RESUMO
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
Chromatin undergoes extensive reprogramming during immune cell differentiation. Here we report the repression of controlled histone H3 amino terminus proteolytic cleavage (H3ΔN) during monocyte-to-macrophage development. This abundant histone mark in human peripheral blood monocytes is catalyzed by neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase and proteinase 3. NSPs are repressed as monocytes mature into macrophages. Integrative epigenomic analysis reveals widespread H3ΔN distribution across the genome in a monocytic cell line and primary monocytes, which becomes largely undetectable in fully differentiated macrophages. H3ΔN is enriched at permissive chromatin and actively transcribed genes. Simultaneous NSP depletion in monocytic cells results in H3ΔN loss and further increase in chromatin accessibility, which likely primes the chromatin for gene expression reprogramming. Importantly, H3ΔN is reduced in monocytes from patients with systemic juvenile idiopathic arthritis, an autoinflammatory disease with prominent macrophage involvement. Overall, we uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.
Assuntos
Artrite Juvenil/imunologia , Diferenciação Celular/imunologia , Epigênese Genética/imunologia , Histonas/metabolismo , Leucócitos Mononucleares/metabolismo , Macrófagos/imunologia , Adolescente , Artrite Juvenil/sangue , Artrite Juvenil/genética , Sistemas CRISPR-Cas/genética , Catepsina G/genética , Catepsina G/metabolismo , Diferenciação Celular/genética , Núcleo Celular/metabolismo , Criança , Pré-Escolar , Cromatina/metabolismo , Ensaios Enzimáticos , Epigenômica , Feminino , Técnicas de Inativação de Genes , Humanos , Células Jurkat , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Leucócitos Mononucleares/imunologia , Macrófagos/metabolismo , Masculino , Mieloblastina/genética , Mieloblastina/metabolismo , Cultura Primária de Células , Proteólise , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células THP-1 , Adulto JovemRESUMO
Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.
Assuntos
Dissulfiram/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas de Ligação a Fosfato/antagonistas & inibidores , Piroptose/efeitos dos fármacos , Sepse/tratamento farmacológico , Animais , Caspase 1/genética , Caspase 1/metabolismo , Inibidores de Caspase/farmacologia , Caspases/metabolismo , Caspases Iniciadoras/genética , Caspases Iniciadoras/metabolismo , Linhagem Celular Tumoral , Dissulfiram/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Feminino , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Lipossomos , Camundongos , Mutagênese Sítio-Dirigida , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Piroptose/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sepse/imunologia , Células Sf9 , SpodopteraRESUMO
Sexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell. The sperm-restricted single-pass transmembrane protein HAP2-GCS1 has been postulated to function in membrane merger. Its presence in the major eukaryotic taxa-animals, plants, and protists (including important human pathogens like Plasmodium)-suggests that many eukaryotic organisms share a common gamete fusion mechanism. Here, we report combined bioinformatic, biochemical, mutational, and X-ray crystallographic studies on the unicellular alga Chlamydomonas reinhardtii HAP2 that reveal homology to class II viral membrane fusion proteins. We further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion. These results demonstrate that HAP2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion, indicating a way to block Plasmodium transmission and highlighting the impact of virus-cell genetic exchanges on the evolution of eukaryotic life.
Assuntos
Chlamydomonas/metabolismo , Proteínas de Fusão de Membrana/química , Proteínas de Plantas/química , Plasmodium/metabolismo , Proteínas de Protozoários/química , Sequência de Aminoácidos , Evolução Biológica , Chlamydomonas/citologia , Cristalografia por Raios X , Células Germinativas/química , Células Germinativas/metabolismo , Proteínas de Fusão de Membrana/genética , Proteínas de Fusão de Membrana/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plasmodium/citologia , Domínios Proteicos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
The VEGF-A isoforms play a crucial role in vascular development, and the VEGF signaling pathway is a clinically validated therapeutic target for several pathological conditions. Alternative mRNA splicing leads to the generation of multiple VEGF-A isoforms, including VEGF165. A recent study reported the presence of another isoform, VEGF-Ax, arising from programmed readthrough translation. Compared to VEGF165, VEGF-Ax has a 22-amino-acid extension in the COOH terminus and has been reported to function as a negative regulator of VEGF signaling in endothelial cells, with potent anti-angiogenic effects. Here, we show that, contrary to the earlier report, VEGF-Ax stimulates endothelial cell mitogenesis, angiogenesis, as well as vascular permeability. Accordingly, VEGF-Ax induces phosphorylation of key tyrosine residues in VEGFR-2. Notably, VEGF-Ax was less potent than VEGF165, consistent with its impaired binding to the VEGF co-receptor neuropilin-1.
Assuntos
Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular , Processamento Alternativo , Sequência de Aminoácidos , Indutores da Angiogênese/farmacologia , Inibidores da Angiogênese/farmacologia , Animais , Permeabilidade Capilar/genética , Permeabilidade Capilar/fisiologia , Quimiotaxia/efeitos dos fármacos , Clonagem Molecular , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Cobaias , Células HEK293 , Humanos , Camundongos , Mitógenos/farmacologia , Mitose/efeitos dos fármacos , Mitose/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Neuropilina-1/metabolismo , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Tirosina/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
MicroRNA (miRNA) maturation is initiated by Microprocessor composed of RNase III DROSHA and its cofactor DGCR8, whose fidelity is critical for generation of functional miRNAs. To understand how Microprocessor recognizes pri-miRNAs, we here reconstitute human Microprocessor with purified recombinant proteins. We find that Microprocessor is an â¼364 kDa heterotrimeric complex of one DROSHA and two DGCR8 molecules. Together with a 23-amino acid peptide from DGCR8, DROSHA constitutes a minimal functional core. DROSHA serves as a "ruler" by measuring 11 bp from the basal ssRNA-dsRNA junction. DGCR8 interacts with the stem and apical elements through its dsRNA-binding domains and RNA-binding heme domain, respectively, allowing efficient and accurate processing. DROSHA and DGCR8, respectively, recognize the basal UG and apical UGU motifs, which ensure proper orientation of the complex. These findings clarify controversies over the action mechanism of DROSHA and allow us to build a general model for pri-miRNA processing.
Assuntos
MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/química , Ribonuclease III/química , Sequência de Bases , Dimerização , Humanos , MicroRNAs/genética , Dados de Sequência Molecular , Motivos de Nucleotídeos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
The nuclear pore complex (NPC) arose in evolution as the cell's largest and most versatile transport channel. Current models for selective transport mediated by NPCs are focused on properties of intrinsically disordered regions of nucleoporins that bind transport factors. In contrast, structured regions are considered to provide static anchoring sites for the disordered regions without affecting transport factor binding. Here, we demonstrate allosteric coupling between a structured domain of a channel nucleoporin (Nup58) and its neighboring disordered domain in interaction with another channel nucleoporin (Nup54) and a transport factor (Kapß1). Analysis of multiple equilibria showed that multivalent interactions of Kapß1 with the disordered domains of Nup58 stabilize the neighboring structured domain associated with Nup54, shifting conformational equilibria from homo-oligomers to hetero-oligomers. Based on these and previous crystallographic results, a quantitative framework was established to describe constriction and dilation of the central channel as a function of transport factor occupancy.
Assuntos
Transporte Ativo do Núcleo Celular , Mamíferos/metabolismo , Poro Nuclear/metabolismo , Regulação Alostérica , Animais , Humanos , Mamíferos/genética , Poro Nuclear/química , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
Epstein-Barr virus (EBV) represents a major global health problem. Though it is associated with infectious mononucleosis and â¼200,000 cancers annually worldwide, a vaccine is not available. The major target of immunity is EBV glycoprotein 350/220 (gp350) that mediates attachment to B cells through complement receptor 2 (CR2/CD21). Here, we created self-assembling nanoparticles that displayed different domains of gp350 in a symmetric array. By focusing presentation of the CR2-binding domain on nanoparticles, potent neutralizing antibodies were elicited in mice and non-human primates. The structurally designed nanoparticle vaccine increased neutralization 10- to 100-fold compared to soluble gp350 by targeting a functionally conserved site of vulnerability, improving vaccine-induced protection in a mouse model. This rational approach to EBV vaccine design elicited potent neutralizing antibody responses by arrayed presentation of a conserved viral entry domain, a strategy that can be applied to other viruses.
Assuntos
Vacinas contra Herpesvirus/química , Vacinas contra Herpesvirus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Cristalografia por Raios X , Desenho de Fármacos , Feminino , Herpesvirus Humano 4 , Vacinas contra Herpesvirus/genética , Vacinas contra Herpesvirus/isolamento & purificação , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Nanopartículas/ultraestrutura , Receptores de Complemento 3d/química , Receptores de Complemento 3d/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The deployment of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to eradicate the coronavirus disease 2019 (COVID-19) pandemic. Many licensed vaccines confer protection by inducing long-lived plasma cells (LLPCs) and memory B cells (MBCs), cell types canonically generated during germinal center (GC) reactions. Here, we directly compared two vaccine platforms-mRNA vaccines and a recombinant protein formulated with an MF59-like adjuvant-looking for their abilities to quantitatively and qualitatively shape SARS-CoV-2-specific primary GC responses over time. We demonstrated that a single immunization with SARS-CoV-2 mRNA, but not with the recombinant protein vaccine, elicited potent SARS-CoV-2-specific GC B and T follicular helper (Tfh) cell responses as well as LLPCs and MBCs. Importantly, GC responses strongly correlated with neutralizing antibody production. mRNA vaccines more efficiently induced key regulators of the Tfh cell program and influenced the functional properties of Tfh cells. Overall, this study identifies SARS-CoV-2 mRNA vaccines as strong candidates for promoting robust GC-derived immune responses.
Assuntos
Anticorpos Neutralizantes/metabolismo , Linfócitos B/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , Centro Germinativo/imunologia , SARS-CoV-2/fisiologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas Sintéticas/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Células Cultivadas , Epitopos , Humanos , Ativação Linfocitária , Polissorbatos , RNA Viral/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Esqualeno , Vacinação , Vacinas de mRNARESUMO
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
Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.
Assuntos
Antígenos CD/química , Proteínas do Tecido Nervoso/química , Peptídeos/química , Receptores Acoplados a Proteínas G/química , Receptores de Peptídeos/química , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Sítios de Ligação , Células COS , Chlorocebus aethiops , Cristalografia por Raios X , Expressão Gênica , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteólise , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
How are E3 ubiquitin ligases configured to match substrate quaternary structures? Here, by studying the yeast GID complex (mutation of which causes deficiency in glucose-induced degradation of gluconeogenic enzymes), we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryoelectron microscopy (cryo-EM) structures show that, to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two minimally functional GID E3s assemble into the 20-protein Chelator-GIDSR4, which resembles an organometallic supramolecular chelate. The Chelator-GIDSR4 assembly avidly binds multiple Fbp1 degrons so that multiple Fbp1 protomers are simultaneously ubiquitylated at lysines near the allosteric and substrate binding sites. Importantly, key structural and biochemical features, including capacity for supramolecular assembly, are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical, and cell biological data, we propose that higher-order E3 ligase assembly generally enables multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Moléculas de Adesão Celular/química , Frutose-Bifosfatase/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Complexos Multienzimáticos/química , Proteínas de Saccharomyces cerevisiae/química , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sítios de Ligação , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Microscopia Crioeletrônica , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/metabolismo , Expressão Gênica , Gluconeogênese/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células K562 , Cinética , Modelos Moleculares , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Regiões Promotoras Genéticas , 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 , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Células Sf9 , Spodoptera , Homologia Estrutural de Proteína , Especificidade por Substrato , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
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
Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.
Assuntos
Moléculas de Adesão Celular/química , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/química , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/química , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Humanos , Camundongos , Modelos Moleculares , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/antagonistas & inibidores , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Receptores da Família Eph/antagonistas & inibidores , Receptores da Família Eph/química , Receptores da Família Eph/genética , Receptores da Família Eph/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Spodoptera , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
When converging replication forks meet during replication termination, the CMG (Cdc45-MCM2-7-GINS) helicase is polyubiquitylated by CRL2Lrr1 and unloaded from chromatin by the p97 ATPase. Here, we investigate the signal that triggers CMG unloading in Xenopus egg extracts using single-molecule and ensemble approaches. We show that converging CMGs pass each other and keep translocating at the same speed as before convergence, whereafter they are rapidly and independently unloaded. When CMG unloading is blocked, diverging CMGs do not support DNA synthesis, indicating that after bypass CMGs encounter the nascent lagging strands of the converging fork and then translocate along double-stranded DNA (dsDNA). However, translocation on dsDNA is not required for CMG's removal from chromatin because in the absence of nascent strand synthesis, converging CMGs are still unloaded. Moreover, recombinant CMG added to nuclear extract undergoes ubiquitylation and disassembly in the absence of any DNA, and DNA digestion triggers CMG ubiquitylation at stalled replication forks. Our findings suggest that DNA suppresses CMG ubiquitylation during elongation and that this suppression is relieved when CMGs converge, leading to CMG unloading.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA , Proteínas de Xenopus/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , DNA/química , DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitinação , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Liquid granules rich in intrinsically disordered proteins and RNA play key roles in critical cellular functions such as RNA processing and translation. Many details of the mechanism via which this occurs remain to be elucidated. Motivated by the lacuna in the field and by the prospects of developing de novo artificial granules that provide extrinsic control of translation, we report a bottom-up approach to engineer ribonucleoprotein granules composed of a recombinant RNA-binding IDP that exhibits phase behavior in water. We developed a kinetic model to illustrate that these granules inhibit translation through reversible or irreversible sequestration of mRNA. Within monodisperse droplets capable of transcription and translation, we experimentally demonstrate temporal inhibition of translation by using designer IDPs that exhibit tunable phase behavior. This work lays the foundation for developing artificial granules that promise to further our mechanistic understanding of their naturally occurring counterparts.
Assuntos
Células Artificiais/metabolismo , Grânulos Citoplasmáticos/genética , Proteínas Intrinsicamente Desordenadas/genética , Peptidomiméticos/metabolismo , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Sequência de Aminoácidos , Células Artificiais/citologia , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Elastina/química , Elastina/genética , Elastina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Biológicos , Peptidomiméticos/química , Transição de Fase , Plasmídeos/genética , Plasmídeos/metabolismo , Biossíntese de Proteínas , Engenharia de Proteínas/métodos , RNA/genética , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismoRESUMO
Abnormal processing of stressed replication forks by nucleases can cause fork collapse, genomic instability, and cell death. Despite its importance, it is poorly understood how the cell properly controls nucleases to prevent detrimental fork processing. Here, we report a signaling pathway that controls the activity of exonuclease Exo1 to prevent aberrant fork resection during replication stress. Our results indicate that replication stress elevates intracellular Ca2+ concentration ([Ca2+]i), leading to activation of CaMKK2 and the downstream kinase 5' AMP-activated protein kinase (AMPK). Following activation, AMPK directly phosphorylates Exo1 at serine 746 to promote 14-3-3 binding and inhibit Exo1 recruitment to stressed replication forks, thereby avoiding unscheduled fork resection. Disruption of this signaling pathway results in excessive ssDNA, chromosomal instability, and hypersensitivity to replication stress inducers. These findings reveal a link between [Ca2+]i and the replication stress response as well as a function of the Ca2+-CaMKK2-AMPK signaling axis in safeguarding fork structure to maintain genome stability.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Cálcio/metabolismo , Enzimas Reparadoras do DNA/genética , Reparo do DNA , Replicação do DNA , Exodesoxirribonucleases/genética , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Sinalização do Cálcio/genética , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Cromatina/química , Cromatina/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Exodesoxirribonucleases/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/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.