Proteolytic modulation of toxoplasma invasion proteins

描述(申请人提供)：专性细胞内原生动物弓形虫是普通人群后葡萄膜炎、免疫缺陷患者脑炎和先天性感染儿童认知障碍的主要原因。我们试图了解弓形虫是如何准备和实现细胞入侵的。虽然人们已经认识到许多弓形虫入侵蛋白在进入顶端分泌细胞器的过程中经历了蛋白水解性成熟，但对所涉及的蛋白水解酶以及成熟发生的地点和原因知之甚少。根据初步的免疫定位、基因敲除和抑制研究，以及蛋白酶专一性分析和对可能底物的体外处理，我们假设弓形虫使用组织蛋白酶来激活和稳定新的多囊内体(MVE)中的侵袭蛋白。为了验证这一假设，我们将：(1)鉴定CPL基因敲除寄生虫中的缺陷和补偿机制；(2)通过遗传和抑制研究证实组织蛋白在ProMIC成熟过程中的重叠作用；以及(3)测试“锁定和加载”假说，即蛋白质降解成熟稳定ProMIC蛋白复合体。这项工作将阐明分泌蛋白运输和蛋白水解性调节蛋白功能的基本机制，这些机制可能与相关的顶复合体寄生虫和其他利用调节分泌的真核细胞共享。

DESCRIPTION (provided by applicant): The obligate intracellular protozoan Toxoplasma gondii is a leading cause of posterior uveitis in the general population, encephalitis in immunodeficient individuals, and cognitive impairment in congenitally infected children. We seek to understand how Toxoplasma prepares for and achieves cell invasion. Although it has been well recognized that many Toxoplasma invasion proteins undergo proteolytic maturation en route to apical secretory organelles, little is known about the proteases involved or where and why maturation occurs. We hypothesize that T. gondii uses a cathepsin proteases to proteolytically activate and stabilize invasion proteins within a novel multivesicular endosome (MVE), based on preliminary immunolocalization, gene knockout, and inhibitor studies, along with protease specificity analysis and in vitro processing of a putative substrate. To test this hypothesis we will: (1) Characterize defects and compensatory mechanisms in CPL knockout parasites; (2) Confirm the overlapping roles of cathepsins in proMIC maturation by genetic and inhibitor studies; and (3) Test the "lock and load" hypothesis that proteolytic maturation stabilizes proMIC protein complexes. This work will illuminate basic mechanisms of secretory protein trafficking and proteolytic modulation of protein function that may be shared with related apicomplexan parasites and other eukaryotic cells that utilize regulated secretion.