SECRETION AND PROTEIN TARGETING IN AFRICAN TRYPANOSOMES

这项提议的广泛目标是：i)确定非洲锥虫分泌运输的基本机制，以及ii)调查这些寄生虫特有的分泌贩运方面。非洲锥虫在人类(昏睡病)和牛(Nagana)中都会引起疾病。它们通过反复改变VSGs(可变表面糖蛋白)的表达来避免宿主的免疫反应；主要的糖基磷脂酰肌醇(GPI)锚定抗原。因此，必需表面蛋白的分泌性运输，如VSG，对于锥虫作为寄生虫的成功至关重要。这项提案的相互关联的目标包含在三个具体目标中。首先，对两个相关的ATPase的锥体同系物进行生化表征，这两个ATPase对于整个分泌途径中运输小泡与靶膜的融合是必不可少的。这两种ATPase分别是VCP(Valosin-Containing Protein)和NSF(NEM-Sensitive Fusion Protein)。这些蛋白的显性负ATPase突变体将在转基因布鲁氏锥虫中工程和表达，导致自由运输小泡的积累，并提高突变的ATPase与提供囊泡对接和融合特异性的膜受体的结合。这将允许直接分析分泌囊泡的组成和货物，并识别锥虫体内分泌室的特定膜标记。其次，将研究GPI锚在正向分泌运输中的作用。防止添加GPI显著降低VSG在锥虫体内的转运速度，导致VSG在内质网中积累。GPI锚点调节分泌运输的充分性将通过将GPI添加到其他分泌货物蛋白中来评估。这些嵌合记者运输速度的加快将证实GPI锚在锥体分泌途径的最早部分扮演着积极的前向运输信号的角色。然后，将设计实验来询问GPI依赖的转运是用受体介导的模型解释得最好，还是用脂质微域模型来解释。第三，从转基因原环锥虫表面释放重组VSG的金属蛋白水解酶将被生化鉴定。这种释放模拟了血流锥体在分化过程中VSG的周转，因此该酶可能在VSG的正常代谢中发挥重要作用。为了解决这个问题，在分化过程中蛋白酶的表达将被破坏，从而对VSG释放和整个分化过程的影响将被确定。总体而言，这些研究将提供对锥虫基本分泌过程的更多了解，并将阐明分泌在锥虫病发病机制中的作用以及锥虫在生命周期分化过程中细胞表面结构的重组。

The Broad Goals of this proposal are: i) to define the basic machinery of secretory transport in African trypanosomes, and ii) to investigate aspects of secretory trafficking that are unique to these parasites. African trypanosomes cause disease in both humans (sleeping sickness) and cattle (nagana). They avoid the host immune response be repeatedly changing the expression of VSGs (variant surface glycoprotein); the major glycosylphosphatidyl-inositol (GPI) anchored antigens. Consequently, the secretory trafficking of essential surface proteins, such as VSG, is critical to the success of trypanosomes as parasites. The interrelated goals of this proposal are contained in three Specific Aims. First, trypanosomal homologs of two related ATPases that are essential for the fusion of transport vesicles with target membranes throughout the secretory pathway will be biochemically characterized. These ATPases are VCP (valosin-containing protein) and NSF (NEM-sensitive fusion protein). Dominant negative ATPase mutants of these proteins will be engineered and expressed in transgenic T. brucei, resulting in accumulation of free transport vesicles and elevated association of the mutant ATPases with the membrane receptors that provide specificity to vesicle docking and fusion. This will allow direct analysis of the composition and cargo of secretory vesicles, and the identification of specific membrane markers for secretory compartments in trypanosomes. Second, the role of GPI anchors in forward secretory transport will be investigated. Prevention of GPI addition dramatically reduces the rate of VSG transport in trypanosomes, leading to accumulation of VSG in the endoplasmic reticulum. The sufficiency of GPI anchors to mediate secretory transport will be assessed by engineering GPI addition to other secretory cargo proteins. Acceleration of the rate of transport of these chimeric reporters will establish that GPI anchors act as positive forward transport signals in the earliest part of the trypanosomal secretory pathway. Experiments will then be designed to ask if GPI-dependent transport is best explained by a receptor-mediated or a lipid microdomain model. Third, a metalloprotease that releases recombinant VSG from the surface of transgenic procyclic trypanosomes will be biochemically characterized. This release mimics the turnover of VSG from bloodstream trypanosomes during differentiation; thus this enzyme may play an important role in the normal metabolism of VSG. To address this issue the expression of the protease during differentiation will be disrupted and the resulting effects on VSG release, and the entire differentiation process, will be determined. Overall, these studies will provide a greater understanding of essential secretory processes in trypanosomes and will illuminate the role of secretion in both the pathogenesis of trypanosomiasis and the reorganization of trypanosomal cell surface architecture during life cycle differentiation.