Regulation of Plasmodium falciparum transcription

恶性疟原虫转录的调控

• 批准号: 8223300
• 负责人: Manuel Llinas
• 金额: $ 39.1万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8223300
• 关键词: Affect; Antimalarials; Binding; Binding Sites; Biochemistry; Biological Assay; Cell Nucleus; Cessation of life; ChIP-on-chip; Clinical; Collection; Complex; Confocal Microscopy; Culicidae; DNA; DNA Binding; DNA Binding Domain; DNA Microarray Chip; DNA-Binding Proteins; DNA-Protein Interaction; Data; Detection; Development; Disease; Drug Delivery Systems; Drug resistance; Environment; Erythrocytes; Euchromatin; Eukaryota; Evolution; Exhibits; Family; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genome; Goals; Growth; Health; Hepatocyte; Hour; Human; In Vitro; Individual; Infection; Intervention; Knock-out; Libraries; Ligands; Malaria; Measures; Methods; Modeling; Modification; Molecular; Molecular Biology; Nuclear; Nuclear Localization Signal; Oligonucleotides; Organism; Parasites; Pattern; Peptides; Phase; Plants; Plasmids; Plasmodium; Plasmodium falciparum; Protein Binding; Proteins; Recombinants; Regulation; Research; Role; Signal Transduction; Specificity; Staging; Tertiary Protein Structure; Time; Transcript; Transcription Factor AP-2 Alpha; Transcriptional Regulation; Work; base; chromatin immunoprecipitation; functional genomics; in vivo; novel; novel therapeutic intervention; nucleocytoplasmic transport; plant growth/development; promoter; tool; transcription factor

DESCRIPTION (provided by applicant): The long-term goals of the proposed work are to understand the molecular mechanisms involved in transcriptional regulation in the malaria-causing parasite Plasmodium falciparum. During the malaria infection, the red blood cell stage of Plasmodium development is responsible for all of the clinical manifestations of this disease. This developmental stage is characterized by a cyclical 48-hour growth and replication phase that must be exquisitely regulated at the transcriptional level to maximize developmental efficiency and fidelity. The mechanisms used by the parasite to achieve this high level of transcriptional control remain unclear, although there is mounting evidence that transcription is strictly regulated in this organism. Our hypothesis is that a recently identified set of putative transcriptional regulators - the Apicomplexan AP2 (ApiAP2) proteins - are a major family of key transcriptional regulators during parasite development. The role of ApiAP2 proteins as transcriptional regulators will be analyzed using biochemistry, molecular biology and functional genomics tools. The first approach will determine the DNA recognition sequences for ApiAP2 proteins in vitro using two complementary methods. One method is a PCR-based selective enrichment of ligands by exponential enrichment (SELEX) using recombinant ApiAP2 proteins and a random library of putative DNA binding sites. Alternatively, a protein binding microarray (PBM) will be used to hybridize ApiAP2 proteins to a comprehensive collection of double stranded oligonucleotides. The second approach will be to establish the in vivo role of ApiAP2 proteins by 1) modulating transcriptional levels of these putative regulators and 2) generating knockout parasite lines. The effects of these genetic modifications will be assayed globally using DNA microarrays to detect changes in gene expression. Next, the binding of ApiAP2 proteins to DNA will be measured in vivo by chromatin immunoprecipitation with DNA microarray detection (ChIP-chip). These results will define the gene sets directly regulated by the ApiAP2 proteins and will be compared to the in vitro binding results in Aim 2 as well as to prior gene expression data to establish a model for stage-specific transcriptional regulation during parasite development. Finally, we plan to establish and experimentally verify a peptide- based signaling motif that will allow us to predict the active nuclear localization of Plasmodial proteins. The ApiAP2 proteins are the first large family of putative transcription factor domains identified in the genome of P. falciparum and hold great promise for antimalarial intervention since there are no mammalian counterparts to these proteins. PUBLIC HEALTH RELEVANCE: Malaria is a major worldwide disease caused by the Plasmodium parasite with over half a billion cases annually. With the sharp rise in drug-resistant parasites, the challenge is to identify and characterize novel drug targets for anti-malarial strategies. Our research plan will investigate the mechanisms underlying the regulation of gene expression during parasite development as a potential avenue for new therapeutic interventions.

描述（由申请人提供）：拟议工作的长期目标是了解引起疟疾的寄生虫恶性疟原虫中涉及转录调控的分子机制。在疟疾感染期间，疟原虫发育的红细胞阶段负责这种疾病的所有临床表现。这个发育阶段的特征是一个周期性的48小时生长和复制阶段，必须在转录水平上进行精细调控，以最大限度地提高发育效率和保真度。寄生虫实现这种高水平转录控制的机制尚不清楚，尽管有越来越多的证据表明，转录在这种生物体中受到严格调控。我们的假设是，最近确定的一组假定的转录调节因子-顶复门AP 2（ApiAP 2）蛋白-是寄生虫发育过程中的关键转录调节因子的一个主要家族。ApiAP 2蛋白作为转录调节因子的作用将使用生物化学，分子生物学和功能基因组学工具进行分析。第一种方法将使用两种互补方法在体外确定ApiAP 2蛋白的DNA识别序列。一种方法是使用重组ApiAP 2蛋白和推定的DNA结合位点的随机文库通过指数富集（SELEX）的基于PCR的配体选择性富集。或者，蛋白质结合微阵列（PBM）将用于使ApiAP 2蛋白质与双链寡核苷酸的综合集合杂交。第二种方法将是通过1）调节这些推定的调节物的转录水平和2）产生敲除寄生虫系来建立ApiAP 2蛋白的体内作用。这些遗传修饰的影响将使用DNA微阵列在全球范围内进行分析，以检测基因表达的变化。接下来，ApiAP 2蛋白与DNA的结合将通过染色质免疫沉淀与DNA微阵列检测（ChIP-芯片）在体内测量。这些结果将定义由ApiAP 2蛋白直接调控的基因组，并将与Aim 2中的体外结合结果以及先前的基因表达数据进行比较，以建立寄生虫发育期间阶段特异性转录调控的模型。最后，我们计划建立并实验验证一个基于肽的信号基序，这将使我们能够预测疟原虫蛋白的活性核定位。ApiAP 2蛋白是在恶性疟原虫基因组中鉴定的第一个推定的转录因子结构域的大家族，并且对于抗疟干预具有很大的希望，因为这些蛋白没有哺乳动物对应物。公共卫生相关性：疟疾是由疟原虫寄生虫引起的一种主要的世界性疾病，每年有超过5亿例病例。随着抗药性寄生虫的急剧增加，挑战是识别和表征抗疟疾策略的新型药物靶标。我们的研究计划将研究寄生虫发育过程中基因表达调控的潜在机制，作为新的治疗干预措施的潜在途径。