Secretory cargo selection by p24 transmembrane proteins in African Trypanosomes

描述（由申请人提供）：本提案的目的是评估原生动物寄生虫布鲁氏锥虫中负责适当定位毒力因子的内源性分泌机制。布氏t虫引起非洲人非洲锥虫病（HAT），在撒哈拉以南非洲36个国家流行，每年造成数万人死亡。该病不经治疗一律致命，现有的治疗方案毒性很大，难以实施，而且没有疫苗。布氏体通过被感染采采蝇的叮咬传播到宿主血液中，寄生虫在感染期间的成功依赖于两种基本的毒力成分，这两种成分都是布氏体分泌系统的产物。第一种是表面定位的糖基磷脂酰肌醇（GPI）锚定的变异表面糖蛋白（VSG）外壳。第二种是寄生虫溶酶体，这是一种消化细胞器，可以处理内吞的营养因子并降解宿主的免疫复合物。正确靶向VSG到细胞表面和细胞器成分到溶酶体是布鲁氏杆菌发病的核心。尽管在感染过程中起着至关重要的作用，但布鲁氏杆菌的分泌和膜运输机制仍未被广泛研究。所有分泌货物（与最终目的地无关）所遇到的第一个贩运步骤是在COPII（外壳蛋白II）包被的囊泡中从内质网出口。在其他真核生物中，跨膜蛋白p24家族的成员形成特征不佳的异质复合物，介导COPII囊泡出芽过程中的货物选择。特别是，所描述的两种定义明确的p24复合物（酵母和哺乳动物）结合并选择成熟的gpi锚定蛋白用于ER退出。生物信息学分析鉴定了布鲁氏体基因组中p24家族的8个成员（TbERP1-8），我们初步假设它们可能参与了GPI- VSG的贩运。通过RNAi沉默分别评估TbERP1-8。似乎没有一个是必要的，只有TbERP2沉默导致中度生长缺陷。此外，TbERP1-8沉默细胞系在VSG运输或大量分泌方面没有延迟。然而，TbERP1、TbERP2或TbERP8的沉默足以导致两种内源性溶酶体蛋白p67和TbCatL的前高尔基延迟。此外，沉默TbERP1、TbERP2或TbERP8导致内质网驻留蛋白分泌增加，提示其在蛋白靶向中具有次要调节作用。在这些敲低实验中观察到的平行表型表明，Erp1、Erp2和/或Erp8在内质网退出过程中发挥作用，并且可能包含一个具有新型货物特异性的p24复合体。这是本提案的目标，以充分表征这些候选者，并描述的亚基身份和货物特异性的功能TbErp复合物在布鲁塞。

DESCRIPTION (provided by applicant): The goal of this proposal is to evaluate the endogenous secretory machinery responsible for the proper localization of virulence factors in the protozoan parasite Trypanosoma brucei. T. brucei causes African Human African trypanosomiasis (HAT), endemic to 36 countries of sub-Saharan Africa and responsible for tens of thousands of deaths annually. The disease is uniformly fatal without treatment, available treatment regimens are highly toxic and difficult to administer, and no vaccines exist. T. brucei i transmitted to the host bloodstream via the bite of an infected tsetse fly, and the success of the parasite during infection relies on two essential virulence components that are both products of the T. brucei secretory system. The first is the surface- localized glycosylphosphatidylinositol (GPI)-anchored Variant Surface Glycoprotein (VSG) coat. The second is the parasite lysosome, a digestive organelle that processes endocytosed nutritional factors and degrades lytic host immune complexes. The proper targeting of VSG to the cell surface and organellar constituents to the lysosome is central to the pathogenesis of T. brucei. Despite its crucial role during infection, the T. brucei secretory and endomembrane trafficking machinery remain widely uncharacterized. The first trafficking step encountered by all secretory cargo (independent of their final destinations) is exit from the ER in COPII (coat protein II)-coated vesicles. In other eukaryotes, members of the p24 family of transmembrane proteins form poorly characterized heteromeric complexes that mediate cargo selection during COPII vesicle budding. In particular, the two well-defined p24 complexes described (yeast & mammals) bind and select mature GPI-anchored proteins for ER exit. Bioinformatic analyses identified 8 members of the p24 family in the T. brucei genome (TbERP1-8), and we initially hypothesized that they might be involved in GPI- VSG trafficking. TbERP1-8 were evaluated individually via RNAi silencing. None appear to be essential, and only TbERP2 silencing caused a moderate growth defect. Additionally, the TbERP1-8 silenced cell lines exhibited no delays in VSG trafficking or in bulk secretion. However, silencing of TbERP1, TbERP2 or TbERP8 was sufficient to cause dramatic pre-Golgi delays in the forward trafficking of two endogenous lysosomal proteins, p67 and TbCatL. Additionally, silencing of TbERP1, TbERP2 or TbERP8 caused increased secretion of an ER resident protein, suggestive of a secondary regulatory role in protein targeting. The parallel phenotypes observed in these knockdown experiments suggest that Erp1, Erp2, and/or Erp8 function during ER exit and may comprise a p24 complex with novel cargo specificity(s). It is the goal of this proposal to fully characterize these candidates, and to describe the subunit identity and cargo specificity of the functional TbErp complex in T. brucei.