RESUMEN
Detection of cytosolic DNA is a central element of the innate immunity system against viral infection. The Ku heterodimer, a component of the NHEJ pathway of DNA repair in the nucleus, functions as DNA sensor that detects dsDNA of viruses that replicate in the cytoplasm. Vaccinia virus expresses two proteins, C4 and C16, that inactivate DNA sensing and enhance virulence. The structural basis for this is unknown. Here we determine the structure of the C16 - Ku complex using cryoEM. Ku binds dsDNA by a preformed ring but C16 sterically blocks this access route, abrogating binding to a dsDNA end and its insertion into DNA-PK, thereby averting signalling into the downstream innate immunity system. C4 replicates these activities using a domain with 54% identity to C16. Our results reveal how vaccinia virus subverts the capacity of Ku to recognize viral DNA.
Asunto(s)
Proteínas de Unión al ADN , Virus Vaccinia , Virus Vaccinia/genética , Proteínas de Unión al ADN/metabolismo , Autoantígeno Ku/metabolismo , ADN/metabolismo , Proteína Quinasa Activada por ADN/metabolismoRESUMEN
Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo-electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/fisiología , Especificidad por Sustrato/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/fisiología , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos/metabolismo , Aminoácidos Neutros/metabolismo , Transporte Biológico/fisiología , Microscopía por Crioelectrón/métodos , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Células HeLa , Humanos , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR+ metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis.
Asunto(s)
Vesículas Extracelulares , Melanoma , Animales , Células Endoteliales/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Linfangiogénesis/fisiología , Metástasis Linfática , Melanoma/metabolismo , Ratones , Proteínas del Tejido Nervioso , Receptores de Factor de Crecimiento Nervioso/genética , Microambiente TumoralRESUMEN
Neuroblastoma (NB) is the most common extracranial pediatric solid cancer originating from undifferentiated neural crest cells. NB cells express EZH2 and GLI1 genes that are known to maintain the undifferentiated phenotype of cancer stem cells (CSC) in NB. Recent studies suggest that tumor-derived extracellular vesicles (EVs) can regulate the transformation of surrounding cells into CSC by transferring tumor-specific molecules they contain. However, the horizontal transfer of EVs molecules in NB remains largely unknown. We report the analysis of NB-derived EVs in bioengineered models of NB that are based on a collagen 1/hyaluronic acid scaffold designed to mimic the native tumor niche. Using these models, we observed an enrichment of GLI1 and EZH2 mRNAs in NB-derived EVs. As a consequence of the uptake of NB-derived EVs, the host cells increased the expression levels of GLI1 and EZH2. These results suggest the alteration of the expression profile of stromal cells through an EV-based mechanism, and point the GLI1 and EZH2 mRNAs in the EV cargo as diagnostic biomarkers in NB.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Transferencia de Gen Horizontal , Neuroblastoma/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismo , Biomarcadores , Diferenciación Celular , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/genética , Vesículas Extracelulares , Humanos , Células Madre Mesenquimatosas , Microscopía Electrónica de Rastreo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células del Estroma , Ingeniería de Tejidos , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Congenital microcephaly (MCPH) is a neurodevelopmental disease associated with mutations in genes encoding proteins involved in centrosomal and chromosomal dynamics during mitosis. Detailed MCPH pathogenesis at the cellular level is still elusive, given the diversity of MCPH genes and lack of comparative in vivo studies. By generating a series of CRISPR/Cas9-mediated genetic KOs, we report here that - whereas defects in spindle pole proteins (ASPM, MCPH5) result in mild MCPH during development - lack of centrosome (CDK5RAP2, MCPH3) or centriole (CEP135, MCPH8) regulators induces delayed chromosome segregation and chromosomal instability in neural progenitors (NPs). Our mouse model of MCPH8 suggests that loss of CEP135 results in centriole duplication defects, TP53 activation, and cell death of NPs. Trp53 ablation in a Cep135-deficient background prevents cell death but not MCPH, and it leads to subcortical heterotopias, a malformation seen in MCPH8 patients. These results suggest that MCPH in some MCPH patients can arise from the lack of adaptation to centriole defects in NPs and may lead to architectural defects if chromosomally unstable cells are not eliminated during brain development.
Asunto(s)
Centriolos/genética , Inestabilidad Cromosómica , Microcefalia/genética , Células-Madre Neurales/patología , Animales , Encéfalo/citología , Encéfalo/patología , Sistemas CRISPR-Cas/genética , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centriolos/patología , Modelos Animales de Enfermedad , Embrión de Mamíferos , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Microcefalia/patología , Microscopía Electrónica de Transmisión , Imagen Molecular , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/ultraestructura , Cultivo Primario de Células , Imagen de Lapso de Tiempo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Due to their capability to transport chemicals or proteins into target cells, cell-penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine-rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic lateral sclerosis (ALS) as a model of an arginine-rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA- and DNA-binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids, such as RNA transcription, translation, splicing and degradation, or DNA replication and repair, is impaired by the presence of the CPPs. Interestingly, the effects of (PR)n are fully mimicked by protamine, a small arginine-rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA- and DNA-binding factors from chromatin and mRNA accounts for the toxicity of arginine-rich CPPs, including those that have been recently associated with the onset of ALS.
Asunto(s)
Arginina/genética , Péptidos de Penetración Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ARN/genética , Esclerosis Amiotrófica Lateral/genética , Línea Celular Tumoral , Cromatina/genética , ADN/genética , Células HeLa , Histonas/genética , Humanos , Ácidos Nucleicos/genética , ARN/genética , Empalme del ARN/genética , ARN Mensajero/genética , Espermatogénesis/genéticaRESUMEN
Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1, and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón , ADN Helicasas/metabolismo , ARN Helicasas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , Proteínas Portadoras/genética , Clonación Molecular , ADN Helicasas/genética , Regulación Enzimológica de la Expresión Génica , Células HEK293 , Humanos , ARN Helicasas/genéticaRESUMEN
Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes, regulating important cellular processes, including cell migration, proliferation, and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo-electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to undergo a large conformational change and interact itself with the membrane in an orientation that places the active site toward the membrane. In this conformation, the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and resulting cellular activity including cancer cell invasion, while stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage-independent manner. Together, our data provide structural details of a key membrane bound state of FAK that is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediated FAK activation and signaling at focal adhesions.
Asunto(s)
Proteínas Aviares/química , Proteína-Tirosina Quinasas de Adhesión Focal/química , Membranas/química , Multimerización de Proteína , Animales , Proteínas Aviares/metabolismo , Pollos , Activación Enzimática , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Células HEK293 , Humanos , Membranas/enzimología , Relación Estructura-ActividadRESUMEN
Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The assessment of surrogate markers of tumor progression in circulating extracellular vesicles could be a powerful non-invasive approach in this setting. We have characterized extracellular vesicles purified from the lymphatic drainage also known as exudative seroma (ES) of stage III melanoma patients obtained after lymphadenectomy. Proteomic analysis showed that seroma-derived exosomes are enriched in proteins resembling melanoma progression. In addition, we found that the BRAFV600E mutation can be detected in ES-derived extracellular vesicles and its detection correlated with patients at risk of relapse.
Asunto(s)
Progresión de la Enfermedad , Vesículas Extracelulares/metabolismo , Exudados y Transudados/metabolismo , Melanoma/genética , Melanoma/metabolismo , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Seroma/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Estudios de Cohortes , Supervivencia sin Enfermedad , Drenaje , Exosomas/metabolismo , Femenino , Humanos , Metástasis Linfática , Masculino , Melanoma/patología , Persona de Mediana Edad , Proteómica/métodos , Neoplasias Cutáneas/patologíaRESUMEN
The topology and dynamics of the scanning ribosomal 43S pre-initiation complex (PIC) bound to mRNA and initiation factors (eIFs) are probably the least understood aspects of translation initiation in eukaryotes. Recently, we described a trapping mechanism in alphavirus that stalls the PIC during scanning of viral mRNA. Using this model, we were able to snapshot for the first time the eIF4A helicase bound to mRNA in a 48S initiation complex assembled in vitro. This interaction was only detected in the presence of the natural stem loop structure (DLP) located downstream from the AUG in viral mRNA that promoted stalling of the PIC, suggesting that DLP stability was enough to jam the helicase activity of eIF4A in a fraction of assembled 48S complexes. However, a substantial proportion of DLP mRNA molecules were effectively unwound by eIF4A in vitro, an activity that alphaviruses counteract in infected cells by excluding eIF4A from viral factories. Our data indicated that eIF4A-mRNA contact occurred in (or near) the ES6S region of the 40S subunit, suggesting that incoming mRNA sequences penetrate through the ES6S region during the scanning process. We propose a topological model of the scanning PIC and how some viruses have exploited this topology to translate their mRNAs with fewer eIF requirements.
Asunto(s)
Alphavirus/genética , Factor 4A Eucariótico de Iniciación/química , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/química , ARN Viral/química , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Animales , Línea Celular , Factor 4A Eucariótico de Iniciación/metabolismo , Modelos Moleculares , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismoRESUMEN
CAD, the multifunctional protein initiating and controlling de novo biosynthesis of pyrimidines in animals, self-assembles into â¼1.5 MDa hexamers. The structures of the dihydroorotase (DHO) and aspartate transcarbamoylase (ATC) domains of human CAD have been previously determined, but we lack information on how these domains associate and interact with the rest of CAD forming a multienzymatic unit. Here, we prove that a construct covering human DHO and ATC oligomerizes as a dimer of trimers and that this arrangement is conserved in CAD-like from fungi, which holds an inactive DHO-like domain. The crystal structures of the ATC trimer and DHO-like dimer from the fungus Chaetomium thermophilum confirm the similarity with the human CAD homologs. These results demonstrate that, despite being inactive, the fungal DHO-like domain has a conserved structural function. We propose a model that sets the DHO and ATC complex as the central element in the architecture of CAD.
Asunto(s)
Aspartato Carbamoiltransferasa/química , Aspartato Carbamoiltransferasa/metabolismo , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/química , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/metabolismo , Dihidroorotasa/química , Dihidroorotasa/metabolismo , Aspartato Carbamoiltransferasa/genética , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Carbamoil Fosfato/química , Carbamoil Fosfato/metabolismo , Chaetomium/enzimología , Cristalografía por Rayos X , Dihidroorotasa/genética , Humanos , Microscopía Electrónica , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Dominios Proteicos , Multimerización de Proteína , Pirimidinas/biosíntesisRESUMEN
Telomeres are specific DNA-protein structures found at both ends of eukaryotic chromosomes that protect the genome from degradation and from being recognized as double-stranded breaks. In vertebrates, telomeres are composed of tandem repeats of the TTAGGG sequence that are bound by a six-subunit complex called shelterin. Molecular mechanisms of telomere functions remain unknown in large part due to lack of structural data on shelterins, shelterin complex, and its interaction with the telomeric DNA repeats. TRF1 is one of the best studied shelterin components; however, the molecular architecture of the full-length protein remains unknown. We have used single-particle electron microscopy to elucidate the structure of TRF1 and its interaction with telomeric DNA sequence. Our results demonstrate that full-length TRF1 presents a molecular architecture that assists its interaction with telometic DNA and at the same time makes TRFH domains accessible to other TRF1 binding partners. Furthermore, our studies suggest hypothetical models on how other proteins as TIN2 and tankyrase contribute to regulate TRF1 function.
Asunto(s)
ADN/química , Secuencias Repetidas en Tándem , Telómero/química , Proteína 1 de Unión a Repeticiones Teloméricas/química , Animales , ADN/metabolismo , Ratones , Dominios Proteicos , Células Sf9 , Spodoptera , Tanquirasas/química , Tanquirasas/genética , Tanquirasas/metabolismo , Telómero/metabolismo , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/química , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismoRESUMEN
DNA replication is a key biological process that involves different protein complexes whose assembly is rigorously regulated in a successive order. One of these complexes is a replicative hexameric helicase, the MCM complex, which is essential for the initiation and elongation phases of replication. After the assembly of a double heterohexameric MCM2-7 complex at replication origins in G1, the 2 heterohexamers separate from each other and associate with Cdc45 and GINS proteins in a CMG complex that is capable of unwinding dsDNA during S phase. Here, we have reconstituted and characterized the purified human MCM2-7 (hMCM2-7) hexameric complex by co-expression of its 6 different subunits in insect cells. The conformational variability of the complex has been analyzed by single particle electron microscopy in the presence of different nucleotide analogs and DNA. The interaction with nucleotide stabilizes the complex while DNA introduces conformational changes in the hexamer inducing a cylindrical shape. Our studies suggest that the assembly of GINS and Cdc45 to the hMCM2-7 hexamer would favor conformational changes on the hexamer bound to ssDNA shifting the cylindrical shape of the complex into a right-handed spiral conformation as observed in the CMG complex bound to DNA.
Asunto(s)
ADN/metabolismo , Proteínas de Mantenimiento de Minicromosoma/química , Nucleótidos/metabolismo , Proteínas Recombinantes/química , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , ADN/química , Humanos , Imagenología Tridimensional , Proteínas de Mantenimiento de Minicromosoma/aislamiento & purificación , Proteínas de Mantenimiento de Minicromosoma/ultraestructura , Modelos Moleculares , Nucleótidos/química , Conformación Proteica , Estabilidad Proteica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
During translation initiation, eukaryotic initiation factor 2 (eIF2) delivers the Met-tRNA to the 40S ribosomal subunit to locate the initiation codon (AUGi) of mRNA during the scanning process. Stress-induced eIF2 phosphorylation leads to a general blockade of translation initiation and represents a key antiviral pathway in mammals. However, some viral mRNAs can initiate translation in the presence of phosphorylated eIF2 via stable RNA stem-loop structures (DLP; Downstream LooP) located in their coding sequence (CDS), which promote 43S preinitiation complex stalling on the initiation codon. We show here that during the scanning process, DLPs of Alphavirus mRNA become trapped in ES6S region (680-914 nt) of 18S rRNA that are projected from the solvent side of 40S subunit. This trapping can lock the progress of the 40S subunit on the mRNA in a way that places the upstream initiator AUGi on the P site of 40S subunit, obviating the participation of eIF2. Notably, the DLP structure is released from 18S rRNA upon 60S ribosomal subunit joining, suggesting conformational changes in ES6Ss during the initiation process. These novel findings illustrate how viral mRNA is threaded into the 40S subunit during the scanning process, exploiting the topology of the 40S subunit solvent side to enhance its translation in vertebrate hosts.
Asunto(s)
Alphavirus/genética , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/genética , ARN Viral/genética , Aedes , Alphavirus/metabolismo , Animales , Secuencia de Bases , Línea Celular , Codón Iniciador , Cricetinae , Regulación Viral de la Expresión Génica , Secuencias Invertidas Repetidas , Modelos Moleculares , Estabilidad del ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Ribosómico 18S/química , ARN Ribosómico 18S/fisiología , ARN Viral/química , ARN Viral/metabolismo , Ribosomas/fisiologíaRESUMEN
Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca(2+) are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca(2+) signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca(2+)-dependent recruitment of the pyrabactin resistance 1/PYR1-like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca(2+) sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca(2+)-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress.
Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Multimerización de Proteína , Transducción de Señal , Ácido Abscísico/farmacología , Proteínas de Arabidopsis/química , Sitios de Unión , Calorimetría , Membrana Celular/efectos de los fármacos , Cristalografía por Rayos X , Modelos Biológicos , Fenotipo , Fosfolípidos/química , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Soluciones , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
MuB is an ATP-dependent DNA-binding protein that regulates the activity of MuA transposase and delivers the target DNA for transposition of phage Mu. Mechanistic insight into MuB function is limited to its AAA+ ATPase module, which upon ATP binding assembles into helical filaments around the DNA. However, the structure and function of the flexible N-terminal domain (NTD) appended to the AAA+ module remains uncharacterized. Here we report the solution structure of MuB NTD determined by NMR spectroscopy. The structure reveals a compact domain formed by four α-helices connected by short loops, and confirms the presence of a helix-turn-helix motif. High structural similarity and sequence homology with λ repressor-like DNA-binding domains suggest a possible role of MuB NTD in DNA binding. We also demonstrate that the NTD directly mediates the ability of MuB to establish filament-filament interactions. These findings lead us to a model in which the NTD interacts with the AAA+ spirals and perhaps also with the DNA bound within the filament, favoring MuB polymerization and filament clustering. We propose that the MuB NTD-dependent filament interactions might be an effective mechanism to bridge distant DNA regions during Mu transposition.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas Virales/química , Sitios de Unión , ADN/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase (NRTK) with key roles in integrating growth and cell matrix adhesion signals, and FAK is a major driver of invasion and metastasis in cancer. Cell adhesion via integrin receptors is well known to trigger FAK signaling, and many of the players involved are known; however, mechanistically, FAK activation is not understood. Here, using a multidisciplinary approach, including biochemical, biophysical, structural, computational, and cell biology approaches, we provide a detailed view of a multistep activation mechanism of FAK initiated by phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Interestingly, the mechanism differs from canonical NRTK activation and is tailored to the dual catalytic and scaffolding function of FAK. We find PI(4,5)P2 induces clustering of FAK on the lipid bilayer by binding a basic region in the regulatory 4.1, ezrin, radixin, moesin homology (FERM) domain. In these clusters, PI(4,5)P2 induces a partially open FAK conformation where the autophosphorylation site is exposed, facilitating efficient autophosphorylation and subsequent Src recruitment. However, PI(4,5)P2 does not release autoinhibitory interactions; rather, Src phosphorylation of the activation loop in FAK results in release of the FERM/kinase tether and full catalytic activation. We propose that PI(4,5)P2 and its generation in focal adhesions by the enzyme phosphatidylinositol 4-phosphate 5-kinase type Iγ are important in linking integrin signaling to FAK activation.
Asunto(s)
Proteína-Tirosina Quinasas de Adhesión Focal/química , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Fosfatidilinositol 4,5-Difosfato/farmacología , Adenosina Trifosfato/farmacología , Regulación Alostérica/efectos de los fármacos , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Biocatálisis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Análisis por Conglomerados , Activación Enzimática/efectos de los fármacos , Transferencia Resonante de Energía de Fluorescencia , Proteína-Tirosina Quinasas de Adhesión Focal/ultraestructura , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Transducción de Señal/efectos de los fármacos , Familia-src Quinasas/metabolismoRESUMEN
DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of them is the replicative helicase, which is required for initiation and elongation phases. A MCM helicase found as a prophage in the genome of Bacillus cereus is fused with a primase domain constituting an integrative arrangement of two essential activities for replication. We have isolated this helicase-primase complex (BcMCM) showing that it can bind DNA and displays not only helicase and primase but also DNA polymerase activity. Using single-particle electron microscopy and 3D reconstruction, we obtained structures of BcMCM using ATPγS or ADP in the absence and presence of DNA. The complex depicts the typical hexameric ring shape. The dissection of the unwinding mechanism using site-directed mutagenesis in the Walker A, Walker B, arginine finger and the helicase channels, suggests that the BcMCM complex unwinds DNA following the extrusion model similarly to the E1 helicase from papillomavirus.
Asunto(s)
Proteínas Bacterianas/química , ADN Helicasas/química , ADN Primasa/química , Adenosina Difosfato/química , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Bacillus cereus/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , ADN/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Helicasas/ultraestructura , ADN Primasa/genética , ADN Primasa/metabolismo , ADN Primasa/ultraestructura , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleótidos/metabolismo , Modelos Moleculares , Mutación , Estructura Terciaria de ProteínaRESUMEN
Essential cell division protein FtsZ is an assembling GTPase which directs the cytokinetic ring formation in dividing bacterial cells. FtsZ shares the structural fold of eukaryotic tubulin and assembles forming tubulin-like protofilaments, but does not form microtubules. Two puzzling problems in FtsZ assembly are the nature of protofilament association and a possible mechanism for nucleated self-assembly of single-stranded protofilaments above a critical FtsZ concentration. We assembled two-dimensional arrays of FtsZ on carbon supports, studied linear polymers of FtsZ with cryo-electron microscopy of vitrified unsupported solutions, and formulated possible polymerization models. Nucleated self-assembly of FtsZ from Escherichia coli with GTP and magnesium produces flexible filaments 4-6 nm-wide, only compatible with a single protofilament. This agrees with previous scanning transmission electron microscopy results and is supported by recent cryo-electron tomography studies of two bacterial cells. Observations of double-stranded FtsZ filaments in negative stain may come from protofilament accretion on the carbon support. Preferential protofilament cyclization does not apply to FtsZ assembly. The apparently cooperative polymerization of a single protofilament with identical intermonomer contacts is explained by the switching of one inactive monomer into the active structure preceding association of the next, creating a dimer nucleus. FtsZ behaves as a cooperative linear assembly machine.