RESUMEN
Necroptosis is a regulated form of necrosis, with the dying cell rupturing and releasing intracellular components that can trigger an innate immune response. Toll-like receptor 3 and 4 agonists, tumor necrosis factor, certain viral infections, or the T cell receptor can trigger necroptosis if the activity of the protease caspase-8 is compromised. Necroptosis signaling is modulated by the kinase RIPK1 and requires the kinase RIPK3 and the pseudokinase MLKL. Either RIPK3 deficiency or RIPK1 inhibition confers resistance in various animal disease models, suggesting that inflammation caused by necroptosis contributes to tissue damage and that inhibitors of these kinases could have therapeutic potential. Recent studies have revealed unexpected complexity in the regulation of cell death programs by RIPK1 and RIPK3 with the possibility that necroptosis is but one mechanism by which these kinases promote inflammation.
Asunto(s)
Regulación de la Expresión Génica , Necrosis/genética , Proteínas Quinasas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Animales , Apoptosis , Caspasa 8/genética , Caspasa 8/inmunología , Perfilación de la Expresión Génica , Humanos , Inmunidad Innata , Inflamación , Necrosis/inmunología , Necrosis/patología , Proteínas Quinasas/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Transducción de Señal , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Conventional protein kinase C (cPKC) isozymes tune the signaling output of cells, with loss-of-function somatic mutations associated with cancer and gain-of-function germline mutations identified in neurodegeneration. PKC with impaired autoinhibition is removed from the cell by quality-control mechanisms to prevent the accumulation of aberrantly active enzyme. Here, we examine how a highly conserved residue in the C1A domain of cPKC isozymes permits quality-control degradation when mutated to histidine in cancer (PKCß-R42H) and blocks down-regulation when mutated to proline in the neurodegenerative disease spinocerebellar ataxia (PKCγ-R41P). Using FRET-based biosensors, we determined that mutation of R42 to any residue, including lysine, resulted in reduced autoinhibition as indicated by higher basal activity and faster agonist-induced plasma membrane translocation. R42 is predicted to form a stabilizing salt bridge with E655 in the C-tail and mutation of E655, but not neighboring E657, also reduced autoinhibition. Western blot analysis revealed that whereas R42H had reduced stability, the R42P mutant was stable and insensitive to activator-induced ubiquitination and down-regulation, an effect previously observed by deletion of the entire C1A domain. Molecular dynamics (MD) simulations and analysis of stable regions of the domain using local spatial pattern (LSP) alignment suggested that P42 interacts with Q66 to impair mobility and conformation of one of the ligand-binding loops. Additional mutation of Q66 to the smaller asparagine (R42P/Q66N), to remove conformational constraints, restored degradation sensitivity. Our results unveil how disease-associated mutations of the same residue in the C1A domain can toggle between gain- or loss-of-function of PKC.
Asunto(s)
Neoplasias , Enfermedades Neurodegenerativas , Humanos , Isoenzimas/metabolismo , Enfermedades Neurodegenerativas/genética , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Mutación , Neoplasias/genéticaRESUMEN
BACKGROUND: Approximately one in five breast cancers are driven by amplification and overexpression of the human epidermal growth factor receptor 2 (HER2) receptor kinase, and HER2-enriched (HER2E) is one of four major transcriptional subtypes of breast cancer. We set out to understand the genomics of HER2 amplification independent of subtype, and the underlying drivers and biology of HER2E tumors. METHODS: We investigated published genomic data from 3155 breast tumors and 5391 non-breast tumors. RESULTS: HER2 amplification is a distinct driver event seen in all breast cancer subtypes, rather than a subtype marker, with major characteristics restricted to amplification and overexpression of HER2 and neighboring genes. The HER2E subtype has a distinctive transcriptional landscape independent of HER2A that reflects androgen receptor signaling as replacement for estrogen receptor (ER)-driven tumorigenesis. HER2 amplification is also an event in 1.8% of non-breast tumors. CONCLUSIONS: These discoveries reveal therapeutic opportunities for combining anti-HER2 therapy with anti-androgen agents in breast cancer, and highlight the potential for broader therapeutic use of HER2 inhibitors.
Asunto(s)
Neoplasias de la Mama/genética , Carcinogénesis/genética , Receptor ErbB-2/genética , Receptores de Estrógenos/genética , Biomarcadores de Tumor/genética , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Receptor ErbB-2/antagonistas & inhibidores , Receptores Androgénicos/genética , Receptores de Estrógenos/antagonistas & inhibidoresRESUMEN
Organisms that protect their germ-cell lineages from damage often do so at considerable cost: limited metabolic resources become partitioned away from maintenance of the soma, leaving the ageing somatic tissues to navigate survival amid an environment containing damaged and poorly functioning proteins. Historically, experimental paradigms that limit reproductive investment result in lifespan extension. We proposed that germline-deficient animals might exhibit heightened protection from proteotoxic stressors in somatic tissues. We find that the forced re-investment of resources from the germ line to the soma in Caenorhabditis elegans results in elevated somatic proteasome activity, clearance of damaged proteins and increased longevity. This activity is associated with increased expression of rpn-6, a subunit of the 19S proteasome, by the FOXO transcription factor DAF-16. Ectopic expression of rpn-6 is sufficient to confer proteotoxic stress resistance and extend lifespan, indicating that rpn-6 is a candidate to correct deficiencies in age-related protein homeostasis disorders.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Longevidad/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Estrés Fisiológico/fisiología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Separación Celular , Femenino , Factores de Transcripción Forkhead , Regulación de la Expresión Génica , Células Germinativas/citología , Células Germinativas/metabolismo , Respuesta al Choque Térmico/genética , Homeostasis/efectos de la radiación , Longevidad/genética , Longevidad/efectos de la radiación , Masculino , Mutación/genética , Estrés Oxidativo/fisiología , Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/genética , Estrés Fisiológico/efectos de la radiación , Factores de Transcripción/metabolismo , Rayos UltravioletaRESUMEN
Specialized metabolic enzymes biosynthesize chemicals of ecological importance, often sharing a pedigree with primary metabolic enzymes. However, the lineage of the enzyme chalcone isomerase (CHI) remained unknown. In vascular plants, CHI-catalysed conversion of chalcones to chiral (S)-flavanones is a committed step in the production of plant flavonoids, compounds that contribute to attraction, defence and development. CHI operates near the diffusion limit with stereospecific control. Although associated primarily with plants, the CHI fold occurs in several other eukaryotic lineages and in some bacteria. Here we report crystal structures, ligand-binding properties and in vivo functional characterization of a non-catalytic CHI-fold family from plants. Arabidopsis thaliana contains five actively transcribed genes encoding CHI-fold proteins, three of which additionally encode amino-terminal chloroplast-transit sequences. These three CHI-fold proteins localize to plastids, the site of de novo fatty-acid biosynthesis in plant cells. Furthermore, their expression profiles correlate with those of core fatty-acid biosynthetic enzymes, with maximal expression occurring in seeds and coinciding with increased fatty-acid storage in the developing embryo. In vitro, these proteins are fatty-acid-binding proteins (FAPs). FAP knockout A. thaliana plants show elevated α-linolenic acid levels and marked reproductive defects, including aberrant seed formation. Notably, the FAP discovery defines the adaptive evolution of a stereospecific and catalytically 'perfected' enzyme from a non-enzymatic ancestor over a defined period of plant evolution.
Asunto(s)
Arabidopsis/química , Biocatálisis , Evolución Molecular , Ácidos Grasos/metabolismo , Liasas Intramoleculares/química , Liasas Intramoleculares/metabolismo , Pliegue de Proteína , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cristalografía por Rayos X , Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/deficiencia , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Liasas Intramoleculares/deficiencia , Liasas Intramoleculares/genética , Ligandos , Modelos Moleculares , Fenotipo , Unión Proteica , Estereoisomerismo , Ácido alfa-Linolénico/metabolismoRESUMEN
Although targeting cancer metabolism is a promising therapeutic strategy, clinical success will depend on an accurate diagnostic identification of tumor subtypes with specific metabolic requirements. Through broad metabolite profiling, we successfully identified three highly distinct metabolic subtypes in pancreatic ductal adenocarcinoma (PDAC). One subtype was defined by reduced proliferative capacity, whereas the other two subtypes (glycolytic and lipogenic) showed distinct metabolite levels associated with glycolysis, lipogenesis, and redox pathways, confirmed at the transcriptional level. The glycolytic and lipogenic subtypes showed striking differences in glucose and glutamine utilization, as well as mitochondrial function, and corresponded to differences in cell sensitivity to inhibitors of glycolysis, glutamine metabolism, lipid synthesis, and redox balance. In PDAC clinical samples, the lipogenic subtype associated with the epithelial (classical) subtype, whereas the glycolytic subtype strongly associated with the mesenchymal (QM-PDA) subtype, suggesting functional relevance in disease progression. Pharmacogenomic screening of an additional â¼ 200 non-PDAC cell lines validated the association between mesenchymal status and metabolic drug response in other tumor indications. Our findings highlight the utility of broad metabolite profiling to predict sensitivity of tumors to a variety of metabolic inhibitors.
Asunto(s)
Adenocarcinoma/clasificación , Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/clasificación , Carcinoma Ductal Pancreático/metabolismo , Metaboloma , Metabolómica , Adenocarcinoma/genética , Adenocarcinoma/patología , Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Proliferación Celular , Glucosa/metabolismo , Glutamina/metabolismo , Glucólisis/genética , Humanos , Concentración 50 Inhibidora , Lipogénesis/genética , Mesodermo/metabolismo , Mesodermo/patología , Metaboloma/genética , Reproducibilidad de los Resultados , Transcripción GenéticaRESUMEN
Activating AMPK or inactivating calcineurin slows ageing in Caenorhabditis elegans and both have been implicated as therapeutic targets for age-related pathology in mammals. However, the direct targets that mediate their effects on longevity remain unclear. In mammals, CREB-regulated transcriptional coactivators (CRTCs) are a family of cofactors involved in diverse physiological processes including energy homeostasis, cancer and endoplasmic reticulum stress. Here we show that both AMPK and calcineurin modulate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC. We demonstrate that CRTC-1 is a direct AMPK target, and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK or deactivating calcineurin decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of CRH-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in ageing. Together, these findings indicate a novel role for CRTCs and CREB in determining lifespan downstream of AMPK and calcineurin, and illustrate the molecular mechanisms by which an evolutionarily conserved pathway responds to low energy to increase longevity.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Calcineurina/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Longevidad/fisiología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Envejecimiento/metabolismo , Envejecimiento/fisiología , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/biosíntesis , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Inhibidores de la Calcineurina , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/biosíntesis , Regulación hacia Abajo , Metabolismo Energético , Activación Enzimática , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Longevidad/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Transactivadores/química , Transactivadores/deficiencia , Transactivadores/genética , Factores de Transcripción/biosíntesis , Transcripción GenéticaRESUMEN
ATR and Chk1 are two key protein kinases in the replication checkpoint. Activation of ATR-Chk1 has been extensively investigated, but checkpoint termination and replication fork restart are less well understood. Here, we report that DNA damage not only activates Chk1, but also exposes a degron-like region at the carboxyl terminus of Chk1 to an Fbx6-containing SCF (Skp1-Cul1-F box) E3 ligase, which mediates the ubiquitination and degradation of Chk1 and, in turn, terminates the checkpoint. The protein levels of Chk1 and Fbx6 showed an inverse correlation in both cultured cancer cells and in human breast tumor tissues. Further, we show that low levels of Fbx6 and consequent impairment of replication stress-induced Chk1 degradation are associated with cancer cell resistance to the chemotherapeutic agent, camptothecin. We propose that Fbx6-dependent Chk1 degradation contributes to S phase checkpoint termination and that a defect in this mechanism might increase tumor cell resistance to certain anticancer drugs.
Asunto(s)
Daño del ADN , Replicación del ADN , Neoplasias/enzimología , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Ligasas SKP Cullina F-box/metabolismo , Estrés Fisiológico , Antineoplásicos Fitogénicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada , Camptotecina/farmacología , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Activación Enzimática , Humanos , Lisina , Neoplasias/genética , Neoplasias/patología , Fosforilación , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Interferencia de ARN , Proteínas Ligasas SKP Cullina F-box/genética , Factores de Tiempo , UbiquitinaciónRESUMEN
Sponges are an ancient group of animals that diverged from other metazoans over 600 million years ago. Here we present the draft genome sequence of Amphimedon queenslandica, a demosponge from the Great Barrier Reef, and show that it is remarkably similar to other animal genomes in content, structure and organization. Comparative analysis enabled by the sequencing of the sponge genome reveals genomic events linked to the origin and early evolution of animals, including the appearance, expansion and diversification of pan-metazoan transcription factor, signalling pathway and structural genes. This diverse 'toolkit' of genes correlates with critical aspects of all metazoan body plans, and comprises cell cycle control and growth, development, somatic- and germ-cell specification, cell adhesion, innate immunity and allorecognition. Notably, many of the genes associated with the emergence of animals are also implicated in cancer, which arises from defects in basic processes associated with metazoan multicellularity.
Asunto(s)
Evolución Molecular , Genoma/genética , Poríferos/genética , Animales , Apoptosis/genética , Adhesión Celular/genética , Ciclo Celular/genética , Polaridad Celular/genética , Proliferación Celular , Genes/genética , Genómica , Humanos , Inmunidad Innata/genética , Modelos Biológicos , Neuronas/metabolismo , Fosfotransferasas/química , Fosfotransferasas/genética , Filogenia , Poríferos/anatomía & histología , Poríferos/citología , Poríferos/inmunología , Análisis de Secuencia de ADN , Transducción de Señal/genéticaRESUMEN
As the volume of data relating to proteins increases, researchers rely more and more on the analysis of published data, thus increasing the importance of good access to these data that vary from the supplemental material of individual articles, all the way to major reference databases with professional staff and long-term funding. Specialist protein resources fill an important middle ground, providing interactive web interfaces to their databases for a focused topic or family of proteins, using specialized approaches that are not feasible in the major reference databases. Many are labors of love, run by a single lab with little or no dedicated funding and there are many challenges to building and maintaining them. This perspective arose from a meeting of several specialist protein resources and major reference databases held at the Wellcome Trust Genome Campus (Cambridge, UK) on August 11 and 12, 2014. During this meeting some common key challenges involved in creating and maintaining such resources were discussed, along with various approaches to address them. In laying out these challenges, we aim to inform users about how these issues impact our resources and illustrate ways in which our working together could enhance their accuracy, currency, and overall value.
Asunto(s)
Bases de Datos de Proteínas/normas , Anotación de Secuencia Molecular , Proteínas , Curaduría de DatosRESUMEN
BACKGROUND: Many cancer cells show distorted epigenetic landscapes. The Cancer Genome Atlas (TCGA) project profiles thousands of tumors, allowing the discovery of somatic alterations in the epigenetic machinery and the identification of potential cancer drivers among members of epigenetic protein families. METHODS: We integrated mutation, expression, and copy number data from 5943 tumors from 13 cancer types to train a classification model that predicts the likelihood of being an oncogene (OG), tumor suppressor (TSG) or neutral gene (NG). We applied this predictor to epigenetic regulator genes (ERGs), and used differential expression and correlation network analysis to identify dysregulated ERGs along with co-expressed cancer genes. Furthermore, we quantified global proteomic changes by mass spectrometry after EZH2 inhibition. RESULTS: Mutation-based classifiers uncovered the OG-like profile of DNMT3A and TSG-like profiles for several ERGs. Differential gene expression and correlation network analyses revealed that EZH2 is the most significantly over-expressed ERG in cancer and is co-regulated with a cell cycle network. Proteomic analysis showed that EZH2 inhibition induced down-regulation of cell cycle regulators in lymphoma cells. CONCLUSIONS: Using classical driver genes to train an OG/TSG predictor, we determined the most predictive features at the gene level. Our predictor uncovered one OG and several TSGs among ERGs. Expression analyses elucidated multiple dysregulated ERGs including EZH2 as member of a co-expressed cell cycle network.
Asunto(s)
Biología Computacional , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Neoplasias/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Genes Supresores de Tumor , Humanos , Oncogenes , Complejo Represivo Polycomb 2/genética , Proteoma/genéticaRESUMEN
Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.
Asunto(s)
Janus Quinasa 2/química , Janus Quinasa 2/clasificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Receptor ErbB-3/química , Receptor ErbB-3/clasificación , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Insectos , Janus Quinasa 2/genética , Datos de Secuencia Molecular , Unión Proteica/fisiología , Receptor ErbB-3/genéticaRESUMEN
Cardiolipin is a glycerophospholipid found predominantly in the mitochondrial membranes of eukaryotes and in bacterial membranes. Cardiolipin interacts with protein complexes and plays pivotal roles in cellular energy metabolism, membrane dynamics, and stress responses. We recently identified the mitochondrial phosphatase, PTPMT1, as the enzyme that converts phosphatidylglycerolphosphate (PGP) to phosphatidylglycerol, a critical step in the de novo biosynthesis of cardiolipin. Upon examination of PTPMT1 evolutionary distribution, we found a PTPMT1-like phosphatase in the bacterium Rhodopirellula baltica. The purified recombinant enzyme dephosphorylated PGP in vitro. Moreover, its expression restored cardiolipin deficiency and reversed growth impairment in a Saccharomyces cerevisiae mutant lacking the yeast PGP phosphatase, suggesting that it is a bona fide PTPMT1 ortholog. When ectopically expressed, this bacterial PGP phosphatase was localized in the mitochondria of yeast and mammalian cells. Together, our results demonstrate the conservation of function between bacterial and mammalian PTPMT1 orthologs.
Asunto(s)
Bacterias/enzimología , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Fosfatidilgliceroles/química , Monoéster Fosfórico Hidrolasas/química , Secuencia de Aminoácidos , Animales , Cardiolipinas/química , Secuencia Conservada , Drosophila melanogaster , Prueba de Complementación Genética , Lípidos/química , Ratones , Mitocondrias/metabolismo , Modelos Biológicos , Modelos Genéticos , Datos de Secuencia Molecular , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans.
Asunto(s)
Células Eucariotas/metabolismo , Genoma/genética , Filogenia , Animales , Adhesión Celular , Secuencia Conservada , Células Eucariotas/clasificación , Células Eucariotas/citología , Evolución Molecular , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Especiación Genética , Proteínas Hedgehog/química , Proteínas Hedgehog/genética , Humanos , Intrones/genética , Fosfotirosina/metabolismo , Estructura Terciaria de Proteína/genética , Receptores Notch/química , Receptores Notch/genética , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
PTPMT1 (PTP localized to the Mitochondrion 1) is a member of the protein tyrosine phosphatase superfamily that is localized exclusively to the mitochondrion. We recently reported that PTPMT1 dephosphorylates phosphatidylglycerol phosphate, an essential intermediate of cardiolipin biosynthesis. To gain further insights into the molecular basis of PTPMT1 function, we determined the crystal structures of the phosphatase domain of PTPMT1. PTPMT1 exhibits a canonical protein tyrosine phosphatase domain fold, resembling many dual-specificity phosphatases such as phosphatase and tensin homolog and vaccinia H1-related phosphatase. We also determined the structure of the catalytically inactive phosphatase in complex with a surrogate substrate, phosphatidylinositol 5-phosphate, which sheds light on the substrate recognition and specificity of PTPMT1. Comparison of the apo and substrate-bound structures of PTPMT1 suggests that it undergoes significant conformational change during catalysis, and we further demonstrated that an evolutionarily conserved EEYE loop is important for its activity.
Asunto(s)
Modelos Moleculares , Fosfohidrolasa PTEN/química , Fosfohidrolasa PTEN/genética , Conformación Proteica , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cardiolipinas/biosíntesis , Cromatografía de Afinidad , Cristalización , Humanos , Funciones de Verosimilitud , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Estructura Molecular , Mutación/genética , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Recent advances in sequencing technologies have greatly increased the identification of mutations in cancer genomes. However, it remains a significant challenge to identify cancer-driving mutations, since most observed missense changes are neutral passenger mutations. Various computational methods have been developed to predict the effects of amino acid substitutions on protein function and classify mutations as deleterious or benign. These include approaches that rely on evolutionary conservation, structural constraints, or physicochemical attributes of amino acid substitutions. Here we review existing methods and further examine eight tools: SIFT, PolyPhen2, Condel, CHASM, mCluster, logRE, SNAP, and MutationAssessor, with respect to their coverage, accuracy, availability and dependence on other tools. RESULTS: Single nucleotide polymorphisms with high minor allele frequencies were used as a negative (neutral) set for testing, and recurrent mutations from the COSMIC database as well as novel recurrent somatic mutations identified in very recent cancer studies were used as positive (non-neutral) sets. Conservation-based methods generally had moderately high accuracy in distinguishing neutral from deleterious mutations, whereas the performance of machine learning based predictors with comprehensive feature spaces varied between assessments using different positive sets. MutationAssessor consistently provided the highest accuracies. For certain combinations metapredictors slightly improved the performance of included individual methods, but did not outperform MutationAssessor as stand-alone tool. CONCLUSIONS: Our independent assessment of existing tools reveals various performance disparities. Cancer-trained methods did not improve upon more general predictors. No method or combination of methods exceeds 81% accuracy, indicating there is still significant room for improvement for driver mutation prediction, and perhaps more sophisticated feature integration is needed to develop a more robust tool.
Asunto(s)
Sustitución de Aminoácidos/genética , Biología Computacional/métodos , Mutación Missense/genética , Neoplasias/genética , Programas Informáticos , Secuencia de Aminoácidos , Bases de Datos Genéticas , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Curva ROC , Sensibilidad y EspecificidadRESUMEN
The mushroom Coprinopsis cinerea is a classic experimental model for multicellular development in fungi because it grows on defined media, completes its life cycle in 2 weeks, produces some 10(8) synchronized meiocytes, and can be manipulated at all stages in development by mutation and transformation. The 37-megabase genome of C. cinerea was sequenced and assembled into 13 chromosomes. Meiotic recombination rates vary greatly along the chromosomes, and retrotransposons are absent in large regions of the genome with low levels of meiotic recombination. Single-copy genes with identifiable orthologs in other basidiomycetes are predominant in low-recombination regions of the chromosome. In contrast, paralogous multicopy genes are found in the highly recombining regions, including a large family of protein kinases (FunK1) unique to multicellular fungi. Analyses of P450 and hydrophobin gene families confirmed that local gene duplications drive the expansions of paralogous copies and the expansions occur in independent lineages of Agaricomycotina fungi. Gene-expression patterns from microarrays were used to dissect the transcriptional program of dikaryon formation (mating). Several members of the FunK1 kinase family are differentially regulated during sexual morphogenesis, and coordinate regulation of adjacent duplications is rare. The genomes of C. cinerea and Laccaria bicolor, a symbiotic basidiomycete, share extensive regions of synteny. The largest syntenic blocks occur in regions with low meiotic recombination rates, no transposable elements, and tight gene spacing, where orthologous single-copy genes are overrepresented. The chromosome assembly of C. cinerea is an essential resource in understanding the evolution of multicellularity in the fungi.
Asunto(s)
Cromosomas Fúngicos/genética , Coprinus/genética , Evolución Molecular , Secuencia de Bases , Mapeo Cromosómico , Coprinus/citología , Coprinus/crecimiento & desarrollo , Sistema Enzimático del Citocromo P-450/genética , Cartilla de ADN/genética , Proteínas Fúngicas/genética , Duplicación de Gen , Genoma Fúngico , Meiosis/genética , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Proteínas Quinasas/genética , ARN de Hongos/genética , Recombinación Genética , Retroelementos/genéticaRESUMEN
Conventional protein kinase C (PKC) isozymes tune the signaling output of cells, with loss-of-function somatic mutations associated with cancer and gain-of-function germline mutations identified in neurodegeneration. PKC with impaired autoinhibition is removed from the cell by quality-control mechanisms to prevent accumulation of aberrantly active enzyme. Here, we examine how a single residue in the C1A domain of PKCß, arginine 42 (R42), permits quality-control degradation when mutated to histidine in cancer (R42H) and blocks downregulation when mutated to proline in the neurodegenerative disease spinocerebellar ataxia (R42P). Using FRET-based biosensors, we determined that mutation of R42 to any residue, including lysine, resulted in reduced autoinhibition as indicated by higher basal activity and faster agonist-induced plasma membrane translocation. R42 is predicted to form a stabilizing salt bridge with E655 in the C-tail and mutation of E655, but not neighboring E657, also reduced autoinhibition. Western blot analysis revealed that whereas R42H had reduced stability, the R42P mutant was stable and insensitive to activator-induced ubiquitination and downregulation, an effect previously observed by deletion of the entire C1A domain. Molecular dynamics (MD) simulations and analysis of stable regions of the domain using local spatial pattern (LSP) alignment suggested that P42 interacts with Q66 to impair mobility and conformation of one of the ligand-binding loops. Additional mutation of Q66 to the smaller asparagine (R42P/Q66N), to remove conformational constraints, restored degradation sensitivity to that of WT. Our results unveil how disease-associated mutations of the same residue in the C1A domain can toggle between gain- or loss-of-function of PKC.
RESUMEN
A new family of adenylyltransferases, defined by the presence of a Fic domain, was recently discovered to catalyze the addition of adenosine monophosphate (AMP) to Rho GTPases (Yarbrough, M. L., Li, Y., Kinch, L. N., Grishin, N. V., Ball, H. L., and Orth, K. (2009) Science 323, 269-272; Worby, C. A., Mattoo, S., Kruger, R. P., Corbeil, L. B., Koller, A., Mendez, J. C., Zekarias, B., Lazar, C., and Dixon, J. E. (2009) Mol. Cell 34, 93-103). This adenylylation event inactivates Rho GTPases by preventing them from binding to their downstream effectors. We reported that the Fic domain(s) of the immunoglobulin-binding protein A (IbpA) from the pathogenic bacterium Histophilus somni adenylylates mammalian Rho GTPases, RhoA, Rac1, and Cdc42, thereby inducing host cytoskeletal collapse, which allows H. somni to breach alveolar barriers and cause septicemia. The IbpA-mediated adenylylation occurs on a functionally critical tyrosine in the switch 1 region of these GTPases. Here, we conduct a detailed characterization of the IbpA Fic2 domain and compare its activity with other known Fic adenylyltransferases, VopS (Vibrio outer protein S) from the bacterial pathogen Vibrio parahaemolyticus and the human protein HYPE (huntingtin yeast interacting protein E; also called FicD). We also included the Fic domains of the secreted protein, PfhB2, from the opportunistic pathogen Pasteurella multocida, in our analysis. PfhB2 shares a common domain architecture with IbpA and contains two Fic domains. We demonstrate that the PfhB2 Fic domains also possess adenylyltransferase activity that targets the switch 1 tyrosine of Rho GTPases. Comparative kinetic and phylogenetic analyses of IbpA-Fic2 with the Fic domains of PfhB2, VopS, and HYPE reveal important aspects of their specificities for Rho GTPases and nucleotide usage and offer mechanistic insights for determining nucleotide and substrate specificities for these enzymes. Finally, we compare the evolutionary lineages of Fic proteins with those of other known adenylyltransferases.
Asunto(s)
Proteínas Bacterianas/química , Evolución Molecular , Proteínas de la Membrana/química , Nucleotidiltransferasas/química , Pasteurellaceae/enzimología , Proteínas Bacterianas/genética , Humanos , Proteínas de la Membrana/genética , Nucleotidiltransferasas/genética , Pasteurellaceae/genética , Estructura Terciaria de Proteína , Vibrio parahaemolyticus/enzimología , Vibrio parahaemolyticus/genéticaRESUMEN
Chemical modulation of histone deacetylase (HDAC) activity by HDAC inhibitors (HDACi) is an increasingly important approach for modifying the etiology of human disease. Loss-of-function diseases arise as a consequence of protein misfolding and degradation, which lead to system failures. The DeltaF508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) results in the absence of the cell surface chloride channel and a loss of airway hydration, leading to the premature lung failure and reduced lifespan responsible for cystic fibrosis. We now show that the HDACi suberoylanilide hydroxamic acid (SAHA) restores surface channel activity in human primary airway epithelia to levels that are 28% of those of wild-type CFTR. Biological silencing of all known class I and II HDACs reveals that HDAC7 plays a central role in restoration of DeltaF508 function. We suggest that the tunable capacity of HDACs can be manipulated by chemical biology to counter the onset of cystic fibrosis and other human misfolding disorders.