RESUMO
Beneficial mutations that arise in an evolving asexual population may compete or interact in ways that alter the overall rate of adaptation through mechanisms such as clonal or functional interference. The application of multiple selective pressures simultaneously may allow for a greater number of adaptive mutations, increasing the opportunities for competition between selectively advantageous alterations, and thereby reducing the rate of adaptation. We evolved a strain of Saccharomyces cerevisiae that could not produce its own histidine or uracil for ~500 generations under one or three selective pressures: limitation of the concentration of glucose, histidine, and/or uracil in the media. The rate of adaptation was obtained by measuring evolved relative fitness using competition assays. Populations evolved under a single selective pressure showed a statistically significant increase in fitness on those pressures relative to the ancestral strain, but the populations evolved on all three pressures did not show a statistically significant increase in fitness over the ancestral strain on any single pressure. Simultaneously limiting three essential nutrients for a population of S. cerevisiae effectively slows the rate of evolution on any one of the three selective pressures applied, relative to the single selective pressure cases. We identify possible mechanisms for fitness changes seen between populations evolved on one or three limiting nutrient pressures by high-throughput sequencing. Adding multiple selective pressures to evolving disease like cancer and infectious diseases could reduce the rate of adaptation and thereby may slow disease progression, prolong drug efficacy and prevent deaths.
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MYCN amplification and 11q deletion are two inversely correlated prognostic factors of poor outcome in neuroblastoma. Here we identify common variants at 11q22.2 within MMP20 that associate with neuroblastoma cases harboring 11q deletion (rs10895322), using GWAS in 113 European-American cases and 5109 ancestry-matched controls. The association is replicated in 44 independent cases and 1902 controls. Our study yields novel insights into the genetic underpinnings of neuroblastoma, demonstrating that the inherited common variants reported contribute to the origin of intra-tumor genetic heterogeneity in neuroblastoma.Chromosomal abnormalities such as 11q deletion are associated with poor prognosis in neuroblastoma. Here, the authors perform a genome-wide association study and identify an association between a variant within a Matrix metalloproteinase (MMP) gene member, MMP20, and 11q-deletion subtype neuroblastoma.
Assuntos
Deleção Cromossômica , Metaloproteinase 20 da Matriz/genética , Neuroblastoma/genética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 11 , Estudo de Associação Genômica Ampla , Humanos , Locos de Características Quantitativas , Sequenciamento do ExomaRESUMO
Bromodomain and extraterminal motif (BET) proteins are pharmacologic targets for the treatment of diverse diseases, yet the roles of individual BET family members remain unclear. We find that BRD2, but not BRD4, co-localizes with the architectural/insulator protein CCCTC-binding factor (CTCF) genome-wide. CTCF recruits BRD2 to co-bound sites whereas BRD2 is dispensable for CTCF occupancy. Disruption of a CTCF/BRD2-occupied element positioned between two unrelated genes enables regulatory influence to spread from one gene to another, suggesting that CTCF and BRD2 form a transcriptional boundary. Accordingly, single-molecule mRNA fluorescence in situ hybridization (FISH) reveals that, upon site-specific CTCF disruption or BRD2 depletion, expression of the two genes becomes increasingly correlated. HiC shows that BRD2 depletion weakens boundaries co-occupied by CTCF and BRD2, but not those that lack BRD2. These findings indicate that BRD2 supports boundary activity, and they raise the possibility that pharmacologic BET inhibitors can influence gene expression in part by perturbing domain boundary function.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Proteínas Repressoras/metabolismo , Transcrição Gênica , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Sistemas CRISPR-Cas , Linhagem Celular , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Edição de Genes/métodos , Hibridização in Situ Fluorescente , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Imagem Individual de Molécula/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
BACKGROUND: Red blood cell (RBC) alloimmunization is a concern for patients who receive multiple or chronic transfusions. Alloimmunization prevalence in transfused patients with bone marrow failure syndrome (BMFS) is unknown. This study aimed to determine physician practice for RBC antigen matching, immunization rates, and antibody specificities in patients with BMFS. STUDY DESIGN AND METHODS: The clinical records of all patients with BMFS seen at the Children's Hospital of Philadelphia between 2001 and 2015 were reviewed. Immunization rate was determined per 100 units transfused. RESULTS: ABO/D, C, E, and K (CEK) RBC matching was requested for 21.8% of patients. A total of 3782 RBC units were transfused to 87 patients, of which 2551 (67.5%) were CEK matched and 1231 (32.5%) were ABO/D only matched. The majority of units transfused to patients on a chronic transfusion regimen were CEK matched (89.6% of 2728 units). No anti-C, -E, or -K antibodies formed in any patient during the 14-year study period. Two alloantibodies and two autoantibodies formed, resulting in a rate of 0.05 alloantibodies and 0.05 autoantibodies per 100 units transfused. The prevalence of alloimmunization was 2.3%. CONCLUSION: The rate and prevalence of RBC alloimmunization were low in patients with BMFS. CEK matching avoided alloimmunization to these antigens in chronically transfused patients.
Assuntos
Anemia Aplástica/terapia , Doenças da Medula Óssea/terapia , Transfusão de Eritrócitos/efeitos adversos , Eritrócitos/imunologia , Hemoglobinúria Paroxística/terapia , Isoanticorpos/sangue , Adolescente , Anemia Aplástica/complicações , Autoanticorpos/sangue , Antígenos de Grupos Sanguíneos/imunologia , Tipagem e Reações Cruzadas Sanguíneas , Doenças da Medula Óssea/complicações , Transtornos da Insuficiência da Medula Óssea , Criança , Pré-Escolar , Hemoglobinúria Paroxística/complicações , Humanos , Lactente , Recém-Nascido , Prevalência , Estudos Retrospectivos , Adulto JovemRESUMO
S-Acylation, S-glutathionylation, S-nitrosylation, and S-sulfenylation are prominent, chemically distinct modifications that regulate protein function, redox sensing, and trafficking. Although the biological significance of these modifications is increasingly appreciated, their integration in the proteome remains unknown. Novel mass spectrometry-based technologies identified 2,596 predominately unique sites in 1,319 mouse liver proteins under physiological conditions. Structural analysis localized the modifications in unique, evolutionary conserved protein segments, outside commonly annotated functional regions. Contrary to expectations, propensity for modification did not correlate with biophysical properties that regulate cysteine reactivity. However, the in vivo chemical reactivity is fine-tuned for specificity, demonstrated by the nominal complementation between the four modifications and quantitative proteomics which showed that a reduction in S-nitrosylation is not correlated with increased S-glutathionylation. A comprehensive survey uncovered clustering of modifications within biologically related protein networks. The data provide the first evidence for the occurrence of distinct, endogenous protein networks that undergo redox signaling through specific cysteine modifications.
Assuntos
Cisteína/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/química , Proteoma/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Espectrometria de Massas , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mapeamento de Peptídeos , Mapas de Interação de Proteínas , Proteômica/métodosRESUMO
Nitric oxide (NO) is a signaling intermediate during glutamatergic neurotransmission in the central nervous system (CNS). NO signaling is in part accomplished through cysteine S-nitrosylation, a posttranslational modification by which NO regulates protein function and signaling. In our investigation of the protein targets and functional impact of S-nitrosylation in the CNS under physiological conditions, we identified 269 S-nitrosocysteine residues in 136 proteins in the wild-type mouse brain. The number of sites was significantly reduced in the brains of mice lacking endothelial nitric oxide synthase (eNOS(-/-)) or neuronal nitric oxide synthase (nNOS(-/-)). In particular, nNOS(-/-) animals showed decreased S-nitrosylation of proteins that participate in the glutamate/glutamine cycle, a metabolic process by which synaptic glutamate is recycled or oxidized to provide energy. (15)N-glutamine-based metabolomic profiling and enzymatic activity assays indicated that brain extracts from nNOS(-/-) mice converted less glutamate to glutamine and oxidized more glutamate than those from mice of the other genotypes. GLT1 [also known as EAAT2 (excitatory amino acid transporter 2)], a glutamate transporter in astrocytes, was S-nitrosylated at Cys(373) and Cys(561) in wild-type and eNOS(-/-) mice, but not in nNOS(-/-) mice. A form of rat GLT1 that could not be S-nitrosylated at the equivalent sites had increased glutamate uptake compared to wild-type GLT1 in cells exposed to an S-nitrosylating agent. Thus, NO modulates glutamatergic neurotransmission through the selective, nNOS-dependent S-nitrosylation of proteins that govern glutamate transport and metabolism.
Assuntos
Encéfalo/metabolismo , Cisteína/metabolismo , Ácido Glutâmico/metabolismo , Óxido Nítrico/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Cromatografia Líquida , Cisteína/análogos & derivados , Cisteína/genética , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Ratos , S-Nitrosotióis/metabolismo , Espectrometria de Massas em TandemRESUMO
We report on whole-exome sequencing (WES) of 213 melanomas. Our analysis established NF1, encoding a negative regulator of RAS, as the third most frequently mutated gene in melanoma, after BRAF and NRAS. Inactivating NF1 mutations were present in 46% of melanomas expressing wild-type BRAF and RAS, occurred in older patients and showed a distinct pattern of co-mutation with other RASopathy genes, particularly RASA2. Functional studies showed that NF1 suppression led to increased RAS activation in most, but not all, melanoma cases. In addition, loss of NF1 did not predict sensitivity to MEK or ERK inhibitors. The rebound pathway, as seen by the induction of phosphorylated MEK, occurred in cells both sensitive and resistant to the studied drugs. We conclude that NF1 is a key tumor suppressor lost in melanomas, and that concurrent RASopathy gene mutations may enhance its role in melanomagenesis.
Assuntos
Exoma , Melanoma/genética , Neurofibromina 1/genética , Neoplasias Cutâneas/genética , Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Concentração Inibidora 50 , Estimativa de Kaplan-Meier , Perda de Heterozigosidade , Masculino , Melanoma/tratamento farmacológico , Melanoma/etiologia , Mutação de Sentido Incorreto , Análise de Sequência de RNA , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/etiologia , Luz Solar/efeitos adversos , Células Tumorais Cultivadas , Proteínas ras/genéticaRESUMO
Inhibitors of bromodomain and extraterminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned about how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. Pharmacologic BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, BETs promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPR-Cas9-mediated genomic engineering and shRNA approaches, we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.
Assuntos
Células Eritroides/metabolismo , Regulação da Expressão Gênica , Hematopoese/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Ligação a RNA/fisiologia , Animais , Células Cultivadas , Fator de Transcrição GATA1/genética , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/químicaRESUMO
Exome sequencing in cancer is a powerful tool for identifying mutational events across the coding region of human genes. Here, we describe computational methods that use exome sequencing reads from cancer samples to identify somatic single nucleotide variants (SNVs), copy number alterations, and short insertions and deletions (InDels). We further describe analytical methods to generate lists of driver genes with more mutational events than expected by chance.
Assuntos
Biologia Computacional , Exoma , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias/genética , Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Humanos , Mutação INDEL , Perda de Heterozigosidade , Mutação , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BRAF inhibitors improve melanoma patient survival, but resistance invariably develops. Here we report the discovery of a novel BRAF mutation that confers resistance to PLX4032 employing whole-exome sequencing of drug-resistant BRAF(V600K) melanoma cells. We further describe a new screening approach, a genome-wide piggyBac mutagenesis screen that revealed clinically relevant aberrations (N-terminal BRAF truncations and CRAF overexpression). The novel BRAF mutation, a Leu505 to His substitution (BRAF(L505H) ), is the first resistance-conferring second-site mutation identified in BRAF mutant cells. The mutation replaces a small nonpolar amino acid at the BRAF-PLX4032 interface with a larger polar residue. Moreover, we show that BRAF(L505H) , found in human prostate cancer, is itself a MAPK-activating, PLX4032-resistant oncogenic mutation. Lastly, we demonstrate that the PLX4032-resistant melanoma cells are sensitive to novel, next-generation BRAF inhibitors, especially the 'paradox-blocker' PLX8394, supporting its use in clinical trials for treatment of melanoma patients with BRAF-mutations.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sulfonamidas/farmacologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Elementos de DNA Transponíveis/genética , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/enzimologia , Melanoma/patologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Insercional/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , VemurafenibRESUMO
A common goal of tumor sequencing projects is finding genes whose mutations are selected for during tumor development. This is accomplished by choosing genes that have more non-synonymous mutations than expected from an estimated background mutation frequency. While this background frequency is unknown, it can be estimated using both the observed synonymous mutation frequency and the non-synonymous to synonymous mutation ratio. The synonymous mutation frequency can be determined across all genes or in a gene-specific manner. This choice introduces an interesting trade-off. A gene-specific frequency adjusts for an underlying mutation bias, but is difficult to estimate given missing synonymous mutation counts. Using a genome-wide synonymous frequency is more robust, but is less suited for adjusting biases. Studying four evaluation criteria for identifying genes with high non-synonymous mutation burden (reflecting preferential selection of expressed genes, genes with mutations in conserved bases, genes with many protein interactions, and genes that show loss of heterozygosity), we find that the gene-specific synonymous frequency is superior in the gene expression and protein interaction tests. In conclusion, the use of the gene-specific synonymous mutation frequency is well suited for assessing a gene's non-synonymous mutation burden.
Assuntos
Biologia Computacional/métodos , Genes Neoplásicos , Melanoma/genética , Modelos Genéticos , Taxa de Mutação , Perfilação da Expressão Gênica , Humanos , Perda de Heterozigosidade , Melanoma/metabolismo , Mutação/genética , Mutação/efeitos da radiação , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1(P29S)) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1(P29S) showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.
Assuntos
Exoma/genética , Melanoma/genética , Mutação , Neoplasias Cutâneas/genética , Proteínas rac1 de Ligação ao GTP/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Análise Mutacional de DNA , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Masculino , Melanoma/epidemiologia , Pessoa de Meia-Idade , Modelos Moleculares , Mutação/fisiologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de DNA , Neoplasias Cutâneas/epidemiologia , Neoplasias Uveais/epidemiologia , Neoplasias Uveais/genética , Proteínas rac1 de Ligação ao GTP/químicaRESUMO
Over the course of HIV infection, virus replication is facilitated by the phosphorylation of HIV proteins by human ERK1 and ERK2 mitogen-activated protein kinases (MAPKs). MAPKs are known to phosphorylate their substrates by first binding with them at a docking site. Docking site interactions could be viable drug targets because the sequences guiding them are more specific than phosphorylation consensus sites. In this study we use multiple bioinformatics tools to discover candidate MAPK docking site motifs on HIV proteins known to be phosphorylated by MAPKs, and we discuss the possibility of targeting docking sites with drugs. Using sequence alignments of HIV proteins of different subtypes, we show that MAPK docking patterns previously described for human proteins appear on the HIV matrix, Tat, and Vif proteins in a strain dependent manner, but are absent from HIV Rev and appear on all HIV Nef strains. We revise the regular expressions of previously annotated MAPK docking patterns in order to provide a subtype independent motif that annotates all HIV proteins. One revision is based on a documented human variant of one of the substrate docking motifs, and the other reduces the number of required basic amino acids in the standard docking motifs from two to one. The proposed patterns are shown to be consistent with in silico docking between ERK1 and the HIV matrix protein. The motif usage on HIV proteins is sufficiently different from human proteins in amino acid sequence similarity to allow for HIV specific targeting using small-molecule drugs.