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.

尽管多年来一直认为疟原虫是疟疾的致病药物， 没有序列特异性的转录因子，我们实验室的工作和其他人现在已经有 证明它们具有有限数量的此类调节蛋白。 Apicomplexan AP2 （APIAP2）蛋白质的起源很像植物，现在被广泛认为是单一专业 DNA结合蛋白家族在疟原虫基因组中鉴定出来。我们的中心假设是 APIAP2蛋白家族的成员是整个整个整个发展的主要监管机构 疟疾寄生虫的完整生命周期。 APIAP2蛋白的大小高度可变，并且包含 一到三个AP2 DNA结合域的任何位置。我们已经测量了DNA结合 来自疟原虫的所有AP2结构域的特异性表明了一系列DNA序列 特殊性。通过确定这些DNA基序的全基因组发生，我们可以开始 预测某些APIAP2蛋白的功能作用。但是，详细的机械研究 确认这些预测一直在滞后。该建议旨在定义一个的功能作用 与血液阶段至关重要的过程相关的APIAP2蛋白的子集 调节红细胞入侵，对配子细胞生成和配子细胞的承诺 分化和成熟。首先，我们将定义PFAP2-G的转录调节网络 （PF3D7_1222600），我们已证明是性阶段的主管 承诺。我们还将研究与配子细胞有关 开发（PF3D7_1317200，PF3D7_1408200）定义它们在配子细胞调节中的作用 分化。接下来，我们建议表征PF3D7_1007700的作用，我们预计将是 红细胞侵袭的调节剂。由于PF3D7_1007700包含三个AP2 DNA结合域，因此 每个具有独特的DNA结合特异性，这将使我们能够剖析单个AP2的贡献 该蛋白（和其他APIAP2蛋白的功能）的域。最后，我们有 确定了APIAP2蛋白质中一个新的保守域，并将开始努力以表征 该领域的结构和功能以及试图在其中定义反式激活区域 蛋白质。为了实现我们的目标，我们将使用既定技术的组合，包括 寄生虫，生物化学和全基因组方法的基因操纵。了解 恶性疟原虫中转录调节过程的分子机制将非常重要 研究寄生虫开发的所需工具，还为新的靶向靶标提供了治疗目标 旨在阻止寄生虫成熟的干预策略。 APIAP2蛋白严格发现 Apicomplexan寄生虫，因此是新抗疟疾疗法的有吸引力的候选者。