RESUMO
Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.
Assuntos
Redes Reguladoras de Genes , Análise de Célula Única , Animais , Feminino , Humanos , Camundongos , Córtex Cerebral/metabolismo , Córtex Cerebral/citologia , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Vetores Genéticos/metabolismo , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Neurônios/citologia , Análise de Célula Única/métodos , Transcriptoma/genética , Linhagem Celular , Transcrição GênicaRESUMO
The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.
Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , Imunidade nas Mucosas , Administração Intranasal , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , COVID-19/virologia , Vacinas contra COVID-19/imunologia , Citocinas/sangue , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Vetores Genéticos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Testes de Neutralização , Nucleocapsídeo/genética , Nucleocapsídeo/imunologia , Nucleocapsídeo/metabolismo , Pan troglodytes , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
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.
Assuntos
Hemiterpenos/biossíntese , Hevea/metabolismo , Látex/biossíntese , Proteínas de Plantas/química , Borracha/química , Transferases/química , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hemiterpenos/química , Hemiterpenos/metabolismo , Hevea/química , Hevea/genética , Látex/química , Látex/metabolismo , Modelos Moleculares , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Borracha/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Terpenos/química , Terpenos/metabolismo , Transferases/genética , Transferases/metabolismoRESUMO
Transforming the vast knowledge from genetics, biochemistry, and structural biology into detailed molecular descriptions of biological processes inside cells remains a major challenge-one in sore need of better imaging technologies. For example, transcription involves the complex interplay between RNA polymerase II (Pol II), regulatory factors (RFs), and chromatin, but visualizing these dynamic molecular transactions in their native intracellular milieu remains elusive. Here, we zoom into single tagged genes using nanoscopy techniques, including an active target-locking, ultra-sensitive system that enables single-molecule detection in addressable sub-diffraction volumes, within crowded intracellular environments. We image, track, and quantify Pol II with single-molecule resolution, unveiling its dynamics during the transcription cycle. Further probing multiple functionally linked events-RF-chromatin interactions, Pol II dynamics, and nascent transcription kinetics-reveals detailed operational parameters of gene-regulatory mechanisms hitherto-unseen in vivo. Our approach sets the stage for single-molecule studies of complex molecular processes in live cells.
Assuntos
Nanotecnologia , RNA Polimerase II/metabolismo , Imagem Individual de Molécula/métodos , Transcrição Gênica , Linhagem Celular Tumoral , Cromatina/metabolismo , Genes Reporter , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Cinética , Mutagênese , RNA Polimerase II/genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Imagem com Lapso de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Homeostatic control of core body temperature is essential for survival. Temperature is sensed by specific neurons, in turn eliciting both behavioral (i.e., locomotion) and physiologic (i.e., thermogenesis, vasodilatation) responses. Here, we report that a population of GABAergic (Vgat-expressing) neurons in the dorsolateral portion of the dorsal raphe nucleus (DRN), hereafter DRNVgat neurons, are activated by ambient heat and bidirectionally regulate energy expenditure through changes in both thermogenesis and locomotion. We find that DRNVgat neurons innervate brown fat via a descending projection to the raphe pallidus (RPa). These neurons also densely innervate ascending targets implicated in the central regulation of energy expenditure, including the hypothalamus and extended amygdala. Optogenetic stimulation of different projection targets reveals that DRNVgat neurons are capable of regulating thermogenesis through both a "direct" descending pathway through the RPa and multiple "indirect" ascending pathways. This work establishes a key regulatory role for DRNVgat neurons in controlling energy expenditure.
Assuntos
Metabolismo Energético , Neurônios GABAérgicos/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Mapeamento Encefálico , Clozapina/análogos & derivados , Clozapina/farmacologia , Núcleo Dorsal da Rafe/metabolismo , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Optogenética , Temperatura , TermogêneseRESUMO
Directed evolution, artificial selection toward designed objectives, is routinely used to develop new molecular tools and therapeutics. Successful directed molecular evolution campaigns repeatedly test diverse sequences with a designed selective pressure. Unicellular organisms and their viral pathogens are exceptional for this purpose and have been used for decades. However, many desirable targets of directed evolution perform poorly or unnaturally in unicellular backgrounds. Here, we present a system for facile directed evolution in mammalian cells. Using the RNA alphavirus Sindbis as a vector for heredity and diversity, we achieved 24-h selection cycles surpassing 10-3 mutations per base. Selection is achieved through genetically actuated sequences internal to the host cell, thus the system's name: viral evolution of genetically actuating sequences, or "VEGAS." Using VEGAS, we evolve transcription factors, GPCRs, and allosteric nanobodies toward functional signaling endpoints each in less than 1 weeks' time.
Assuntos
Evolução Molecular Direcionada/métodos , Regulação Alostérica , Sequência de Aminoácidos , Animais , Transferência Ressonante de Energia de Fluorescência , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Mutação , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Alinhamento de Sequência , Sindbis virus/genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.
Assuntos
Proteoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Animais , Antígenos de Neoplasias/metabolismo , Sistemas CRISPR-Cas/genética , Biologia Computacional/métodos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Leupeptinas/farmacologia , Fases de Leitura Aberta/genética , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Proteólise , Proteoma/genética , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismoRESUMO
Gene replacement using adeno-associated virus (AAV) vectors is a promising therapeutic approach for many diseases1,2. However, this therapeutic modality is challenged by the packaging capacity of AAVs (approximately 4.7 kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14 kilobase messenger RNA. Here we developed a new method for expressing large dystrophins by utilizing the protein trans-splicing mechanism mediated by split inteins. We identified several split intein pairs that efficiently join two or three fragments to generate a large midi-dystrophin or the full-length protein. We show that delivery of two or three AAVs into dystrophic mice results in robust expression of large dystrophins and significant physiological improvements compared with micro-dystrophins. Moreover, using the potent myotropic AAVMYO4, we demonstrate that low total doses (2 × 1013 viral genomes per kg) are sufficient to express large dystrophins in striated muscles body-wide with significant physiological corrections in dystrophic mice. Our data show a clear functional superiority of large dystrophins over micro-dystrophins that are being tested in clinical trials. This method could benefit many patients with Duchenne or Becker muscular dystrophy, regardless of genotype, and could be adapted to numerous other disorders caused by mutations in large genes that exceed the AAV capacity.
Assuntos
Distrofina , Terapia Genética , Inteínas , Distrofia Muscular de Duchenne , Processamento de Proteína , Animais , Humanos , Masculino , Camundongos , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animais de Doenças , Distrofina/biossíntese , Distrofina/deficiência , Distrofina/genética , Distrofina/metabolismo , Terapia Genética/métodos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Inteínas/genética , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/metabolismo , Processamento de Proteína/genéticaRESUMO
Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, in vivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13acrRNA in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with in vitro biochemical and in vivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.
Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Endodesoxirribonucleases/química , Leptotrichia/genética , RNA Guia de Cinetoplastídeos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Leptotrichia/metabolismo , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Clivagem do RNA , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) facilitates arrestin binding and receptor desensitization. Although this process can be regulated by Ca2+-binding proteins such as calmodulin (CaM) and recoverin, the molecular mechanisms are poorly understood. Here, we report structural, computational, and biochemical analysis of a CaM complex with GRK5, revealing how CaM shapes GRK5 response to calcium. The CaM N and C domains bind independently to two helical regions at the GRK5 N and C termini to inhibit GPCR phosphorylation, though only the C domain interaction disrupts GRK5 membrane association, thereby facilitating cytoplasmic translocation. The CaM N domain strongly activates GRK5 via ordering of the amphipathic αN-helix of GRK5 and allosteric disruption of kinase-RH domain interaction for phosphorylation of cytoplasmic GRK5 substrates. These results provide a framework for understanding how two functional effects, GRK5 activation and localization, can cooperate under control of CaM for selective substrate targeting by GRK5.
Assuntos
Cálcio/metabolismo , Calmodulina/química , Quinase 5 de Receptor Acoplado a Proteína G/química , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Calmodulina/genética , Calmodulina/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Quinase 5 de Receptor Acoplado a Proteína G/genética , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Cinética , Simulação de Dinâmica Molecular , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Células Sf9 , Spodoptera , Especificidade por Substrato , TermodinâmicaRESUMO
The activation of cap-dependent translation in eukaryotes requires multisite, hierarchical phosphorylation of 4E-BP by the 1 MDa kinase mammalian target of rapamycin complex 1 (mTORC1). To resolve the mechanism of this hierarchical phosphorylation at the atomic level, we monitored by NMR spectroscopy the interaction of intrinsically disordered 4E binding protein isoform 1 (4E-BP1) with the mTORC1 subunit regulatory-associated protein of mTOR (Raptor). The N-terminal RAIP motif and the C-terminal TOR signaling (TOS) motif of 4E-BP1 bind separate sites in Raptor, resulting in avidity-based tethering of 4E-BP1. This tethering orients the flexible central region of 4E-BP1 toward the mTORC1 kinase site for phosphorylation. The structural constraints imposed by the two tethering interactions, combined with phosphorylation-induced conformational switching of 4E-BP1, explain the hierarchy of 4E-BP1 phosphorylation by mTORC1. Furthermore, we demonstrate that mTORC1 recognizes both free and eIF4E-bound 4E-BP1, allowing rapid phosphorylation of the entire 4E-BP1 pool and efficient activation of translation. Finally, our findings provide a mechanistic explanation for the differential rapamycin sensitivity of the 4E-BP1 phosphorylation sites.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Ciclo Celular/química , Fator de Iniciação 4E em Eucariotos/química , Alvo Mecanístico do Complexo 1 de Rapamicina/química , Proteína Regulatória Associada a mTOR/química , Serina-Treonina Quinases TOR/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaetomium/química , Chaetomium/genética , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/metabolismo , Transdução de Sinais , Homologia Estrutural de Proteína , Especificidade por Substrato , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Rho is a general transcription termination factor playing essential roles in RNA polymerase (RNAP) recycling, gene regulation, and genomic stability in most bacteria. Traditional models of transcription termination postulate that hexameric Rho loads onto RNA prior to contacting RNAP and then translocates along the transcript in pursuit of the moving RNAP to pull RNA from it. Here, we report the cryoelectron microscopy (cryo-EM) structures of two termination process intermediates. Prior to interacting with RNA, Rho forms a specific "pre-termination complex" (PTC) with RNAP and elongation factors NusA and NusG, which stabilize the PTC. RNA exiting RNAP interacts with NusA before entering the central channel of Rho from the distal C-terminal side of the ring. We map the principal interactions in the PTC and demonstrate their critical role in termination. Our results support a mechanism in which the formation of a persistent PTC is a prerequisite for termination.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fatores de Alongamento de Peptídeos/química , Fatores de Transcrição/química , Terminação da Transcrição Genética , Fatores de Elongação da Transcrição/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Gene delivery vehicles based on adeno-associated viruses (AAVs) are enabling increasing success in human clinical trials, and they offer the promise of treating a broad spectrum of both genetic and non-genetic disorders. However, delivery efficiency and targeting must be improved to enable safe and effective therapies. In recent years, considerable effort has been invested in creating AAV variants with improved delivery, and computational approaches have been increasingly harnessed for AAV engineering. In this review, we discuss how computationally designed AAV libraries are enabling directed evolution. Specifically, we highlight approaches that harness sequences outputted by next-generation sequencing (NGS) coupled with machine learning (ML) to generate new functional AAV capsids and related regulatory elements, pushing the frontier of what vector engineering and gene therapy may achieve.
Assuntos
Dependovirus , Técnicas de Transferência de Genes , Dependovirus/genética , Humanos , Terapia Genética/métodos , Vetores Genéticos/metabolismo , Engenharia Genética , Animais , Biologia Computacional/métodosRESUMO
The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH3-ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH3-ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.
Assuntos
Acetilcolina/química , Carbacol/química , Isoxazóis/química , Pilocarpina/química , Piridinas/química , Compostos de Amônio Quaternário/química , Receptor Muscarínico M2/química , Tiadiazóis/química , Acetilcolina/metabolismo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Carbacol/metabolismo , Clonagem Molecular , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Isoxazóis/metabolismo , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Pilocarpina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Piridinas/metabolismo , Compostos de Amônio Quaternário/metabolismo , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Termodinâmica , Tiadiazóis/metabolismoRESUMO
CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas18-Cas24-Csn28, referred to as monomer, contains â¼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA â¼30-bp duplexes within the channel. A filamentous form also comprises Cas18-Cas24-Csn28 units (typically 2-6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.
Assuntos
Proteína 9 Associada à CRISPR/química , Sistemas CRISPR-Cas , DNA Intergênico/química , DNA/química , Streptococcus thermophilus/genética , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , DNA/genética , DNA/metabolismo , DNA Intergênico/genética , DNA Intergênico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus thermophilus/metabolismo , Especificidade por SubstratoRESUMO
Ryanodine receptors (RyRs) are intracellular Ca2+ release channels controlling essential cellular functions. RyRs are targeted by cyclic AMP (cAMP)-dependent protein kinase A (PKA), a controversial regulation implicated in disorders ranging from heart failure to Alzheimer's. Using crystal structures, we show that the phosphorylation hotspot domain of RyR2 embraces the PKA catalytic subunit, with an extensive interface not seen in PKA complexes with peptides. We trapped an intermediary open-form PKA bound to the RyR2 domain and an ATP analog, showing that PKA can engage substrates in an open form. Phosphomimetics or prior phosphorylation at nearby sites in RyR2 either enhance or reduce the activity of PKA. Finally, we show that a phosphomimetic at S2813, a well-known target site for calmodulin-dependent kinase II, induces the formation of an alpha helix in the phosphorylation domain, resulting in increased interactions and PKA activity. This shows that the different phosphorylation sites in RyR2 are not independent.
Assuntos
Cálcio/química , Proteínas Quinases Dependentes de AMP Cíclico/química , AMP Cíclico/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Animais , Sítios de Ligação , Cálcio/metabolismo , Clonagem Molecular , Cristalografia por Raios X , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Camundongos , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
FANCA is a component of the Fanconi anemia (FA) core complex that activates DNA interstrand crosslink repair by monoubiquitination of FANCD2. Here, we report that purified FANCA protein catalyzes bidirectional single-strand annealing (SA) and strand exchange (SE) at a level comparable to RAD52, while a disease-causing FANCA mutant, F1263Δ, is defective in both activities. FANCG, which directly interacts with FANCA, dramatically stimulates its SA and SE activities. Alternatively, FANCB, which does not directly interact with FANCA, does not stimulate this activity. Importantly, five other patient-derived FANCA mutants also exhibit deficient SA and SE, suggesting that the biochemical activities of FANCA are relevant to the etiology of FA. A cell-based DNA double-strand break (DSB) repair assay demonstrates that FANCA plays a direct role in the single-strand annealing sub-pathway (SSA) of DSB repair by catalyzing SA, and this role is independent of the canonical FA pathway and RAD52.
Assuntos
Reparo do DNA por Junção de Extremidades , Reparo de Erro de Pareamento de DNA , DNA/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Reparo de DNA por Recombinação , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação G da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Mariposas , Osteoblastos/citologia , Osteoblastos/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.
Assuntos
DNA/química , Histonas/química , Proteína 1 de Modelagem do Nucleossomo/química , Nucleossomos/ultraestrutura , Animais , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Radical Hidroxila/química , Modelos Moleculares , Proteína 1 de Modelagem do Nucleossomo/genética , Proteína 1 de Modelagem do Nucleossomo/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Concentração Osmolar , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
Aberrant proteins can be deleterious to cells and are cleared by the ubiquitin-proteasome system. A group of C-end degrons that are recognized by specific cullin-RING ubiquitin E3 ligases (CRLs) has recently been identified in some of these abnormal polypeptides. Here, we report three crystal structures of a CRL2 substrate receptor, KLHDC2, in complex with the diglycine-ending C-end degrons of two early-terminated selenoproteins and the N-terminal proteolytic fragment of USP1. The E3 recognizes the degron peptides in a similarly coiled conformation and cradles their C-terminal diglycine with a deep surface pocket. By hydrogen bonding with multiple backbone carbonyls of the peptides, KLHDC2 further locks in the otherwise degenerate degrons with a compact interface and unexpected high affinities. Our results reveal the structural mechanism by which KLHDC2 recognizes the simplest C-end degron and suggest a functional necessity of the E3 to tightly maintain the low abundance of its select substrates.
Assuntos
Antígenos de Neoplasias/química , Glicilglicina/química , Selenoproteínas/química , Proteases Específicas de Ubiquitina/química , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicilglicina/metabolismo , Células HEK293 , Humanos , Cinética , Simulação de Acoplamento Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismo , Spodoptera , Especificidade por Substrato , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismoRESUMO
Permeabilization of the outer mitochondrial membrane by pore-forming Bcl2 proteins is a crucial step for the induction of apoptosis. Despite a large set of data suggesting global conformational changes within pro-apoptotic Bak during pore formation, high-resolution structural details in a membrane environment remain sparse. Here, we used NMR and HDX-MS (Hydrogen deuterium exchange mass spectrometry) in lipid nanodiscs to gain important high-resolution structural insights into the conformational changes of Bak at the membrane that are dependent on a direct activation by BH3-only proteins. Furthermore, we determined the first high-resolution structure of the Bak transmembrane helix. Upon activation, α-helix 1 in the soluble domain of Bak dissociates from the protein and adopts an unfolded and dynamic potentially membrane-bound state. In line with this finding, comparative protein folding experiments with Bak and anti-apoptotic BclxL suggest that α-helix 1 in Bak is a metastable structural element contributing to its pro-apoptotic features. Consequently, mutagenesis experiments aimed at stabilizing α-helix 1 yielded Bak variants with delayed pore-forming activity. These insights will contribute to a better mechanistic understanding of Bak-mediated membrane permeabilization.