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.

弓形虫和相关寄生虫给社会带来巨大的健康和经济负担。不幸的是，针对这些寄生虫的药物有限，而且治疗方法往往有毒，耐药性是一个严峻的挑战。因此，新疗法的发现是当务之急。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，弓形虫编码许多哺乳动物细胞中不存在的植物样蛋白质。为了利用这一独特功能，我们重点研究了含有蛋白磷酸酶 TgPPKL 的 kelch 结构域，其最接近的同源物是植物磷酸酶 BSU1。我们已经证明，TgPPKL 存在于寄生虫的细胞质中，并且在寄生虫分裂过程中与新生细胞骨架结构相关。我们已经证明 PPKL 对于寄生虫的生存至关重要，并且条件性敲低会导致异常的寄生虫分裂。 PPKL 的植物同源物 BSU1 是特征最明确的植物信号传导途径之一油菜素类固醇级联的核心。油菜素类固醇会激活级联反应，其中包括激活 BSU1，进而使激酶 BIN2 在高度保守的酪氨酸处去磷酸化，使其失活。当磷酸化时，BIN2 通过直接磷酸化使转录因子失活。有趣的是，弓形虫还具有 BIN2 的密切同源物，其保守酪氨酸被磷酸化。我们已经证明，TgBIN2 位于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，我们已经证明 TgBIN2 与包括两个植物样 AP2 转录因子的转录调节因子复合物相互作用，这表明，作为其植物同源物，它参与调节基因表达。总的来说，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，并且 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型信号通路的一部分协同作用。我们的首要目标是在超微结构水平上分析 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。第二个目标是，我们将通过生成和表征敲低菌株并鉴定 TgBIN2 底物来关注 TgBIN2 的功能。这些研究将结合最先进的细胞生物学、蛋白质组学和分子遗传学方法来阐明这两种独特且重要的信号蛋白的功能。同时，我们的实验将阐明弓形虫中独特的信号通路，该通路由植物油菜素类固醇通路的同系物驱动。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节。