RESUMO
The incidence and related death of hepatocellular carcinoma (HCC) have increased over the past decades. However, the molecular mechanisms underlying HCC pathogenesis are not fully understood. Long noncoding RNA (lncRNA) RP11-495P10.1 has been proven to be closely associated with the progression of prostate cancer, but its role and specific mechanism in HCC are still unknown. Here, we identify that RP11-495P10.1 is highly expressed in HCC tissues and cells and contributes to the proliferation of HCC cells. Moreover, this study demonstrates that RP11-495P10.1 affects the proliferation of HCC by negatively regulating the expression of nuclear receptor subfamily 4 group a member 3 (NR4A3). Glycometabolism reprogramming is one of the main characteristics of tumor cells. In this study, we discover that RP11-495P10.1 regulates glycometabolism reprogramming by changing the expression of pyruvate dehydrogenase kinase 1 (PDK1) and pyruvate dehydrogenase (PDH), thus contributing to the proliferation of HCC cells. Furthermore, knockdown of RP11-495P10.1 increases enrichment of H3K27Ac in the promoter of NR4A3 by promoting the activity of PDH and the production of acetyl-CoA, which leads to the increased transcription of NR4A3. Altogether, RP11-495P10.1 promotes HCC cell proliferation by regulating the reprogramming of glucose metabolism and acetylation of the NR4A3 promoter via the PDK1/PDH axis, which provides an lncRNA-oriented therapeutic strategy for the diagnosis and treatment of HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , RNA Longo não Codificante , Receptores de Esteroides , Humanos , Masculino , Acetilação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Glucose , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Complexo Piruvato Desidrogenase/metabolismoRESUMO
This chapter is an introduction to phosphoinositide 3-kinases (PI3K), with class I PI3Ks as the central focus. First, the various PI3K isoforms in class I are presented with emphasis on their overall structure, subunits, subunit constitutive domains, domain-domain interactions, and functional relevance. This structural analysis is followed by a comprehensive history of seminal investigations into PI3K activity. Next, we highlight the divergent roles of the isoforms: PI3Kα, PI3Kß, PI3Kδ, and PI3Kγ. This section details signaling pathways in which these PI3K isoforms are involved, including the key upstream regulators of PI3K activity and some downstream cellular effects. Nodes of the PI3K pathway are also presented. Inhibitors of some isoforms are discussed to give an overview of the basis of some immunotherapies that are being used to target cell signaling. Finally, the chapter ends with a discussion of the dysregulation of PI3Ks in diseases including APDS, asthma, arthritis, and oncogenic mutations.
Assuntos
Fosfatidilinositol 3-Quinases , Transdução de Sinais , Biologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Transdução de Sinais/fisiologiaRESUMO
AIMS: The occurrence of alcoholic liver injury is related to the oxidative stress. Bacteria for alleviating alcoholic related liver injury have received widespread attention. Study aims to investigate the alleviated efficacy of Lactiplantibacillus plantarum (L. plantarum) P101 on alcohol-induced liver injury and its potential mechanism. METHODS AND RESULTS: The model of alcoholic liver injury was obtained according to the NIAAA method and the mice were treated with L. plantarum P101 (108 CFU.mice-1). Results showed that treatment of L. plantarum P101 could significantly improve liver function and antioxidant capacity. Furthermore, L. plantarum P101 significantly up-regulated Nuclear factor erythroid 2-related factor (Nrf2) and its target molecule, Hemeoxygenase 1 (HO-1), by promoting nuclear translocation of Nrf2. Moreover, inflammatory factors and pro-apoptotic protein (Caspase3) levels were significantly decreased in mice treated with L. plantarum P101. CONCLUSIONS: This study confirmed that the beneficial effect of L. plantarum P101 supplement was achieved via regulating Nrf2/HO-1 antioxidant pathway, and alleviated alcoholic liver injury.
Assuntos
Antioxidantes , Fator 2 Relacionado a NF-E2 , Animais , Camundongos , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/farmacologia , Fígado , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/farmacologia , Estresse Oxidativo , Lactobacillaceae/químicaRESUMO
Cyclophosphamide causes side effects in cancer patients, including hepatotoxicity. Probiotics have recently emerged as potential approaches for the administration of many diseases. This study aimed to evaluate the protective effects of Lactiplantibacillus plantarum P101 against cyclophosphamide-induced liver injury and elucidate the underlying mechanism. In this study, Lactiplantibacillus plantarum P101 or Lactobacillus rhamnosus GG were pre-administered to mice with varying duration (1 week, 2 weeks, and 3 weeks) before being intraperitoneally injected with cyclophosphamide at a dose of 30 mg/kg/day for 7 days to induce liver injury. Results demonstrated that cyclophosphamide-induced liver injury was characterized by histopathological disorders, including irregular central venous shape and hepatic vascular rupture, as well as a severe inflammation response and oxidative stress. The administration of probiotics for 3 weeks exerted the most significant improvements in alleviating liver injury, oxidative stress, and inflammation when compared to the shorter intervention duration. Notably, Lactiplantibacillus plantarum P101 exhibited more pronounced effects than Lactobacillus rhamnosus GG. Furthermore, Lactiplantibacillus plantarum P101 enhanced the antioxidant defense system by activating the Nrf2/ARE signaling pathway, ultimately alleviating hepatotoxicity and hepatocyte apoptosis. In conclusion, this study highlighted the potential of Lactiplantibacillus plantarum P101 to alleviate cyclophosphamide-induced hepatotoxicity.
Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Lacticaseibacillus rhamnosus , Animais , Camundongos , Ciclofosfamida/toxicidade , Inflamação , Fator 2 Relacionado a NF-E2 , Transdução de SinaisRESUMO
Macrophage migration inhibitory factor (MIF) is a master regulator of proinflammatory cytokines and plays pathological roles when not properly regulated in rheumatoid arthritis, lupus, atherosclerosis, asthma and cancer. Unlike canonical cytokines, MIF has vestigial keto-enol tautomerase activity. Most of the current MIF inhibitors were screened for the inhibition of this enzymatic activity. However, only some of the enzymatic inhibitors inhibit receptor-mediated biological functions of MIF, such as cell recruitment, through an unknown molecular mechanism. The goal of this study was to understand the molecular basis underlying the pharmacological inhibition of biological functions of MIF. Here, we demonstrate how the structural changes caused upon inhibitor binding translate into the alteration of MIF-induced downstream signalling. Macrophage migration inhibitory factor activates phosphoinositide 3-kinases (PI3Ks) that play a pivotal role in immune cell recruitment in health and disease. There are several different PI3K isoforms, but little is known about how they respond to MIF. We demonstrate that MIF up-regulates the expression of Class IB PI3Ks in leucocytes. We also demonstrate that MIF tautomerase active site inhibitors down-regulate the expression of Class IB PI3Ks as well as leucocyte recruitment in vitro and in vivo. Finally, based on our MIF:inhibitor complex crystal structures, we hypothesize that the reduction in Class IB PI3K expression occurs because of the displacement of Pro1 towards the second loop of MIF upon inhibitor binding, which results in increased flexibility of the loop 2 and sub-optimal MIF binding to its receptors. These results will provide molecular insights for fine-tuning the biological functions of MIF.
Assuntos
Inibidores Enzimáticos/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Células HL-60 , Humanos , Macrófagos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Class IB phosphoinositide 3-kinases γ (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110γ monoclonal antibody [mAb(A)p110γ] to block PI3Kγ enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110γ interface, hydrogen-deuterium exchange coupled to MS (HDX-MS) measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110γ, which was accompanied by conformational changes in the helical domain harbouring the Gßγ-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kγ by the antibody. p87-p110γ showed a significantly reduced Gßγ-mediated phospholipid recruitment as compared with p101-p110γ. Concomitantly, in the presence of mAb(A)p110γ, Gßγ did not bind to p87-p110γ. These data correlated with the ability of the antibody to block Gßγ-stimulated lipid kinase activity of p87-p110γ 30-fold more potently than p101-p110γ. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gßγ-dependent regulation of p101 in PI3Kγ activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kγ variants downstream of GPCRs.
Assuntos
Anticorpos Monoclonais Murinos/química , Classe Ib de Fosfatidilinositol 3-Quinase , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Animais , Classe Ib de Fosfatidilinositol 3-Quinase/química , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Medição da Troca de Deutério , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Células Sf9 , SpodopteraRESUMO
Class IB phosphoinositide 3-kinase γ (PI3Kγ) comprises a single catalytic p110γ subunit, which binds to two non-catalytic subunits, p87 or p101, and controls a plethora of fundamental cellular responses. The non-catalytic subunits are assumed to be redundant adaptors for Gßγ enabling G-protein-coupled receptor-mediated regulation of PI3Kγ. Growing experimental data provide contradictory evidence. To elucidate the roles of the non-catalytic subunits in determining the specificity of PI3Kγ, we tested the impact of p87 and p101 in heterodimeric p87-p110γ and p101-p110γ complexes on the modulation of PI3Kγ activity in vitro and in living cells. RT-PCR, biochemical, and imaging data provide four lines of evidence: (i) specific expression patterns of p87 and p101, (ii) up-regulation of p101, providing the basis to consider p87 as a protein forming a constitutively and p101 as a protein forming an inducibly expressed PI3Kγ, (iii) differences in basal and stimulated enzymatic activities, and (iv) differences in complex stability, all indicating apparent diversity within class IB PI3Kγ. In conclusion, expression and activities of PI3Kγ are modified differently by p87 and p101 in vitro and in living cells, arguing for specific regulatory roles of the non-catalytic subunits in the differentiation of PI3Kγ signaling pathways.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Multimerização Proteica/fisiologia , Transdução de Sinais/fisiologia , Animais , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Feminino , Células HEK293 , Humanos , Masculino , Células Sf9 , Spodoptera , Especificidade por Substrato/fisiologiaRESUMO
Prion diseases are progressive disorders that affect the central nervous system leading to memory loss, personality changes, ataxia and neurodegeneration. In humans, these disorders include Creutzfeldt-Jakob disease, kuru and Gerstmann-Straüssler-Scheinker (GSS) syndrome, the latter being a dominantly inherited prion disease associated with missense mutations in the gene that codes for the prion protein. The exact mechanism by which mutant prion proteins affect the central nervous system and cause neurological disease is not well understood. We have generated an inducible model of GSS disease in Drosophila melanogaster by temporally expressing a misfolded form of the murine prion protein in cholinergic neurons. Flies accumulating this mutant protein develop motor abnormalities which are associated with electrophysiological defects in cholinergic neurons. We find that, upon blocking the expression of the mutant protein, both behavioral and electrophysiological defects can be reversed. This represents the first case of reversibility reported in a model of genetic prion disease. Additionally, we observe that endogenous mechanisms exist within Drosophila that are capable of clearing the accumulated prion protein.
Assuntos
Doença de Gerstmann-Straussler-Scheinker/genética , Mutação , Príons/genética , Animais , Modelos Animais de Doenças , Drosophila melanogaster/genética , Doença de Gerstmann-Straussler-Scheinker/metabolismo , Doença de Gerstmann-Straussler-Scheinker/fisiopatologia , Modelos Genéticos , Atividade Motora/genética , Príons/metabolismoRESUMO
The decline in male sperm quality caused by multiple factors has become a widespread concern. Alcohol excessive consumption is one of the factors that induce testicular dysfunction. Testicular dysfunction caused by alcohol abuse is related to oxidative stress and inflammation. Probiotics can ameliorate alcohol-induced testicular dysfunction. However, the specific mechanism is not explicit. This study aimed to elucidate the underlying mechanism by which Lactiplantibacillus plantarum P101 ameliorates the alcohol-induced testicular dysfunction. The model of alcohol-induced testicular dysfunction in C57B/6 male mice was established according to the National Institute on Alcohol Abuse and Alcoholism, and Lactiplantibacillus plantarum P101 supplementation was orally administered to mice during the experiment. The results showed that Lactiplantibacillus plantarum P101 promoted androgen production, reduced testis inflammation, and improved testis antioxidant capacity, thereby improving sperm quality and sperm motility and ultimately ameliorating alcohol-induced testicular disorder. Three key metabolite pathways and six key metabolites were identified by metabolome analysis.
Assuntos
Lactobacillus plantarum , Doenças Testiculares , Masculino , Animais , Camundongos , Humanos , Sêmen , Motilidade dos Espermatozoides , Etanol , Doenças Testiculares/induzido quimicamente , Doenças Testiculares/prevenção & controle , Metaboloma , InflamaçãoRESUMO
Mitigating steatosis is essential for delaying the progression of alcoholic liver disease. The effect and mechanism of Lactiplantibacillus plantarum P101 (LP.P101) on alleviating alcohol-induced hepatic lipid accumulation were investigated in our study. The mouse model was constructed by a short-term (10-day)-plus-binge ethanol feeding and gavaged with 108 CFU/mL of LP.P101 daily. Lipid droplet in the liver was significantly reduced by LP.101 intervention on AMPK activation. However, when AMPK was inhibited by dorsomorphin, the levels of related indicators (ALT, TG, etc.) and the expression levels of AMPK and relevant genes in the liver converged to that of the alcohol-fed group. Compared with the alcohol-fed group, LP.P101 reduced the relative abundance of Firmicutes and increased that of Bacteroidetes. Parabacteroides merdae was negatively correlated with lipid accumulation, and unclassified Negativibacillus was negatively associated with AMPK activation. Importantly, LP.P101 modified the compositions of the serum metabolites. The potential biomarker stercobilinogen was positively correlated with AMPK activation and negatively associated with lipid accumulation. This work confirmed that LP.P101 attenuated alcohol-induced hepatic lipid accumulation in mice through AMPK activation, and the alterations in gut microbiota and metabolites may play a significant role on AMPK activation.
RESUMO
PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCß activated by the IgE receptor, and Gßγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gßγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCß helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCß phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCß-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.
Assuntos
Metabolismo dos Lipídeos , Transdução de Sinais , Regulação Alostérica , Transdução de Sinais/fisiologia , Fosforilação , Membrana CelularRESUMO
Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. The p110γ-p84 complex is dynamic compared with p110γ-p101. While p110γ-p101 is robustly recruited by Gßγ subunits, p110γ-p84 is weakly recruited to membranes by Gßγ subunits alone and requires recruitment by Ras to allow for Gßγ activation. We mapped two distinct Gßγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gßγ activation. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated.
Assuntos
Fosfatidilinositol 3-Quinases , Transdução de Sinais , Transdução de Sinais/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Acoplados a Proteínas G , Modelos Moleculares , Fosfatidilinositol 3-QuinaseRESUMO
Mast cells are the major effector cells in immunoglobulin E (IgE)-mediated allergy. The high affinity IgE receptor FcεRI, as well as G protein-coupled receptors (GPCRs) on the mast cell surface signals to phosphoinositide 3-kinase γ (PI3Kγ) to initiate degranulation, cytokine release, and chemotaxis. PI3Kγ is therefore considered as a target for treatment of allergic disorders. However, leukocyte PI3Kγ is key to many functions in innate and adaptive immunity, and attenuation of host defense mechanisms is an expected adverse effect that complicates treatment of chronic illnesses. PI3Kγ operates as a p110γ/p84 or p110γ/p101 complex, where p110γ/p84 requires Ras activation. Here we investigated if modulation of Ras-isoprenylation could target PI3Kγ activity to attenuate PI3Kγ-dependent mast cell responses without impairment of macrophage functions. In murine bone marrow-derived mast cells, GPCR stimulation triggers activation of N-Ras and H-Ras isoforms, which is followed by the phosphorylation of protein kinase B (PKB/Akt) relayed through PI3Kγ. Although K-Ras is normally not activated in Ras wild-type cells, it is able to compensate for genetically deleted N- and H-Ras isoforms. Inhibition of Ras isoprenylation with farnesyltransferase inhibitor FTI-277 leads to a significant reduction of mast cell degranulation, cytokine production, and migration. Complementation experiments expressing PI3Kγ adaptor proteins p84 or p101 demonstrated a differential sensitivity towards Ras-inhibition depending on PI3Kγ complex composition. Mast cell responses are exclusively p84-dependent and were effectively controlled by FTI-277. Similar results were obtained when GTP-Ras was inactivated by overexpression of the GAP-domain of Neurofibromin-1 (NF-1). Unlike mast cells, macrophages express p84 and p101 but are p101-dominated and thus remain functional under treatment with FTI-277. Our work demonstrates that p101 and p84 have distinct physiological roles, and that Ras dependence of PI3Kγ signaling differs between cell types. FTI-277 reduces GPCR-activated PI3Kγ responses in p84-expressing but not p101-containing bone marrow derived cells. However, prenylation inhibitors have pleiotropic effects beyond Ras and non-tolerable side-effects that disfavor further clinical validation. Statins are, however, clinically well-established drugs that have previously been proposed to block mast cell degranulation by interference with protein prenylation. We show here that Simvastatin inhibits mast cell degranulation, but that this does not occur via Ras-PI3Kγ pathway alterations.