RESUMO
ß2-glycoprotein I (ß2GPI) is an abundant multidomain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as antiphospholipid syndrome (APS). Previous studies have shown that ß2GPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to ß2GPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate ß2GPI's structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of ß2GPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating ß2GPI's physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.
Assuntos
Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/imunologia , Autoanticorpos/imunologia , Proteínas Recombinantes/metabolismo , beta 2-Glicoproteína I/metabolismo , Regulação Alostérica , Anticorpos Antifosfolipídeos/sangue , Síndrome Antifosfolipídica/patologia , Autoanticorpos/sangue , Linhagem Celular , Cristalografia por Raios X/métodos , Humanos , Oxirredução , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , beta 2-Glicoproteína I/química , beta 2-Glicoproteína I/imunologia , beta 2-Glicoproteína I/isolamento & purificaçãoRESUMO
ß2-Glycoprotein I (ß2-GPI) is an abundant plasma glycoprotein with unknown physiological function and is currently recognized as the main target of antiphospholipid Abs responsible for complement activation and vascular thrombosis in patients with antiphospholipid syndrome (APS). In this study, we provide evidence that mannose-binding lectin (MBL) binds to ß2-GPI in Ca++ and a dose-dependent manner and that this interaction activates complement and promotes complement-dependent thrombin generation. Surprisingly, a significant binding was observed between MBL and isolated domains II and IV of ß2-GPI, whereas the carbohydrate chains, domain I and domain V, were not involved in the interaction, documenting a noncanonical binding mode between MBL and ß2-GPI. Importantly, this interaction may occur on endothelial cells because binding of MBL to ß2-GPI was detected on the surface of HUVECs, and colocalization of MBL with ß2-GPI was observed on the endothelium of a biopsy specimen of a femoral artery from an APS patient. Because ß2-GPI-mediated MBL-dependent thrombin generation was increased after priming the endothelium with TNF-α, our data suggests that this mechanism could play an important yet unrecognized role under physiological conditions and may be upregulated in pathological situations. Moreover, the complement activation and the procoagulant effects of the ß2-GPI/MBL complex may contribute to amplify similar activities of anti-ß2-GPI Abs in APS and possibly act independently of Abs, raising the issue of developing appropriate therapies to avoid recurrences and disability in patients at risk for these clinical conditions.
Assuntos
Ativação do Complemento/imunologia , Lectina de Ligação a Manose/metabolismo , Trombina/metabolismo , beta 2-Glicoproteína I/metabolismo , Síndrome Antifosfolipídica/imunologia , Síndrome Antifosfolipídica/metabolismo , Cálcio/metabolismo , Linhagem Celular , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Endotélio/imunologia , Endotélio/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Lectina de Ligação a Manose/imunologia , Ligação Proteica/imunologia , Trombina/imunologia , Trombose/imunologia , Trombose/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , beta 2-Glicoproteína I/imunologiaRESUMO
ß2-Glycoprotein I (ß2GPI) is an abundant plasma protein displaying phospholipid-binding properties. Because it binds phospholipids, it is a target of antiphospholipid antibodies (aPLs) in antiphospholipid syndrome (APS), a life-threatening autoimmune thrombotic disease. Indeed, aPLs prefer membrane-bound ß2GPI to that in solution. ß2GPI exists in two almost equally populated redox states: oxidized, in which all the disulfide bonds are formed, and reduced, in which one or more disulfide bonds are broken. Furthermore, ß2GPI can adopt multiple conformations (i.e. J-elongated, S-twisted, and O-circular). While strong evidence indicates that the J-form is the structure bound to aPLs, which conformation exists and predominates in solution remains controversial, and so is the conformational pathway leading to the bound state. Here, we report that human recombinant ß2GPI purified under native conditions is oxidized. Moreover, under physiological pH and salt concentrations, this oxidized form adopts a J-elongated, flexible conformation, not circular or twisted, in which the N-terminal domain I (DI) and the C-terminal domain V (DV) are exposed to the solvent. Consistent with this model, binding kinetics and mutagenesis experiments revealed that in solution the J-form interacts with negatively charged liposomes and with MBB2, a monoclonal anti-DI antibody that recapitulates most of the features of pathogenic aPLs. We conclude that the preferential binding of aPLs to phospholipid-bound ß2GPI arises from the ability of its preexisting J-form to accumulate on the membranes, thereby offering an ideal environment for aPL binding. We propose that targeting the J-form of ß2GPI provides a strategy to block pathogenic aPLs in APS.
Assuntos
Anticorpos Antifosfolipídeos/química , Síndrome Antifosfolipídica , beta 2-Glicoproteína I/química , Animais , Anticorpos Antifosfolipídeos/metabolismo , Cricetinae , Células HEK293 , Humanos , Cinética , Mutagênese , Domínios Proteicos , beta 2-Glicoproteína I/metabolismoRESUMO
We determined the role of carboxyl-terminal regulation of NOPR (nociceptin, orphanin FQ receptor) signaling and function. We mutated C-terminal serine and threonine residues and examined their role in NOPR trafficking, homologous desensitization, and arrestin-dependent MAPK signaling. The NOPR agonist, nociceptin, caused robust NOPR-YFP receptor internalization, peaking at 30 min. Mutation of serine 337, 346, and 351, had no effect on NOPR internalization. However, mutation of C-terminal threonine 362, serine 363, and threonine 365 blocked nociceptin-induced internalization of NOPR. Furthermore, point mutation of only Ser-363 was sufficient to block NOPR internalization. Homologous desensitization of NOPR-mediated calcium channel blockade and inhibition of cAMP were also shown to require Ser-363. Additionally, NOPR internalization was absent when GRK3, and Arrestin3 were knocked down using siRNA, but not when GRK2 and Arrestin2 were knocked down. We also found that nociceptin-induced NOPR-mediated JNK but not ERK signaling requires Ser-363, GRK3, and Arrestin3. Dominant-positive Arrestin3 but not Arrestin2 was sufficient to rescue NOPR-S363A internalization and JNK signaling. These findings suggest that NOPR function may be regulated by GRK3 phosphorylation of Ser-363 and Arrestin3 and further demonstrates the complex nature of G-protein-dependent and -independent signaling in opioid receptors.
Assuntos
Arrestinas/metabolismo , MAP Quinase Quinase 4/metabolismo , Peptídeos Opioides/metabolismo , Receptores Opioides/metabolismo , Transdução de Sinais/fisiologia , Arrestinas/genética , Quinase 3 de Receptor Acoplado a Proteína G/genética , Quinase 3 de Receptor Acoplado a Proteína G/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , MAP Quinase Quinase 4/genética , Peptídeos Opioides/genética , Fosforilação/fisiologia , Transporte Proteico/fisiologia , Receptores Opioides/genética , Receptor de Nociceptina , NociceptinaRESUMO
Ret signaling is critical for formation of the enteric nervous system (ENS) because Ret activation promotes ENS precursor survival, proliferation, and migration and provides trophic support for mature enteric neurons. Although these roles are well established, we now provide evidence that increasing levels of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) in mice causes alterations in ENS structure and function that are critically dependent on the time and location of increased GDNF availability. This is demonstrated using two different strains of transgenic mice and by injecting newborn mice with GDNF. Furthermore, because different subclasses of ENS precursors withdraw from the cell cycle at different times during development, increases in GDNF at specific times alter the ratio of neuronal subclasses in the mature ENS. In addition, we confirm that esophageal neurons are GDNF responsive and demonstrate that the location of GDNF production influences neuronal process projection for NADPH diaphorase-expressing, but not acetylcholinesterase-, choline acetyltransferase-, or tryptophan hydroxylase-expressing, small bowel myenteric neurons. We further demonstrate that changes in GDNF availability influence intestinal function in vitro and in vivo. Thus, changes in GDNF expression can create a wide variety of alterations in ENS structure and function and may in part contribute to human motility disorders.
Assuntos
Sistema Nervoso Entérico/embriologia , Sistema Nervoso Entérico/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Neurônios/metabolismo , Células-Tronco/metabolismo , Animais , Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células , Sistema Nervoso Entérico/citologia , Esôfago/embriologia , Esôfago/inervação , Motilidade Gastrointestinal/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Plexo Mientérico/citologia , Plexo Mientérico/embriologia , Plexo Mientérico/metabolismo , NADPH Desidrogenase/metabolismo , Neuritos/metabolismo , Neuritos/ultraestrutura , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Neurônios/citologia , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacosRESUMO
Anti-phosphatidylserine/prothrombin (aPS/PT) antibodies are often detected in patients with antiphospholipid syndrome (APS), but how aPS/PT engage prothrombin at the molecular level remains unknown. Here, the antigenic determinants of immunoglobulin G aPS/PT were investigated in 24 triple-positive APS patients at high risk of thrombosis by using prothrombin mutants biochemically trapped in closed and open conformations, and relevant fragments spanning the entire length of prothrombin. Two novel unexpected findings emerged from these studies. First, we discovered that some aPS/PT are unique among other anti-prothrombin antibodies insofar as they efficiently recognize prothrombin in solution after a conformational change requiring exposure of fragment-1 to the solvent. Second, we identified and characterized 2 previously unknown subpopulations of aPS/PT, namely type I and type II, which engage fragment-1 of prothrombin at different epitopes and with different mechanisms. Type I target a discontinuous density-dependent epitope, whereas type II engage the C-terminal portion of the Gla-domain, which remains available for binding even when prothrombin is bound to the phospholipids. Based on these findings, APS patients positive for aPS/PT were classified into 2 groups, group A and group B, according to their autoantibody profile. Group A contains mostly type I antibodies whereas group B contains both type I and type II antibodies. In conclusion, this study offers a first encouraging step toward unveiling the heterogeneity of anti-prothrombin antibodies in correlation with thrombosis, shedding new light on the mechanisms of antigen-autoantibody recognition in APS.
Assuntos
Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/imunologia , Epitopos/imunologia , Protrombina/imunologia , Trombose/imunologia , Feminino , Humanos , Masculino , Domínios ProteicosRESUMO
Coagulation factor II, or prothrombin, is a multi-domain glycoprotein that is essential for life and a key target of anticoagulant therapy. In plasma, prothrombin circulates in two forms at equilibrium, "closed" (~80%) and "open" (~20%), brokered by the flexibility of the linker regions. Its structure remained elusive until recently when our laboratory solved the first X-ray crystal structure of the zymogen locked in the predominant closed form. Because of this technical breakthrough, fascinating aspects of the biology of prothrombin have started to become apparent, and with this, novel and important questions arise. Here, we examine the significance of the "closed"/"open" equilibrium in the context of the mechanism of thrombin generation. Further, we discuss the potential translational opportunities for the development of next-generation anticoagulants that arise from this discovery. By providing a structural overview of each alternative conformation, this minireview also offers a relevant example of modern structural biology and establishes a practical workflow to elucidate the structural features of analogous clotting and complement factors.
RESUMO
The endogenous neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is secreted by both neuronal and non-neuronal cells in the brain and spinal cord, in response to pathological conditions such as stroke, seizures, chronic inflammatory and neuropathic pain. PACAP has been shown to exert various neuromodulatory and neuroprotective effects. However, direct influence of PACAP on the function of intrinsically excitable ion channels that are critical to both hyperexcitation as well as cell death, remain largely unexplored. The major dendritic K(+) channel Kv4.2 is a critical regulator of neuronal excitability, back-propagating action potentials in the dendrites, and modulation of synaptic inputs. We identified, cloned and characterized the downstream signaling originating from the activation of three PACAP receptor (PAC1) isoforms that are expressed in rodent hippocampal neurons that also exhibit abundant expression of Kv4.2 protein. Activation of PAC1 by PACAP leads to phosphorylation of Kv4.2 and downregulation of channel currents, which can be attenuated by inhibition of either PKA or ERK1/2 activity. Mechanistically, this dynamic downregulation of Kv4.2 function is a consequence of reduction in the density of surface channels, without any influence on the voltage-dependence of channel activation. Interestingly, PKA-induced effects on Kv4.2 were mediated by ERK1/2 phosphorylation of the channel at two critical residues, but not by direct channel phosphorylation by PKA, suggesting a convergent phosphomodulatory signaling cascade. Altogether, our findings suggest a novel GPCR-channel signaling crosstalk between PACAP/PAC1 and Kv4.2 channel in a manner that could lead to neuronal hyperexcitability.
Assuntos
Dendritos/efeitos dos fármacos , Neurônios/citologia , Neurotransmissores/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Canais de Potássio Shal/metabolismo , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/citologia , Humanos , Masculino , Camundongos , Mutagênese/genética , Mutação/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Ratos , Ratos Sprague-Dawley , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Optogenetics has provided a revolutionary approach to dissecting biological phenomena. However, the generation and use of optically active GPCRs in these contexts is limited and it is unclear how well an opsin-chimera GPCR might mimic endogenous receptor activity. Here we show that a chimeric rhodopsin/ß2 adrenergic receptor (opto-ß2AR) is similar in dynamics to endogenous ß2AR in terms of: cAMP generation, MAP kinase activation and receptor internalization. In addition, we develop and characterize a novel toolset of optically active, functionally selective GPCRs that can bias intracellular signalling cascades towards either G-protein or arrestin-mediated cAMP and MAP kinase pathways. Finally, we show how photoactivation of opto-ß2AR in vivo modulates neuronal activity and induces anxiety-like behavioural states in both fiber-tethered and wireless, freely moving animals when expressed in brain regions known to contain ß2ARs. These new GPCR approaches enhance the utility of optogenetics and allow for discrete spatiotemporal control of GPCR signalling in vitro and in vivo.
Assuntos
Optogenética , Quinases de Receptores Adrenérgicos beta/metabolismo , Animais , Ansiedade/enzimologia , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismoRESUMO
Optogenetics is now a widely accepted tool for spatiotemporal manipulation of neuronal activity. However, a majority of optogenetic approaches use binary on/off control schemes. Here, we extend the optogenetic toolset by developing a neuromodulatory approach using a rationale-based design to generate a Gi-coupled, optically sensitive, mu-opioid-like receptor, which we term opto-MOR. We demonstrate that opto-MOR engages canonical mu-opioid signaling through inhibition of adenylyl cyclase, activation of MAPK and G protein-gated inward rectifying potassium (GIRK) channels and internalizes with kinetics similar to that of the mu-opioid receptor. To assess in vivo utility, we expressed a Cre-dependent viral opto-MOR in RMTg/VTA GABAergic neurons, which led to a real-time place preference. In contrast, expression of opto-MOR in GABAergic neurons of the ventral pallidum hedonic cold spot led to real-time place aversion. This tool has generalizable application for spatiotemporal control of opioid signaling and, furthermore, can be used broadly for mimicking endogenous neuronal inhibition pathways.
Assuntos
Analgésicos Opioides/farmacologia , Comportamento Animal/efeitos dos fármacos , Neurônios GABAérgicos/efeitos dos fármacos , Optogenética , Receptores Opioides mu/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Células Cultivadas , Neurônios GABAérgicos/metabolismo , Ratos , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/fisiologiaRESUMO
Hirschsprung disease (distal intestinal aganglionosis, HSCR) is a multigenic disorder with incomplete penetrance, variable expressivity, and a strong male gender bias. Recent studies demonstrated that these genetic patterns arise because gene interactions determine whether enteric nervous system (ENS) precursors successfully proliferate and migrate into the distal bowel. We now demonstrate that male gender bias in the extent of distal intestinal aganglionosis occurs in mice with Ret dominant-negative mutations (RetDN) that mimic human HSCR. We hypothesized that male gender bias could result from reduced expression of a gene already known to be essential for ENS development. Using quantitative real-time polymerase chain reaction (PCR) we demonstrated reduced levels of endothelin converting enzyme-1 and endothelin-3 mRNA in the male mouse bowel at the time that ENS precursors migrate into the colon. Other HSCR-associated genes are expressed at comparable levels in male and female mice. Testosterone and Mullerian inhibiting substance had no deleterious effect on ENS precursor development, but adding EDN3 peptide to E11.5 male RetDN heterozygous mouse gut explants in organ culture significantly increased the rate of ENS precursor migration through the bowel.