Secretory Protein Trafficking and Folding in African Trypanosomes

这项建议的主要目标是了解非洲人分泌蛋白运输的基本过程， 锥虫，特别是糖基磷脂酰肌醇（GPI）膜锚。这些过程 传统上在酵母和哺乳动物中进行了研究，但复杂的遗传策略的可用性， 布氏锥虫为真核分泌细胞生物学的研究提供了一个有效的替代系统。 重要的是，两个特征使非洲锥虫特别适合研究。首先，锥虫是 在撒哈拉以南非洲对人类和兽医产生高影响的病原体。世界卫生组织估计， 36个国家的人有感染人类非洲锥虫（HAT）的风险。很少有药物 可用，其中最好的（依氟鸟氨酸）是昂贵的，需要一个困难的方案，其中最坏的是， （美拉胂醇）杀死高达10%的接受者。如果不进行干预，感染总是致命的，接种疫苗不是一种预防措施。 这是一种选择，迫切需要新的治疗方法。第二，古代的系统发育地位 锥虫及其独特的分泌结构，确保了相对于"标准"的新结果。 将获得真核模型系统。锥虫病发病机制的关键是GPI- 血流阶段的锚定变体表面糖蛋白（VSG）和相关转铁蛋白受体（TfR） 的生命周期。了解它们如何被转运到细胞表面，以及GPIs在其中的作用 贩运，是至关重要的了解寄生虫的一半宿主-寄生虫的关系。这项建议是 旨在研究这些过程。具体目标都是直接从以前的进展中产生的。 融资期。首先，我们已经确定了一个p24膜蛋白基因家族作为推定的负荷受体 用于从ER排出分泌性货物，包括VSG（目的#1）。其次，我们已经证明，非- 通过ERAD（ER相关降解）监测和降解功能性GPI锚定蛋白 途径（目标#2）。第三，我们开发了一种原位修饰TfR异源二聚体亚基的策略 （ESAG6/ESAG7），这使我们能够检验GPI效价调节肿瘤进展/稳定性的假设。 后高尔基体途径内的蛋白质（目标#3）。这些结果将推动更深入的研究分泌 贩运锥虫阐明基本锥虫细胞生物学的关键方面。我们相信 这项工作可能有助于为未来的药物开发奠定基础，并将不仅突出差异， 而且也是真核生物进化过程中常见的细胞生物学过程的相似性。

The broad goal of this proposal is to understand basic processes of secretory protein transport in African trypanosomes, with special regard to glycosylphosphatidylinositol (GPI) membrane anchors. These processes have been traditionally studied in yeast and mammals, but the availability of sophisticated genetic strategies in Trypanosoma brucei provides a potent alternative system to study eukaryotic secretory cell biology. Importantly, two features make African trypanosomes especially relevant for study. First, trypanosomes are high impact human and veterinary pathogens in sub-Saharan Africa. The WHO estimates that >60 million people in 36 countries are at risk of acquiring Human African Trypanosomiasis (HAT). Few drugs are available, the best of which (eflornithine) is expensive and requires a difficult regimen, the worst of which (melarsoprol) kills up to 10% of recipients. Infection is always fatal without intervention, vaccination is not an option, and there is a critical need for new therapies. Second, the ancient phylogenetic status of trypanosomes, and their unique secretory architecture, ensure that novel results relative to the `standard' eukaryotic model systems will be obtained. The lynchpins of pathogenesis in trypanosomiasis are the GPI- anchored variant surface glycoprotein (VSG), and related transferrin receptor (TfR), of the bloodstream stage of the life cycle. Understanding how they are transported to the cell surface, and the role of GPIs in this trafficking, are critical to understanding the parasite half of the host-parasite relationship. This proposal is designed to investigate these processes. The Specific Aims all derive directly from progress from the previous funding period. First, we have identified a gene family of p24 membrane proteins as putative loading receptors for exit of secretory cargo, including VSG, from the ER (Aim #1). Second, we have demonstrated that non- functional GPI-anchored proteins are monitored and degraded by the ERAD (ER-Associated Degradation) pathway (Aim #2). Third, we have developed a strategy for in situ modification of the TfR heterodimer subunits (ESAG6/ESAG7) that allows us to test the hypothesis that GPI valence regulates progression/stability of proteins within post-Golgi pathways (Aim #3). These results will drive deeper investigations of secretory trafficking in trypanosomes to illuminate critical aspects of basic trypanosome cell biology. It is our belief that this work may help lay the foundation for future drug development, and will highlight not just the differences, but also the similarities of cell biological processes common to the full range of eukaryotic evolution.