Secretion and Protein Targeting in African Trypanosomes

描述（由申请人提供）：本申请的广泛目标是使用内源性表面蛋白（VSG和原环蛋白）作为报告基因表征非洲锥虫中分泌运输的途径。锥虫在人类和家畜中引起疾病，并且它们通过改变VSG（血流阶段的主要糖基磷脂酰肌醇（GPI）锚定抗原）的表达来避免宿主免疫应答。因此，必需外壳蛋白（如VSG）的适当运输对寄生虫的成功至关重要。这项申请的相互关联的目标都源于在当前供资期间提出的意见。首先，VSG和其他报告者的贩运严重依赖于GPI锚，导致我们提出GPI价假说：GPI锚的数量调节分泌途径中的进展和命运。第二，分化期间的表面涂层交换（VSG的释放和原环素的表达）涉及旧VSG涂层的蛋白水解和GP 1水解。蛋白水解是由一种新的表面金属蛋白酶介导的，GPI水解意外地涉及内源性细胞质GPI-PLC的表面表达。目的#1将测试“GPI效价假说”，即GPI效价控制血流阶段寄生虫分泌途径内膜蛋白的进展、目的地和最终稳定性。目的#2将评估膜脂筏作为价假说的潜在机制的作用。目的#3将生物化学鉴定金属蛋白酶（可能是新鉴定的MSP基因家族的成员），并将研究其机制（硫代酰化？）调节细胞质GPI-PLC的表面表达。总之，这些研究将阐明锥虫的基本分泌过程及其在锥虫病发病机制中的作用。

DESCRIPTION (provided by applicant): The broad goal of this application is to characterize pathways of secretory trafficking in African trypanosomes using endogenous surface proteins (VSG and procyclin) as reporters. Trypanosomes cause disease in humans and livestock, and they avoid host immune responses by changing the expression of VSGs, the major glycosylphosphatidyl-inositol (GPI) anchored antigens of the bloodstream stage. Thus, proper trafficking of essential coat proteins, such as VSG, is critical to the success of the parasite. The interrelated Aims of this application all stem from observations made in the current funding period. First, trafficking of VSG and other reporters is critically dependent on GPI anchors, leading us to propose the GPI Valence Hypothesis: number of GPI anchors regulates progression and fate in the secretory pathway. Second, surface coat exchange (release of VSG and expression of procyclin) during differentiation involves both proteolysis and GPl hydrolysis of the old VSG coat. Proteolysis is mediated by a novel surface metalloprotease, and GPI hydrolysis unexpectedly involves surface expression of endogenous cytoplasmic GPI-PLC. Aim #1 will test the 'GPI Valence Hypothesis', that GPI valence controls progression, destination, and ultimately stability of membrane proteins within the secretory pathway of bloodstream stage parasites. Aim #2 will assess the role of membrane lipid rafts as the underlying mechanism of the Valence Hypothesis. Aim #3 will biochemically identify the metalloprotease (possibly a member of the newly identified MSP gene family), and will investigate the mechanism (thioacylation?) regulating surface expression of cytoplasmic GPI-PLC. Overall, these studies will illuminate essential secretory processes in trypanosomes and the role they play in the pathogenesis of trypanosomiasis.