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。这些过程主要在脊椎动物组织培养细胞和酵母中被定义。然而，复杂的遗传策略操作布氏锥虫的可用性提供了一个有效的系统，在其中研究一般的细胞生物学。更重要的是，两个特征使非洲锥虫对研究特别有吸引力。首先，锥虫是撒哈拉以南非洲地区影响人类和兽医的病原体。世界卫生组织目前估计，36个国家的6000万人面临感染人类非洲锥虫病的风险。只有少数药物用于治疗，其中最好的（依氟鸟氨酸）价格昂贵，需要一个困难的方案，其中最差的（美拉胂醇）杀死高达10%的接受者。如果不进行干预，感染不可避免地是致命的，由于疫苗接种不是一种选择，因此迫切需要开发新药。第二，锥虫与其他真核生物在遗传学上的古老关系确保了无论获得什么结果，都可能因为它们与标准的“高等”真核系统的共同性而感兴趣，因为它们与标准的“高等”真核系统的区别。这些寄生虫的发病机制的关键是生命周期的血流阶段的糖基磷脂酰肌醇（GPI）锚定的变异表面糖蛋白（VSG）。了解VSG和相关的转铁蛋白受体如何被运输到细胞表面并维持在细胞表面，以及GPI锚在适当运输中的作用，对于了解寄生虫的宿主-寄生虫关系至关重要。本竞争性续期申请中的所有具体目标都直接来自当前资助期内取得的进展。首先，我们已经证实GPI效价调节分泌途径内蛋白质的进展和最终稳定性。在目标#1中，我们将继续这些研究，通过定义GPI-负报告分子到溶酶体的默认途径，并通过研究天然转铁蛋白受体的明显异常行为。其次，我们已经证明，VSG选择性地加载到COPII囊泡ER退出。在目标#2中，我们将研究锥虫早期分泌途径中GPI依赖性靶向机制。第三，在我们对寄生虫脂质在GPI依赖性贩运中的作用的研究中，我们发现鞘磷脂合成在血流阶段锥虫中是必不可少的，与其他动质体原生动物相反，它们根本不合成鞘磷脂。在目标#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，从而完成对一个独特的鞘脂环化酶家族的表征。这些研究将拓宽我们对所有真核生物中基本细胞生物学过程的了解，并为靶向鞘脂途径用于新药开发奠定基础。 公共卫生相关性：本研究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。锥虫是撒哈拉以南非洲地区影响人类和兽医的病原体。这些研究将拓宽我们对所有真核生物中基本细胞生物学过程的认识，并将为靶向鞘脂途径用于新药开发奠定基础

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