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1.
Blood Adv ; 5(9): 2412-2425, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33956058

RESUMO

Advances in cancer genomics have revealed genomic classes of acute myeloid leukemia (AML) characterized by class-defining mutations, such as chimeric fusion genes or in genes such as NPM1, MLL, and CEBPA. These class-defining mutations frequently synergize with internal tandem duplications in FLT3 (FLT3-ITDs) to drive leukemogenesis. However, ∼20% of FLT3-ITD-positive AMLs bare no class-defining mutations, and mechanisms of leukemic transformation in these cases are unknown. To identify pathways that drive FLT3-ITD mutant AML in the absence of class-defining mutations, we performed an insertional mutagenesis (IM) screening in Flt3-ITD mice, using Sleeping Beauty transposons. All mice developed acute leukemia (predominantly AML) after a median of 73 days. Analysis of transposon insertions in 38 samples from Flt3-ITD/IM leukemic mice identified recurrent integrations at 22 loci, including Setbp1 (20/38), Ets1 (11/38), Ash1l (8/38), Notch1 (8/38), Erg (7/38), and Runx1 (5/38). Insertions at Setbp1 led exclusively to AML and activated a transcriptional program similar, but not identical, to those of NPM1-mutant and MLL-rearranged AMLs. Guide RNA targeting of Setbp1 was highly detrimental to Flt3ITD/+/Setbp1IM+, but not to Flt3ITD/+/Npm1cA/+, AMLs. Also, analysis of RNA-sequencing data from hundreds of human AMLs revealed that SETBP1 expression is significantly higher in FLT3-ITD AMLs lacking class-defining mutations. These findings propose that SETBP1 overexpression collaborates with FLT3-ITD to drive a subtype of human AML. To identify genetic vulnerabilities of these AMLs, we performed genome-wide CRISPR-Cas9 screening in Flt3ITD/+/Setbp1IM+ AMLs and identified potential therapeutic targets, including Kdm1a, Brd3, Ezh2, and Hmgcr. Our study gives new insights into epigenetic pathways that can drive AMLs lacking class-defining mutations and proposes therapeutic approaches against such cases.


Assuntos
Leucemia Mieloide Aguda , Doença Aguda , Animais , Proteínas de Ligação a DNA , Histona-Lisina N-Metiltransferase , Leucemia Mieloide Aguda/genética , Camundongos , Mutação , Proteínas Nucleares/genética
3.
Genome Biol ; 21(1): 181, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32727536

RESUMO

BACKGROUND: Glioma is the most common intrinsic brain tumor and also occurs in the spinal cord. Activating EGFR mutations are common in IDH1 wild-type gliomas. However, the cooperative partners of EGFR driving gliomagenesis remain poorly understood. RESULTS: We explore EGFR-mutant glioma evolution in conditional mutant mice by whole-exome sequencing, transposon mutagenesis forward genetic screening, and transcriptomics. We show mutant EGFR is sufficient to initiate gliomagenesis in vivo, both in the brain and spinal cord. We identify significantly recurrent somatic alterations in these gliomas including mutant EGFR amplifications and Sub1, Trp53, and Tead2 loss-of-function mutations. Comprehensive functional characterization of 96 gliomas by genome-wide piggyBac insertional mutagenesis in vivo identifies 281 known and novel EGFR-cooperating driver genes, including Cdkn2a, Nf1, Spred1, and Nav3. Transcriptomics confirms transposon-mediated effects on expression of these genes. We validate the clinical relevance of new putative tumor suppressors by showing these are frequently altered in patients' gliomas, with prognostic implications. We discover shared and distinct driver mutations in brain and spinal gliomas and confirm in vivo differential tumor suppressive effects of Pten between these tumors. Functional validation with CRISPR-Cas9-induced mutations in novel genes Tead2, Spred1, and Nav3 demonstrates heightened EGFRvIII-glioma cell proliferation. Chemogenomic analysis of mutated glioma genes reveals potential drug targets, with several investigational drugs showing efficacy in vitro. CONCLUSION: Our work elucidates functional driver landscapes of EGFR-mutant gliomas, uncovering potential therapeutic strategies, and provides new tools for functional interrogation of gliomagenesis.


Assuntos
Neoplasias do Sistema Nervoso Central/genética , Elementos de DNA Transponíveis , Receptores ErbB/genética , Genes erbB , Glioma/genética , Animais , Carcinogênese , Receptores ErbB/metabolismo , Instabilidade Genômica , Humanos , Camundongos Transgênicos , Terapia de Alvo Molecular , Mutagênese Insercional , Neoplasias Experimentais , Proteínas do Tecido Nervoso , Sequenciamento Completo do Exoma
4.
Blood ; 135(4): 269-273, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31697828

RESUMO

Although acquisition of leukemia-associated somatic mutations by 1 or more hematopoietic stem cells is inevitable with advancing age, its consequences are highly variable, ranging from clinically silent clonal hematopoiesis (CH) to leukemic progression. To investigate the influence of heritable factors on CH, we performed deep targeted sequencing of blood DNA from 52 monozygotic (MZ) and 27 dizygotic (DZ) twin pairs (aged 70-99 years). Using this highly sensitive approach, we identified CH (variant allele frequency ≥0.5%) in 62% of individuals. We did not observe higher concordance for CH within MZ twin pairs as compared with that within DZ twin pairs, or to that expected by chance. However, we did identify 2 MZ pairs in which both twins harbored identical rare somatic mutations, suggesting a shared cell of origin. Finally, in 3 MZ twin pairs harboring mutations in the same driver genes, serial blood samples taken 4 to 5 years apart showed substantial twin-to-twin variability in clonal trajectories. Our findings propose that the inherited genome does not exert a dominant influence on the behavior of adult CH and provide evidence that CH mutations may be acquired in utero.


Assuntos
Hematopoese , Leucemia/genética , Mutação , Gêmeos/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Doenças em Gêmeos/genética , Feminino , Humanos , Masculino , Gêmeos Dizigóticos/genética , Gêmeos Monozigóticos/genética
6.
PLoS Comput Biol ; 15(11): e1007337, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31682597

RESUMO

Gene expression governs cell fate, and is regulated via a complex interplay of transcription factors and molecules that change chromatin structure. Advances in sequencing-based assays have enabled investigation of these processes genome-wide, leading to large datasets that combine information on the dynamics of gene expression, transcription factor binding and chromatin structure as cells differentiate. While numerous studies focus on the effects of these features on broader gene regulation, less work has been done on the mechanisms of gene-specific transcriptional control. In this study, we have focussed on the latter by integrating gene expression data for the in vitro differentiation of murine ES cells to macrophages and cardiomyocytes, with dynamic data on chromatin structure, epigenetics and transcription factor binding. Combining a novel strategy to identify communities of related control elements with a penalized regression approach, we developed individual models to identify the potential control elements predictive of the expression of each gene. Our models were compared to an existing method and evaluated using the existing literature and new experimental data from embryonic stem cell differentiation reporter assays. Our method is able to identify transcriptional control elements in a gene specific manner that reflect known regulatory relationships and to generate useful hypotheses for further testing.


Assuntos
Diferenciação Celular/genética , Ensaios de Triagem em Larga Escala/métodos , Elementos Reguladores de Transcrição/genética , Animais , Diferenciação Celular/fisiologia , Cromatina/metabolismo , Bases de Dados Genéticas , Epigênese Genética , Epigenômica , Regulação da Expressão Gênica/genética , Genoma , Macrófagos/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/metabolismo , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo
7.
Infect Immun ; 87(2)2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30510101

RESUMO

Toxoplasma gondii is associated with physiological effects in the host. Dysregulation of catecholamines in the central nervous system has previously been observed in chronically infected animals. In the study described here, the noradrenergic system was found to be suppressed with decreased levels of norepinephrine (NE) in brains of infected animals and in infected human and rat neural cells in vitro The mechanism responsible for the NE suppression was found to be downregulation of dopamine ß-hydroxylase (DBH) gene expression, encoding the enzyme that synthesizes norepinephrine from dopamine, with downregulation observed in vitro and in infected brain tissue, particularly in the dorsal locus coeruleus/pons region. The downregulation was sex specific, with males expressing reduced DBH mRNA levels whereas females were unchanged. Rather, DBH expression correlated with estrogen receptor in the female rat brains for this estrogen-regulated gene. DBH silencing was not a general response of neurons to infection, as human cytomegalovirus did not downregulate DBH expression. The noradrenergic-linked behaviors of sociability and arousal were altered in chronically infected animals, with a high correlation between DBH expression and infection intensity. A decrease in DBH expression in noradrenergic neurons can elevate dopamine levels, which provides a possible explanation for mixed observations of changes in this neurotransmitter with infection. Decreased NE is consistent with the loss of coordination and motor impairments associated with toxoplasmosis. Further, the altered norepinephrine synthesis observed here may, in part, explain behavioral effects of infection and associations with mental illness.


Assuntos
Catecolaminas/metabolismo , Doenças do Sistema Nervoso Central/parasitologia , Dopamina beta-Hidroxilase/metabolismo , Norepinefrina/metabolismo , Toxoplasmose/metabolismo , Animais , Encéfalo/metabolismo , Doenças do Sistema Nervoso Central/metabolismo , Dopamina/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica , Camundongos , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Ratos
8.
Nat Commun ; 9(1): 5378, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30568163

RESUMO

We recently identified the splicing kinase gene SRPK1 as a genetic vulnerability of acute myeloid leukemia (AML). Here, we show that genetic or pharmacological inhibition of SRPK1 leads to cell cycle arrest, leukemic cell differentiation and prolonged survival of mice transplanted with MLL-rearranged AML. RNA-seq analysis demonstrates that SRPK1 inhibition leads to altered isoform levels of many genes including several with established roles in leukemogenesis such as MYB, BRD4 and MED24. We focus on BRD4 as its main isoforms have distinct molecular properties and find that SRPK1 inhibition produces a significant switch from the short to the long isoform at the mRNA and protein levels. This was associated with BRD4 eviction from genomic loci involved in leukemogenesis including BCL2 and MYC. We go on to show that this switch mediates at least part of the anti-leukemic effects of SRPK1 inhibition. Our findings reveal that SRPK1 represents a plausible new therapeutic target against AML.


Assuntos
Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular , Diferenciação Celular , Cromatina/metabolismo , Epigênese Genética , Células HL-60 , Hematopoese , Humanos , Células K562 , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA
9.
Nat Med ; 24(8): 1167-1177, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30013198

RESUMO

Mutations in the gene encoding isocitrate dehydrogenase 2 (IDH2) occur in several types of cancer, including acute myeloid leukemia (AML). In model systems, mutant IDH2 causes hematopoietic differentiation arrest. Enasidenib, a selective small-molecule inhibitor of mutant IDH2, produces a clinical response in 40% of treated patients with relapsed/refractory AML by promoting leukemic cell differentiation. Here, we studied the clonal basis of response and acquired resistance to enasidenib treatment. Using sequential patient samples, we determined the clonal structure of hematopoietic cell populations at different stages of differentiation. Before therapy, IDH2-mutant clones showed variable differentiation arrest. Enasidenib treatment promoted hematopoietic differentiation from either terminal or ancestral mutant clones; less frequently, treatment promoted differentiation of nonmutant cells. Analysis of paired diagnosis/relapse samples did not identify second-site mutations in IDH2 at relapse. Instead, relapse arose by clonal evolution or selection of terminal or ancestral clones, thus highlighting multiple bypass pathways that could potentially be targeted to restore differentiation arrest. These results show how mapping of clonal structure in cell populations at different stages of differentiation can reveal the response and evolution of clones during treatment response and relapse.


Assuntos
Aminopiridinas/uso terapêutico , Isocitrato Desidrogenase/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Triazinas/uso terapêutico , Aminopiridinas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Clonais , Estudos de Coortes , Hematopoese , Humanos , Imunofenotipagem , Isocitrato Desidrogenase/metabolismo , Mutação/genética , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Triazinas/farmacologia
10.
Cell Rep ; 23(13): 3905-3919, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29949773

RESUMO

In metazoans, tissues experiencing proteotoxic stress induce "transcellular chaperone signaling" (TCS) that activates molecular chaperones, such as hsp-90, in distal tissues. How this form of inter-tissue communication is mediated to upregulate systemic chaperone expression and whether it can be utilized to protect against protein misfolding diseases remain open questions. Using C. elegans, we identified key components of a systemic stress signaling pathway that links the innate immune response with proteostasis maintenance. We show that mild perturbation of proteostasis in the neurons or the intestine activates TCS via the GATA zinc-finger transcription factor PQM-1. PQM-1 coordinates neuron-activated TCS via the innate immunity-associated transmembrane protein CLEC-41, whereas intestine-activated TCS depends on the aspartic protease ASP-12. Both TCS pathways can induce hsp-90 in muscle cells and facilitate amelioration of Aß3-42-associated toxicity. This may have powerful implications for the treatment of diseases related to proteostasis dysfunction.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteostase/fisiologia , Transativadores/metabolismo , Peptídeos beta-Amiloides/toxicidade , Animais , Animais Geneticamente Modificados/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Choque Térmico HSP90/genética , Fatores de Transcrição de Choque Térmico/antagonistas & inibidores , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Imunidade Inata/fisiologia , Fatores Imunológicos/genética , Fatores Imunológicos/metabolismo , Mucosa Intestinal/metabolismo , Músculos/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/toxicidade , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Temperatura , Transativadores/antagonistas & inibidores , Transativadores/genética
11.
Blood ; 130(17): 1911-1922, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-28835438

RESUMO

NPM1 mutations define the commonest subgroup of acute myeloid leukemia (AML) and frequently co-occur with FLT3 internal tandem duplications (ITD) or, less commonly, NRAS or KRAS mutations. Co-occurrence of mutant NPM1 with FLT3-ITD carries a significantly worse prognosis than NPM1-RAS combinations. To understand the molecular basis of these observations, we compare the effects of the 2 combinations on hematopoiesis and leukemogenesis in knock-in mice. Early effects of these mutations on hematopoiesis show that compound Npm1cA/+;NrasG12D/+ or Npm1cA;Flt3ITD share a number of features: Hox gene overexpression, enhanced self-renewal, expansion of hematopoietic progenitors, and myeloid differentiation bias. However, Npm1cA;Flt3ITD mutants displayed significantly higher peripheral leukocyte counts, early depletion of common lymphoid progenitors, and a monocytic bias in comparison with the granulocytic bias in Npm1cA/+;NrasG12D/+ mutants. Underlying this was a striking molecular synergy manifested as a dramatically altered gene expression profile in Npm1cA;Flt3ITD , but not Npm1cA/+;NrasG12D/+ , progenitors compared with wild-type. Both double-mutant models developed high-penetrance AML, although latency was significantly longer with Npm1cA/+;NrasG12D/+ During AML evolution, both models acquired additional copies of the mutant Flt3 or Nras alleles, but only Npm1cA/+;NrasG12D/+ mice showed acquisition of other human AML mutations, including IDH1 R132Q. We also find, using primary Cas9-expressing AMLs, that Hoxa genes and selected interactors or downstream targets are required for survival of both types of double-mutant AML. Our results show that molecular complementarity underlies the higher frequency and significantly worse prognosis associated with NPM1c/FLT3-ITD vs NPM1/NRAS-G12D-mutant AML and functionally confirm the role of HOXA genes in NPM1c-driven AML.


Assuntos
Leucemia Mieloide Aguda/genética , Mutação/genética , Proteínas Nucleares/genética , Alelos , Animais , Diferenciação Celular , Autorrenovação Celular , Sobrevivência Celular/genética , Progressão da Doença , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Células-Tronco Multipotentes/metabolismo , Mielopoese , Proteínas Nucleares/metabolismo , Penetrância , Fenótipo , Fatores de Transcrição/genética , Transcriptoma/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo
12.
Methods Mol Biol ; 1552: 1-12, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28224487

RESUMO

A number of real-world systems have common underlying patterns among them and deducing these patterns is important for us in order to understand the environment around us. These patterns in some instances are apparent upon observation while in many others especially those found in nature are well hidden. Moreover, the inherent stochasticity in these systems introduces sufficient noise that we need models capable to handling it in order to decipher the underlying pattern. Hidden Markov model (HMM) is a probabilistic model that is frequently used for studying the hidden patterns in an observed sequence or sets of observed sequences. Since its conception in the late 1960s it has been extensively applied in biology to capture patterns in various disciplines ranging from small DNA and protein molecules, their structure and architecture that forms the basis of life to multicellular levels such as movement analysis in humans. This chapter aims at a gentle introduction to the theory of HMM, the statistical problems usually associated with HMMs and their uses in biology.


Assuntos
Algoritmos , Biologia Computacional/métodos , Cadeias de Markov , Análise de Sequência de DNA/métodos , Humanos
13.
Mol Cancer ; 15(1): 76, 2016 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-27887606

RESUMO

BACKGROUND: Transcriptional regulation of gene expression is essential for cellular differentiation and function, and defects in the process are associated with cancer. The ENCODE project has mapped potential regulatory sites across the complete genome in many cell types, and these regions have been shown to harbour many of the somatic mutations that occur in cancer cells, suggesting that their effects may drive cancer initiation and development. The ENCODE data suggests a very large number of regulatory sites, and methods are needed to identify those that are most relevant and to connect them to the genes that they control. METHODS: Predictive models of gene expression were developed by integrating the ENCODE data for regulation, including transcription factor binding and DNase1 hypersensitivity, with RNA-seq data for gene expression. A penalized regression method was used to identify the most predictive potential regulatory sites for each transcript. Known cancer somatic mutations from the COSMIC database were mapped to potential regulatory sites, and we examined differences in the mapping frequencies associated with sites chosen in regulatory models and other (rejected) sites. The effects of potential confounders, for example replication timing, were considered. RESULTS: Cancer somatic mutations preferentially occupy those regulatory regions chosen in our models as most predictive of gene expression. CONCLUSION: Our methods have identified a significantly reduced set of regulatory sites that are enriched in cancer somatic mutations and are more predictive of gene expression. This has significance for the mechanistic interpretation of cancer mutations, and the understanding of genetic regulation.


Assuntos
Mutação , Neoplasias/genética , Sequências Reguladoras de Ácido Nucleico , Sítios de Ligação , Mapeamento Cromossômico , Biologia Computacional/métodos , Bases de Dados de Ácidos Nucleicos , Desoxirribonuclease I/metabolismo , Regulação da Expressão Gênica , Humanos , Neoplasias/metabolismo , Ligação Proteica , Sítio de Iniciação de Transcrição
14.
Dev Cell ; 36(5): 572-87, 2016 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-26923725

RESUMO

Metazoan development involves the successive activation and silencing of specific gene expression programs and is driven by tissue-specific transcription factors programming the chromatin landscape. To understand how this process executes an entire developmental pathway, we generated global gene expression, chromatin accessibility, histone modification, and transcription factor binding data from purified embryonic stem cell-derived cells representing six sequential stages of hematopoietic specification and differentiation. Our data reveal the nature of regulatory elements driving differential gene expression and inform how transcription factor binding impacts on promoter activity. We present a dynamic core regulatory network model for hematopoietic specification and demonstrate its utility for the design of reprogramming experiments. Functional studies motivated by our genome-wide data uncovered a stage-specific role for TEAD/YAP factors in mammalian hematopoietic specification. Our study presents a powerful resource for studying hematopoiesis and demonstrates how such data advance our understanding of mammalian development.


Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Animais , Linhagem da Célula/fisiologia , Proteínas de Homeodomínio/metabolismo , Camundongos , Ligação Proteica/genética , Fatores de Transcrição/metabolismo
15.
EMBO J ; 31(22): 4318-33, 2012 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-23064151

RESUMO

Cell fate decisions during haematopoiesis are governed by lineage-specific transcription factors, such as RUNX1, SCL/TAL1, FLI1 and C/EBP family members. To gain insight into how these transcription factors regulate the activation of haematopoietic genes during embryonic development, we measured the genome-wide dynamics of transcription factor assembly on their target genes during the RUNX1-dependent transition from haemogenic endothelium (HE) to haematopoietic progenitors. Using a Runx1-/- embryonic stem cell differentiation model expressing an inducible Runx1 gene, we show that in the absence of RUNX1, haematopoietic genes bind SCL/TAL1, FLI1 and C/EBPß and that this early priming is required for correct temporal expression of the myeloid master regulator PU.1 and its downstream targets. After induction, RUNX1 binds to numerous de novo sites, initiating a local increase in histone acetylation and rapid global alterations in the binding patterns of SCL/TAL1 and FLI1. The acquisition of haematopoietic fate controlled by Runx1 therefore does not represent the establishment of a new regulatory layer on top of a pre-existing HE program but instead entails global reorganization of lineage-specific transcription factor assemblies.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Epigênese Genética/fisiologia , Hematopoese/fisiologia , Acetilação , Animais , Sequência de Bases , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Células-Tronco Embrionárias/fisiologia , Epigênese Genética/genética , Hematopoese/genética , Histonas/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Fatores de Transcrição/fisiologia
16.
PLoS Comput Biol ; 8(5): e1002505, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22615547

RESUMO

There are many well-known examples of proteins with low sequence similarity, adopting the same structural fold. This aspect of sequence-structure relationship has been extensively studied both experimentally and theoretically, however with limited success. Most of the studies consider remote homology or "sequence conservation" as the basis for their understanding. Recently "interaction energy" based network formalism (Protein Energy Networks (PENs)) was developed to understand the determinants of protein structures. In this paper we have used these PENs to investigate the common non-covalent interactions and their collective features which stabilize the TIM barrel fold. We have also developed a method of aligning PENs in order to understand the spatial conservation of interactions in the fold. We have identified key common interactions responsible for the conservation of the TIM fold, despite high sequence dissimilarity. For instance, the central beta barrel of the TIM fold is stabilized by long-range high energy electrostatic interactions and low-energy contiguous vdW interactions in certain families. The other interfaces like the helix-sheet or the helix-helix seem to be devoid of any high energy conserved interactions. Conserved interactions in the loop regions around the catalytic site of the TIM fold have also been identified, pointing out their significance in both structural and functional evolution. Based on these investigations, we have developed a novel network based phylogenetic analysis for remote homologues, which can perform better than sequence based phylogeny. Such an analysis is more meaningful from both structural and functional evolutionary perspective. We believe that the information obtained through the "interaction conservation" viewpoint and the subsequently developed method of structure network alignment, can shed new light in the fields of fold organization and de novo computational protein design.


Assuntos
Modelos Químicos , Modelos Moleculares , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/ultraestrutura , Sequência de Aminoácidos , Simulação por Computador , Transferência de Energia , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína
17.
Biophys J ; 99(11): 3704-15, 2010 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21112295

RESUMO

The three-dimensional structure of a protein is formed and maintained by the noncovalent interactions among the amino-acid residues of the polypeptide chain. These interactions can be represented collectively in the form of a network. So far, such networks have been investigated by considering the connections based on distances between the amino-acid residues. Here we present a method of constructing the structure network based on interaction energies among the amino-acid residues in the protein. We have investigated the properties of such protein energy-based networks (PENs) and have shown correlations to protein structural features such as the clusters of residues involved in stability, formation of secondary and super-secondary structural units. Further we demonstrate that the analysis of PENs in terms of parameters such as hubs and shortest paths can provide a variety of biologically important information, such as the residues crucial for stabilizing the folded units and the paths of communication between distal residues in the protein. Finally, the energy regimes for different levels of stabilization in the protein structure have clearly emerged from the PEN analysis.


Assuntos
Aminoácidos/metabolismo , Proteínas/química , Proteínas/metabolismo , Regulação Alostérica , Sítios de Ligação , Humanos , Ligantes , Simulação de Dinâmica Molecular , Muramidase/química , Domínios PDZ , Ligação Proteica , Termodinâmica
18.
BMC Bioinformatics ; 11 Suppl 1: S49, 2010 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-20122223

RESUMO

BACKGROUND: Thermophilic proteins sustain themselves and function at higher temperatures. Despite their structural and functional similarities with their mesophilic homologues, they show enhanced stability. Various comparative studies at genomic, protein sequence and structure levels, and experimental works highlight the different factors and dominant interacting forces contributing to this increased stability. METHODS: In this comparative structure based study, we have used interaction energies between amino acids, to generate structure networks called as Protein Energy Networks (PENs). These PENs are used to compute network, sub-graph, and node specific parameters. These parameters are then compared between the thermophile-mesophile homologues. RESULTS: The results show an increased number of clusters and low energy cliques in thermophiles as the main contributing factors for their enhanced stability. Further more, we see an increase in the number of hubs in thermophiles. We also observe no community of electrostatic cliques forming in PENs. CONCLUSION: In this study we were able to take an energy based network approach, to identify the factors responsible for enhanced stability of thermophiles, by comparative analysis. We were able to point out that the sub-graph parameters are the prominent contributing factors. The thermophiles have a better-packed hydrophobic core. We have also discussed how thermophiles, although increasing stability through higher connectivity retains conformational flexibility, from a cliques and communities perspective.


Assuntos
Biologia Computacional/métodos , Proteínas/química , Modelos Moleculares , Conformação Proteica , Temperatura , Termodinâmica
19.
Biochemistry ; 47(42): 11110-7, 2008 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-18826253

RESUMO

The irreversible dodecamerization of native Dps trimers from Mycobacterium smegmatis, in vitro, is known to be directly associated with the bimodal function of this protein. Hence it is important to explore this pathway at the molecular level. Two types of trimers, Trimer A (tA) and Trimer B (tB), can be derived from the dodecamer due to the inherent 3-fold symmetry of the spherical crystal structure. These derived trimers were expressed as protein structure graphs (PSGs) using the computed interaction strength among the residues. Interface clusters which were identified from PSGs allowed us to convincingly predict E146 and F47 for further mutation studies. Various single and double mutants were constructed and characterized. We were finally able to generate a single mutant F47E impaired in dodecamerization and a double mutant E146AF47E as native monomer in solution. These two observed results suggest that the two trimers are important for dodecamerization and that the residues selected are important for the structural stability of the protein in vitro.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Mycobacterium smegmatis/química , Mycobacterium smegmatis/genética , Substituição de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
20.
PLoS Comput Biol ; 4(9): e1000170, 2008 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-18773096

RESUMO

Protein-DNA interactions are crucial for many cellular processes. Now with the increased availability of structures of protein-DNA complexes, gaining deeper insights into the nature of protein-DNA interactions has become possible. Earlier, investigations have characterized the interface properties by considering pairwise interactions. However, the information communicated along the interfaces is rarely a pairwise phenomenon, and we feel that a global picture can be obtained by considering a protein-DNA complex as a network of noncovalently interacting systems. Furthermore, most of the earlier investigations have been carried out from the protein point of view (protein-centric), and the present network approach aims to combine both the protein-centric and the DNA-centric points of view. Part of the study involves the development of methodology to investigate protein-DNA graphs/networks with the development of key parameters. A network representation provides a holistic view of the interacting surface and has been reported here for the first time. The second part of the study involves the analyses of these graphs in terms of clusters of interacting residues and the identification of highly connected residues (hubs) along the protein-DNA interface. A predominance of deoxyribose-amino acid clusters in beta-sheet proteins, distinction of the interface clusters in helix-turn-helix, and the zipper-type proteins would not have been possible by conventional pairwise interaction analysis. Additionally, we propose a potential classification scheme for a set of protein-DNA complexes on the basis of the protein-DNA interface clusters. This provides a general idea of how the proteins interact with the different components of DNA in different complexes. Thus, we believe that the present graph-based method provides a deeper insight into the analysis of the protein-DNA recognition mechanisms by throwing more light on the nature and the specificity of these interactions.


Assuntos
Simulação por Computador , DNA/química , Modelos Moleculares , Proteínas/química , Sítios de Ligação , Biologia Computacional , Gráficos por Computador , Proteínas de Ligação a DNA/química , Bases de Dados de Ácidos Nucleicos , Bases de Dados de Proteínas , Substâncias Macromoleculares/química , Estrutura Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Estrutura Secundária de Proteína
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