Homologs of brassinosteroid signaling proteins in Toxoplasma gondii regulate parasite division

弓形虫中油菜素类固醇信号蛋白的同源物调节寄生虫分裂

基本信息

批准号：
10312866
负责人：
Gustavo A Arrizabalaga
金额：
$ 23.78万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-12 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10312866
关键词：
Ablation Abscisic Acid Address Affect Apicomplexa Attention Biological Biology Calcium Signaling Cell Nucleus Cellular biology Complex Cytoplasm DNA Data Drug Targeting Economic Burden Enzymes Eukaryota Event Family Gene Expression Genes Genetic Transcription Genome Goals Growth Growth and Development function Health Heart Homologous Gene Horizontal Gene Transfer Immunocompromised Host In Vitro Individual Knowledge Label Laboratories Light Mammalian Cell Molecular Genetics Parasites Pathogenicity Pathway interactions Pharmaceutical Preparations Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Plant Growth Regulators Plant Proteins Plants Plasmodium falciparum Plastids Process Protein phosphatase Proteins Proteomics Regulation Research Resistance Role Signal Pathway Signal Transduction Signaling Protein Societies Structure Testing Toxoplasma Toxoplasma gondii Tyrosine Tyrosine Phosphorylation Work base biological adaptation to stress calcium-dependent protein kinase combat drug development enhancer-binding protein AP-2 experimental study genetic approach health economics in vivo knock-down member new therapeutic target novel novel therapeutics obligate intracellular parasite phosphoproteomics programs receptor response transcription factor

项目摘要

Toxoplasma gondii and related parasites, exert great health and economic burden on society. Unfortunately, drugs against these parasites are limited, and treatments are often toxic and resistance is a serious challenge. Accordingly, the discovery of novel therapeutics is a priority. As these are obligate intracellular parasites, identifying novel therapeutic targets requires a thorough understanding of events and proteins that are unique to the parasite. Interestingly, Toxoplasma encodes for numerous plant-like proteins that are absent in mammalian cells. To exploit this unique feature, we have focused on the kelch domain containing protein phosphatase TgPPKL, which closest homolog is the plant phosphatase BSU1. We have shown that TgPPKL is in the cytoplasm of the parasite and that it associates with nascent cytoskeletal structures during parasite division. We have shown that PPKL is essential for parasite survival and conditional knockdown results in aberrant parasite division. PPKL’s plant homolog, BSU1, is central to one of the best characterized plant signaling pathways, the brassinosteroid cascade. Brassinosteroid activates a cascade that includes activation of BSU1, which in turns dephosphorylates the kinase BIN2 at a highly conserved tyrosine, inactivating it. When phosphorylated, BIN2 inactivates transcription factors through direct phosphorylation. Interestingly, Toxoplasma also has a close homologue of BIN2 that is phosphorylated at the conserved tyrosine. We have shown that TgBIN2 is in the nucleus of non-dividing parasites, but accumulates in the cytoplasm during division. Importantly, we have shown that TgBIN2 interacts with a complex of transcription regulators including two plant-like AP2 transcription factors, suggesting that, as its plant homologue, it is involved in regulating gene expression. In total, our preliminary data show that expression and localization of both Toxoplasma PPKL and BIN2 change according to the division cycle, knockdown of PPKL affects parasite division, and BIN2 interacts with transcription factors. Accordingly, we hypothesize that TgPPKL and TgBIN2 act in concert as part of a novel signaling pathway to regulate parasite division and structure. Our first aim will be to analyze the effect of TgPPKL disruption at the ultrastructural level, test for a physical and functional interaction between TgPPKL and TgBIN2, and identify signaling proteins upstream of TgPPKL. In a second aim we will focus on TgBIN2’s function by generating and characterizing a knockdown strain and identifying TgBIN2 substrates. These studies will combine state of the art cell biology, proteomic and molecular genetic approaches to elucidate the function of these two unique and essential signaling proteins. In conjunction, our experiments will elucidate a unique signaling pathway in Toxoplasma that is driven by homologs of the plant brassinosteroid pathway. This work will undoubtedly uncover proteins that can be exploited as drug targets and will shed light on the regulation of parasite division.

弓形虫弓形虫及相关寄生虫，对社会施加了巨大的健康和经济焚烧。不幸的是，针对这些寄生虫的药物是有限的，治疗通常是有毒的，抵抗是一个严重的挑战。根据这些是强制性的细胞内寄生虫，识别新型的治疗靶标需要对寄生虫独有的事件和蛋白质有透彻的了解。有趣的是，弓形虫编码在哺乳动物细胞中不存在的许多植物样蛋白质。为了利用这一独特的特征，我们专注于含有蛋白质磷酸酶TGPPKL的Kelch结构域，最接近的同源物是植物磷酸酶BSU1。我们已经表明，TGPPKL位于寄生虫的细胞质中，并且它与寄生虫分裂期间的新生细胞骨架结构相关。我们已经表明，PPKL对于寄生虫存活至关重要，有条件的敲低导致异常的寄生虫分裂。 PPKL的植物同源物BSU1是特征性最佳的植物信号通路之一的核心核心固醇级联。黄铜固醇激活了包括BSU1激活的级联反应，该级联激活在高度保守的酪氨酸下脱磷酸化激酶BIN2，使其灭活。当磷酸化时，BIN2通过直接磷酸化会使转录因子失活。有趣的是，弓形虫还具有BIN2的紧密同源物，该同源物在配置的酪氨酸处被磷酸化。我们已经表明，tgbin2位于非分散寄生虫的核中，但在分裂过程中积聚在细胞质中。重要的是，我们已经表明TGBIN2与转录调节剂的复合物相互作用，包括两个类似植物的AP2转录因子，这表明作为其植物同源物，它参与了控制基因表达。总的来说，我们的初步数据表明，根据分裂周期，弓形虫PPKL和BIN2的表达和定位变化，ppkl的敲低会影响寄生虫分裂，而BIN2与转录因子相互作用。根据，我们假设TGPPKL和TGBIN2协同起作用，是调节寄生虫分裂和结构的新信号通路的一部分。我们的第一个目的是分析在超微结构水平上TGPPKL破坏的影响，测试TGPPKL和TGBIN2之间的物理和功能相互作用，并鉴定TGPPKL上游的信号蛋白。在第二个目标中，我们将通过产生和表征敲低菌株并识别TGBIN2底物来关注TGBIN2的功能。这些研究将结合ART细胞生物学，蛋白质组学和分子遗传学方法的状态，以阐明这两种独特和必不可少的信号蛋白的功能。结合起来，我们的实验将阐明由植物胸腺素途径的同源物驱动的弓形虫中的独特信号通路。这项工作无疑会发现可以作为药物靶标探索的蛋白质，并将阐明寄生虫分裂的调节。