RESUMO
Chronic hypoxia causes pulmonary hypertension (PH), vascular remodeling, right ventricular (RV) hypertrophy, and cardiac failure. Protein kinase G Iα (PKGIα) is susceptible to oxidation, forming an interprotein disulfide homodimer associated with kinase targeting involved in vasodilation. Here we report increased disulfide PKGIα in pulmonary arteries from mice with hypoxic PH or lungs from patients with pulmonary arterial hypertension. This oxidation is likely caused by oxidants derived from NADPH oxidase-4, superoxide dismutase 3, and cystathionine γ-lyase, enzymes that were concomitantly increased in these samples. Indeed, products that may arise from these enzymes, including hydrogen peroxide, glutathione disulfide, and protein-bound persulfides, were increased in the plasma of hypoxic mice. Furthermore, low-molecular-weight hydropersulfides, which can serve as "superreductants" were attenuated in hypoxic tissues, consistent with systemic oxidative stress and the oxidation of PKGIα observed. Inhibiting cystathionine γ-lyase resulted in decreased hypoxia-induced disulfide PKGIα and more severe PH phenotype in wild-type mice, but not in Cys42Ser PKGIα knock-in (KI) mice that are resistant to oxidation. In addition, KI mice also developed potentiated PH during hypoxia alone. Thus, oxidation of PKGIα is an adaptive mechanism that limits PH, a concept further supported by polysulfide treatment abrogating hypoxia-induced RV hypertrophy in wild-type, but not in the KI, mice. Unbiased transcriptomic analysis of hypoxic lungs before structural remodeling identified up-regulation of endothelial-to-mesenchymal transition pathways in the KI compared with wild-type mice. Thus, disulfide PKGIα is an intrinsic adaptive mechanism that attenuates PH progression not only by promoting vasodilation but also by limiting maladaptive growth and fibrosis signaling.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Hipertensão Pulmonar/patologia , Hipóxia/complicações , Artéria Pulmonar/patologia , Adulto , Animais , Linhagem Celular , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Dissulfetos/química , Feminino , Fibrose , Técnicas de Introdução de Genes , Humanos , Hipertensão Pulmonar/sangue , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/prevenção & controle , Hipóxia/sangue , Hipóxia/tratamento farmacológico , Pulmão/irrigação sanguínea , Pulmão/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Oxidantes/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Sulfetos/administração & dosagem , Sulfetos/sangue , Sulfetos/metabolismo , Regulação para Cima , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
Singlet molecular oxygen (1O2) has well-established roles in photosynthetic plants, bacteria and fungi1-3, but not in mammals. Chemically generated 1O2 oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine4, whereas enzymatic oxidation of tryptophan to N-formylkynurenine is catalysed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 15. Under inflammatory conditions, this haem-containing enzyme is expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure6. However, whether indoleamine 2,3-dioxygenase 1 forms 1O2 and whether this contributes to blood pressure control have remained unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of 1O2. We observed that in the presence of hydrogen peroxide, the enzyme generates 1O2 and that this is associated with the stereoselective oxidation of L-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophan-derived hydroperoxide acts in vivo as a signalling molecule, inducing arterial relaxation and decreasing blood pressure; this activity is dependent on Cys42 of protein kinase G1α. Our findings demonstrate a pathophysiological role for 1O2 in mammals through formation of an amino acid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions.
Assuntos
Pressão Sanguínea/fisiologia , Inflamação/sangue , Inflamação/fisiopatologia , Oxigênio Singlete/metabolismo , Vasodilatadores/metabolismo , Animais , Linhagem Celular , Proteína Quinase Dependente de GMP Cíclico Tipo I/antagonistas & inibidores , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Cisteína/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/química , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Inflamação/enzimologia , Masculino , Oxirredução/efeitos dos fármacos , Ratos , Transdução de Sinais , Oxigênio Singlete/química , Triptofano/química , Triptofano/metabolismoRESUMO
BACKGROUND: Thoracic aortic aneurysm and dissection (TAAD) is a common condition associated with high mortality. It is predominantly inherited in an autosomal dominant manner with reduced penetrance and variable expression. The genetic basis of the majority of TAAD cases remains unknown. CASE PRESENTATION: We described a 53 years old male presented with abdominal aortic dissection as well as aortic tortuosity. To investigate the genetic basis of the clinical presentation, whole-exome sequencing was performed. Exome sequencing identified a de novo heterozygous undescribed mutation in the PRKG1 gene (NM_001098512.2: c.1108 G > A), predicted to cause the missense change p.Gly370Ser in the ATP binding motif of the protein. This mutation was not reported in the dbSNP, 1000 Genome Project, and Exome sequencing databases. Furthermore, the Glycine370 residue of PRKG1 is highly conserved among various species and it is predicted to be damaging by multiple in silico programs, suggesting that this substitution may cause a major disruption of protein function. To our knowledge, this is the second reported mutation locus of PRKG1 accounting for the disease. CONCLUSIONS: Our study expands the mutation spectrum of PRKG1 and clinical phenotype of mutation-carriers. Screening for PRKG1 mutations should be considered in patients with unexplained aortic disease, and identification of the causative gene will aid in individualized, gene-tailored management.
Assuntos
Aneurisma da Aorta Torácica/genética , Dissecção Aórtica/genética , Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Exoma , Mutação de Sentido Incorreto , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Dissecção Aórtica/diagnóstico por imagem , Dissecção Aórtica/metabolismo , Dissecção Aórtica/patologia , Aneurisma da Aorta Torácica/diagnóstico por imagem , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/patologia , Angiografia por Tomografia Computadorizada , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Expressão Gênica , Genes Dominantes , Humanos , Masculino , Pessoa de Meia-Idade , Domínios e Motivos de Interação entre Proteínas , Sequenciamento do ExomaRESUMO
The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type Iα isoform can be constitutively activated by oxidizing conditions. However, the amino acid residues implicated in this phenomenon are not fully elucidated. To investigate the molecular basis for this mechanism, we studied the effects of oxidation using recombinant WT, truncated, and mutant constructs of PKG I. Using an in vitro assay, we observed that oxidation with hydrogen peroxide (H2O2) resulted in constitutive, cGMP-independent activation of PKG Iα. PKG Iα C42S and a truncation construct that does not contain Cys-42 (Δ53) were both constitutively activated by H2O2 In contrast, oxidation of PKG Iα C117S maintained its cGMP-dependent activation characteristics, although oxidized PKG Iα C195S did not. To corroborate these results, we also tested the effects of our constructs on the PKG Iα-specific substrate, the large conductance potassium channel (KCa 1.1). Application of WT PKG Iα activated by either cGMP or H2O2 increased the open probabilities of the channel. Neither cGMP nor H2O2 activation of PKG Iα C42S significantly increased channel open probabilities. Moreover, cGMP-stimulated PKG Iα C117S increased KCa 1.1 activity, but this effect was not observed under oxidizing conditions. Finally, we observed that PKG Iα C42S caused channel flickers, indicating dramatically altered KCa 1.1 channel characteristics compared with channels exposed to WT PKG Iα. Cumulatively, these results indicate that constitutive activation of PKG Iα proceeds through oxidation of Cys-117 and further suggest that the formation of a sulfur acid is necessary for this phenotype.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , GMP Cíclico/metabolismo , Cisteína/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Cisteína/química , Modelos Moleculares , Óxido Nítrico/metabolismo , Oxirredução , Fosforilação , Conformação Proteica , Homologia de SequênciaRESUMO
Activation of protein kinase G (PKG) Iα in nociceptive neurons induces long-term hyperexcitability that causes chronic pain. Recently, a derivative of the fungal metabolite balanol, N46, has been reported to inhibit PKG Iα with high potency and selectivity and attenuate thermal hyperalgesia and osteoarthritic pain. Here we determined co-crystal structures of the PKG Iα C-domain and cAMP-dependent protein kinase (PKA) Cα, each bound with N46, at 1.98 Å and 2.65 Å, respectively. N46 binds the active site with its external phenyl ring, specifically interacting with the glycine-rich loop and the αC helix. Phe-371 at the PKG Iα glycine-rich loop is oriented parallel to the phenyl ring of N46, forming a strong π-stacking interaction, whereas the analogous Phe-54 in PKA Cα rotates 30° and forms a weaker interaction. Structural comparison revealed that steric hindrance between the preceding Ser-53 and the propoxy group of the phenyl ring may explain the weaker interaction with PKA Cα. The analogous Gly-370 in PKG Iα, however, causes little steric hindrance with Phe-371. Moreover, Ile-406 on the αC helix forms a hydrophobic interaction with N46 whereas its counterpart in PKA, Thr-88, does not. Substituting these residues in PKG Iα with those in PKA Cα increases the IC50 values for N46, whereas replacing these residues in PKA Cα with those in PKG Iα reduces the IC50, consistent with our structural findings. In conclusion, our results explain the structural basis for N46-mediated selective inhibition of human PKG Iα and provide a starting point for structure-guided design of selective PKG Iα inhibitors.
Assuntos
Azepinas/química , Azepinas/farmacologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/antagonistas & inibidores , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Hidroxibenzoatos/química , Hidroxibenzoatos/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Humanos , Modelos Moleculares , Fosforilação , Conformação ProteicaRESUMO
Despite the mechanisms for endogenous nitroxyl (HNO) production and action being incompletely understood, pharmacological donors show broad therapeutic promise and are in clinical trials. Mass spectrometry and site-directed mutagenesis showed that chemically distinct HNO donors 1-nitrosocyclohexyl acetate or Angeli's salt induced disulfides within cGMP-dependent protein kinase I-alpha (PKGIα), an interdisulfide between Cys42 of the two identical subunits of the kinase and a previously unobserved intradisulfide between Cys117 and Cys195 in the high affinity cGMP-binding site. Kinase activity was monitored in cells transfected with wildtype (WT), Cys42Ser or Cys117/195Ser PKGIα that cannot form the inter- or intradisulfide, respectively. HNO enhanced WT kinase activity, an effect significantly attenuated in inter- or intradisulfide-deficient PKGIα. To investigate whether the intradisulfide modulates cGMP binding, real-time imaging was performed in vascular smooth muscle cells expressing a FRET-biosensor comprising the cGMP-binding sites of PKGIα. HNO induced FRET changes similar to those elicited by an increase of cGMP, suggesting that intradisulfide formation is associated with activation of PKGIα. Intradisulfide formation in PKGIα correlated with enhanced HNO-mediated vasorelaxation in mesenteric arteries in vitro and arteriolar dilation in vivo in mice. HNO induces intradisulfide formation in PKGIα, inducing the same effect as cGMP binding, namely kinase activation and thus vasorelaxation.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , GMP Cíclico/metabolismo , Dissulfetos/metabolismo , Mutagênese Sítio-Dirigida , Óxidos de Nitrogênio/farmacologia , Animais , Domínio Catalítico , Células Cultivadas , Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Cisteína/genética , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Masculino , Espectrometria de Massas , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , OxirreduçãoRESUMO
Cyclic GMP analogs, 8-Br, 8-pCPT, and PET-cGMP, have been widely used for characterizing cellular functions of cGMP-dependent protein kinase (PKG) I and II isotypes. However, interpreting results obtained using these analogs has been difficult due to their low isotype specificity. Additionally, each isotype has two binding sites with different cGMP affinities and analog selectivities, making understanding the molecular basis for isotype specificity of these compounds even more challenging. To determine isotype specificity of cGMP analogs and their structural basis, we generated the full-length regulatory domains of PKG I and II isotypes with each binding site disabled, determined their affinities for these analogs, and obtained cocrystal structures of both isotypes bound with cGMP analogs. Our affinity and activation measurements show that PET-cGMP is most selective for PKG I, whereas 8-pCPT-cGMP is most selective for PKG II. Our structures of cyclic nucleotide binding (CNB) domains reveal that the B site of PKG I is more open and forms a unique π/π interaction through Arg285 at ß4 with the PET moiety, whereas the A site of PKG II has a larger ß5/ß6 pocket that can better accommodate the bulky 8-pCPT moiety. Our structural and functional results explain the selectivity of these analogs for each PKG isotype and provide a starting point for the rational design of isotype selective activators.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo II/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , GMP Cíclico/análogos & derivados , Tionucleotídeos/metabolismo , Cristalografia por Raios X , GMP Cíclico/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Proteína Quinase Dependente de GMP Cíclico Tipo II/química , Humanos , Modelos Moleculares , Conformação Proteica , Especificidade por SubstratoRESUMO
Nitrovasodilators relax vascular smooth-muscle cells in part by modulating the interaction of the C-terminal coiled-coil domain (CC) and/or the leucine zipper (LZ) domain of the myosin light-chain phosphatase component, myosin-binding subunit (MBS), with the N-terminal LZ domain of protein kinase G (PKG)-Iα. Despite the importance of vasodilation in cardiovascular homeostasis and therapy, our structural understanding of the MBS CC interaction with LZ PKG-1α has remained limited. Here, we report the 3D NMR solution structure of homodimeric CC MBS in which amino acids 932-967 form a coiled-coil of two monomeric α-helices in parallel orientation. We found that the structure is stabilized by non-covalent interactions, with dominant contributions from hydrophobic residues at a and d heptad positions. Using NMR chemical-shift perturbation (CSP) analysis, we identified a subset of hydrophobic and charged residues of CC MBS (localized within and adjacent to the C-terminal region) contributing to the dimer-dimer interaction interface between homodimeric CC MBS and homodimeric LZ PKG-Iα. 15N backbone relaxation NMR revealed the dynamic features of the CC MBS interface residues identified by NMR CSP. Paramagnetic relaxation enhancement- and CSP-NMR-guided HADDOCK modeling of the dimer-dimer interface of the heterotetrameric complex exhibits the involvement of non-covalent intermolecular interactions that are localized within and adjacent to the C-terminal regions of each homodimer. These results deepen our understanding of the binding restraints of this CC MBS·LZ PKG-Iα low-affinity heterotetrameric complex and allow reevaluation of the role(s) of myosin light-chain phosphatase partner polypeptides in regulation of vascular smooth-muscle cell contractility.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Zíper de Leucina , Miosinas/química , Animais , Dicroísmo Circular , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Camundongos , Simulação de Dinâmica Molecular , Músculo Liso Vascular/citologia , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Ultravioleta , Eletricidade EstáticaRESUMO
The R-diastereomer of phosphorothioate analogs of cGMP, Rp-cGMPS, is one of few known inhibitors of cGMP-dependent protein kinase I (PKG I); however, its mechanism of inhibition is currently not fully understood. Here, we determined the crystal structure of the PKG Iß cyclic nucleotide-binding domain (PKG Iß CNB-B), considered a 'gatekeeper' for cGMP activation, bound to Rp-cGMPS at 1.3 Å. Our structural and NMR data show that PKG Iß CNB-B bound to Rp-cGMPS displays an apo-like structure with its helical domain in an open conformation. Comparison with the cAMP-dependent protein kinase regulatory subunit (PKA RIα) showed that this conformation resembles the catalytic subunit-bound inhibited state of PKA RIα more closely than the apo or Rp-cAMPS-bound conformations. These results suggest that Rp-cGMPS inhibits PKG I by stabilizing the inactive conformation of CNB-B.
Assuntos
Apoenzimas/química , Apoenzimas/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Tionucleotídeos/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , GMP Cíclico/química , Proteína Quinase Dependente de GMP Cíclico Tipo I/antagonistas & inibidores , Estabilidade Enzimática , Cinética , Espectroscopia de Ressonância Magnética , Conformação Proteica , Domínios Proteicos , Estereoisomerismo , Tionucleotídeos/químicaRESUMO
Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/química , GMP Cíclico/metabolismo , Simulação de Acoplamento Molecular , Sítios de Ligação , Cristalografia por Raios X , GMP Cíclico/química , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Multimerização ProteicaRESUMO
Sildenafil, an inhibitor of the cGMP-degrading phosphodiesterase 5 that is used to treat erectile dysfunction, has been linked to an increased risk of melanoma. Here, we have examined the potential connection between cGMP-dependent signaling cascades and melanoma growth. Using a combination of biochemical assays and real-time monitoring of melanoma cells, we report a cGMP-dependent growth-promoting pathway in murine and human melanoma cells. We document that C-type natriuretic peptide (CNP), a ligand of the membrane-bound guanylate cyclase B, enhances the activity of cGMP-dependent protein kinase I (cGKI) in melanoma cells by increasing the intracellular levels of cGMP. Activation of this cGMP pathway promotes melanoma cell growth and migration in a p44/42 MAPK-dependent manner. Sildenafil treatment further increases intracellular cGMP concentrations, potentiating activation of this pathway. Collectively, our data identify this cGMP-cGKI pathway as the link between sildenafil usage and increased melanoma risk.
Assuntos
GMP Cíclico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Citrato de Sildenafila/farmacologia , Animais , Butadienos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Feminino , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Peptídeo Natriurético Tipo C/toxicidade , Nitrilas/farmacologia , Inibidores da Fosfodiesterase 5/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Citrato de Sildenafila/uso terapêutico , Transplante HomólogoRESUMO
Protein kinase G (PKG) is a major receptor of cGMP and controls signaling pathways often distinct from those regulated by cAMP. Hence, the selective activation of PKG by cGMP versus cAMP is critical. However, the mechanism of cGMP-versus-cAMP selectivity is only limitedly understood. Although the C-terminal cyclic nucleotide-binding domain B of PKG binds cGMP with higher affinity than cAMP, the intracellular concentrations of cAMP are typically higher than those of cGMP, suggesting that the cGMP-versus-cAMP selectivity of PKG is not controlled uniquely through affinities. Here, we show that cAMP is a partial agonist for PKG, and we elucidate the mechanism for cAMP partial agonism through the comparative NMR analysis of the apo, cGMP-, and cAMP-bound forms of the PKG cyclic nucleotide-binding domain B. We show that although cGMP activation is adequately explained by a two-state conformational selection model, the partial agonism of cAMP arises from the sampling of a third, partially autoinhibited state.
Assuntos
AMP Cíclico/química , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , GMP Cíclico/química , Modelos Moleculares , Humanos , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
cGMP-dependent protein kinase (PKG) Iα is a central regulator of smooth muscle tone and vasorelaxation. The N-terminal leucine zipper (LZ) domain dimerizes and targets PKG Iα by interacting with G-kinase-anchoring proteins. The PKG Iα LZ contains C42 that is known to form a disulfide bond upon oxidation and to activate PKG Iα. To understand the molecular details of the PKG Iα LZ and C42-C42' disulfide bond, we determined crystal structures of the PKG Iα wild-type (WT) LZ and C42L LZ. Our data demonstrate that the C42-C42' disulfide bond dramatically stabilizes PKG Iα and that the C42L mutant mimics the oxidized WT LZ structurally.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Cistina/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Estabilidade Enzimática , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Temperatura de TransiçãoRESUMO
Podocytes play a fundamental role in regulating glomerular permeability to albumin. This mechanism is disrupted in the course of diabetes. Both insulin and high glucose concentrations enhance the permeability of podocytes to albumin by stimulating oxygen free radical production, primarily by NAD(P)H oxidase-4 (NOX4), and by activating protein kinase G, isoform Iα (PKGIα). However, no study has investigated the combined effects of insulin and high glucose concentration. Here, we investigated the effects of applying insulin (INS, 300 nM) and high glucose (HG, 30 mM), both separately and combined, for 5 days, on cultured rat podocyte permeability to albumin. We measured podocyte permeability with a transmembrane albumin flux assay. We measured NOX4 and PKGIα mRNA expression with real-time PCR. We used Western blots to evaluate protein expression levels of NOX4, PKGIα, the myosin-binding subunit of myosin phosphatase 1, and myosin light chain. We found that INS and HG had a synergistic effect on podocyte permeability to albumin, and this synergy was not dependent on NOX4 or PKGIα. These results suggested that the combined action of INS and HG may exacerbate glomerular dysfunction in diabetes.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Podócitos/metabolismo , Albumina Sérica/metabolismo , Animais , Células Cultivadas , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Feminino , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Permeabilidade , Multimerização Proteica , Ratos , Ratos WistarRESUMO
Nitroxyl (HNO), the reduced and protonated form of nitric oxide (NO·), confers unique physiological effects including vasorelaxation and enhanced cardiac contractility. These features have spawned current pharmaceutical development of HNO donors as heart failure therapeutics. HNO interacts with selective redox sensitive cysteines to effect signaling but is also proposed to activate soluble guanylate cyclase (sGC) in vitro to induce vasodilation and potentially enhance contractility. Here, we tested whether sGC stimulation is required for these HNO effects in vivo and if HNO also modifies a redox-sensitive cysteine (C42) in protein kinase G-1α to control vasorelaxation. Intact mice and isolated arteries lacking the sGC-ß subunit (sGCKO, results in full sGC deficiency) or expressing solely a redox-dead C42S mutant protein kinase G-1α were exposed to the pure HNO donor, CXL-1020. CXL-1020 induced dose-dependent systemic vasodilation while increasing contractility in controls; however, vasodilator effects were absent in sGCKO mice whereas contractility response remained. The CXL-1020 dose reversing 50% of preconstricted force in aortic rings was ≈400-fold greater in sGCKO than controls. Cyclic-GMP and cAMP levels were unaltered in myocardium exposed to CXL-1020, despite its inotropic-vasodilator activity. In protein kinase G-1α(C42S) mice, CXL-1020 induced identical vasorelaxation in vivo and in isolated aortic and mesenteric vessels as in littermate controls. In both groups, dilation was near fully blocked by pharmacologically inhibiting sGC. Thus, sGC and cGMP-dependent signaling are necessary and sufficient for HNO-induced vasodilation in vivo but are not required for positive inotropic action. Redox modulation of protein kinase G-1α is not a mechanism for HNO-mediated vasodilation.
Assuntos
Cardiotônicos/farmacologia , Guanilato Ciclase/fisiologia , Óxidos de Nitrogênio/farmacologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Vasodilatação/fisiologia , Animais , Aorta/efeitos dos fármacos , GMP Cíclico/fisiologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Proteína Quinase Dependente de GMP Cíclico Tipo I/deficiência , Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Cisteína/química , Guanilato Ciclase/deficiência , Guanilato Ciclase/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/fisiopatologia , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Óxido Nítrico/fisiologia , Doadores de Óxido Nítrico/farmacologia , Oxirredução , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Sistemas do Segundo Mensageiro/fisiologia , Guanilil Ciclase Solúvel , Sulfonamidas/farmacologia , Vasodilatação/efeitos dos fármacosRESUMO
High selectivity of cyclic-nucleotide binding (CNB) domains for cAMP and cGMP are required for segregating signaling pathways; however, the mechanism of selectivity remains unclear. To investigate the mechanism of high selectivity in cGMP-dependent protein kinase (PKG), we determined a room-temperature joint X-ray/neutron (XN) structure of PKG Iß CNB-B, a domain 200-fold selective for cGMP over cAMP, bound to cGMP (2.2 Å), and a low-temperature X-ray structure of CNB-B with cAMP (1.3 Å). The XN structure directly describes the hydrogen bonding interactions that modulate high selectivity for cGMP, while the structure with cAMP reveals that all these contacts are disrupted, explaining its low affinity for cAMP.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Ativadores de Enzimas/química , Nêutrons , Espalhamento de Radiação , Animais , AMP Cíclico/química , GMP Cíclico/química , Desenho de Fármacos , Ativação Enzimática , Humanos , Ligação de HidrogênioRESUMO
Although cGMP-dependent protein kinase type I (cGKI) is an important mediator of cGMP signaling and upcoming drug target, its in vivo-biochemistry is not well understood. Many studies showed that purified cGKI autophosphorylates multiple sites at its N-terminus. Autophosphorylation might be involved in kinase activation, but it is unclear whether this happens also in intact cells. To study cGKI autophosphorylation in vitro and in vivo, we have generated phospho-specific antisera against major in vitro-autophosphorylation sites of the cGKI isoforms, cGKIα and cGKIß. These antisera detected specifically and with high sensitivity phospho-cGKIα (Thr58), phospho-cGKIα (Thr84), or phospho-cGKIß (Thr56/Ser63/Ser79). Using these antisera, we show that ATP-induced autophosphorylation of cGKI in purified preparations and cell extracts did neither require nor induce an enzyme conformation capable of substrate heterophosphorylation; it was even inhibited by pre-incubation with cGMP. Interestingly, phospho-cGKI species were not detectable in intact murine cells and tissues, both under basal conditions and after induction of cGKI catalytic activity. We conclude that N-terminal phosphorylation, although readily induced in vitro, is not required for the catalytic activity of cGKIα and cGKIß in vivo. These results will also inform screening strategies to identify novel cGKI modulators.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Domínios e Motivos de Interação entre Proteínas , Animais , Catálise , Bovinos , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Proteína Quinase Dependente de GMP Cíclico Tipo I/imunologia , Soros Imunes/imunologia , Camundongos , Fosforilação , Isoformas de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The nitration of tyrosine to 3-nitrotyrosine is an oxidative modification of tyrosine by nitric oxide and is associated with many diseases, and targeting of protein kinase G (PKG)-I represents a potential therapeutic strategy for pulmonary hypertension and chronic pain. The direct assignment of tyrosine residues of PKG-I has remained to be made due to the low sensitivity of the current proteomic approach. In order to assign modified tyrosine residues of PKG-I, we nitrated purified PKG-Iα expressed in insect Sf9 cells by use of peroxynitrite in vitro and analyzed the trypsin-digested fragments by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Among the 21 tyrosine residues of PKG-Iα, 16 tyrosine residues were assigned in 13 fragments; and six tyrosine residues were nitrated, those at Y71, Y141, Y212, Y336, Y345, and Y567, in the peroxynitrite-treated sample. Single mutation of tyrosine residues at Y71, Y212, and Y336 to phenylalanine significantly reduced the nitration of PKG-Iα; and four mutations at Y71, Y141, Y212, and Y336 (Y4F mutant) reduced it additively. PKG-Iα activity was inhibited by peroxynitrite in a concentration-dependent manner from 30 µM to 1 mM, and this inhibition was attenuated in the Y4F mutant. These results demonstrated that PKG-Iα was nitrated at multiple tyrosine residues and that its activity was reduced by nitration of these residues.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Fragmentos de Peptídeos/análise , Proteínas Recombinantes/química , Tirosina/análogos & derivados , Tirosina/química , Animais , Baculoviridae/genética , Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Expressão Gênica , Humanos , Cinética , Mutação , Nitratos/química , Óxido Nítrico/química , Ácido Peroxinitroso , Fenilalanina/química , Fenilalanina/genética , Proteínas Recombinantes/genética , Células Sf9 , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Spodoptera , Espectrometria de Massas em Tandem , Tripsina/química , Tirosina/genéticaRESUMO
The androgen receptor (AR) pathway plays critical roles in controlling differentiation and proliferation of prostate epithelial cells. We previously identified a novel AR cofactor, p44/WDR77, which specifically enhances AR transcriptional activity in the prostate gland and prostate cancer. To further elucidate p44/WDR77's role in the AR signaling pathway, we conducted a yeast two-hybrid screening and identified cGMP-dependent protein kinase (PKG) as a p44/WDR77-interacting protein. Further investigation by lusiferase assay and kinase assay demonstrated that PKG-Iß physically interacted with and phosphorylated both p44 and AR and enhanced AR transactivity in synergy with p44 in an androgen- and cGMP-dependent manner. Furthermore, PKG1ß expression promoted p44/WDR77 nuclear translocation and inhibited prostate cancer cell growth via G1 cell cycle arrest. Our findings characterize PKG as a novel regulator of AR-mediated transcription by enhancing AR cofactor p44/WDR77's function, which provide a novel mechanism for the growth regulation of prostate cancer cells by the androgen signaling.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Regulação Neoplásica da Expressão Gênica , Fragmentos de Peptídeos/metabolismo , Receptores Androgênicos/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Ciclo Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Humanos , Masculino , Camundongos Endogâmicos C57BL , Fosforilação , Regiões Promotoras Genéticas/genética , Próstata/crescimento & desenvolvimento , Próstata/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/metabolismo , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Cyclic guanosine monophosphate (cGMP) and cyclic AMP (cAMP)-dependent protein kinases (PKG and PKA) are closely related homologs, and the cyclic nucleotide specificity of each kinase is crucial for keeping the two signaling pathways segregated, but the molecular mechanism of cyclic nucleotide selectivity is unknown. Here, we report that the PKG Iß C-terminal cyclic nucleotide binding domain (CNB-B) is highly selective for cGMP binding, and we have solved crystal structures of CNB-B with and without bound cGMP. These structures, combined with a comprehensive mutagenic analysis, allowed us to identify Leu296 and Arg297 as key residues that mediate cGMP selectivity. In addition, by comparing the cGMP bound and unbound structures, we observed large conformational changes in the C-terminal helices in response to cGMP binding, which were stabilized by recruitment of Tyr351 as a "capping residue" for cGMP. The observed rearrangements of the C-terminal helices provide a mechanical insight into release of the catalytic domain and kinase activation.