RESUMO
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.
Assuntos
Citocinas/metabolismo , Imunoterapia/tendências , Animais , Citocinas/genética , Humanos , Imunidade Humoral , Imunomodulação , Multimerização Proteica , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/imunologiaRESUMO
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.
Assuntos
Sítio Alostérico , Microscopia Crioeletrônica , Simulação de Dinâmica Molecular , Receptores Nicotínicos , Transdução de Sinais , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Regulação Alostérica , Humanos , Animais , Cristalografia por Raios X , Sítios de Ligação , Conformação Proteica , Ligantes , Modelos Moleculares , Multimerização Proteica , Agonistas Nicotínicos/química , Agonistas Nicotínicos/farmacologia , Agonistas Nicotínicos/metabolismoRESUMO
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.
Assuntos
COVID-19/imunologia , COVID-19/virologia , Imunidade/imunologia , SARS-CoV-2/imunologia , Células T Auxiliares Foliculares/imunologia , Vacinação , Vacinas Sintéticas/imunologia , Vacinas de mRNA/imunologia , Adulto , Linfócitos B/imunologia , Vacina BNT162/imunologia , COVID-19/sangue , Células Clonais , Estudos de Coortes , Citocinas/metabolismo , Feminino , Centro Germinativo/imunologia , Cadeias beta de HLA-DP/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Células Jurkat , Linfonodos/metabolismo , Masculino , Pessoa de Meia-Idade , Peptídeos/química , Peptídeos/metabolismo , Multimerização Proteica , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
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.
Assuntos
Butirofilinas , Receptores de Antígenos de Linfócitos T gama-delta , Butirofilinas/metabolismo , Butirofilinas/imunologia , Butirofilinas/química , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Humanos , Ligação Proteica , Multimerização Proteica , Antígenos CD/metabolismo , Antígenos CD/imunologia , Antígenos CD/química , Linfócitos T/imunologia , Linfócitos T/metabolismo , Cristalografia por Raios X , Ativação Linfocitária/imunologia , Modelos Moleculares , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/metabolismoRESUMO
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.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Sequência de Aminoácidos , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/ultraestrutura , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
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.
Assuntos
Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Modelos Moleculares , Mutação/genética , Quinases Relacionadas a NIMA/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/isolamento & purificação , Proteína 3 que Contém Domínio de Pirina da Família NLR/ultraestrutura , Nigericina/farmacologia , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Rede trans-Golgi/metabolismoRESUMO
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.
Assuntos
Arginina/análogos & derivados , Condensados Biomoleculares/metabolismo , Proteínas do Complexo SMN/química , Proteínas do Complexo SMN/metabolismo , Animais , Arginina/metabolismo , Núcleo Celular/metabolismo , Corpos Enovelados/metabolismo , Drosophila melanogaster/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligantes , Metilação , Camundongos , Modelos Biológicos , Células NIH 3T3 , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Ribonucleoproteínas Nucleares Pequenas/metabolismoRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Bases , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , Mutação/genética , Motivos de Nucleotídeos/genética , Transição de Fase , Mutação Puntual/genética , Poli A/metabolismo , Ligação Proteica , Multimerização Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de SequênciaRESUMO
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.
Assuntos
Fenômenos Eletrofisiológicos , Transportadores de Sulfato/metabolismo , Ânions , Sítios de Ligação , Cloretos/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Humanos , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Domínios Proteicos , Multimerização Proteica , Estabilidade Proteica , Ácido Salicílico/metabolismo , Homologia Estrutural de Proteína , Transportadores de Sulfato/química , Transportadores de Sulfato/ultraestruturaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Resistência à Doença/imunologia , Imunidade Vegetal , Transdução de Sinais , Animais , Morte Celular , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Ácido Glutâmico/metabolismo , Bicamadas Lipídicas/metabolismo , Oócitos/metabolismo , Células Vegetais/metabolismo , Multimerização Proteica , Protoplastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Imagem Individual de Molécula , Vacúolos/metabolismo , XenopusRESUMO
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.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Chlamydomonas/metabolismo , Multimerização Proteica , Synechocystis/metabolismo , Tilacoides/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Membrana Celular/metabolismo , Chlamydomonas/ultraestrutura , Microscopia Crioeletrônica , Proteínas de Fluorescência Verde/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Luz , Lipídeos/química , Modelos Moleculares , Nucleotídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estresse Fisiológico/efeitos da radiação , Synechocystis/ultraestrutura , Tilacoides/ultraestruturaRESUMO
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.
Assuntos
Capsídeo/metabolismo , Dependovirus/metabolismo , Evolução Molecular Direcionada , Técnicas de Transferência de Genes , Músculo Esquelético/metabolismo , Sequência de Aminoácidos , Animais , Capsídeo/química , Células Cultivadas , Modelos Animais de Doenças , Células HEK293 , Humanos , Integrinas/metabolismo , Macaca fascicularis , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/terapia , Multimerização Proteica , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/uso terapêutico , RNA Guia de Cinetoplastídeos/metabolismo , Recombinação Genética/genética , Especificidade da Espécie , TransgenesRESUMO
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.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores de Dopamina D1/metabolismo , Transdução de Sinais , Regulação Alostérica , Sítio Alostérico , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Catecóis/metabolismo , Microscopia Crioeletrônica , Fenoldopam/química , Fenoldopam/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Multimerização Proteica , Receptores de Dopamina D1/química , Receptores de Dopamina D1/ultraestrutura , Receptores de Dopamina D2/metabolismo , Homologia Estrutural de ProteínaRESUMO
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.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Ligadas por GPI/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Oncogênicas/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/química , Movimento Celular , Receptor DCC/deficiência , Receptor DCC/genética , Proteínas Ligadas por GPI/química , Cones de Crescimento/fisiologia , Humanos , Ventrículos Laterais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Neurônios/citologia , Neurônios/metabolismo , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
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).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Multimerização Proteica , Piroptose , Transdução de Sinais , Aminoácidos/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular , Testes Genéticos , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Macrófagos/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas de Ligação a Fosfato/química , Domínios Proteicos , RNA Guia de Cinetoplastídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
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.
Assuntos
Anticorpos Neutralizantes/imunologia , Linfócitos B/metabolismo , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/química , Anticorpos Antivirais/sangue , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/metabolismo , Reações Antígeno-Anticorpo , Linfócitos B/citologia , Linfócitos B/virologia , COVID-19/patologia , COVID-19/virologia , Microscopia Crioeletrônica , Cristalografia por Raios X , Perfilação da Expressão Gênica , Humanos , Imunoglobulina A/imunologia , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Domínios Proteicos/imunologia , Multimerização Proteica , Estrutura Quaternária de Proteína , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
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.
Assuntos
Coenzimas/química , Manganês/química , Oxigênio/química , Complexo de Proteína do Fotossistema II/química , Água/química , Coenzimas/metabolismo , Cristalografia por Raios X , Expressão Gênica , Lasers , Manganês/metabolismo , Modelos Moleculares , Oxirredução , Oxigênio/metabolismo , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Teoria Quântica , Termodinâmica , Thermosynechococcus/química , Thermosynechococcus/enzimologia , Água/metabolismoRESUMO
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.
Assuntos
Proteínas Amiloidogênicas/química , Glicoproteínas da Zona Pelúcida/química , Zigoto/metabolismo , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Animais , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/ultraestrutura , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Oócitos/ultraestrutura , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Zona Pelúcida/metabolismo , Zona Pelúcida/ultraestrutura , Glicoproteínas da Zona Pelúcida/genética , Glicoproteínas da Zona Pelúcida/metabolismo , Zigoto/crescimento & desenvolvimento , Zigoto/ultraestruturaRESUMO
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.
Assuntos
Sistemas CRISPR-Cas/efeitos dos fármacos , Edição de Genes/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Virais/genética , Vírus/genética , Archaea/genética , Archaea/imunologia , Archaea/virologia , Bactérias/genética , Bactérias/imunologia , Bactérias/virologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Coevolução Biológica , Proteínas Associadas a CRISPR/antagonistas & inibidores , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , DNA/antagonistas & inibidores , DNA/química , DNA/genética , DNA/metabolismo , Clivagem do DNA/efeitos dos fármacos , Endodesoxirribonucleases/antagonistas & inibidores , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Humanos , Modelos Moleculares , Família Multigênica , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Virais/farmacologia , Vírus/metabolismo , Vírus/patogenicidadeRESUMO
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.