Dissecting the role of ApiAP2 proteins in transcriptional regulation during Plasmodium falciparum development

剖析 ApiAP2 蛋白在恶性疟原虫发育过程中转录调控中的作用

• 批准号: 9158624
• 负责人: Manuel Llinas
• 金额: $ 38.33万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-11 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9158624
• 关键词: Affect; Affinity Chromatography; Antimalarials; Binding; Binding Sites; Biochemistry; Blood; C-terminal; Cells; Cessation of life; ChIP-seq; Child; Clinical; Complex; Computer Retrieval of Information on Scientific Projects Database; Culicidae; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; DNA-Binding Proteins; Development; Disease; Erythrocytes; Family; Future; Gene Targeting; Genome; Genomics; Goals; Hour; Human; Individual; Infection; Intervention; Invaded; Link; Malaria; Measures; Molecular; Parasites; Pathogenesis; Plants; Plasmodium; Plasmodium falciparum; Population; Pregnant Women; Process; Protein Family; Proteins; Publishing; Regulation; Research; Resolution; Risk; Role; Sexual Development; Specificity; Staging; Structure; Symptoms; System; Techniques; Testing; Time; Transactivation; Transcription Factor AP-2 Alpha; Transcriptional Regulation; Transgenic Organisms; Work; base; genetic manipulation; genetic regulatory protein; genome-wide; in vivo; insight; knock-down; member; new therapeutic target; novel vaccines; parasite invasion; pathogen; programs; protein complex; protein function; tool; transcription factor; transcriptome; transmission process

Although for many years it was thought that Plasmodium parasites, the causative agents of malaria, were devoid of sequence-specific transcription factors, work from our lab and others has now demonstrated that they possess a limited number of such regulatory proteins. The apicomplexan AP2 (ApiAP2) proteins, which are plant-like in their origin, are now widely considered to be the single major family of DNA binding proteins identified in the Plasmodium genome. Our central hypothesis is that members of the ApiAP2 protein family are the major regulators of development throughout the complete lifecycle of the malaria parasite. ApiAP2 proteins are highly variable in size and contain anywhere from one to three AP2 DNA binding domains. We have measured the DNA binding specificities for all AP2 domains from Plasmodium demonstrating a broad array of DNA sequence specificities. By determining the genome-wide occurrence of these DNA motifs, we can begin to predict the functional roles for some ApiAP2 proteins. However, detailed mechanistic studies to confirm these predictions have been lagging. This proposal seeks to define the functional role for a subset of ApiAP2 proteins associated with processes critical to blood-stage development including the regulation of red blood cell invasion, commitment to gametocytogenesis and gametocyte differentiation and maturation. First, we will define the transcriptional regulatory network of PfAP2-G (PF3D7_1222600), which we have demonstrated to be a master regulator of sexual stage commitment. We will also investigate two additional ApiAP2 proteins implicated in gametocyte development (PF3D7_1317200, PF3D7_1408200) to define their role in the regulation of gametocyte differentiation. We next propose to characterize the role of PF3D7_1007700, which we predict to be a regulator of red blood cell invasion. Since PF3D7_1007700 contains three AP2 DNA binding domains, each with unique DNA binding specificity, this will allow us to dissect the contribution of individual AP2 domains to the function of this protein (and other ApiAP2 proteins by analogy). Lastly, we have identified a new conserved domain in ApiAP2 proteins and will initiate efforts to characterize the structure and function of this domain as well as seeking to define transactivation regions within these proteins. To accomplish our goals, we will use a combination of established techniques, including genetic manipulation of the parasite, biochemistry, and genome-wide approaches. Understanding the molecular mechanisms of transcriptional regulatory processes in P. falciparum will provide critically needed tools for studying parasite development, and also presents therapeutic targets for new intervention strategies aimed at blocking parasite maturation. ApiAP2 proteins are strictly found in Apicomplexan parasites and therefore are attractive candidates for new antimalarial therapies.

尽管多年来人们一直认为疟疾的病原体--疟原虫， 缺乏序列特异的转录因子，我们实验室和其他实验室的工作现在已经 证明它们拥有数量有限的这种调节蛋白。顶端复合体AP2 (ApiAP2)蛋白在起源上类似于植物，现在被广泛认为是单一的主要 在疟原虫基因组中发现的DNA结合蛋白家族。我们的中心假设是 ApiAP2蛋白家族的成员是整个过程中发育的主要调节因子。 疟疾寄生虫的完整生命周期。ApiAP2蛋白在大小上高度可变，并含有 一个到三个AP2DNA结合域的任何地方。我们已经测量了DNA结合 疟原虫所有AP2结构域的特异性显示了广泛的DNA序列 具体细节。通过确定这些DNA基序在全基因组范围内的出现情况，我们可以开始 预测一些ApiAP2蛋白的功能。然而，详细的机制研究 证实这些预测是滞后的。这项提案旨在定义一个 ApiAP2蛋白的一个子集，与血液阶段发育的关键过程相关，包括 红细胞侵入、配子体发生和配子体发育的调控 分化和成熟。首先，我们将定义PfAP2-G的转录调控网络 (PF3D7_1222600)，我们已经证明它是性阶段的主要调节者 承诺。我们还将研究与配子体有关的另外两种ApiAP2蛋白 研究进展(PF3D7_1317200、PF3D7_1408200)以确定它们在配子体调节中的作用 差异化。接下来，我们建议表征PF3D7_1007700的作用，我们预测它是一个 红细胞入侵的调节剂。由于PF3D7_1007700包含三个AP2DNA结合域， 每一个都具有独特的DNA结合特异性，这将使我们能够剖析单个AP2的贡献 该蛋白的功能结构域(以及类似的其他ApiAP2蛋白)。最后，我们有 在ApiAP2蛋白中发现了一个新的保守结构域，并将开始研究 该结构域的结构和功能，以及寻求在这些区域内定义反式激活区域 蛋白质。为了实现我们的目标，我们将使用一系列成熟的技术，包括 寄生虫的遗传操作、生物化学和全基因组方法。了解 恶性疟原虫转录调控过程的分子机制将提供关键 研究寄生虫发展所需的工具，并提出了新的治疗目标 旨在阻止寄生虫成熟的干预策略。ApiAP2蛋白严格存在于 Apicomplexan寄生虫，因此是新的抗疟疾疗法的有吸引力的候选者。