Regulation of Plasmodium falciparum transcription

恶性疟原虫转录的调控

基本信息

批准号：
8013635
负责人：
Manuel Llinas
金额：
$ 39.06万
依托单位：
PRINCETON UNIVERSITY
依托单位国家：
美国
项目类别：
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/8013635
关键词：
Affect Antimalarials Binding Binding Sites Biochemistry Biological Assay Cell Nucleus Cessation of life 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

项目摘要

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转录调控所涉及的分子机制。在疟疾感染期间，疟原虫发育的红细胞阶段是导致该疾病所有临床表现的原因。该发育阶段的特点是周期性的 48 小时生长和复制阶段，必须在转录水平上进行精细调节，以最大限度地提高发育效率和保真度。尽管越来越多的证据表明转录在这种生物体中受到严格调控，但寄生虫用于实现这种高水平转录控制的机制仍不清楚。我们的假设是，最近发现的一组假定的转录调节因子——Apicomplexan AP2 (ApiAP2) 蛋白——是寄生虫发育过程中关键转录调节因子的一个主要家族。将使用生物化学、分子生物学和功能基因组学工具来分析 ApiAP2 蛋白作为转录调节因子的作用。第一种方法将使用两种互补的方法在体外确定 ApiAP2 蛋白的 DNA 识别序列。一种方法是使用重组 ApiAP2 蛋白和推定 DNA 结合位点的随机文库，通过指数富集 (SELEX) 进行基于 PCR 的配体选择性富集。或者，蛋白质结合微阵列 (PBM) 将用于将 ApiAP2 蛋白质与双链寡核苷酸的全面集合杂交。第二种方法是通过 1) 调节这些假定调节因子的转录水平和 2) 生成敲除寄生虫系来确定 ApiAP2 蛋白的体内作用。这些基因修饰的影响将在全球范围内使用 DNA 微阵列进行分析，以检测基因表达的变化。接下来，将通过染色质免疫沉淀和 DNA 微阵列检测（ChIP 芯片）在体内测量 ApiAP2 蛋白与 DNA 的结合。这些结果将定义由 ApiAP2 蛋白直接调控的基因集，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，以建立寄生虫发育过程中阶段特异性转录调控的模型。最后，我们计划建立并通过实验验证基于肽的信号基序，这将使我们能够预测疟原虫蛋白的活性核定位。 ApiAP2 蛋白是在恶性疟原虫基因组中发现的第一个假定转录因子结构域大家族，由于这些蛋白在哺乳动物中没有对应物，因此在抗疟干预方面具有广阔的前景。公共卫生相关性：疟疾是由疟原虫寄生虫引起的世界范围内的一种主要疾病，每年有超过 5 亿例病例。随着耐药寄生虫的急剧增加，挑战在于识别和表征抗疟策略的新药物靶点。我们的研究计划将调查寄生虫发育过程中基因表达调控的机制，作为新治疗干预的潜在途径。