Regulation of Plasmodium falciparum transcription

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负责这种疾病的所有临床表现。这一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控，以最大限度地提高发育效率和保真度。寄生虫用来实现如此高水平转录控制的机制尚不清楚，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。我们的假设是，最近发现的一组假定的转录调控因子-Apicomplexan AP2(ApiAP2)蛋白-是寄生虫发育过程中关键转录调控因子的主要家族。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的ApiAP2蛋白和一个假定的DNA结合位点的随机文库，通过指数富集法(SELEX)基于PCR的选择性配体富集法。或者，将使用蛋白质结合微阵列(PBM)将ApiAP2蛋白质杂交到一个全面的双链寡核苷酸集合中。第二种方法是通过1)调节这些假定的调节子的转录水平和2)产生敲除寄生虫系来确定ApiAP2蛋白在体内的作用。这些基因修改的效果将使用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，将通过染色质免疫沉淀结合DNA微阵列检测(CHIP-CHIP)在体内检测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，我们计划建立并实验验证一个基于多肽的信号基序，它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转录因子结构域的大家族，由于这些蛋白在哺乳动物中没有对应的结构域，因此在抗疟疾干预方面具有很大的前景。公共卫生意义：疟疾是由疟原虫引起的一种主要世界性疾病，每年有超过5亿例病例。随着抗药性寄生虫的急剧上升，挑战是为抗疟疾战略确定和表征新的药物靶点。我们的研究计划将调查寄生虫发育过程中基因表达调节的潜在机制，作为新的治疗干预的潜在途径。

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.