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: 9271152
• 负责人: Manuel Llinas
• 金额: $ 38.29万
• 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/9271152
• 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; Genetic Transcription; Genome; Genomics; Goals; Hour; Human; Individual; Infection; Intervention; Invaded; Link; Malaria; Measures; Molecular; Morphology; Parasites; Pathogenesis; Periodicity; Plants; Plasmodium; Plasmodium falciparum; Population; Pregnant Women; Process; Protein Family; Proteins; Publishing; Regulation; Research; Resolution; Risk; Role; Sexual Development; Specificity; Structure; Symptoms; System; Techniques; Testing; Time; Transactivation; Transcription Factor AP-2 Alpha; Transcriptional Regulation; Transgenic Organisms; Vaccines; Work; base; genetic manipulation; genetic regulatory protein; genome-wide; in vivo; insight; knock-down; member; new therapeutic target; novel; 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.

尽管多年来人们认为疟疾的病原体——疟原虫寄生虫， 缺乏序列特异性转录因子，我们实验室和其他人的工作现在已经 证明它们拥有数量有限的此类调节蛋白。 apicomplexan AP2 (ApiAP2) 蛋白起源与植物相似，现在被广泛认为是唯一的主要蛋白。 在疟原虫基因组中鉴定出的 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发育过程的主要调节因子 疟疾寄生虫的完整生命周期。 ApiAP2 蛋白的大小变化很大，并且含有 1 到 3 个 AP2 DNA 结合域。我们测量了 DNA 结合 疟原虫所有 AP2 结构域的特异性，展示了广泛的 DNA 序列 特殊性。通过确定这些 DNA 基序在全基因组范围内的出现，我们可以开始 预测某些 ApiAP2 蛋白的功能作用。然而，详细的机制研究 证实这些预测已经滞后。该提案旨在定义以下机构的功能角色： ApiAP2 蛋白的子集与血液阶段发育的关键过程相关，包括 调节红细胞侵袭、配子细胞发生和配子细胞 分化和成熟。首先，我们将定义PfAP2-G的转录调控网络 （PF3D7_1222600），我们已证明它是性阶段的主要调节器 承诺。我们还将研究与配子细胞有关的另外两种 ApiAP2 蛋白 开发（PF3D7_1317200，PF3D7_1408200）来定义它们在配子体调节中的作用 差异化。接下来我们建议描述 PF3D7_1007700 的角色，我们预测它是 红细胞侵袭的调节因子。由于 PF3D7_1007700 包含三个 AP2 DNA 结合域， 每个都具有独特的 DNA 结合特异性，这将使我们能够剖析单个 AP2 的贡献 该蛋白的功能域（以及其他 ApiAP2 蛋白，以此类推）。最后，我们有 在 ApiAP2 蛋白中发现了一个新的保守结构域，并将开始努力表征 该结构域的结构和功能，以及寻求定义这些区域内的反式激活区域 蛋白质。为了实现我们的目标，我们将结合使用现有的技术，包括 寄生虫的基因操作、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的 研究寄生虫发育所需的工具，并且还提出了新的治疗靶点 旨在阻止寄生虫成熟的干预策略。 ApiAP2 蛋白严格存在于 因此，顶复门寄生虫是新的抗疟疗法的有吸引力的候选者。