RESUMO
Since their discovery, nicotinic acetylcholine receptors (nAChRs) have been extensively studied to understand their function, as well as the consequence of alterations leading to disease states. Importantly, these receptors represent pharmacological targets to treat a number of neurological and neurodegenerative disorders. Nevertheless, their therapeutic value has been limited by the absence of high-resolution structures that allow for the design of more specific and effective drugs. This article offers a comprehensive review of five decades of research pursuing high-resolution structures of nAChRs. We provide a historical perspective, from initial structural studies to the most recent X-ray and cryogenic electron microscopy (Cryo-EM) nAChR structures. We also discuss the most relevant structural features that emerged from these studies, as well as perspectives in the field.
Assuntos
Doenças do Sistema Nervoso/metabolismo , Receptores Nicotínicos/química , Animais , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Modelos Moleculares , Terapia de Alvo Molecular , Doenças do Sistema Nervoso/tratamento farmacológico , Conformação Proteica , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismoRESUMO
For a long time, traditional purification and extraction methods for the native Torpedo californica nicotinic acetylcholine receptor in lipid-like detergent complex (nAChR-DC) have compromised its purity, functionality and X-ray structural studies possibility. The dataset presented in this article provide a characterization of the Torpedo californica nAChR-DC purified using a sequential purification processes developed in our laboratory [1]. This purification takes in consideration all of the physicochemical and functional requirements stablished by several researchers for the past three decades for the nAChR. These requirements were addressed in order to preserve the stability and functionality of nAChR-DC while ensuring the highest degree of protein purity. We focused on the effect of cholesteryl hemisuccinate (CHS) supplementation on nAChR conformational changes during the purification process. Data from the size exclusion chromatography of the nAChR-DC supplemented with CHS in concentrations ranging from 0.01 mM, 0.1 mM, 0.2 mM and 0.5 mM consistently demonstrated that 0.5 mM CHS affects receptor stability via disassemble of the pentameric oligomer. However, 0.2 mM CHS produced negligible nAChR-DC subunit disruption. The purified nAChR-DC has been characterized by circular dichroism (CD) and fluorescence recovery after photobleaching (FRAP), in order to assess its stability. The CD data was recorded in the wavelength range of 190-250 nm, showed that CHS induce a âº-helix to ß-sheet transition of the nAChR-DC. The nAChR-LFC-16 delipidation with Methyl-ß-Cyclodextrin decreased the percentage of α-helix and increased the ß-sheet antiparallel secondary structure and levels the percentage of turns to that of the nAChR-DC without CHS treatment. Additionally, the stability of the nAChR-DC supplemented with CHS and incorporated into lipid cubic phase (LCP) was monitored for a period of 30 days by means of FRAP. The LCP-FRAP data allowed to establish possible optimal crystallization conditions for the development of crystals from purified nAChR-conjugated to α-Bungarotoxin, Alexa Fluor ™ 488 (α-BTX) in order to obtain a high-resolution atomic structure by X-ray diffraction.
RESUMO
Over the past 10 years we have been developing a multi-attribute analytical platform that allows for the preparation of milligram amounts of functional, high-pure, and stable Torpedo (muscle-type) nAChR detergent complexes for crystallization purpose. In the present work, we have been able to significantly improve and optimize the purity and yield of nicotinic acetylcholine receptors in detergent complexes (nAChR-DC) without compromising stability and functionality. We implemented new methods in the process, such as analysis and rapid production of samples for future crystallization preparations. Native nAChR was extracted from the electric organ of Torpedo californica using the lipid-like detergent LysoFos Choline 16 (LFC-16), followed by three consecutive steps of chromatography purification. We evaluated the effect of cholesteryl hemisuccinate (CHS) supplementation during the affinity purification steps of nAChR-LFC-16 in terms of receptor secondary structure, stability and functionality. CHS produced significant changes in the degree of ß-secondary structure, these changes compromise the diffusion of the nAChR-LFC-16 in lipid cubic phase. The behavior was reversed by Methyl-ß-Cyclodextrin treatment. Also, CHS decreased acetylcholine evoked currents of Xenopus leavis oocyte injected with nAChR-LFC-16 in a concentration-dependent manner. Methyl-ß-Cyclodextrin treatment do not reverse functionality, however column delipidation produced a functional protein similar to nAChR-LFC-16 without CHS treatment.
Assuntos
Ésteres do Colesterol/química , Proteínas de Peixes/química , Receptores Nicotínicos/química , Acetilcolina/farmacologia , Animais , Detergentes/química , Potenciais Evocados/efeitos dos fármacos , Proteínas de Peixes/isolamento & purificação , Proteínas de Peixes/metabolismo , Oócitos/fisiologia , Conformação Proteica em Folha beta , Receptores Nicotínicos/isolamento & purificação , Receptores Nicotínicos/metabolismo , Torpedo/metabolismo , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo , beta-Ciclodextrinas/químicaRESUMO
Currently, there are no specific therapies to treat HIV-1 associated neurocognitive disorders (HAND). The HIV-1 envelope, gp120, induces neuropathological changes similar to those in HAND patients; furthermore, it triggers an upregulation of the α7-nicotinic acetylcholine receptor (α7-nAChR), facilitating intracellular calcium overload and neuronal cell death. Using a gp120IIIB-transgenic mouse (gp120-tgm) model, we demonstrate that α7-nAChRs are upregulated on striatal neurons. Activation of α7-nAChRs leads to an increase in both intracellular calcium and percentage of apoptotic cells, which can be abrogated by antagonizing the receptor, suggesting a role for α7-nAChRs in gp120-induced neurotoxicity. Moreover, we demonstrate for the first time that gp120-tgm have learning deficiencies on a striatum-dependent behavioral task. They also show locomotor deficiencies, which improved with α7-nAChR antagonists, further supporting a role for this receptor in gp120-induced neurotoxicity. Together, these results uncover a new mechanism through which gp120-induced modulation of α7-nAChRs in the striatum can contribute to HAND development.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Transtornos Neurocognitivos/metabolismo , Neurônios/metabolismo , Síndromes Neurotóxicas/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Morte Celular/fisiologia , Corpo Estriado/metabolismo , Feminino , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Nicotínicos/metabolismoRESUMO
This study compares the lipid composition, including individual phospholipid molecular species of solubilized nAChR detergent complexes (nAChR-DCs) with those of the bulk lipids from their source, Torpedo californica (Tc) electric tissue. This lipidomic analysis revealed seventy-seven (77) phospholipid species in the Tc tissue. Analysis of affinity-purified nAChR-DCs prepared with C-12 to C-16 phospholipid analog detergents alkylphosphocholine (FC) and lysofoscholine (LFC) demonstrated that nAChR-DCs prepared with FC12, LFC14, and LFC16 contained >60 phospholipids/nAChR, which was more than twice of those prepared with FC14, FC16, and LFC12. Significantly, all the nAChR-DCs lacked ethanolamine and anionic phospholipids, contained only four cholesterol molecules, and a limited number of phospholipid molecular species per nAChR. Upon incorporation into oocytes, FC12 produce significant functionality, whereas LFC14 and LFC16 nAChR-DCs displayed an increased functionality as compared to the crude Tc membrane. All three nAChR-DCs displayed different degrees of alterations in macroscopic activation and desensitization kinetics.
Assuntos
Detergentes/química , Lipídeos/química , Receptores Nicotínicos/química , Acetilcolina/farmacologia , Animais , Catálise , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Órgão Elétrico/metabolismo , Eletrodos , Hidrólise , Lipídeos/isolamento & purificação , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Torpedo , XenopusRESUMO
Despite the recent advances in antiretroviral therapy, human immunodeficiency virus type 1 (HIV-1) remains a global health threat. HIV-1 affects the central nervous system by releasing viral proteins that trigger neuronal death and neuroinflammation, and promotes alterations known as HIV-associated neurocognitive disorders (HAND). This disorder is not fully understood, and no specific treatments are available. Recently, we demonstrated that the HIV-1 envelope protein gp120IIIB induces a functional upregulation of the α7-nicotinic acetylcholine receptor (α7) in neuronal cells. Furthermore, this upregulation promotes cell death that can be abrogated with receptor antagonists, suggesting that α7 may play an important role in the development of HAND. The partial duplication of the gene coding for the α7, known as CHRFAM7A, negatively regulates α7 expression but its role in HIV infection has not been studied. Hence, we studied both CHRNA7 and CHRFAM7A regulation patterns in various gp120IIIB in vitro conditions. In addition, we measured CHRNA7 and CHRFAM7A expression levels in postmortem brain samples from patients suffering from different stages of HAND. Our results demonstrate the induction of CHRNA7 expression accompanied by a significant downregulation of CHRFAM7A in neuronal cells when exposed to pathophysiological concentrations of gp120IIIB. Our results suggest a dysregulation of CHRFAM7A and CHRNA7 expressions in the basal ganglia from postmortem brain samples of HIV+ subjects and expand the current knowledge about the consequences of HIV infection in the brain.
Assuntos
Complexo AIDS Demência/genética , Encéfalo/virologia , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Interações Hospedeiro-Patógeno , Receptor Nicotínico de Acetilcolina alfa7/genética , Complexo AIDS Demência/metabolismo , Complexo AIDS Demência/patologia , Complexo AIDS Demência/virologia , Adulto , Autopsia , Gânglios da Base/metabolismo , Gânglios da Base/patologia , Gânglios da Base/virologia , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp120 do Envelope de HIV/farmacologia , HIV-1/metabolismo , HIV-1/patogenicidade , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Antiretroviral therapy partially restores the immune system and markedly increases life expectancy of HIV-infected patients. However, antiretroviral therapy does not restore full health. These patients suffer from poorly understood chronic inflammation that causes a number of AIDS and non-AIDS complications. Here we show that chronic inflammation in HIV+ patients may be due to the disruption of the cholinergic anti-inflammatory pathway by HIV envelope protein gp120IIIB. Our results demonstrate that HIV gp120IIIB induces α7 nicotinic acetylcholine receptor (α7) upregulation and a paradoxical proinflammatory phenotype in macrophages, as activation of the upregulated α7 is no longer capable of inhibiting the release of proinflammatory cytokines. Our results demonstrate that disruption of the cholinergic-mediated anti-inflammatory response can result from an HIV protein. Collectively, these findings suggest that HIV tampering with a natural strategy to control inflammation could contribute to a crucial, unresolved problem of HIV infection: chronic inflammation.
RESUMO
Lys49 phospholipase A2 (PLA2) homologues present in crotalid snake venoms lack enzymatic activity, yet they induce skeletal muscle necrosis by a membrane permeabilizing mechanism whose details are only partially understood. The present study evaluated the effect of altering the membrane cholesterol content on the cytolytic activity of myotoxin II, a Lys49 PLA2 isolated from the venom of Bothrops asper, using the myogenic cell line C2C12 as a model target. Cell membrane cholesterol depletion by methyl-ß-cyclodextrin (MßCD) treatment enhanced the cytolytic action of myotoxin II, as well as of its bioactive C-terminal synthetic peptide p(115-129) . Conversely, cell membrane cholesterol enrichment by preformed cholesterol-MßCD complexes reduced the cytolytic effect of myotoxin II. The toxic actions of myotoxin I, a catalytically active PLA2 from the same venom, as well as of the cytolytic peptide melittin from bee venom, also increased in cholesterol-depleted cells. Although physical and functional changes resulting from variations in membrane cholesterol are complex, these findings suggest that membrane fluidity could be a relevant parameter to explain the observed modulation of the cytolytic mechanism of myotoxin II, possibly influencing bilayer penetration. In concordance, the cytolytic effect of myotoxin II decreased in direct proportion to lower temperature, a physical factor that affects membrane fluidity. In conclusion, physicochemical properties that depend on membrane cholesterol content significantly influence the cytolytic mechanism of myotoxin II, reinforcing the concept that the primary site of action of Lys49 PLA2 myotoxins is the plasma membrane.
Assuntos
Membrana Celular/efeitos dos fármacos , Colesterol/metabolismo , Venenos de Crotalídeos/química , Fosfolipases A2 do Grupo II/farmacologia , Proteínas de Répteis/farmacologia , Animais , Bothrops , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Fosfolipases A2 do Grupo II/toxicidade , Lactato Desidrogenases/efeitos dos fármacos , Lactato Desidrogenases/metabolismo , Lisina , Meliteno/farmacologia , Camundongos , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas de Répteis/toxicidade , Temperatura , beta-Ciclodextrinas/farmacologiaRESUMO
Cholesterol modulates the plasmalemma's biophysical properties and influences the function and trafficking of membrane proteins. A fundamental phenomenon that remains obscure is how the plasmalemma's lipid composition regulates the activatable pool of membrane receptors. An outstanding model to study this phenomenon is the nicotinic acetylcholine receptor (nAChR), since the nAChR activatable pool has been estimated to be but a small fraction of the receptors present in the plasmalemma. Studies on the effect of cholesterol depletion in the function of the Torpedo californica nAChR, using the lipid-exposed nAChR mutation (alpha C418W) that produces a congenital myasthenic syndrome (CMS), demonstrated that cholesterol depletion causes a remarkable increase in the alpha C418W nAChR's macroscopic current whereas not in the wild-type (WT). A variety of approaches were used to define the mechanism responsible for the cholesterol depletion mediated-increase in the alpha C418W nAChR's macroscopic current. The present study suggests that a substantial fraction of the alpha C418W nAChRs is located in caveolin-1-positive domains, "trapped" in a non-activatable state, and that membrane cholesterol depletion results in the relocation of these receptors to the activatable pool. Co-fractionation and co-immunoprecipitation of the alpha C418W nAChR and the membrane raft protein caveolin-1 (cav1) support the notion that interactions at lipid-exposed domains regulate the partition of the receptor into membrane raft microdomains. These results have potential implications as a novel mechanism to fine-tune cholinergic transmission in the nervous system and in the pathogenesis associated to the alpha C418W nAChR.