RESUMEN
Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many neuropsychiatric disorders including depression, anxiety, and substance abuse. How psychedelics mediate their actions-both therapeutic and hallucinogenic-are not understood, although activation of the 5-HT2A serotonin receptor (HTR2A) is key. To gain molecular insights into psychedelic actions, we determined the active-state structure of HTR2A bound to 25-CN-NBOH-a prototypical hallucinogen-in complex with an engineered Gαq heterotrimer by cryoelectron microscopy (cryo-EM). We also obtained the X-ray crystal structures of HTR2A complexed with the arrestin-biased ligand LSD or the inverse agonist methiothepin. Comparisons of these structures reveal determinants responsible for HTR2A-Gαq protein interactions as well as the conformational rearrangements involved in active-state transitions. Given the potential therapeutic actions of hallucinogens, these findings could accelerate the discovery of more selective drugs for the treatment of a variety of neuropsychiatric disorders.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Alucinógenos/química , Receptor de Serotonina 5-HT2A/química , Receptor de Serotonina 5-HT2A/metabolismo , Animales , Microscopía por Crioelectrón , Cristalografía por Rayos X , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Expresión Génica , Células HEK293 , Alucinógenos/farmacología , Alucinógenos/uso terapéutico , Humanos , Ligandos , Dietilamida del Ácido Lisérgico/química , Dietilamida del Ácido Lisérgico/farmacología , Metiotepina/química , Metiotepina/metabolismo , Modelos Químicos , Mutación , Conformación Proteica en Hélice alfa , Receptor de Serotonina 5-HT2A/genética , Proteínas Recombinantes , Serotonina/metabolismo , SpodopteraRESUMEN
Drugs selectively targeting CB2 hold promise for treating neurodegenerative disorders, inflammation, and pain while avoiding psychotropic side effects mediated by CB1. The mechanisms underlying CB2 activation and signaling are poorly understood but critical for drug design. Here we report the cryo-EM structure of the human CB2-Gi signaling complex bound to the agonist WIN 55,212-2. The 3D structure reveals the binding mode of WIN 55,212-2 and structural determinants for distinguishing CB2 agonists from antagonists, which are supported by a pair of rationally designed agonist and antagonist. Further structural analyses with computational docking results uncover the differences between CB2 and CB1 in receptor activation, ligand recognition, and Gi coupling. These findings are expected to facilitate rational structure-based discovery of drugs targeting the cannabinoid system.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Receptor Cannabinoide CB2/química , Transducción de Señal , Animales , Sitios de Unión , Células CHO , Agonistas de Receptores de Cannabinoides/síntesis química , Agonistas de Receptores de Cannabinoides/farmacología , Antagonistas de Receptores de Cannabinoides/síntesis química , Antagonistas de Receptores de Cannabinoides/farmacología , Cricetinae , Cricetulus , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/antagonistas & inhibidores , Receptor Cannabinoide CB2/metabolismo , Células Sf9 , SpodopteraRESUMEN
Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.
Asunto(s)
Agonistas de Receptores de Cannabinoides/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Receptor Cannabinoide CB1/química , Receptor Cannabinoide CB2/química , Transducción de Señal , Regulación Alostérica , Sitio Alostérico , Animales , Células CHO , Agonistas de Receptores de Cannabinoides/química , Cannabinoides/química , Cannabinoides/farmacología , Línea Celular Tumoral , Colesterol/química , Colesterol/farmacología , Cricetinae , Cricetulus , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Simulación de Dinámica Molecular , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Células Sf9 , SpodopteraRESUMEN
Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10- to 100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.
Asunto(s)
Betacoronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Pandemias/prevención & control , Neumonía Viral/prevención & control , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Diseño Universal , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/química , COVID-19 , Vacunas contra la COVID-19 , Línea Celular Tumoral , Chlorocebus aethiops , Infecciones por Coronavirus/virología , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Coronavirus del Síndrome Respiratorio de Oriente Medio/química , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/virología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/inmunología , Receptores Virales/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , SARS-CoV-2 , Células Sf9 , Organismos Libres de Patógenos Específicos , Spodoptera , Transfección , Vacunación/métodos , Células Vero , Vacunas ViralesRESUMEN
The hyperpolarization-activated cyclic nucleotide-gated (HCN) channel is a voltage-gated cation channel that mediates neuronal and cardiac pacemaker activity. The HCN channel exhibits reversed voltage dependence, meaning it closes with depolarization and opens with hyperpolarization. Different from Na+, Ca2+, and Kv1-Kv7 channels, the HCN channel does not have domain-swapped voltage sensors. We introduced a reversible, metal-mediated cross bridge into the voltage sensors to create the chemical equivalent of a hyperpolarized conformation and determined the structure using cryoelectron microscopy (cryo-EM). Unlike the depolarized HCN channel, the S4 helix is displaced toward the cytoplasm by two helical turns. Near the cytoplasm, the S4 helix breaks into two helices, one running parallel to the membrane surface, analogous to the S4-S5 linker of domain-swapped voltage-gated channels. These findings suggest a basis for allosteric communication between voltage sensors and the gate in this kind of channel. They also imply that voltage sensor movements are not the same in all voltage-gated channels.
Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/química , Activación del Canal Iónico , Animales , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Potenciales de la Membrana , Conformación Proteica en Hélice alfa , Células Sf9 , SpodopteraRESUMEN
Voltage-gated sodium (NaV) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state exists only at deeply negative membrane potentials. To stabilize the resting conformation, we inserted voltage-shifting mutations and introduced a disulfide crosslink in the VS of the ancestral bacterial sodium channel NaVAb. Here, we present a cryo-EM structure of the resting state and a complete voltage-dependent gating mechanism. The S4 segment of the VS is drawn intracellularly, with three gating charges passing through the transmembrane electric field. This movement forms an elbow connecting S4 to the S4-S5 linker, tightens the collar around the S6 activation gate, and prevents its opening. Our structure supports the classical "sliding helix" mechanism of voltage sensing and provides a complete gating mechanism for voltage sensor function, pore opening, and activation-gate closure based on high-resolution structures of a single sodium channel protein.
Asunto(s)
Potenciales de Acción/fisiología , Membrana Externa Bacteriana/metabolismo , Escherichia coli/metabolismo , Activación del Canal Iónico/fisiología , Canales de Sodio Activados por Voltaje/metabolismo , Animales , Línea Celular , Microscopía por Crioelectrón , Cristalografía por Rayos X , Mutación , Conformación Proteica en Hélice alfa , Sodio/metabolismo , Spodoptera/citología , Canales de Sodio Activados por Voltaje/químicaRESUMEN
P2X receptors are trimeric, non-selective cation channels activated by extracellular ATP. The P2X7 receptor subtype is a pharmacological target because of involvement in apoptotic, inflammatory, and tumor progression pathways. It is the most structurally and functionally distinct P2X subtype, containing a unique cytoplasmic domain critical for the receptor to initiate apoptosis and not undergo desensitization. However, lack of structural information about the cytoplasmic domain has hindered understanding of the molecular mechanisms underlying these processes. We report cryoelectron microscopy structures of full-length rat P2X7 receptor in apo and ATP-bound states. These structures reveal how one cytoplasmic element, the C-cys anchor, prevents desensitization by anchoring the pore-lining helix to the membrane with palmitoyl groups. They show a second cytoplasmic element with a unique fold, the cytoplasmic ballast, which unexpectedly contains a zinc ion complex and a guanosine nucleotide binding site. Our structures provide first insights into the architecture and function of a P2X receptor cytoplasmic domain.
Asunto(s)
Lipoilación , Receptores Purinérgicos P2X7/química , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Microscopía por Crioelectrón , Guanosina/metabolismo , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Receptores Purinérgicos P2X7/metabolismo , Células Sf9 , Spodoptera , Xenopus , Zinc/metabolismoRESUMEN
Numerous interventions are in clinical development for respiratory syncytial virus (RSV) infection, including small molecules that target viral transcription and replication. These processes are catalyzed by a complex comprising the RNA-dependent RNA polymerase (L) and the tetrameric phosphoprotein (P). RSV P recruits multiple proteins to the polymerase complex and, with the exception of its oligomerization domain, is thought to be intrinsically disordered. Despite their critical roles in RSV transcription and replication, structures of L and P have remained elusive. Here, we describe the 3.2-Å cryo-EM structure of RSV L bound to tetrameric P. The structure reveals a striking tentacular arrangement of P, with each of the four monomers adopting a distinct conformation. The structure also rationalizes inhibitor escape mutants and mutations observed in live-attenuated vaccine candidates. These results provide a framework for determining the molecular underpinnings of RSV replication and transcription and should facilitate the design of effective RSV inhibitors.
Asunto(s)
Fosfoproteínas/ultraestructura , ARN Polimerasa Dependiente del ARN/ultraestructura , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/enzimología , Proteínas Virales/ultraestructura , Acetatos/química , Animales , Antivirales/química , Antivirales/uso terapéutico , Dominio Catalítico , Microscopía por Crioelectrón , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Quinolinas/química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Vacunas contra Virus Sincitial Respiratorio/química , Células Sf9 , Spodoptera , Proteínas Virales/química , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Mdn1 is an essential AAA (ATPase associated with various activities) protein that removes assembly factors from distinct precursors of the ribosomal 60S subunit. However, Mdn1's large size (â¼5,000 amino acid [aa]) and its limited homology to other well-studied proteins have restricted our understanding of its remodeling function. Here, we present structures for S. pombe Mdn1 in the presence of AMPPNP at up to â¼4 Å or ATP plus Rbin-1, a chemical inhibitor, at â¼8 Å resolution. These data reveal that Mdn1's MIDAS domain is tethered to its ring-shaped AAA domain through an â¼20 nm long structured linker and a flexible â¼500 aa Asp/Glu-rich motif. We find that the MIDAS domain, which also binds other ribosome-assembly factors, docks onto the AAA ring in a nucleotide state-specific manner. Together, our findings reveal how conformational changes in the AAA ring can be directly transmitted to the MIDAS domain and thereby drive the targeted release of assembly factors from ribosomal 60S-subunit precursors.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/química , Simulación de Dinámica Molecular , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/enzimología , Secuencias de Aminoácidos , Animales , Sitios de Unión , Microscopía por Crioelectrón , Biogénesis de Organelos , Unión Proteica , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Células Sf9 , SpodopteraRESUMEN
During cell division, mitotic motors organize microtubules in the bipolar spindle into either polar arrays at the spindle poles or a "nematic" network of aligned microtubules at the spindle center. The reasons for the distinct self-organizing capacities of dynamic microtubules and different motors are not understood. Using in vitro reconstitution experiments and computer simulations, we show that the human mitotic motors kinesin-5 KIF11 and kinesin-14 HSET, despite opposite directionalities, can both organize dynamic microtubules into either polar or nematic networks. We show that in addition to the motor properties the natural asymmetry between microtubule plus- and minus-end growth critically contributes to the organizational potential of the motors. We identify two control parameters that capture system composition and kinetic properties and predict the outcome of microtubule network organization. These results elucidate a fundamental design principle of spindle bipolarity and establish general rules for active filament network organization.
Asunto(s)
Cinesinas/metabolismo , Microtúbulos/metabolismo , Simulación de Dinámica Molecular , Huso Acromático/metabolismo , Animales , Humanos , Cinesinas/química , Microtúbulos/química , Células Sf9 , Huso Acromático/química , SpodopteraRESUMEN
Signaling across cellular membranes, the 826 human G protein-coupled receptors (GPCRs) govern a wide range of vital physiological processes, making GPCRs prominent drug targets. X-ray crystallography provided GPCR molecular architectures, which also revealed the need for additional structural dynamics data to support drug development. Here, nuclear magnetic resonance (NMR) spectroscopy with the wild-type-like A2A adenosine receptor (A2AAR) in solution provides a comprehensive characterization of signaling-related structural dynamics. All six tryptophan indole and eight glycine backbone 15N-1H NMR signals in A2AAR were individually assigned. These NMR probes provided insight into the role of Asp522.50 as an allosteric link between the orthosteric drug binding site and the intracellular signaling surface, revealing strong interactions with the toggle switch Trp 2466.48, and delineated the structural response to variable efficacy of bound drugs across A2AAR. The present data support GPCR signaling based on dynamic interactions between two semi-independent subdomains connected by an allosteric switch at Asp522.50.
Asunto(s)
Regulación Alostérica , Receptor de Adenosina A2A/química , Transducción de Señal , Agonistas del Receptor de Adenosina A2/química , Agonistas del Receptor de Adenosina A2/farmacología , Sitio Alostérico , Animales , Simulación del Acoplamiento Molecular , Pichia , Unión Proteica , Receptor de Adenosina A2A/metabolismo , Células Sf9 , SpodopteraRESUMEN
N-methyl-D-aspartate receptors (NMDARs) play essential roles in memory formation, neuronal plasticity, and brain development, with their dysfunction linked to a range of disorders from ischemia to schizophrenia. Zinc and pH are physiological allosteric modulators of NMDARs, with GluN2A-containing receptors inhibited by nanomolar concentrations of divalent zinc and by excursions to low pH. Despite the widespread importance of zinc and proton modulation of NMDARs, the molecular mechanism by which these ions modulate receptor activity has proven elusive. Here, we use cryoelectron microscopy to elucidate the structure of the GluN1/GluN2A NMDAR in a large ensemble of conformations under a range of physiologically relevant zinc and proton concentrations. We show how zinc binding to the amino terminal domain elicits structural changes that are transduced though the ligand-binding domain and result in constriction of the ion channel gate.
Asunto(s)
Complejos Multiproteicos/química , Protones , Receptores de N-Metil-D-Aspartato/química , Zinc/química , Regulación Alostérica , Animales , Microscopía por Crioelectrón , Concentración de Iones de Hidrógeno , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Dominios Proteicos , Ratas , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Células Sf9 , Spodoptera , Zinc/metabolismoRESUMEN
The immune system can mount T cell responses against tumors; however, the antigen specificities of tumor-infiltrating lymphocytes (TILs) are not well understood. We used yeast-display libraries of peptide-human leukocyte antigen (pHLA) to screen for antigens of "orphan" T cell receptors (TCRs) expressed on TILs from human colorectal adenocarcinoma. Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A∗02:01 library. Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2. These results show that the exposed recognition surface of MHC-bound peptides accessible to the TCR contains sufficient structural information to enable the reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity. This finding underscores the surprising specificity of TCRs for their cognate antigens and enables the facile indentification of tumor antigens through unbiased screening.
Asunto(s)
Adenocarcinoma/inmunología , Antígenos de Neoplasias/inmunología , Neoplasias Colorrectales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Anciano , Animales , Antígenos de Neoplasias/química , Línea Celular Tumoral , Células Cultivadas , Células HEK293 , Antígenos HLA-A/química , Antígenos HLA-A/inmunología , Humanos , Masculino , Persona de Mediana Edad , Biblioteca de Péptidos , Células Sf9 , SpodopteraRESUMEN
The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics.
Asunto(s)
Simulación del Acoplamiento Molecular , Receptores Opioides kappa/química , Analgésicos/química , Analgésicos/farmacología , Animales , Sitios de Unión , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Morfinanos/química , Morfinanos/farmacología , Unión Proteica , Estabilidad Proteica , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Células Sf9 , SpodopteraRESUMEN
The multidrug resistance protein MRP1 is an ATP-driven pump that confers resistance to chemotherapy. Previously, we have shown that intracellular substrates are recruited to a bipartite binding site when the transporter rests in an inward-facing conformation. A key question remains: how are high-affinity substrates transferred across the membrane and released outside the cell? Using electron cryomicroscopy, we show here that ATP binding opens the transport pathway to the extracellular space and reconfigures the substrate-binding site such that it relinquishes its affinity for substrate. Thus, substrate is released prior to ATP hydrolysis. With this result, we now have a complete description of the conformational cycle that enables substrate transfer in a eukaryotic ABC exporter.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Adenosina Trifosfato/metabolismo , Leucotrieno C4/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/química , Animales , Sitios de Unión , Células HEK293 , Humanos , Leucotrieno C4/química , Unión Proteica , Multimerización de Proteína , Células Sf9 , SpodopteraRESUMEN
Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Trastornos Congénitos de Glicosilación/metabolismo , Inhibidores Enzimáticos/farmacología , N-Acetilglucosaminiltransferasas/química , Animales , Antibióticos Antituberculosos/química , Sitios de Unión , Trastornos Congénitos de Glicosilación/genética , Inhibidores Enzimáticos/química , Femenino , Células HEK293 , Células Hep G2 , Humanos , Metabolismo de los Lípidos , Ratones , Simulación del Acoplamiento Molecular , Mutación , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Unión Proteica , Células Sf9 , Spodoptera , Tunicamicina/química , Tunicamicina/farmacología , Uridina Difosfato Ácido Glucurónico/química , Uridina Difosfato Ácido Glucurónico/metabolismoRESUMEN
Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.
Asunto(s)
Disulfiram/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Piroptosis/efectos de los fármacos , Sepsis/tratamiento farmacológico , Animales , Caspasa 1/genética , Caspasa 1/metabolismo , Inhibidores de Caspasas/farmacología , Caspasas/metabolismo , Caspasas Iniciadoras/genética , Caspasas Iniciadoras/metabolismo , Línea Celular Tumoral , Disulfiram/uso terapéutico , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Femenino , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/inmunología , Liposomas , Ratones , Mutagénesis Sitio-Dirigida , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Piroptosis/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sepsis/inmunología , Células Sf9 , SpodopteraRESUMEN
Cytoplasmic dynein-1 binds dynactin and cargo adaptor proteins to form a transport machine capable of long-distance processive movement along microtubules. However, it is unclear why dynein-1 moves poorly on its own or how it is activated by dynactin. Here, we present a cryoelectron microscopy structure of the complete 1.4-megadalton human dynein-1 complex in an inhibited state known as the phi-particle. We reveal the 3D structure of the cargo binding dynein tail and show how self-dimerization of the motor domains locks them in a conformation with low microtubule affinity. Disrupting motor dimerization with structure-based mutagenesis drives dynein-1 into an open form with higher affinity for both microtubules and dynactin. We find the open form is also inhibited for movement and that dynactin relieves this by reorienting the motor domains to interact correctly with microtubules. Our model explains how dynactin binding to the dynein-1 tail directly stimulates its motor activity.
Asunto(s)
Dineínas Citoplasmáticas/química , Complejos Multiproteicos/química , Animales , Microscopía por Crioelectrón , Dineínas Citoplasmáticas/metabolismo , Dineínas Citoplasmáticas/ultraestructura , Dimerización , Complejo Dinactina/química , Complejo Dinactina/metabolismo , Humanos , Ratones , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Moleculares , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Células Sf9 , Spodoptera , PorcinosRESUMEN
Inflammasome sensors detect pathogen- and danger-associated molecular patterns and promote inflammation and pyroptosis1. NLRP1 was the first inflammasome sensor to be described, and its hyperactivation is linked to autoinflammatory disease and cancer2-6. However, the mechanism underlying the activation and regulation of NLRP1 has not been clearly elucidated4,7,8. Here we identify ubiquitously expressed endogenous thioredoxin (TRX) as a binder of NLRP1 and a suppressor of the NLRP1 inflammasome. The cryo-electron microscopy structure of human NLRP1 shows NLRP1 bound to Spodoptera frugiperda TRX. Mutagenesis studies of NLRP1 and human TRX show that TRX in the oxidized form binds to the nucleotide-binding domain subdomain of NLRP1. This observation highlights the crucial role of redox-active cysteines of TRX in NLRP1 binding. Cellular assays reveal that TRX suppresses NLRP1 inflammasome activation and thus negatively regulates NLRP1. Our data identify the TRX system as an intrinsic checkpoint for innate immunity and provide opportunities for future therapeutic intervention in NLRP1 inflammasome activation targeting this system.
Asunto(s)
Inflamasomas , Proteínas NLR , Tiorredoxinas , Humanos , Microscopía por Crioelectrón , Inflamasomas/metabolismo , Proteínas NLR/antagonistas & inhibidores , Proteínas NLR/química , Proteínas NLR/metabolismo , Proteínas NLR/ultraestructura , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Spodoptera , Proteínas de Insectos , Oxidación-Reducción , Cisteína/metabolismo , Inmunidad InnataRESUMEN
Voltage-gated sodium channels initiate electrical signals and are frequently targeted by deadly gating-modifier neurotoxins, including tarantula toxins, which trap the voltage sensor in its resting state. The structural basis for tarantula-toxin action remains elusive because of the difficulty of capturing the functionally relevant form of the toxin-channel complex. Here, we engineered the model sodium channel NaVAb with voltage-shifting mutations and the toxin-binding site of human NaV1.7, an attractive pain target. This mutant chimera enabled us to determine the cryoelectron microscopy (cryo-EM) structure of the channel functionally arrested by tarantula toxin. Our structure reveals a high-affinity resting-state-specific toxin-channel interaction between a key lysine residue that serves as a "stinger" and penetrates a triad of carboxyl groups in the S3-S4 linker of the voltage sensor. By unveiling this high-affinity binding mode, our studies establish a high-resolution channel-docking and resting-state locking mechanism for huwentoxin-IV and provide guidance for developing future resting-state-targeted analgesic drugs.