Hsa-miR-874-3p inhibits the expression of the RGS4-1 isoform by targeting +853-+859.
Assuntos
Regiões 3' não Traduzidas , MicroRNAs , Isoformas de Proteínas , Proteínas RGS , Humanos , Proteínas RGS/genética , Proteínas RGS/metabolismo , MicroRNAs/genética , Células HEK293 , Isoformas de Proteínas/genética , Regiões 3' não Traduzidas/genética , Esquizofrenia/genética , Esquizofrenia/metabolismo , Córtex Pré-Frontal/metabolismo , Linhagem Celular TumoralRESUMO
BACKGROUND: Esophageal cancer is one of the most common malignant tumors in the world. It is urgent to prevent the development and progression of esophageal cancer. Cancer stem cells (CSCs) were reported to have the ability to initiate tumorigenesis, and reducing the stem cell-like characteristics of tumors is an important strategy to inhibit the occurrence and development of tumors. miRNAs are key regulators of the stemness of cancer. Here, we aimed to investigate the role and regulatory mechanism of miR-191-3p in the stemness properties of esophageal cancer cells. METHODS: Esophageal cancer cells with stable expression of miR-191-3p were established by lentivirus system. CCK-8 assay, transwell assay, wound healing assay were used to evaluate the effect of miR-191-3p on proliferation and metastasis of esophageal cancer cells. The expression of stemness-related markers (NANOG, OCT4, SOX2), ALDH activity, sphere-forming assay and subcutaneous tumor model in nude mice were performed to evaluate the stemness properties of esophageal cancer cells in vitro and in vivo. Dual-luciferase reporter assay was used to verify the molecular mechanism. RESULT: Here we found that overexpression of miR-191-3p promoted the stemness properties of esophageal cancer cells in vitro and in vivo, including increasing esophageal cancer cell proliferation and metastasis ability, the expression of stemness-related markers NANOG, OCT4, and SOX2, ALDH activity, the number of spheres formed and tumor growth. Bioinformatic analysis and dual-luciferase assay demonstrated that regulator of G protein signaling 1 (RGS1) was the directed target gene of miR-191-3p and attenuated the promotion effect of miR-191-3p on the stemness of esophageal cancer cells. Furthermore, we found that RGS1 knockdown activated the PI3K/AKT pathway by negatively regulating CXCR4 to promote the stemness of esophageal cancer cells. CONCLUSIONS: Our findings revealed that RGS1 targeted by miR-191-3p inhibited the stemness of esophageal cancer cells by suppressing the CXCR4/PI3K/AKT pathway, which provide potential prognostic markers and therapeutic targets in the future.
Assuntos
Neoplasias Esofágicas , Camundongos Nus , MicroRNAs , Células-Tronco Neoplásicas , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Proteínas RGS , Receptores CXCR4 , Transdução de Sinais , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica , Camundongos Endogâmicos BALB C , MicroRNAs/metabolismo , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Proteínas RGS/metabolismo , Proteínas RGS/genéticaRESUMO
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
BACKGROUND: Diabetic nephropathy (DN) is a major contributor to chronic kidney disease. This study aims to identify immune biomarkers and potential therapeutic drugs in DN. METHODS: We analyzed two DN microarray datasets (GSE96804 and GSE30528) for differentially expressed genes (DEGs) using the Limma package, overlapping them with immune-related genes from ImmPort and InnateDB. LASSO regression, SVM-RFE, and random forest analysis identified four hub genes (EGF, PLTP, RGS2, PTGDS) as proficient predictors of DN. The model achieved an AUC of 0.995 and was validated on GSE142025. Single-cell RNA data (GSE183276) revealed increased hub gene expression in epithelial cells. CIBERSORT analysis showed differences in immune cell proportions between DN patients and controls, with the hub genes correlating positively with neutrophil infiltration. Molecular docking identified potential drugs: cysteamine, eltrombopag, and DMSO. And qPCR and western blot assays were used to confirm the expressions of the four hub genes. RESULTS: Analysis found 95 and 88 distinctively expressed immune genes in the two DN datasets, with 14 consistently differentially expressed immune-related genes. After machine learning algorithms, EGF, PLTP, RGS2, PTGDS were identified as the immune-related hub genes associated with DN. In addition, the mRNA and protein levels of them were obviously elevated in HK-2 cells treated with glucose for 24 h, as well as their mRNA expressions in kidney tissues of mice with DN. CONCLUSION: This study identified 4 hub immune-related genes (EGF, PLTP, RGS2, PTGDS), as well as their expression profiles and the correlation with immune cell infiltration in DN.
Assuntos
Nefropatias Diabéticas , Aprendizado de Máquina , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/tratamento farmacológico , Humanos , Animais , Camundongos , Proteínas RGS/genética , Perfilação da Expressão Gênica , Simulação de Acoplamento Molecular , AlgoritmosRESUMO
Regulator of G-protein signaling (RGS) proteins exhibit GTPase-accelerating protein activities to govern G-protein function. In the rice blast fungus Magnaporthe oryzae, there is a family of at least eight RGS and RGS-like proteins (MoRgs1 to MoRgs8), each exhibiting distinct or shared functions in the growth, appressorium formation, and pathogenicity. MoRgs3 recently emerged as one of the crucial regulators that senses intracellular oxidation during appressorium formation. To explore this unique regulatory mechanism of MoRgs3, we identified the nucleoside diphosphate kinase MoNdk1 that interacts with MoRgs3. MoNdk1 phosphorylates MoRgs3 under induced intracellular reactive oxygen species levels, and MoRgs3 phosphorylation is required for appressorium formation and pathogenicity. In addition, we showed that MoRgs3 phosphorylation determines its interaction with MoCrn1, a coronin-like actin-binding protein homolog, which regulates MoRgs3 internalization. Finally, we provided evidence demonstrating that MoRgs3 functions in MoMagA-mediated cAMP signaling to regulate normal appressorium induction. By revealing a novel signal perception mechanism, our studies highlighted the complexity of regulation during the appressorium function and pathogenicity of the blast fungus. IMPORTANCE: We report that MoRgs3 becomes phosphorylated in an oxidative intracellular environment during the appressorium formation stage. We found that this phosphorylation is carried out by MoNdk1, a nucleoside diphosphate kinase. In addition, this phosphorylation leads to a higher binding affinity between MoRgs3 and MoCrn1, a coronin-like actin-binding protein that was implicated in the endocytic transport of several other RGS proteins of Magnaporthe oryzae. We further found that the internalization of MoRgs3 is indispensable for its GTPase-activating protein function toward the Gα subunit MoMagA. Importantly, we characterized how such cellular regulatory events coincide with cAMP signaling-regulated appressorium formation and pathogenicity in the blast fungus. Our studies uncovered a novel intracellular reactive oxygen species signal-transducing mechanism in a model pathogenic fungus with important basic and applied implications.
Assuntos
AMP Cíclico , Proteínas Fúngicas , Doenças das Plantas , Espécies Reativas de Oxigênio , Transdução de Sinais , Espécies Reativas de Oxigênio/metabolismo , AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Fosforilação , Doenças das Plantas/microbiologia , Oryza/microbiologia , Proteínas RGS/metabolismo , Proteínas RGS/genética , Regulação Fúngica da Expressão Gênica , Núcleosídeo-Difosfato Quinase/metabolismo , Núcleosídeo-Difosfato Quinase/genética , Ascomicetos/genética , Ascomicetos/metabolismo , Ascomicetos/patogenicidade , VirulênciaRESUMO
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
BACKGROUND: ZNF384-fusion (Z-fusion) genes were recently identified in B-cell acute lymphoblastic leukemia (B-ALL) and are frequent in Japanese adult patients. The frequency is about 20% in those with Philadelphia chromosome-negative B-ALL. ZNF384 is a transcription factor and Z-fusion proteins have increased transcriptional activity; however, the detailed mechanisms of leukemogenesis of Z-fusion proteins have yet to be clarified. METHODS: We established three transfectants of cell lines expressing different types of Z-fusion proteins, and analyzed their gene expression profile (GEP) by RNA-seq. We also analyzed the GEP of clinical ALL samples using our previous RNA-seq data of 323 Japanese ALL patients. We selected upregulated genes in both Z-fusion gene-expressing transfectants and Z-fusion gene-positive ALL samples, and investigated the binding of Z-fusion proteins to regulatory regions of the candidate genes by ChIP-qPCR. RESULTS: We selected six commonly upregulated genes. After the investigation by ChIP-qPCR, we finally identified CREB5 and RGS1 as direct and common target genes. RGS1 is an inhibitor of CXCL12-CXCR4 signaling that is required for the homing of hematopoietic progenitor cells to the bone marrow microenvironment and development of B cells. Consistent with this, Z-fusion gene transfectants showed impaired migration toward CXCL12. CONCLUSIONS: We identified CREB5 and RGS1 as direct and common transcriptional targets of Z-fusion proteins. The present results provide novel insight into the aberrant transcriptional regulation by Z-fusion proteins.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Proteínas de Fusão Oncogênica , Proteínas RGS , Humanos , Linhagem Celular Tumoral , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação Leucêmica da Expressão Gênica , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo , TransativadoresRESUMO
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
GPCR-G protein signaling from endosomes plays a crucial role in various physiological and pathological processes. However, the mechanism by which endosomal G protein signaling is terminated remains largely unknown. In this study, we aimed to investigate the regulatory mechanisms involved in terminating the signaling of Gα subunits from endosomes. Through structural analysis and cell-based assays, we have discovered that SNX25, a protein that targets endosomes via its PXA or PXC domain, interacts with regulator of G protein signaling (RGS) proteins (including RGS2, RGS4, RGS8, and RGS17) in a redox-regulated manner. The interaction between SNX25 and these RGS proteins enhances their GTPase-accelerating activity towards Gαi/q and their ability to bind GDP-bound (inactive form) Gαi/q. As a result, SNX25 recruits these RGS proteins to endosomes, leading to the termination of endosomal Gαi/q signaling. Furthermore, we have found that the SNX25/RGS complex also exerts a negative regulatory effect on Gαi/q signaling from the plasma membrane. This is achieved by recruiting Gαi/q to endosomes and preventing its activation on the plasma membrane. Our findings shed light on the previously unknown role of redox-modulated SNX25 in inhibiting Gαi/q signaling, thereby uncovering a novel mechanism for terminating Gαi/q signaling from endosomes. Importantly, this study expands our understanding of the regulation of GPCR-Gαi/q signaling beyond the plasma membrane.
Assuntos
Endossomos , Receptores Acoplados a Proteínas G , Transdução de Sinais , Nexinas de Classificação , Humanos , Endossomos/metabolismo , Oxirredução , Ligação Proteica , Receptores Acoplados a Proteínas G/metabolismo , Proteínas RGS/metabolismo , Proteínas RGS/genética , Nexinas de Classificação/metabolismo , Nexinas de Classificação/genéticaRESUMO
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
Colorectal cancer (CRC) remains a major global cause of cancer-related mortality, lacking effective biomarkers and therapeutic targets. Revealing the critical pathogenic factors of CRC and the underlying mechanisms would offer potential therapeutic strategies for clinical application. G protein signaling (RGS) protein family modulators play essential role within regulating downstream signaling of GPCR receptors, with function in cancers unclear. Our study focused on the expression patterns of RGS proteins in CRC, identifying Regulator of G protein signaling 16 (RGS16) as a prospective diagnostic and therapeutic target. Analyzing 899 CRC tissues revealed elevated RGS16 levels, correlating with clinicopathological features and CRC prognosis by immunohistochemistry (IHC) combined with microarray. We confirmed the elevated RGS16 protein level in CRC, and found that patients with RGS16-high tumors exhibited decreased disease-specific survival (DSS) and disease-free survival (DFS) compared to those with low RGS16 expression. Functional assays demonstrated that RGS16 promoted the CRC progression, knockdown of RGS16 led to significantly increased apoptosis rates of CRC in vitro and in vivo. Notably, we also confirmed these phenotypes of RGS16 in organoids originated from resected primary human CRC tissues. Mechanistically, RGS16 restrained JNK/P38-mediated apoptosis in CRC cells through disrupting the recruitment of TAB2/TAK1 to TRAF6. This study provides insights into addressing the challenges posed by CRC, offering avenues for clinical translation.
Assuntos
Apoptose , Neoplasias Colorretais , MAP Quinase Quinase Quinases , Proteínas RGS , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/genética , Sistema de Sinalização das MAP Quinases , Camundongos Nus , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas RGS/metabolismo , Proteínas RGS/genética , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genéticaRESUMO
Hepatocellular carcinoma (HCC) is one of the most common fatal cancers worldwide, and the identification of novel treatment targets and prognostic biomarkers is urgently needed because of its unsatisfactory prognosis. Regulator of G-protein signaling 19 (RGS19) is a multifunctional protein that regulates the progression of various cancers. However, the specific function of RGS19 in HCC remains unclear. The expression of RGS19 was determined in clinical HCC samples. Functional and molecular biology experiments involving RGS19 were performed to explore the potential mechanisms of RGS19 in HCC. The results showed that the expression of RGS19 is upregulated in HCC tissues and is significantly associated with poor prognosis in HCC patients. RGS19 promotes the proliferation and metastasis of HCC cells in vitro and in vivo. Mechanistically, RGS19, via its RGS domain, stabilizes the MYH9 protein by directly inhibiting the interaction of MYH9 with STUB1, which has been identified as an E3 ligase of MYH9. Moreover, RGS19 activates ß-catenin/c-Myc signaling via MYH9, and RGS19 is also a transcriptional target gene of c-Myc. A positive feedback loop formed by RGS19, MYH9, and the ß-catenin/c-Myc axis was found in HCC. In conclusion, our research revealed that competition between RGS19 and STUB1 is a critical mechanism of MYH9 regulation and that the RGS19/MYH9/ß-catenin/c-Myc feedback loop may represent a promising strategy for HCC therapy.
Assuntos
Carcinoma Hepatocelular , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas , Cadeias Pesadas de Miosina , Proteínas Proto-Oncogênicas c-myc , Proteínas RGS , beta Catenina , Animais , Feminino , Humanos , Masculino , Camundongos , beta Catenina/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proliferação de Células , Retroalimentação Fisiológica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Cadeias Pesadas de Miosina/metabolismo , Cadeias Pesadas de Miosina/genética , Prognóstico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas RGS/metabolismo , Proteínas RGS/genética , Transdução de SinaisRESUMO
Staff and animals in livestock buildings are constantly exposed to fine particulate matter (PM2.5), which affects their respiratory health. However, its exact pathogenic mechanism remains unclear. Regulator of G-protein signaling 2 (RGS2) has been reported to play a regulatory role in pneumonia. The aim of this study was to explore the therapeutic potential of RGS2 in cowshed PM2.5-induced respiratory damage. PM2.5 was collected from a cattle farm, and the alveolar macrophages (NR8383) of the model animal rat were stimulated with different treatment conditions of cowshed PM2.5. The RGS2 overexpression vector was constructed and transfected it into cells. Compared with the control group, cowshed PM2.5 significantly induced a decrease in cell viability and increased the levels of apoptosis and proinflammatory factor expression. Overexpression of RGS2 ameliorated the above-mentioned cellular changes induced by cowshed PM2.5. In addition, PM2.5 has significantly induced intracellular Ca2+ dysregulation. Affinity inhibition of Gq/11 by RGS2 attenuated the cytosolic calcium signaling pathway mediated by PLCß/IP3R. To further investigate the causes and mechanisms of action of differential RGS2 expression, the possible effects of oxidative stress and TLR2/4 activation were investigated. The results have shown that RGS2 expression was not only regulated by oxidative stress-induced nitric oxide during cowshed PM2.5 cells stimulation but the activation of TLR2/4 had also an important inhibitory effect on its protein expression. The present study demonstrates the intracellular Ca2+ regulatory role of RGS2 during cellular injury, which could be a potential target for the prevention and treatment of PM2.5-induced respiratory injury.
Assuntos
Macrófagos Alveolares , Material Particulado , Proteínas RGS , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Animais , Proteínas RGS/genética , Proteínas RGS/metabolismo , Material Particulado/toxicidade , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Ratos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Bovinos , Receptor 2 Toll-Like/metabolismo , Receptor 2 Toll-Like/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Linhagem Celular , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Apoptose/efeitos dos fármacos , Poluentes Atmosféricos/toxicidadeRESUMO
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
AIMS: Ventral pathway circuits are constituted by the interconnected brain areas that are distributed throughout the brain. These brain circuits are primarily involved in processing of object related information in brain. However, their role in object recognition memory (ORM) enhancement remains unknown. Here, we have studied on the implication of these circuits in ORM enhancement and in reversal of ORM deficit in aging. METHODS: The brain areas interconnected to ventral pathway circuits in rat brain were activated by an expression of a protein called regulator of G-protein signaling 14 of 414 amino acids (RGS14414). RGS14414 is an ORM enhancer and therefore used here as a gain-in-function tool. ORM test and immunohistochemistry, lesions, neuronal arborization, and knockdown studies were performed to uncover the novel function of ventral pathway circuits. RESULTS: An activation of each of the brain areas interconnected to ventral pathway circuits individually induced enhancement in ORM; however, same treatment in brain areas not interconnected to ventral pathway circuits produced no effect. Further study in perirhinal cortex (PRh), area V2 of visual cortex and frontal cortex (FrC), which are brain areas that have been shown to be involved in ORM and are interconnected to ventral pathway circuits, revealed that ORM enhancement seen after the activation of any one of the three brain areas was unaffected by the lesions in other two brain areas either individually in each area or even concurrently in both areas. This ORM enhancement in all three brain areas was associated to increase in structural plasticity of pyramidal neurons where more than 2-fold higher dendritic spines were observed. Additionally, we found that an activation of either PRh, area V2, or FrC not only was adequate but also was sufficient for the reversal of ORM deficit in aging rats, and the blockade of RGS14414 activity led to loss in increase in dendritic spine density and failure in reversal of ORM deficit. CONCLUSIONS: These results suggest that brain areas interconnected to ventral pathway circuits facilitate ORM enhancement by an increase in synaptic connectivity between the local brain area circuits and the passing by ventral pathway circuits and an upregulation in activity of ventral pathway circuits. In addition, the finding of the reversal of ORM deficit through activation of an interconnected brain area might serve as a platform for developing not only therapy against memory deficits but also strategies for other brain diseases in which neuronal circuits are compromised.
Assuntos
Encéfalo , Transtornos da Memória , Proteínas RGS , Reconhecimento Psicológico , Animais , Reconhecimento Psicológico/fisiologia , Masculino , Ratos , Proteínas RGS/metabolismo , Proteínas RGS/genética , Vias Neurais , Envelhecimento/fisiologiaRESUMO
BACKGROUND: Pericytes are capillary-associated mural cells involved in the maintenance and stability of the vascular network. Although aging is one of the main risk factors for cardiovascular disease, the consequences of aging on cardiac pericytes are unknown. METHODS: In this study, we have combined single-nucleus RNA sequencing and histological analysis to determine the effects of aging on cardiac pericytes. Furthermore, we have conducted in vivo and in vitro analysis of RGS5 (regulator of G-protein signaling 5) loss of function and finally have performed pericytes-fibroblasts coculture studies to understand the effect of RGS5 deletion in pericytes on the neighboring fibroblasts. RESULTS: Aging reduced the pericyte area and capillary coverage in the murine heart. Single-nucleus RNA sequencing analysis further revealed that the expression of Rgs5 was reduced in cardiac pericytes from aged mice. In vivo and in vitro studies showed that the deletion of RGS5 impaired cardiac function, induced fibrosis, and morphological changes in pericytes characterized by a profibrotic gene expression signature and the expression of different ECM (extracellular matrix) components and growth factors, for example, TGFB2 and PDGFB. Indeed, culturing fibroblasts with the supernatant of RGS5-deficient pericytes induced their activation as evidenced by the increased expression of αSMA (alpha smooth muscle actin) in a TGFß (transforming growth factor beta)2-dependent mechanism. CONCLUSIONS: Our results have identified RGS5 as a crucial regulator of pericyte function during cardiac aging. The deletion of RGS5 causes cardiac dysfunction and induces myocardial fibrosis, one of the hallmarks of cardiac aging.
Assuntos
Fibroblastos , Fibrose , Pericitos , Proteínas RGS , Pericitos/metabolismo , Pericitos/patologia , Animais , Proteínas RGS/genética , Proteínas RGS/metabolismo , Proteínas RGS/deficiência , Fibroblastos/metabolismo , Fibroblastos/patologia , Camundongos , Células Cultivadas , Envelhecimento/metabolismo , Envelhecimento/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Masculino , Técnicas de CoculturaRESUMO
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.