RESUMEN
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.
Asunto(s)
Hemiterpenos/biosíntesis , Hevea/metabolismo , Látex/biosíntesis , Proteínas de Plantas/química , Goma/química , Transferasas/química , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismo , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Hemiterpenos/química , Hemiterpenos/metabolismo , Hevea/química , Hevea/genética , Látex/química , Látex/metabolismo , Modelos Moleculares , Compuestos Organofosforados/química , Compuestos Organofosforados/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Goma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Terpenos/química , Terpenos/metabolismo , Transferasas/genética , Transferasas/metabolismoRESUMEN
Stress granules are condensates of non-translating mRNAs and proteins involved in the stress response and neurodegenerative diseases. Stress granules form in part through intermolecular RNA-RNA interactions, and to better understand how RNA-based condensation occurs, we demonstrate that RNA is effectively recruited to the surfaces of RNA or RNP condensates in vitro. We demonstrate that, through ATP-dependent RNA binding, the DEAD-box protein eIF4A reduces RNA condensation in vitro and limits stress granule formation in cells. This defines a function for eIF4A to limit intermolecular RNA-RNA interactions in cells. These results establish an important role for eIF4A, and potentially other DEAD-box proteins, as ATP-dependent RNA chaperones that limit the condensation of RNA, analogous to the function of proteins like HSP70 in combatting protein aggregates.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4F Eucariótico de Iniciación/metabolismo , ARN Helicasas/metabolismo , ARN de Hongos/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/metabolismo , Células HeLa , Humanos , Microscopía Confocal , Unión Proteica , ARN de Hongos/aislamiento & purificación , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Imagen de Lapso de TiempoRESUMEN
Strategies for installing authentic ADP-ribosylation (ADPr) at desired positions are fundamental for creating the tools needed to explore this elusive post-translational modification (PTM) in essential cellular processes. Here, we describe a phospho-guided chemoenzymatic approach based on the Ser-ADPr writer complex for rapid, scalable preparation of a panel of pure, precisely modified peptides. Integrating this methodology with phage display technology, we have developed site-specific as well as broad-specificity antibodies to mono-ADPr. These recombinant antibodies have been selected and characterized using multiple ADP-ribosylated peptides and tested by immunoblotting and immunofluorescence for their ability to detect physiological ADPr events. Mono-ADPr proteomics and poly-to-mono comparisons at the modification site level have revealed the prevalence of mono-ADPr upon DNA damage and illustrated its dependence on PARG and ARH3. These and future tools created on our versatile chemical biology-recombinant antibody platform have broad potential to elucidate ADPr signaling pathways in health and disease.
Asunto(s)
ADP-Ribosilación , Proteínas Portadoras/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , ADP-Ribosilación/efectos de los fármacos , Secuencia de Aminoácidos , Anticuerpos/metabolismo , Bencimidazoles/farmacología , Línea Celular Tumoral , Técnicas de Visualización de Superficie Celular , Daño del ADN , Glicósido Hidrolasas/metabolismo , Histonas/metabolismo , Humanos , Fosfatos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/química , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Tirosina/metabolismoRESUMEN
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.
Asunto(s)
Artritis Juvenil/inmunología , Diferenciación Celular/inmunología , Epigénesis Genética/inmunología , Histonas/metabolismo , Leucocitos Mononucleares/metabolismo , Macrófagos/inmunología , Adolescente , Artritis Juvenil/sangre , Artritis Juvenil/genética , Sistemas CRISPR-Cas/genética , Catepsina G/genética , Catepsina G/metabolismo , Diferenciación Celular/genética , Núcleo Celular/metabolismo , Niño , Preescolar , Cromatina/metabolismo , Pruebas de Enzimas , Epigenómica , Femenino , Técnicas de Inactivación de Genes , Humanos , Células Jurkat , Elastasa de Leucocito/genética , Elastasa de Leucocito/metabolismo , Leucocitos Mononucleares/inmunología , Macrófagos/metabolismo , Masculino , Mieloblastina/genética , Mieloblastina/metabolismo , Cultivo Primario de Células , Proteolisis , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células THP-1 , Adulto JovenRESUMEN
Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.
Asunto(s)
Disulfiram/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Piroptosis/efectos de los fármacos , Sepsis/tratamiento farmacológico , Animales , Caspasa 1/genética , Caspasa 1/metabolismo , Inhibidores de Caspasas/farmacología , Caspasas/metabolismo , Caspasas Iniciadoras/genética , Caspasas Iniciadoras/metabolismo , Línea Celular Tumoral , Disulfiram/uso terapéutico , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Femenino , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/inmunología , Liposomas , Ratones , Mutagénesis Sitio-Dirigida , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Piroptosis/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sepsis/inmunología , Células Sf9 , SpodopteraRESUMEN
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.
Asunto(s)
Chlamydomonas/metabolismo , Proteínas de la Fusión de la Membrana/química , Proteínas de Plantas/química , Plasmodium/metabolismo , Proteínas Protozoarias/química , Secuencia de Aminoácidos , Evolución Biológica , Chlamydomonas/citología , Cristalografía por Rayos X , Células Germinativas/química , Células Germinativas/metabolismo , Proteínas de la Fusión de la Membrana/genética , Proteínas de la Fusión de la Membrana/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plasmodium/citología , Dominios Proteicos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Neovascularización Fisiológica/fisiología , Factor A de Crecimiento Endotelial Vascular , Empalme Alternativo , Secuencia de Aminoácidos , Inductores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/farmacología , Animales , Permeabilidad Capilar/genética , Permeabilidad Capilar/fisiología , Quimiotaxis/efectos de los fármacos , Clonación Molecular , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Cobayas , Células HEK293 , Humanos , Ratones , Mitógenos/farmacología , Mitosis/efectos de los fármacos , Mitosis/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/genética , Neuropilina-1/metabolismo , Biosíntesis de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Tirosina/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
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.
Asunto(s)
MicroARNs/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/química , Ribonucleasa III/química , Secuencia de Bases , Dimerización , Humanos , MicroARNs/genética , Datos de Secuencia Molecular , Motivos de Nucleótidos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismoRESUMEN
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.
Asunto(s)
Transporte Activo de Núcleo Celular , Mamíferos/metabolismo , Poro Nuclear/metabolismo , Regulación Alostérica , Animales , Humanos , Mamíferos/genética , Poro Nuclear/química , Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Asociadas a CRISPR/química , Sistemas CRISPR-Cas , Endodesoxirribonucleasas/química , Leptotrichia/genética , ARN Guía de Kinetoplastida/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Clonación Molecular , Microscopía por Crioelectrón , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Leptotrichia/metabolismo , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , División del ARN , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Calmodulina/química , Quinasa 5 del Receptor Acoplado a Proteína-G/química , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Calmodulina/genética , Calmodulina/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Quinasa 5 del Receptor Acoplado a Proteína-G/genética , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Cinética , Simulación de Dinámica Molecular , Fosforilación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Células Sf9 , Spodoptera , Especificidad por Sustrato , TermodinámicaRESUMEN
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.
Asunto(s)
Antígenos CD/química , Proteínas del Tejido Nervioso/química , Péptidos/química , Receptores Acoplados a Proteínas G/química , Receptores de Péptidos/química , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Sitios de Unión , Células COS , Chlorocebus aethiops , Cristalografía por Rayos X , Expresión Génica , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteolisis , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Moléculas de Adhesión Celular/química , Fructosa-Bifosfatasa/química , Péptidos y Proteínas de Señalización Intracelular/química , Complejos Multienzimáticos/química , Proteínas de Saccharomyces cerevisiae/química , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Sitios de Unión , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Microscopía por Crioelectrón , Fructosa-Bifosfatasa/genética , Fructosa-Bifosfatasa/metabolismo , Expresión Génica , Gluconeogénesis/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células K562 , Cinética , Modelos Moleculares , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de 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 , Homología Estructural de Proteína , Especificidad por Sustrato , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas de Ciclo Celular/química , Factor 4E Eucariótico de Iniciación/química , Diana Mecanicista del Complejo 1 de la Rapamicina/química , Proteína Reguladora Asociada a mTOR/química , Serina-Treonina Quinasas TOR/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaetomium/química , Chaetomium/genética , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Cinética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Modelos Moleculares , Fosforilación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Reguladora Asociada a mTOR/genética , Proteína Reguladora Asociada a mTOR/metabolismo , Transducción de Señal , Homología Estructural de Proteína , Especificidad por Sustrato , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Many neurodegenerative diseases are characterized by the accumulation of insoluble protein aggregates, including neurofibrillary tangles comprised of tau in Alzheimer's disease and Lewy bodies composed of α-synuclein in Parkinson's disease. Moreover, different pathological proteins frequently codeposit in disease brains. To test whether aggregated α-synuclein can directly cross-seed tau fibrillization, we administered preformed α-synuclein fibrils assembled from recombinant protein to primary neurons and transgenic mice. Remarkably, we discovered two distinct strains of synthetic α-synuclein fibrils that demonstrated striking differences in the efficiency of cross-seeding tau aggregation, both in neuron cultures and in vivo. Proteinase K digestion revealed conformational differences between the two synthetic α-synuclein strains and also between sarkosyl-insoluble α-synuclein extracted from two subgroups of Parkinson's disease brains. We speculate that distinct strains of pathological α-synuclein likely exist in neurodegenerative disease brains and may underlie the tremendous heterogeneity of synucleinopathies.
Asunto(s)
Neuronas/metabolismo , Enfermedad de Parkinson/patología , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Amiloide/química , Amiloide/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Enfermedad de Parkinson/metabolismo , Proteínas Recombinantes/metabolismo , alfa-Sinucleína/químicaRESUMEN
Enzymes or enzyme complexes can be concentrated in different cellular loci to modulate distinct functional processes in response to specific signals. How cells condense and compartmentalize enzyme complexes for spatiotemporally distinct cellular events is not well understood. Here we discover that specific and tight association of GIT1 and ß-Pix, a pair of GTPase regulatory enzymes, leads to phase separation of the complex without additional scaffolding molecules. GIT1/ß-Pix condensates are modular in nature and can be positioned at distinct cellular compartments, such as neuronal synapses, focal adhesions, and cell-cell junctions, by upstream adaptors. Guided by the structure of the GIT/PIX complex, we specifically probed the role of phase separation of the enzyme complex in cell migration and synapse formation. Our study suggests that formation of modular enzyme complex condensates via phase separation can dynamically concentrate limited quantities of enzymes to distinct cellular compartments for specific and optimal signaling.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Animales , Proteínas de Ciclo Celular/química , Proteínas Activadoras de GTPasa/química , Células HEK293 , Células HeLa , Humanos , Ratones , Modelos Moleculares , Paxillin/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismoRESUMEN
Steroid receptors activate gene transcription by recruiting coactivators to initiate transcription of their target genes. For most nuclear receptors, the ligand-dependent activation function domain-2 (AF-2) is a primary contributor to the nuclear receptor (NR) transcriptional activity. In contrast to other steroid receptors, such as ERα, the activation function of androgen receptor (AR) is largely dependent on its ligand-independent AF-1 located in its N-terminal domain (NTD). It remains unclear why AR utilizes a different AF domain from other receptors despite that NRs share similar domain organizations. Here, we present cryoelectron microscopy (cryo-EM) structures of DNA-bound full-length AR and its complex structure with key coactivators, SRC-3 and p300. AR dimerization follows a unique head-to-head and tail-to-tail manner. Unlike ERα, AR directly contacts a single SRC-3 and p300. The AR NTD is the primary site for coactivator recruitment. The structures provide a basis for understanding assembly of the AR:coactivator complex and its domain contributions for coactivator assembly and transcriptional regulation.
Asunto(s)
ADN/química , Proteína p300 Asociada a E1A/metabolismo , Coactivador 3 de Receptor Nuclear/metabolismo , Receptores Androgénicos/metabolismo , Microscopía por Crioelectrón , ADN/metabolismo , Proteína p300 Asociada a E1A/química , Células HEK293 , Humanos , Coactivador 3 de Receptor Nuclear/química , Conformación de Ácido Nucleico , Conformación Proteica , Receptores Androgénicos/química , Receptores Androgénicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
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.
Asunto(s)
Moléculas de Adhesión Celular/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas Receptoras/química , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/química , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Humanos , Ratones , Modelos Moleculares , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/antagonistas & inhibidores , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Receptores de la Familia Eph/antagonistas & inhibidores , Receptores de la Familia Eph/química , Receptores de la Familia Eph/genética , Receptores de la Familia Eph/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Spodoptera , Homología Estructural de Proteína , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN , Proteínas de Xenopus/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , ADN/química , ADN/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitinación , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
Argonaute proteins lie at the heart of the RNA-induced silencing complex (RISC), wherein they use small RNA guides to recognize targets. Initial insight into the architecture of Argonautes came from studies of prokaryotic proteins, revealing a crescent-shaped base made up of the amino-terminal, PAZ, middle, and PIWI domains. The recently reported crystal structure of human Argonaute-2 (hAgo2), the "slicer" in RNA interference, in complex with a mixed population of RNAs derived from insect cells provides insight into the architecture of a eukaryotic Argonaute protein with defined biochemical and biological functions. Here, we report the structure of human Ago2 bound to a physiologically relevant microRNA, microRNA-20a, at 2.2 Å resolution. The miRNA is anchored at both ends by the Mid and PAZ domains and makes several kinks and turns along the binding groove. Interestingly, miRNA binding confers remarkable stability on hAgo2, locking this otherwise flexible enzyme into a stable conformation.