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

• 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
• 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

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.

弓形虫及其相关寄生虫给社会带来了巨大的健康和经济负担。不幸的是，针对这些寄生虫的药物是有限的，治疗往往是有毒的，耐药性是一个严重的挑战。因此，发现新的治疗方法是一个优先事项。由于这些是专性细胞内寄生虫，识别新的治疗靶点需要彻底了解寄生虫特有的事件和蛋白质。有趣的是，弓形虫编码许多在哺乳动物细胞中不存在的植物样蛋白。为了利用这一独特的功能，我们集中在kelch域含有蛋白磷酸酶TgPPKL，最接近的同源物是植物磷酸酶BSU 1。我们已经表明，TgPPKL是在寄生虫的细胞质中，它与寄生虫分裂过程中新生的细胞骨架结构。我们已经表明，PPKL是寄生虫生存和条件敲低的结果在异常寄生虫分裂是必不可少的。PPKL的植物同源物BSU 1是最具特征的植物信号传导途径之一油菜素类固醇级联的核心。BSU 1的激活是一个级联反应，BSU 1的激活反过来使BIN2激酶在一个高度保守的酪氨酸上去磷酸化，使其失活。当BIN2被磷酸化时，BIN2通过直接磷酸化使转录因子失活。有趣的是，弓形虫也具有在保守酪氨酸处磷酸化的BIN2的密切同源物。我们已经表明，TgBIN2是在非分裂寄生虫的细胞核中，但在分裂过程中积累在细胞质中。重要的是，我们已经表明，TgBIN2相互作用的转录调控因子，包括两个植物样的AP2转录因子的复合物，这表明，作为其植物同源物，它参与调节基因表达。总之，我们的初步数据表明，弓形虫PPKL和BIN2的表达和定位根据分裂周期而变化，PPKL的敲低影响寄生虫分裂，BIN2与转录因子相互作用。因此，我们假设TgPPKL和TgBIN2作为调节寄生虫分裂和结构的新信号通路的一部分协同作用。我们的第一个目标将是在超微结构水平上分析TgPPKL破坏的效果，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鉴定TgBIN2底物来关注TgBIN2的功能。这些研究将结合联合收割机最先进的细胞生物学，蛋白质组学和分子遗传学的方法来阐明这两个独特的和必要的信号蛋白的功能。同时，我们的实验将阐明一个独特的信号通路弓形虫是由植物油菜素类固醇途径的同系物。这项工作无疑将揭示可用作药物靶点的蛋白质，并将揭示寄生虫分裂的调控。