RESUMO
Periodontitis (PDS) is a chronic inflammatory disease initiated by a dysbiosis of oral pathogenic bacterial species, such as Porphyromonas gingivalis (Pg). These bacteria can penetrate the bloodstream, releasing various endo and exotoxins that fuel the infection, and stimulate toxic inflammation in different compartments, including the brain. However, the specific mechanisms by which PDS/Pg contribute to brain disorders, such as Alzheimer's disease (AD), remain unclear. This study assessed the effects of Pg's virulence factors - lipopolysaccharide (LPS-Pg) and gingipains (gps) K (Kgp) and Rgp - on SH-SY5Y cells. Our results demonstrated that LPS-Pg activated signaling through the Toll-like receptor (TLR)-2/4 induced a significant downregulation of G protein-coupled receptor kinase 5 (GRK5). Additionally, LPS-Pg stimulation resulted in a robust increase in Tau phosphorylation (pTau) and p53 levels, while causing a marked reduction in Bcl2 and increased cell death compared to unstimulated cells (Ns). LPS-Pg also elevated inducible nitric oxide synthase (iNOS) expression, leading to oxidative damage. In cells overexpressing GRK5 via Adenovirus, LPS-Pg failed to increase iNOS and pTau levels compared to GFP control cells. High GRK5 levels also prevented the nuclear accumulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). Furthermore, the overexpression of a GRK5 mutant form lacking the nuclear localization signal (ΔNLS) nearly abolished LPS-Pg induced p53 and iNOS upregulation. Finally, we tested whether Kgp and Rgp mediated similar effects and our data showed that both gps caused a marked downregulation of GRK5 leading to increased p53 and pTau levels. In conclusion, this study provides further insight into the toxic effects elicited by Pg in cells and suggests that preventing GRK5 deficiency may be a valid strategy to mitigate Pg-induced toxic effects (i.e. cell death, oxidative damage, and Tau hyperphosphorylation) in SH-SY5Y cells, which are typical molecular hallmarks of neurodegenerative disorders.
Assuntos
Quinase 5 de Receptor Acoplado a Proteína G , Lipopolissacarídeos , Porphyromonas gingivalis , Fatores de Virulência , Humanos , Linhagem Celular Tumoral , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/genética , Cisteína Endopeptidases Gingipaínas/metabolismo , Neuroblastoma , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Fosforilação , Porphyromonas gingivalis/patogenicidade , Transdução de Sinais , Proteínas tau/metabolismo , Receptor 2 Toll-Like/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fatores de Virulência/metabolismo , Fatores de Virulência/genéticaRESUMO
GRK5 holds a pivotal role in cellular signaling pathways, with its overexpression in cardiomyocytes, neuronal cells, and tumor cells strongly associated with various chronic degenerative diseases, which highlights the urgent need for potential inhibitors. In this study, multiclass classification-based QSAR models were developed using diverse machine learning algorithms. These models were built from curated compounds with experimentally derived GRK5 inhibitory activity. Additionally, a pharmacophore model was constructed using active compounds from the dataset. Among the models, the SVM-based approach proved most effective and was initially used to screen DrugBank compounds within the applicability domain. Compounds showing significant GRK5 inhibitory potential underwent evaluation for key pharmacophoric features. Prospective compounds were subjected to molecular docking to assess binding affinity towards GRK5's key active site amino acid residues. Stability at the binding site was analyzed through 200 ns molecular dynamics simulations. MM-GBSA analysis quantified individual free energy components contributing to the total binding energy with respect to binding site residues. Metadynamics analysis, including PCA, FEL, and PDF, provided crucial insights into conformational changes of both apo and holo forms of GRK5 at defined energy states. The study identifies DB02844 (S-Adenosyl-1,8-Diamino-3-Thiooctane) and DB13155 (Esculin) as promising GRK5 inhibitors, warranting further in vitro and in vivo validation studies.
Assuntos
Quinase 5 de Receptor Acoplado a Proteína G , Aprendizado de Máquina , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases , Relação Quantitativa Estrutura-Atividade , Quinase 5 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/química , Ligantes , Humanos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Termodinâmica , Ligação Proteica , Sítios de Ligação , Doença Crônica , FarmacóforoRESUMO
G protein-coupled receptors (GPCRs) are key modulators of cell signaling. Multiple GPCRs are present in the heart where they regulate cardiac homeostasis including processes such as myocyte contraction, heart rate and coronary blood flow. GPCRs are pharmacological targets for several cardiovascular disorders including heart failure (HF) such as beta-adrenergic receptor (ßAR) blockers and angiotensin II receptor (AT1R) antagonists. The activity of GPCRs are finely regulated by GPCR kinases (GRKs), which phosphorylate agonist-occupied receptors and start the process of desensitization. Among the seven members of the GRK family, GRK2 and GRK5 are predominantly expressed in the heart, where they exhibit both canonical and non-canonical functions. Both kinases are known to be increased in cardiac pathologies and contribute to pathogenesis through their roles in different cellular compartments. Lowering or inhibiting their actions mediate cardioprotective effects against pathological cardiac growth and failing heart. Therefore, given their importance in cardiac dysfunction, these kinases are drawing attention as promising targets for the treatment of HF, which needs improved therapies. Over the past three decades, broad knowledge on GRK inhibition in HF has been gained by studies using genetically engineered animal models or through gene therapy with peptide inhibitors or using small molecule inhibitors. In this mini review, we summarize the work focusing on GRK2 and GRK5 but also discuss a couple of the non-abundant cardiac subtypes and their multi-functional roles in the normal and diseased heart and the potential and therapeutic targets.
Assuntos
Quinases de Receptores Acoplados a Proteína G , Insuficiência Cardíaca , Animais , Quinases de Receptores Acoplados a Proteína G/genética , Quinases de Receptores Acoplados a Proteína G/metabolismo , Quinases de Receptores Acoplados a Proteína G/uso terapêutico , Quinase 5 de Receptor Acoplado a Proteína G/genética , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Transdução de Sinais , Receptores Acoplados a Proteínas GRESUMO
The development of small-molecule inhibitors targeting G protein-coupled receptor kinase 2 (GRK2) and G protein-coupled receptor kinase 5 (GRK5) for the treatment of chronic degenerative diseases has attracted wide attention. GRK2 and GRK5 can regulate essential physiological processes by phosphorylating G protein-coupled receptor (GPCR). Alterations in the functional levels of GRK2 and GRK5 have been found in a variety of chronic degenerative diseases, such as cardiovascular diseases, neurodegenerative diseases, cancers, type 2 diabetes, and rheumatoid arthritis (RA). Abnormal GRK2 and GRK5 expression contribute to the development of chronic degenerative diseases through environmental molecular mechanisms, making them promising molecular targets for treating chronic degenerative diseases. To date, many novel GRK2 and GRK5 inhibitors have been reported for the treatment of chronic degenerative diseases. We focus on the recent progress of single and dual-target inhibitors of GRK2/GRK5. This review summarizes the structural optimization rationale, structure-activity relationships (SARs), and the latest application in the treatment of chronic degenerative diseases. We believe it will shed light on the future development of small molecule inhibitors of GRK2 and GRK5, as well as the clinical applications in chronic degenerative diseases.
Assuntos
Doenças Cardiovasculares , Diabetes Mellitus Tipo 2 , Doenças Neurodegenerativas , Humanos , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Relação Estrutura-Atividade , Quinase 5 de Receptor Acoplado a Proteína G/metabolismoRESUMO
Triple-negative breast cancer (TNBC) is a malignant tumor that threatens women's health. Exploring novel development-associated biomarkers could help improve the survival rate of TNBC. This study evaluated the significance and mechanism of LINC01315 in TNBC progression aiming to identify a potential biomarker. There were 103 TNBC patients that provided clinical tissues in this study. The expression of LINC01315 was assessed by PCR and its association with clinical data was evaluated by statistical analyses. The in vitro cell experiments were conducted to estimate the biological effect of LINC01315 and its molecular mechanism. A significant upregulation of LINC01315 was observed in TNBC, which was associated with disease development and severity of patients. The upregulation of LINC01315 could be a symptom of the poor prognosis of patients. The knockdown of LINC01315 suppressed the main cellular processes of TNBC progression. Additionally, miR-876-5p was demonstrated to be a target of LINC01315 and regulate the expression of GRK5, through which LINC01315 modulated the progression of TNBC. Upregulated LINC01315 in TNBC indicated the malignant development and poor survival rate of patients. Inhibition of LINC01315 might be a potential therapeutic strategy of TNBC.
Assuntos
Quinase 5 de Receptor Acoplado a Proteína G , MicroRNAs , RNA Longo não Codificante , Neoplasias de Mama Triplo Negativas , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Quinase 5 de Receptor Acoplado a Proteína G/genética , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Signaling-biased ligands acting on G-protein-coupled receptors (GPCRs) differentially activate heterotrimeric G proteins and ß-arrestins. Although a wealth of structural knowledge about signaling bias at the GPCR level exists (preferential engagement of a specific transducer), little is known about the bias at the transducer level (different functions mediated by a single transducer), partly due to a poor understanding of GPCR kinase (GRK)-mediated GPCR phosphorylation. Here, we reveal a unique role of the Gq heterotrimer as a determinant for GRK-subtype selectivity that regulates subsequent ß-arrestin conformation and function. Using the angiotensin II (Ang II) type-1 receptor (AT1R), we show that ß-arrestin recruitment depends on both GRK2/3 and GRK5/6 upon binding of Ang II, but solely on GRK5/6 upon binding of the ß-arrestin-biased ligand TRV027. With pharmacological inhibition or genetic loss of Gq, GRK-subtype selectivity and ß-arrestin functionality by Ang II is shifted to those of TRV027. Single-molecule imaging identifies relocation of AT1R and GRK5, but not GRK2, to an immobile phase under the Gq-inactive, AT1R-stimulated conditions. These findings uncover a previously unappreciated Gq-regulated mechanism that encodes GRK-subtype selectivity and imparts distinct phosphorylation-barcodes directing downstream ß-arrestin functions.
Assuntos
Angiotensina II/farmacologia , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Oligopeptídeos/farmacologia , Receptor Tipo 1 de Angiotensina/metabolismo , beta-Arrestinas/metabolismo , Linhagem Celular , Humanos , Fosforilação , Transdução de Sinais , Vasoconstritores/farmacologiaRESUMO
AIMS: Myocardial infarction (MI) is the most common cause of heart failure (HF) worldwide. G protein-coupled receptor kinase 5 (GRK5) is upregulated in failing human myocardium and promotes maladaptive cardiac hypertrophy in animal models. However, the role of GRK5 in ischemic heart disease is still unknown. In this study, we evaluated whether myocardial GRK5 plays a critical role post-MI in mice and included the examination of specific cardiac immune and inflammatory responses. METHODS AND RESULTS: Cardiomyocyte-specific GRK5 overexpressing transgenic mice (TgGRK5) and non-transgenic littermate control (NLC) mice as well as cardiomyocyte-specific GRK5 knockout mice (GRK5cKO) and wild type (WT) were subjected to MI and, functional as well as structural changes together with outcomes were studied. TgGRK5 post-MI mice showed decreased cardiac function, augmented left ventricular dimension and decreased survival rate compared to NLC post-MI mice. Cardiac hypertrophy and fibrosis as well as fetal gene expression were increased post-MI in TgGRK5 compared to NLC mice. In TgGRK5 mice, GRK5 elevation produced immuno-regulators that contributed to the elevated and long-lasting leukocyte recruitment into the injured heart and ultimately to chronic cardiac inflammation. We found an increased presence of pro-inflammatory neutrophils and macrophages as well as neutrophils, macrophages and T-lymphocytes at 4-days and 8-weeks respectively post-MI in TgGRK5 hearts. Conversely, GRK5cKO mice were protected from ischemic injury and showed reduced early immune cell recruitment (predominantly monocytes) to the heart, improved contractility and reduced mortality compared to WT post-MI mice. Interestingly, cardiomyocyte-specific GRK2 transgenic mice did not share the same phenotype of TgGRK5 mice and did not have increased cardiac leukocyte migration and cytokine or chemokine production post-MI. CONCLUSIONS: Our study shows that myocyte GRK5 has a crucial and GRK-selective role on the regulation of leucocyte infiltration into the heart, cardiac function and survival in a murine model of post-ischemic HF, supporting GRK5 inhibition as a therapeutic target for HF.
Assuntos
Quimiotaxia de Leucócito , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Insuficiência Cardíaca/enzimologia , Leucócitos/metabolismo , Infarto do Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Função Ventricular Esquerda , Animais , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Quinase 5 de Receptor Acoplado a Proteína G/genética , Insuficiência Cardíaca/imunologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Mediadores da Inflamação/metabolismo , Leucócitos/imunologia , Camundongos Knockout , Contração Miocárdica , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , Transdução de Sinais , Volume Sistólico , Transcriptoma , Pressão VentricularRESUMO
Due to extremely poor systemic bioavailability, the mechanism by which curcumin increases energy expenditure remains unelucidated. Accumulating evidence suggests a strong association between the gut microbiota (GM) and energy metabolism. We investigated whether the GM mediates the effects of curcumin on improving energy homeostasis. High-fat diet (HFD)-fed wild type, uncoupling protein 1 (Ucp1) knockout and G protein-coupled membrane receptor 5 (TGR5) knockout mice were treated with curcumin (100 mg kg-1 d-1, p.o.). Curcumin-treated HFD-fed mice displayed decreased body weight gain and augmented cold tolerance due to enhanced adaptive thermogenesis as compared with that in control mice. The anti-obesity effects of curcumin were abolished by Ucp1 knockout. 16S ribosomal DNA sequencing analysis revealed that curcumin restructured the GM in HFD-fed mice. Fecal microbiota transplantation (FMT) and endogenous GM depletion indicated that the GM mediated the enhanced effect of curcumin on Ucp1-dependent thermogenesis. Curcumin altered bile acid (BA) metabolism with increased fractions of circulating deoxycholic acid (DCA) and lithocholic acid (LCA), which are the two most potent ligands for TGR5. Consistently, the enhanced effect of curcumin on Ucp1-dependent thermogenesis was eliminated by TGR5 knockout. Curcumin requires the GM and TGR5 to activate the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway in thermogenic adipose tissue. Here, we demonstrated that the GM mediates the effects of curcumin on enhancing Ucp1-dependent thermogenesis and ameliorating HFD-induced obesity by influencing BA metabolism. We disclosed the potential of nutritional and pharmacologic manipulations of the GM to enhance Ucp1-dependent thermogenesis in the prevention and treatment of obesity.
Assuntos
Curcumina/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Obesidade/tratamento farmacológico , Termogênese/efeitos dos fármacos , Proteína Desacopladora 1/metabolismo , Animais , Fármacos Antiobesidade/farmacologia , Ácidos e Sais Biliares/metabolismo , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético/efeitos dos fármacos , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Aumento de Peso/efeitos dos fármacosRESUMO
We have previously reported that the repeated exposure to cannabinoids upregulates and enhances the activity of serotonin 2A (5-HT2A) and dopamine 2 (D2) receptors and facilitates the formation of D2-5-HT2A receptor heterodimers in the rat prefrontal cortex and two neuronal cell lines. Because the repeated exposure to cannabinoids has been associated with adverse neuropsychiatric disorders, this study investigated the mechanisms that underly the cannabinoid-mediated regulation of D2 receptor expression in a neuronal cell model, CLU213 cells. We initially tested the effects of repeated exposure (72 h) to a non-selective cannabinoid agonist (1 nM CP55940), a selective CB1 receptor agonist (15 nM ACEA), or a selective CB2 receptor drug (1 nM GP1a) on the expression of postsynaptic D2 (D2L) receptors in CLU213 cells. Repeated CP55940, GP1a, or ACEA treatments significantly increased D2L receptor protein levels (99 % ± 7%, 30 % ± 7%, and 39 % ± 5% increases compared with control levels, respectively). Repeated exposure to both GP1a and ACEA increased D2L receptor protein levels by 73 % ± 8%. Interestingly, CP55940 and GP1a, but not ACEA, upregulated D2 mRNA. Using cells that were stably transfected with short-hairpin RNA (shRNA) lentiviral particles targeting CB2 receptors, G protein-coupled receptor kinase 5 (GRK5), and ß-arrestin 2, we found that CB2 receptors regulated D2 expression through a mechanism that is dependent on GRK5, ß-arrestin 2, and extracellular signal-related kinase (ERK)1/2. We also found that repeated exposure to either ACEA or GP1a selectively stimulated the protein and mRNA expression of GRK proteins. ACEA significantly upregulated GRK2 proteins, whereas GP1a upregulated GRK5 protein expression. Our results identified mechanisms associated with the upregulation of D2 receptors in neuronal cells after the repeated exposure to cannabinoids. These data can shed light on the mechanisms that can be targeted to prevent potential adverse effects, while simultaneously determining the therapeutic benefits of cannabinoids.
Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Neurônios/efeitos dos fármacos , Receptor CB2 de Canabinoide/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Linhagem Celular , Dopamina/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Neurônios/metabolismo , Ratos , Receptor CB2 de Canabinoide/agonistas , Regulação para Cima/efeitos dos fármacos , beta-Arrestina 2/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and they are responsible for the transduction of extracellular signals, regulating almost all aspects of mammalian physiology. These receptors are specifically regulated by a family of serine/threonine kinases, called GPCR kinases (GRKs). Given the biological role of GPCRs, it is not surprising that GRKs are also involved in several pathophysiological processes. Particular importance is emerging for GRK5, which is a multifunctional protein, expressed in different cell types, and it has been found located in single or multiple subcellular compartments. For instance, when anchored to the plasma membrane, GRK5 exerts its canonical function, regulating GPCRs. However, under certain conditions (e.g., pro-hypertrophic stimuli), GRK5 translocates to the nucleus of cells where it can interact with non-GPCR-related proteins as well as DNA itself to promote "non-canonical" signaling, including gene transcription. Importantly, due to these actions, several studies have demonstrated that GRK5 has a pivotal role in the pathogenesis of chronic-degenerative disorders. This is true in the cardiac cells, tumor cells, and neurons. For this reason, in this review article, we will inform the readers of the most recent evidence that supports the importance of targeting GRK5 to prevent the development or progression of cancer, cardiovascular, and neurological diseases.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Animais , Quinase 5 de Receptor Acoplado a Proteína G/química , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction (MI), can activate quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor kinase 5 (GRK5) is an important mediator of cardiovascular homeostasis through dampening of GPCR signaling, and is expressed in the heart and up-regulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium-calmodulin (Ca2+-CAM)-dependent manner, promoting hypertrophic gene transcription through activation of nuclear factor of activated T cells (NFAT). Interestingly, NFAT is also involved in fibroblast activation. GRK5 is highly expressed and active in cardiac fibroblasts; however, its pathophysiological role in these crucial cardiac cells is unknown. We demonstrate using adult cardiac fibroblasts that genetic deletion of GRK5 inhibits angiotensin II (AngII)-mediated fibroblast activation. Fibroblast-specific deletion of GRK5 in mice led to decreased fibrosis and cardiac hypertrophy after chronic AngII infusion or after ischemic injury compared to nontransgenic littermate controls (NLCs). Mechanistically, we show that nuclear translocation of GRK5 is involved in fibroblast activation. These data demonstrate that GRK5 is a regulator of fibroblast activation in vitro and cardiac fibrosis in vivo. This adds to previously published data which demonstrate the potential beneficial effects of GRK5 inhibition in the context of cardiac disease.
Assuntos
Fibroblastos/metabolismo , Fibroblastos/patologia , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Miocárdio/patologia , Angiotensina II , Animais , Animais Recém-Nascidos , Cardiomegalia/complicações , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Transdiferenciação Celular , Fibrose , Camundongos Knockout , Modelos Biológicos , Isquemia Miocárdica/complicações , Isquemia Miocárdica/patologia , Isquemia Miocárdica/fisiopatologia , Miofibroblastos/patologia , RatosRESUMO
G protein coupled receptor kinase 5 (GRK5) is localized within the nucleus and moderates functions such as DNA transcription, in addition to its localization at the plasma membrane. In this report, we show that GRK5 modifies the nucleolar stress response activated by the DNA polymerase inhibitor, actinomycin D (ActD). We show an increased sensitivity to the apoptotic effects of ActD on cervical HeLa cells and the breast cancer cell line MDA MB 231 with reduced protein expression of GRK5. We also tested two types of breast cancer cells (MDA MB 231 and MCF7 cells) and found that the rate of response to ActD varied between them because they have innate differences in the protein expression of GRK5. We also found that GRK5 phosphorylates nucleophosmin (NPM1) at T199 before and during the early stages of ActD treatment. Phosphorylation at T199 increases the ability of NPM1 to interact with p14ARF in vitro, which may affect the protein expression levels of p14ARF. We found that the expression levels of p14ARF were lower in the cells transfected with the control shRNA, but higher in cells transfected with GRK5 shRNA. Collectively, this suggests that GRK5 modifies the nucleolar stress response associated with ActD.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Nucléolo Celular/patologia , Dactinomicina/farmacologia , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Neoplasias/patologia , Proteínas Nucleares/metabolismo , Apoptose , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Nucleofosmina , Fosforilação , Ligação Proteica , Células Tumorais CultivadasRESUMO
ß-adrenergic receptors (ß-ARs) play a major role in the physiological regulation of cardiac function through signaling routes tightly controlled by G protein-coupled receptor kinases (GRKs). Although the acute stimulation of ß-ARs and the subsequent production of cyclic AMP (cAMP) have beneficial effects on cardiac function, chronic stimulation of ß-ARs as observed under sympathetic overdrive promotes the development of pathological cardiac remodeling and heart failure (HF), a leading cause of mortality worldwide. This is accompanied by an alteration in cAMP compartmentalization and the activation of the exchange protein directly activated by cAMP 1 (Epac1) signaling. Among downstream signals of ß-ARs, compelling evidence indicates that GRK2, GRK5, and Epac1 represent attractive therapeutic targets for cardiac disease. Here, we summarize the pathophysiological roles of GRK2, GRK5, and Epac1 in the heart. We focus on their signalosome and describe how under pathological settings, these proteins can cross-talk and are part of scaffolded nodal signaling systems that contribute to a decreased cardiac function and HF development.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Remodelação Ventricular , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 5 de Receptor Acoplado a Proteína G/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Humanos , Miocárdio/patologiaRESUMO
G protein-coupled receptor kinases (GRKs), in addition to their role in modulating signal transduction mechanisms associated with activated G protein-coupled receptors (GPCRs), can also interact with many non-GPCR proteins to mediate cellular responses to chemotherapeutics. The rationale for this study is based on the presumption that GRK2 modulates the responses of cancer cells to the chemotherapeutic cisplatin. In this report, we show that GRK2 modulates the responses of cancer cells to cisplatin. Cervical cancer HeLa cells stably transfected with GRK2 shRNA, to decrease GRK2 protein expression, show increased sensitivity to cisplatin. Of interest, these cells also show increased accumulation of NADPH, associating with decreased NADP buildup, at low concentrations of cisplatin tested. These changes in NADPH and NADP levels are also observed in the breast cancer MDA MB 231 cells, which has lower endogenous GRK2 protein expression levels, but not BT549, a breast cancer cell line with higher GRK2 protein expression. This effect of NADPH accumulation may be associated with a decrease in NADPH oxidase 4 (NOX4) protein expression, which is found to correlate with GRK2 protein expression in cancer cells-a relationship which mimics that observed in cardiomyocytes. Furthermore, like in cardiomyocytes, GRK2 and NOX4 interact to form complexes in cancer cells. Collectively, these results suggest that GRK2 interacts with NOX4 to modify cisplatin sensitivity in cancer cells and may also factor into the success of cisplatin-based regimens.
Assuntos
Cisplatino/farmacologia , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 3 de Receptor Acoplado a Proteína G/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Perfilação da Expressão Gênica , Células HeLa , Humanos , Neoplasias/metabolismo , Fosforilação , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
The phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) facilitates arrestin binding and receptor desensitization. Although this process can be regulated by Ca2+-binding proteins such as calmodulin (CaM) and recoverin, the molecular mechanisms are poorly understood. Here, we report structural, computational, and biochemical analysis of a CaM complex with GRK5, revealing how CaM shapes GRK5 response to calcium. The CaM N and C domains bind independently to two helical regions at the GRK5 N and C termini to inhibit GPCR phosphorylation, though only the C domain interaction disrupts GRK5 membrane association, thereby facilitating cytoplasmic translocation. The CaM N domain strongly activates GRK5 via ordering of the amphipathic αN-helix of GRK5 and allosteric disruption of kinase-RH domain interaction for phosphorylation of cytoplasmic GRK5 substrates. These results provide a framework for understanding how two functional effects, GRK5 activation and localization, can cooperate under control of CaM for selective substrate targeting by GRK5.
Assuntos
Cálcio/metabolismo , Calmodulina/química , Quinase 5 de Receptor Acoplado a Proteína G/química , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Calmodulina/genética , Calmodulina/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Quinase 5 de Receptor Acoplado a Proteína G/genética , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Cinética , Simulação de Dinâmica Molecular , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Células Sf9 , Spodoptera , Especificidade por Substrato , TermodinâmicaRESUMO
RATIONALE: Cardiotoxic ß1 adrenergic receptor (ß1AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of ß1AR and organizes a receptor signalosome. OBJECTIVE: We aim to elucidate the dynamics of ß1AR-SAP97 signalosome and its potential role in chronic cardiotoxic ß1AR-CaMKII signaling that contributes to development of heart failure. METHODS AND RESULTS: The integrity of cardiac ß1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine ß1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the ß1AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of ß1AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from ß1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of ß1AR-SAP97 complex and increases in CaMKII activity in hearts. CONCLUSIONS: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac ß1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína 1 Homóloga a Discs-Large/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Insuficiência Cardíaca/enzimologia , Miócitos Cardíacos/enzimologia , Receptores Adrenérgicos beta 1/metabolismo , Animais , Apoptose , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína 1 Homóloga a Discs-Large/genética , Modelos Animais de Doenças , Acoplamento Excitação-Contração , Quinase 5 de Receptor Acoplado a Proteína G/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica , Miócitos Cardíacos/patologia , beta-Arrestina 1/metabolismoRESUMO
Aldosterone (Aldo), when overproduced, is a cardiotoxic hormone underlying heart failure and hypertension. Aldo exerts damaging effects via the mineralocorticoid receptor (MR) but also activates the antiapoptotic G protein-coupled estrogen receptor (GPER) in the heart. G protein-coupled receptor (GPCR)-kinase (GRK)-2 and -5 are the most abundant cardiac GRKs and phosphorylate GPCRs as well as non-GPCR substrates. Herein, we investigated whether they phosphorylate and regulate cardiac MR and GPER. To this end, we used the cardiomyocyte cell line H9c2 and adult rat ventricular myocytes (ARVMs), in which we manipulated GRK5 protein levels via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and GRK2 activity via pharmacological inhibition. We report that GRK5 phosphorylates and inhibits the cardiac MR whereas GRK2 phosphorylates and desensitizes GPER. In H9c2 cardiomyocytes, GRK5 interacts with and phosphorylates the MR upon ß2-adrenergic receptor (AR) activation. In contrast, GRK2 opposes agonist-activated GPER signaling. Importantly, GRK5-dependent MR phosphorylation of the MR inhibits transcriptional activity, since aldosterone-induced gene transcription is markedly suppressed in GRK5-overexpressing cardiomyocytes. Conversely, GRK5 gene deletion augments cardiac MR transcriptional activity. ß2AR-stimulated GRK5 phosphorylates and inhibits the MR also in ARVMs. Additionally, GRK5 is necessary for the protective effects of the MR antagonist drug eplerenone against Aldo-induced apoptosis and oxidative stress in ARVMs. In conclusion, GRK5 blocks the cardiotoxic MR-dependent effects of Aldo in the heart, whereas GRK2 may hinder beneficial effects of Aldo through GPER. Thus, cardiac GRK5 stimulation (e.g., via ß2AR activation) might be of therapeutic value for heart disease treatment via boosting the efficacy of MR antagonists against Aldo-mediated cardiac injury.
Assuntos
Aldosterona/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Miócitos Cardíacos/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transdução de Sinais , Animais , Apoptose , Linhagem Celular , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 5 de Receptor Acoplado a Proteína G/genética , Modelos Biológicos , Estresse Oxidativo , Fosforilação , Ligação Proteica , Ratos , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ativação TranscricionalRESUMO
Tachykinin signaling system is present in both vertebrates and invertebrates, and functions as neuromodulator responsible for the regulation of various physiological processes. In human, the internalization of G protein-coupled receptors has been extensively characterized; however, the insect GPCR internalization has been rarely investigated. Here, we constructed two expression vectors of Bombyx tachykinin-related peptide receptor (BmTKRPR) fused with Enhanced Green Fluorescent Protein (EGFP) at the C-terminal end for direct visualization of receptor expression, localization, and trafficking in cultured mammalian HEK293 and insect Sf21 cells. Our results demonstrated that agonist-activated BmTKRPR underwent rapid internalization in a dose-and time-dependent manner via a clathrin-dependent pathway in both HEK293 and Sf21 cells. Further investigation via RNAi or specific inhibitors, or co-immunoprecipitation demonstrated that agonist-induced BmTKRPR internalization was mediated by PKC, GRK5 and ß-arrestin2/BmKurtz. In addition, we also observed that most of the internalized BmTKRP receptors were recycled to the cell surface via early endosomes upon peptide ligand removal. Our study provides the first in-depth information on mechanisms underlying insect TKRP receptor internalization and perhaps aids in the interpretation of the signaling in the regulation of physiological processes.
Assuntos
Bombyx/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Proteína Quinase C/metabolismo , Receptores de Taquicininas/metabolismo , beta-Arrestina 2/metabolismo , Animais , Endossomos/metabolismo , Células HEK293 , Humanos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Ligantes , Transporte Proteico , Receptores de Taquicininas/genética , Células Sf9 , Transdução de SinaisRESUMO
G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in humans and regulate numerous physiological processes through the activation of heterotrimeric G proteins. GPCR kinases (GRKs) selectively phosphorylate active GPCRs, which promotes arrestin binding, receptor internalization, and initiation of alternative signaling pathways. GRK5 is a representative member of one of three GRK subfamilies that does not need post-translational lipidation or other binding partners to exhibit full activity against GPCRs, rendering it a useful tool for biophysical studies directed at characterizing GRK function. However, recombinant expression of GRK5 has thus far been limited to insect and mammalian systems. Here, we describe the expression of functional GRK5 in E. coli and its purification and biochemical characterization. Bacterially expressed GRK5 is hyperphosphorylated, primarily in regions known to be flexible from prior crystal structures, which slightly decreases its catalytic activity toward receptor substrates. Mutation of a single phosphorylation site, Thr10, restores kinetic parameters to those of GRK5 purified from insect cells. Consequently, bacterial expression will allow for production of GRK5 at a reduced cost and faster pace and would facilitate production of isotopically labeled kinase for NMR studies or for the incorporation of unnatural amino acids.
Assuntos
Trifosfato de Adenosina/química , Quinase 5 de Receptor Acoplado a Proteína G/química , Processamento de Proteína Pós-Traducional , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/genética , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
OBJECTIVE: NF-κB-dependent signaling is an important modulator in osteoarthritis (OA), and G protein-coupled receptor kinase 5 (GRK5) regulates the NF-κB pathway. This study was undertaken to investigate the functional involvement of GRK5 in OA pathogenesis. METHODS: GRK5 expression in normal and OA human knee joints was analyzed immunohistochemically. Gain- or loss-of-function experiments were performed using human and mouse chondrocytes. OA was induced in GRK5-knockout mice by destabilization of the medial meniscus, and histologic examination was performed. OA was also induced in wild-type mice, which were then treated with an intraarticular injection of amlexanox, a selective GRK5 inhibitor, every 5 days for 8 weeks. RESULTS: GRK5 protein expression was increased in human OA cartilage. In vitro, expression levels of OA-related factors and NF-κB transcriptional activation were down-regulated by suppression of the GRK5 gene in human OA chondrocytes (3.49-fold decrease in IL6 [P < 0.01], 2.43-fold decrease in MMP13 [P < 0.01], and 2.66-fold decrease in ADAMTS4 [P < 0.01]). Conversely, GRK5 overexpression significantly increased the expression of OA-related catabolic mediators and NF-κB transcriptional activation. On Western blot analysis, GRK5 deletion reduced IκBα phosphorylation (up to 4.4-fold decrease [P < 0.05]) and decreased p65 nuclear translocation (up to 6.4-fold decrease [P < 0.01]) in mouse chondrocytes. In vivo, both GRK5 deletion and intraarticular amlexanox protected mouse cartilage against OA. CONCLUSION: Our results suggest that GRK5 regulates cartilage degradation through a catabolic response mediated by NF-κB signaling, and is a potential target for OA treatment. Furthermore, amlexanox may be a major compound in relevant drugs.