RESUMEN
Cytokines are secreted or otherwise released polypeptide factors that exert autocrine and/or paracrine actions, with most cytokines acting in the immune and/or hematopoietic system. They are typically pleiotropic, controlling development, cell growth, survival, and/or differentiation. Correspondingly, cytokines are clinically important, and augmenting or attenuating cytokine signals can have deleterious or therapeutic effects. Besides physiological fine-tuning of cytokine signals, altering the nature or potency of the signal can be important in pathophysiological responses and can also provide novel therapeutic approaches. Here, we give an overview of cytokines, their signaling and actions, and the physiological mechanisms and pharmacologic strategies to fine-tune their actions. In particular, the differential utilization of STAT proteins by a single cytokine or by different cytokines and STAT dimerization versus tetramerization are physiological mechanisms of fine-tuning, whereas anticytokine and anticytokine receptor antibodies and cytokines with altered activities, including cytokine superagonists, partial agonists, and antagonists, represent new ways of fine-tuning cytokine signals.
Asunto(s)
Citocinas/metabolismo , Inmunoterapia/tendencias , Animales , Citocinas/genética , Humanos , Inmunidad Humoral , Inmunomodulación , Multimerización de Proteína , Factores de Transcripción STAT/metabolismo , Transducción de Señal/inmunologíaRESUMEN
The nicotinic acetylcholine receptor has served, since its biochemical identification in the 1970s, as a model of an allosteric ligand-gated ion channel mediating signal transition at the synapse. In recent years, the application of X-ray crystallography and high-resolution cryo-electron microscopy, together with molecular dynamic simulations of nicotinic receptors and homologs, have opened a new era in the understanding of channel gating by the neurotransmitter. They reveal, at atomic resolution, the diversity and flexibility of the multiple ligand-binding sites, including recently discovered allosteric modulatory sites distinct from the neurotransmitter orthosteric site, and the conformational dynamics of the activation process as a molecular switch linking these multiple sites. The model emerging from these studies paves the way for a new pharmacology based, first, upon the occurrence of an original mode of indirect allosteric modulation, distinct from a steric competition for a single and rigid binding site, and second, the design of drugs that specifically interact with privileged conformations of the receptor such as agonists, antagonists, and desensitizers. Research on nicotinic receptors is still at the forefront of understanding the mode of action of drugs on the nervous system.
Asunto(s)
Sitio Alostérico , Microscopía por Crioelectrón , Simulación de Dinámica Molecular , Receptores Nicotínicos , Transducción de Señal , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Regulación Alostérica , Humanos , Animales , Cristalografía por Rayos X , Sitios de Unión , Conformación Proteica , Ligandos , Modelos Moleculares , Multimerización de Proteína , Agonistas Nicotínicos/química , Agonistas Nicotínicos/farmacología , Agonistas Nicotínicos/metabolismoRESUMEN
SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine-needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node TFH. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1∗04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.
Asunto(s)
COVID-19/inmunología , COVID-19/virología , Inmunidad/inmunología , SARS-CoV-2/inmunología , Células T Auxiliares Foliculares/inmunología , Vacunación , Vacunas Sintéticas/inmunología , Vacunas de ARNm/inmunología , Adulto , Linfocitos B/inmunología , Vacuna BNT162/inmunología , COVID-19/sangre , Células Clonales , Estudios de Cohortes , Citocinas/metabolismo , Femenino , Centro Germinal/inmunología , Cadenas beta de HLA-DP/inmunología , Humanos , Epítopos Inmunodominantes/inmunología , Células Jurkat , Ganglios Linfáticos/metabolismo , Masculino , Persona de Mediana Edad , Péptidos/química , Péptidos/metabolismo , Multimerización de Proteína , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Butyrophilin (BTN) molecules are emerging as key regulators of T cell immunity; however, how they trigger cell-mediated responses is poorly understood. Here, the crystal structure of a gamma-delta T cell antigen receptor (γδTCR) in complex with BTN2A1 revealed that BTN2A1 engages the side of the γδTCR, leaving the apical TCR surface bioavailable. We reveal that a second γδTCR ligand co-engages γδTCR via binding to this accessible apical surface in a BTN3A1-dependent manner. BTN2A1 and BTN3A1 also directly interact with each other in cis, and structural analysis revealed formation of W-shaped heteromeric multimers. This BTN2A1-BTN3A1 interaction involved the same epitopes that BTN2A1 and BTN3A1 each use to mediate the γδTCR interaction; indeed, locking BTN2A1 and BTN3A1 together abrogated their interaction with γδTCR, supporting a model wherein the two γδTCR ligand-binding sites depend on accessibility to cryptic BTN epitopes. Our findings reveal a new paradigm in immune activation, whereby γδTCRs sense dual epitopes on BTN complexes.
Asunto(s)
Butirofilinas , Receptores de Antígenos de Linfocitos T gamma-delta , Butirofilinas/metabolismo , Butirofilinas/inmunología , Butirofilinas/química , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Humanos , Unión Proteica , Multimerización de Proteína , Antígenos CD/metabolismo , Antígenos CD/inmunología , Antígenos CD/química , Linfocitos T/inmunología , Linfocitos T/metabolismo , Cristalografía por Rayos X , Activación de Linfocitos/inmunología , Modelos Moleculares , Linfocitos Intraepiteliales/inmunología , Linfocitos Intraepiteliales/metabolismoRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) are commonly implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 regulates critical cellular processes at membranous organelles and forms microtubule-based pathogenic filaments, yet the molecular basis underlying these biological roles of LRRK2 remains largely enigmatic. Here, we determined high-resolution structures of full-length human LRRK2, revealing its architecture and key interdomain scaffolding elements for rationalizing disease-causing mutations. The kinase domain of LRRK2 is captured in an inactive state, a conformation also adopted by the most common PD-associated mutation, LRRK2G2019S. This conformation serves as a framework for structure-guided design of conformational specific inhibitors. We further determined the structure of COR-mediated LRRK2 dimers and found that single-point mutations at the dimer interface abolished pathogenic filamentation in cells. Overall, our study provides mechanistic insights into physiological and pathological roles of LRRK2 and establishes a structural template for future therapeutic intervention in PD.
Asunto(s)
Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/química , Secuencia de Aminoácidos , Células HEK293 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/ultraestructura , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de ProteínaRESUMEN
The NACHT-, leucine-rich-repeat- (LRR), and pyrin domain-containing protein 3 (NLRP3) is emerging to be a critical intracellular inflammasome sensor of membrane integrity and a highly important clinical target against chronic inflammation. Here, we report that an endogenous, stimulus-responsive form of full-length mouse NLRP3 is a 12- to 16-mer double-ring cage held together by LRR-LRR interactions with the pyrin domains shielded within the assembly to avoid premature activation. Surprisingly, this NLRP3 form is predominantly membrane localized, which is consistent with previously noted localization of NLRP3 at various membrane organelles. Structure-guided mutagenesis reveals that trans-Golgi network dispersion into vesicles, an early event observed for many NLRP3-activating stimuli, requires the double-ring cages of NLRP3. Double-ring-defective NLRP3 mutants abolish inflammasome punctum formation, caspase-1 processing, and cell death. Thus, our data uncover a physiological NLRP3 oligomer on the membrane that is poised to sense diverse signals to induce inflammasome activation.
Asunto(s)
Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/química , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Humanos , Ratones , Modelos Biológicos , Modelos Moleculares , Mutación/genética , Quinasas Relacionadas con NIMA/genética , Proteína con Dominio Pirina 3 de la Familia NLR/aislamiento & purificación , Proteína con Dominio Pirina 3 de la Familia NLR/ultraestructura , Nigericina/farmacología , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Red trans-Golgi/metabolismoRESUMEN
Biomolecular condensation is a widespread mechanism of cellular compartmentalization. Because the "survival of motor neuron protein" (SMN) is implicated in the formation of three different membraneless organelles (MLOs), we hypothesized that SMN promotes condensation. Unexpectedly, we found that SMN's globular tudor domain was sufficient for dimerization-induced condensation in vivo, whereas its two intrinsically disordered regions (IDRs) were not. Binding to dimethylarginine (DMA) modified protein ligands was required for condensate formation by the tudor domains in SMN and at least seven other fly and human proteins. Remarkably, asymmetric versus symmetric DMA determined whether two distinct nuclear MLOs-gems and Cajal bodies-were separate or "docked" to one another. This substructure depended on the presence of either asymmetric or symmetric DMA as visualized with sub-diffraction microscopy. Thus, DMA-tudor interaction modules-combinations of tudor domains bound to their DMA ligand(s)-represent versatile yet specific regulators of MLO assembly, composition, and morphology.
Asunto(s)
Arginina/análogos & derivados , Condensados Biomoleculares/metabolismo , Proteínas del Complejo SMN/química , Proteínas del Complejo SMN/metabolismo , Animales , Arginina/metabolismo , Núcleo Celular/metabolismo , Cuerpos Enrollados/metabolismo , Drosophila melanogaster/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligandos , Metilación , Ratones , Modelos Biológicos , Células 3T3 NIH , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Ribonucleoproteínas Nucleares Pequeñas/metabolismoRESUMEN
Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Regiones no Traducidas 3'/genética , Secuencia de Bases , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Mutación/genética , Motivos de Nucleótidos/genética , Transición de Fase , Mutación Puntual/genética , Poli A/metabolismo , Unión Proteica , Multimerización de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Eliminación de SecuenciaRESUMEN
Hearing involves two fundamental processes: mechano-electrical transduction and signal amplification. Despite decades of studies, the molecular bases for both remain elusive. Here, we show how prestin, the electromotive molecule of outer hair cells (OHCs) that senses both voltage and membrane tension, mediates signal amplification by coupling conformational changes to alterations in membrane surface area. Cryoelectron microscopy (cryo-EM) structures of human prestin bound with chloride or salicylate at a common "anion site" adopt contracted or expanded states, respectively. Prestin is ensconced within a perimeter of well-ordered lipids, through which it induces dramatic deformation in the membrane and couples protein conformational changes to the bulk membrane. Together with computational studies, we illustrate how the anion site is allosterically coupled to changes in the transmembrane domain cross-sectional area and the surrounding membrane. These studies provide insight into OHC electromotility by providing a structure-based mechanism of the membrane motor prestin.
Asunto(s)
Fenómenos Electrofisiológicos , Transportadores de Sulfato/metabolismo , Aniones , Sitios de Unión , Cloruros/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Humanos , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Dominios Proteicos , Multimerización de Proteína , Estabilidad Proteica , Ácido Salicílico/metabolismo , Homología Estructural de Proteína , Transportadores de Sulfato/química , Transportadores de Sulfato/ultraestructuraRESUMEN
Nucleotide-binding, leucine-rich repeat receptors (NLRs) are major immune receptors in plants and animals. Upon activation, the Arabidopsis NLR protein ZAR1 forms a pentameric resistosome in vitro and triggers immune responses and cell death in plants. In this study, we employed single-molecule imaging to show that the activated ZAR1 protein can form pentameric complexes in the plasma membrane. The ZAR1 resistosome displayed ion channel activity in Xenopus oocytes in a manner dependent on a conserved acidic residue Glu11 situated in the channel pore. Pre-assembled ZAR1 resistosome was readily incorporated into planar lipid-bilayers and displayed calcium-permeable cation-selective channel activity. Furthermore, we show that activation of ZAR1 in the plant cell led to Glu11-dependent Ca2+ influx, perturbation of subcellular structures, production of reactive oxygen species, and cell death. The results thus support that the ZAR1 resistosome acts as a calcium-permeable cation channel to trigger immunity and cell death.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Resistencia a la Enfermedad/inmunología , Inmunidad de la Planta , Transducción de Señal , Animales , Muerte Celular , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Ácido Glutámico/metabolismo , Membrana Dobles de Lípidos/metabolismo , Oocitos/metabolismo , Células Vegetales/metabolismo , Multimerización de Proteína , Protoplastos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Imagen Individual de Molécula , Vacuolas/metabolismo , XenopusRESUMEN
Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Chlamydomonas/metabolismo , Multimerización de Proteína , Synechocystis/metabolismo , Tilacoides/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/ultraestructura , Sitios de Unión , Membrana Celular/metabolismo , Chlamydomonas/ultraestructura , Microscopía por Crioelectrón , Proteínas Fluorescentes Verdes/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Luz , Lípidos/química , Modelos Moleculares , Nucleótidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estrés Fisiológico/efectos de la radiación , Synechocystis/ultraestructura , Tilacoides/ultraestructuraRESUMEN
Replacing or editing disease-causing mutations holds great promise for treating many human diseases. Yet, delivering therapeutic genetic modifiers to specific cells in vivo has been challenging, particularly in large, anatomically distributed tissues such as skeletal muscle. Here, we establish an in vivo strategy to evolve and stringently select capsid variants of adeno-associated viruses (AAVs) that enable potent delivery to desired tissues. Using this method, we identify a class of RGD motif-containing capsids that transduces muscle with superior efficiency and selectivity after intravenous injection in mice and non-human primates. We demonstrate substantially enhanced potency and therapeutic efficacy of these engineered vectors compared to naturally occurring AAV capsids in two mouse models of genetic muscle disease. The top capsid variants from our selection approach show conserved potency for delivery across a variety of inbred mouse strains, and in cynomolgus macaques and human primary myotubes, with transduction dependent on target cell expressed integrin heterodimers.
Asunto(s)
Cápside/metabolismo , Dependovirus/metabolismo , Evolución Molecular Dirigida , Técnicas de Transferencia de Gen , Músculo Esquelético/metabolismo , Secuencia de Aminoácidos , Animales , Cápside/química , Células Cultivadas , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Integrinas/metabolismo , Macaca fascicularis , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/terapia , Miopatías Estructurales Congénitas/patología , Miopatías Estructurales Congénitas/terapia , Multimerización de Proteína , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/uso terapéutico , ARN Guía de Kinetoplastida/metabolismo , Recombinación Genética/genética , Especificidad de la Especie , TransgenesRESUMEN
Dopamine receptors, including D1- and D2-like receptors, are important therapeutic targets in a variety of neurological syndromes, as well as cardiovascular and kidney diseases. Here, we present five cryoelectron microscopy (cryo-EM) structures of the dopamine D1 receptor (DRD1) coupled to Gs heterotrimer in complex with three catechol-based agonists, a non-catechol agonist, and a positive allosteric modulator for endogenous dopamine. These structures revealed that a polar interaction network is essential for catecholamine-like agonist recognition, whereas specific motifs in the extended binding pocket were responsible for discriminating D1- from D2-like receptors. Moreover, allosteric binding at a distinct inner surface pocket improved the activity of DRD1 by stabilizing endogenous dopamine interaction at the orthosteric site. DRD1-Gs interface revealed key features that serve as determinants for G protein coupling. Together, our study provides a structural understanding of the ligand recognition, allosteric regulation, and G protein coupling mechanisms of DRD1.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Receptores de Dopamina D1/metabolismo , Transducción de Señal , Regulación Alostérica , Sitio Alostérico , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Catecoles/metabolismo , Microscopía por Crioelectrón , Fenoldopam/química , Fenoldopam/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/ultraestructura , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Multimerización de Proteína , Receptores de Dopamina D1/química , Receptores de Dopamina D1/ultraestructura , Receptores de Dopamina D2/metabolismo , Homología Estructural de ProteínaRESUMEN
During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a "trimer-of-trimers" super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas Ligadas a GPI/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Oncogénicas/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/química , Movimiento Celular , Receptor DCC/deficiencia , Receptor DCC/genética , Proteínas Ligadas a GPI/química , Conos de Crecimiento/fisiología , Humanos , Ventrículos Laterales/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/química , Neuronas/citología , Neuronas/metabolismo , Proteínas Oncogénicas/química , Proteínas Oncogénicas/genética , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Multimerización de Proteína , Piroptosis , Transducción de Señal , Aminoácidos/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular , Pruebas Genéticas , Humanos , Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Macrófagos/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Proteínas de Unión a Fosfato/química , Dominios Proteicos , ARN Guía de Kinetoplastida/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Monoclonal antibodies (mAbs) are a focus in vaccine and therapeutic design to counteract severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. Here, we combined B cell sorting with single-cell VDJ and RNA sequencing (RNA-seq) and mAb structures to characterize B cell responses against SARS-CoV-2. We show that the SARS-CoV-2-specific B cell repertoire consists of transcriptionally distinct B cell populations with cells producing potently neutralizing antibodies (nAbs) localized in two clusters that resemble memory and activated B cells. Cryo-electron microscopy structures of selected nAbs from these two clusters complexed with SARS-CoV-2 spike trimers show recognition of various receptor-binding domain (RBD) epitopes. One of these mAbs, BG10-19, locks the spike trimer in a closed conformation to potently neutralize SARS-CoV-2, the recently arising mutants B.1.1.7 and B.1.351, and SARS-CoV and cross-reacts with heterologous RBDs. Together, our results characterize transcriptional differences among SARS-CoV-2-specific B cells and uncover cross-neutralizing Ab targets that will inform immunogen and therapeutic design against coronaviruses.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Linfocitos B/metabolismo , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Reacciones Antígeno-Anticuerpo , Linfocitos B/citología , Linfocitos B/virología , COVID-19/patología , COVID-19/virología , Microscopía por Crioelectrón , Cristalografía por Rayos X , Perfilación de la Expresión Génica , Humanos , Inmunoglobulina A/inmunología , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Dominios Proteicos/inmunología , Multimerización de Proteína , Estructura Cuaternaria de Proteína , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismo , Análisis de Secuencia de ARN , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
The investigation of water oxidation in photosynthesis has remained a central topic in biochemical research for the last few decades due to the importance of this catalytic process for technological applications. Significant progress has been made following the 2011 report of a high-resolution X-ray crystallographic structure resolving the site of catalysis, a protein-bound Mn4CaOx complex, which passes through ≥5 intermediate states in the water-splitting cycle. Spectroscopic techniques complemented by quantum chemical calculations aided in understanding the electronic structure of the cofactor in all (detectable) states of the enzymatic process. Together with isotope labeling, these techniques also revealed the binding of the two substrate water molecules to the cluster. These results are described in the context of recent progress using X-ray crystallography with free-electron lasers on these intermediates. The data are instrumental for developing a model for the biological water oxidation cycle.
Asunto(s)
Coenzimas/química , Manganeso/química , Oxígeno/química , Complejo de Proteína del Fotosistema II/química , Agua/química , Coenzimas/metabolismo , Cristalografía por Rayos X , Expresión Génica , Rayos Láser , Manganeso/metabolismo , Modelos Moleculares , Oxidación-Reducción , Oxígeno/metabolismo , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Teoría Cuántica , Termodinámica , Thermosynechococcus/química , Thermosynechococcus/enzimología , Agua/metabolismoRESUMEN
The zona pellucida (ZP) is an extracellular matrix that surrounds all mammalian oocytes, eggs, and early embryos and plays vital roles during oogenesis, fertilization, and preimplantation development. The ZP is composed of three or four glycosylated proteins, ZP1-4, that are synthesized, processed, secreted, and assembled into long, cross-linked fibrils by growing oocytes. ZP proteins have an immunoglobulin-like three-dimensional structure and a ZP domain that consists of two subdomains, ZP-N and ZP-C, with ZP-N of ZP2 and ZP3 required for fibril assembly. A ZP2-ZP3 dimer is located periodically along ZP fibrils that are cross-linked by ZP1, a protein with a proline-rich N terminus. Fibrils in the inner and outer regions of the ZP are oriented perpendicular and parallel to the oolemma, respectively, giving the ZP a multilayered appearance. Upon fertilization of eggs, modification of ZP2 and ZP3 results in changes in the ZP's physical and biological properties that have important consequences. Certain structural features of ZP proteins suggest that they may be amyloid-like proteins.
Asunto(s)
Proteínas Amiloidogénicas/química , Glicoproteínas de la Zona Pelúcida/química , Cigoto/metabolismo , Proteínas Amiloidogénicas/genética , Proteínas Amiloidogénicas/metabolismo , Animales , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/ultraestructura , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Oocitos/ultraestructura , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Zona Pelúcida/metabolismo , Zona Pelúcida/ultraestructura , Glicoproteínas de la Zona Pelúcida/genética , Glicoproteínas de la Zona Pelúcida/metabolismo , Cigoto/crecimiento & desarrollo , Cigoto/ultraestructuraRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.
Asunto(s)
Sistemas CRISPR-Cas/efectos de los fármacos , Edición Génica/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Virales/genética , Virus/genética , Archaea/genética , Archaea/inmunología , Archaea/virología , Bacterias/genética , Bacterias/inmunología , Bacterias/virología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Coevolución Biológica , Proteínas Asociadas a CRISPR/antagonistas & inhibidores , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , ADN/antagonistas & inhibidores , ADN/química , ADN/genética , ADN/metabolismo , División del ADN/efectos de los fármacos , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Humanos , Modelos Moleculares , Familia de Multigenes , Unión Proteica , Multimerización de Proteína/efectos de los fármacos , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Proteínas Virales/farmacología , Virus/metabolismo , Virus/patogenicidadRESUMEN
Natural rubber (NR), principally comprising cis-1,4-polyisoprene, is an industrially important natural hydrocarbon polymer because of its unique physical properties, which render it suitable for manufacturing items such as tires. Presently, industrial NR production depends solely on latex obtained from the Pará rubber tree, Hevea brasiliensis. In latex, NR is enclosed in rubber particles, which are specialized organelles comprising a hydrophobic NR core surrounded by a lipid monolayer and membrane-bound proteins. The similarity of the basic carbon skeleton structure between NR and dolichols and polyprenols, which are found in most organisms, suggests that the NR biosynthetic pathway is related to the polyisoprenoid biosynthetic pathway and that rubber transferase, which is the key enzyme in NR biosynthesis, belongs to the cis-prenyltransferase family. Here, we review recent progress in the elucidation of molecular mechanisms underlying NR biosynthesis through the identification of the enzymes that are responsible for the formation of the NR backbone structure.