Secretion, protein targeting, and lipid metabolism in African Trypanosomes

描述（由申请人提供）：本提案的目的是了解非洲锥虫分泌蛋白转运和脂质代谢的相关过程。这些过程主要是在脊椎动物组织培养细胞和酵母中确定的。然而，复杂的遗传策略的可用性操纵布鲁氏锥虫提供了一个有效的系统，其中研究一般细胞生物学。更重要的是，有两个特征使得非洲锥虫特别值得研究。首先，锥虫是撒哈拉以南非洲地区影响很大的人类和兽医病原体。世卫组织目前估计，36个国家的6000万人面临感染非洲人类锥虫病的风险。只有少数几种药物用于治疗，其中最好的药物（依氟鸟氨酸）价格昂贵，需要一个困难的治疗方案，最坏的药物（美拉胂醇）杀死了高达10%的接受者。如果不进行干预，感染不可避免地是致命的，而且由于不能选择接种疫苗，因此迫切需要开发新药。其次，锥虫与其他真核生物在系统发育上的古老关系确保了无论获得什么结果，它们与标准的“高级”真核生物系统的共性和区别都可能是令人感兴趣的。这些寄生虫发病机制的关键是生命周期血流阶段的糖基磷脂酰肌醇（GPI）锚定的变异体表面糖蛋白（VSG）。了解VSG和相关转铁蛋白受体如何运输到细胞表面并维持在细胞表面，以及GPI锚点在适当运输中的作用，对于理解宿主-寄生虫关系中的寄生虫部分至关重要。本申请竞争性续期的所有具体目标均直接源自当前资助期间所取得的进展。首先，我们已经证实GPI价态调节分泌途径中蛋白质的进展和最终稳定性。在目标1中，我们将通过定义gpi -负报告蛋白到溶酶体的默认途径，并通过调查天然转铁蛋白受体的明显异常行为来继续这些研究。其次，我们已经证明VSG被选择性地装载到COPII囊泡中以供ER退出。在Aim #2中，我们将研究gpi依赖性靶向在锥虫早期分泌途径中的机制。第三，在我们对寄生虫脂质在gpi依赖性运输中的作用的研究中，我们发现鞘磷脂的合成在血流阶段的锥虫中是必不可少的，与其他动着体原生动物相比，它们根本不产生鞘磷脂。在Aim #3中，我们将使用一种新颖且高度创新的无细胞系统来合成膜蛋白，完成我们对独特鞘脂合成酶家族的表征。这些研究将拓宽我们对所有真核生物的基本细胞生物学过程的认识，并为针对鞘脂通路开发新药奠定基础。

DESCRIPTION (provided by applicant): The goal of this proposal is to understand the related processes of secretory protein transport and lipid metabolism in African trypanosomes. These processes have been defined primarily in vertebrate tissue culture cells and yeast. However, the availability of sophisticated genetic strategies for manipulation of Trypanosoma brucei provides a potent system in which to study general cell biology. More importantly, two features make African trypanosomes particularly attractive for study. First, trypanosomes are high impact human and veterinary pathogens in sub-Saharan Africa. The WHO currently estimates that 60 million people in 36 countries are at risk of acquiring Human African Trypanosomiasis. Only a handful of drugs are in use for treatment, 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 inevitably fatal without intervention, and since vaccination is not an option there is a critical need for new drug development. Second, the phylogenetically ancient relationship of trypanosomes to other eukaryotes ensures that whatever results are obtained are as likely to be of interest for their commonality with, as for their distinctness from, standard 'higher' eukaryotic systems. The lynchpin of pathogenesis in these parasites is the glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) of the bloodstream stage of the life cycle. Understanding how VSG, and related transferrin receptor, are transported to and maintained at the cell surface, and the role of GPI anchors in proper trafficking, are critical to understanding the parasite half of the host- parasite relationship. All of the Specific Aims in this application for competitive renewal derive directly from progress made during the current funding period. First, we have confirmed that GPI valence regulates progression and ultimate stability of proteins within the secretory pathway. In Aim #1 we will continue these studies by defining the default route of GPI-minus reporters to the lysosome, and by investigating the apparently aberrant behavior of native transferrin receptor. Second, we have demonstrated that VSG is selectively loaded into COPII vesicles for ER exit. In Aim #2 we will study the machinery of GPI-dependent targeting in the early secretory pathway of trypanosomes. Third, in our studies of the role of parasite lipids in GPI-dependent trafficking we have found that sphingomyelin synthesis is essential in bloodstream stage trypanosomes, in contrast to other kinetoplasted protozoa, which do not make sphingomyelin at all. In Aim #3 we will complete our characterization of a unique family of sphingolipid synthases using a novel and highly innovative cell-free system for synthesis of membrane proteins. These studies will broaden our knowledge of basic cell biological processes in all eukaryotes, and will lay the groundwork for targeting the sphingolipid pathway for novel drug development. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to understand the related processes of secretory protein transport and lipid metabolism in African trypanosomes. Trypanosomes are high impact human and veterinary pathogens in sub-Saharan Africa. These studies will broaden our knowledge of basic cell biological processes in all eukaryotes, and will lay the groundwork for targeting the sphingolipid pathway for novel drug development