RESUMO
Human 12-lipoxygenase (12-LOX) is a key enzyme involved in platelet activation, and the regulation of its activity has been targeted for the treatment of heparin-induced thrombocytopenia. Despite the clinical importance of 12-LOX, the exact mechanisms by which it affects platelet activation are not fully understood, and the lack of structural information has limited drug discovery efforts. In this study, we used single-particle cryo-electron microscopy to determine high-resolution structures (1.7-2.8 Å) of human 12-LOX. Our results showed that 12-LOX can exist in multiple oligomeric states, from monomer to hexamer, which may affect its catalytic activity and membrane association. We also identified different conformations within the 12-LOX dimer, which likely represent different time points in its catalytic cycle. Furthermore, we identified small molecules bound to 12-LOX. The active site of the 12-LOX tetramer was occupied by an endogenous 12-LOX inhibitor, a long-chain acyl coenzyme A. In addition, we found that the 12-LOX hexamer can simultaneously bind to arachidonic acid and ML355, a selective 12-LOX inhibitor that has passed a phase 1 clinical trial for the treatment of heparin-induced thrombocytopenia and received a fast-track designation by the Food and Drug Administration. Overall, our findings provide novel insights into the assembly of 12-LOX oligomers, their catalytic mechanism, and small molecule binding, paving the way for further drug development targeting the 12-LOX enzyme.
Assuntos
Ativação Plaquetária , Trombocitopenia , Estados Unidos , Humanos , Microscopia Crioeletrônica , Ácido Araquidônico/metabolismo , Araquidonato 12-Lipoxigenase/metabolismoRESUMO
The primary means by which ion permeation through potassium channels is controlled, and the key to selective intervention in a range of pathophysiological conditions, is the process by which channels switch between non-conducting and conducting states. Conventionally, this has been explained by a steric mechanism in which the pore alternates between two conformations: a 'closed' state in which the conduction pathway is occluded and an 'open' state in which the pathway is sufficiently wide to accommodate fully hydrated ions. Recently, however, 'non-canonical' mechanisms have been proposed for some classes of K+ channels. The purpose of this review is to illuminate structural and dynamic relationships underpinning permeation control in K+ channels, indicating where additional data might resolve some of the remaining issues.
Assuntos
Canais de Potássio , Potássio , Potássio/metabolismo , Canais de Potássio/metabolismo , Conformação ProteicaRESUMO
The necroptosis pathway is a lytic, pro-inflammatory mode of cell death that is widely implicated in human disease, including renal, pulmonary, gut and skin inflammatory pathologies. The precise mechanism of the terminal steps in the pathway, where the RIPK3 kinase phosphorylates and triggers a conformation change and oligomerization of the terminal pathway effector, MLKL, are only emerging. Here, we structurally identify RIPK3-mediated phosphorylation of the human MLKL activation loop as a cue for MLKL pseudokinase domain dimerization. MLKL pseudokinase domain dimerization subsequently drives formation of elongated homotetramers. Negative stain electron microscopy and modelling support nucleation of the MLKL tetramer assembly by a central coiled coil formed by the extended, ~80 Å brace helix that connects the pseudokinase and executioner four-helix bundle domains. Mutational data assert MLKL tetramerization as an essential prerequisite step to enable the release and reorganization of four-helix bundle domains for membrane permeabilization and cell death.
Assuntos
Proteínas Quinases , Proteína Serina-Treonina Quinases de Interação com Receptores , Humanos , Fosforilação , Necrose , Proteínas Quinases/metabolismo , Dimerização , Morte Celular , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , ApoptoseRESUMO
Almost all interactions and reactions that occur in living organisms involve proteins. The various biological roles of proteins include, but are not limited to, signal transduction, gene transcription, cell death, immune function, structural support, and catalysis of all the chemical reactions that enable organisms to survive. The varied roles of proteins have led to them being dubbed 'the workhorses of all living organisms'. This article discusses the functions of proteins and how protein function is studied in a laboratory setting. In this article, we begin by examining the functions of protein domains, followed by a discussion of some of the major classes of proteins based on their function. We consider protein binding in detail, which is central to protein function. We then examine how protein function can be altered through various mechanisms including post-translational modification, and changes to environment, oligomerisation and mutations. Finally, we consider a handful of the techniques employed in the laboratory to understand and measure the function of proteins.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas , Ligação Proteica , Proteínas/metabolismo , Transdução de SinaisRESUMO
Ion currents through potassium channels are gated. Constriction of the ion conduction pathway at the inner helix bundle, the textbook gate of Kir potassium channels, has been shown to be an ineffective permeation control, creating a rift in our understanding of how these channels are gated. Here we present evidence that anionic lipids act as interactive response elements sufficient to gate potassium conduction. We demonstrate the limiting barrier to K+ permeation lies within the ion conduction pathway and show that this gate is operated by the fatty acyl tails of lipids that infiltrate the conduction pathway via fenestrations in the walls of the pore. Acyl tails occupying a surface groove extending from the cytosolic interface to the conduction pathway provide a potential means of relaying cellular signals, mediated by anionic lipid head groups bound at the canonical lipid binding site, to the internal gate.
Assuntos
Ativação do Canal Iônico , Lipídeos de Membrana/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Potássio/metabolismo , Ânions/química , Ânions/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Transporte de Íons , Lipossomos/química , Lipossomos/metabolismo , Lipídeos de Membrana/química , Simulação de Dinâmica Molecular , Mutação , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/genéticaRESUMO
Potent neutralizing monoclonal antibodies are one of the few agents currently available to treat COVID-19. SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, the generation of a diverse panel of 91 human, neutralizing monoclonal antibodies provides an in-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1. These neutralizing monoclonal antibodies and others that bind analogous epitopes represent potentially useful future anti-SARS-CoV-2 therapeutics.
Assuntos
Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Neutralizantes/imunologia , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/ultraestrutura , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Cricetinae , Microscopia Crioeletrônica/métodos , Epitopos/imunologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Testes de Neutralização , Ligação Proteica/fisiologia , Receptores Virais/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
The canonical mechanistic model explaining potassium channel gating is of a conformational change that alternately dilates and constricts a collar-like intracellular entrance to the pore. It is based on the premise that K+ ions maintain a complete hydration shell while passing between the transmembrane cavity and cytosol, which must be accommodated. To put the canonical model to the test, we locked the conformation of a Kir K+ channel to prevent widening of the narrow collar. Unexpectedly, conduction was unimpaired in the locked channels. In parallel, we employed all-atom molecular dynamics to simulate K+ ions moving along the conduction pathway between the lower cavity and cytosol. During simulations, the constriction did not significantly widen. Instead, transient loss of some water molecules facilitated K+ permeation through the collar. The low free energy barrier to partial dehydration in the absence of conformational change indicates Kir channels are not gated by the canonical mechanism.