Secretory cargo selection by p24 transmembrane proteins in African Trypanosomes

描述(由申请人提供)：本提案的目标是评估负责在原生动物寄生虫布氏锥虫中正确定位毒力因子的内源性分泌机制。布氏毛滴虫导致非洲人类非洲锥虫病(HAT)，该疾病流行于撒哈拉以南非洲的36个国家，每年造成数万人死亡。这种疾病在没有治疗的情况下都是致命的，现有的治疗方案毒性高，难以管理，也不存在疫苗。布氏毛滴虫通过被感染的采采蝇的叮咬传播到宿主的血液中，在感染期间寄生虫的成功依赖于两种基本的毒力成分，这两种成分都是布氏毛滴虫分泌系统的产物。第一种是表面定位的糖基磷脂酰肌醇(GPI)锚定的变体表面糖蛋白(VSG)涂层。第二种是寄生虫溶酶体，这是一种消化细胞器，负责处理内吞的营养因子并降解裂解的宿主免疫复合体。适当的靶向VSG到细胞表面和细胞器成分到溶酶体是布鲁氏毛滴虫致病的核心。尽管布鲁氏毛滴虫在感染过程中起着关键作用，但它的分泌和膜内运输机制仍然很难确定。所有分泌性货物遇到的第一个贩运步骤(与其最终目的地无关)是以COPII(外壳蛋白II)包裹的囊泡离开ER。在其他真核生物中，p24跨膜蛋白家族的成员形成特征不佳的异构体复合体，在COPII囊泡萌发期间介导货物选择。特别是，所描述的两个定义明确的p24复合体(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。生物信息学分析确定了布鲁氏毛滴虫基因组中p24家族的8个成员(TbERP1-8)，我们初步推测他们可能参与了GPI-VSG的运输。通过RNAi沉默来单独评估TbERP1-8。似乎都不是必需的，只有TbERP2沉默导致了适度的生长缺陷。此外，TbERP1-8沉默的细胞系在VSG运输或大量分泌方面没有延迟。然而，沉默TbERP1、TbERP2或TbERP8足以导致两种内源性溶酶体蛋白p67和TbCatL在高尔基体前向转运的显著延迟。此外，沉默TbERP1、TbERP2或TbERP8导致内质网驻留蛋白分泌增加，提示在蛋白质靶向中起二级调节作用。在这些击倒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，并可能包括具有新的货物特异性的p24复合体(S)。本提案的目的是全面描述这些候选基因，并描述布氏锥虫中功能TbErp复合体的亚基身份和货物特异性。

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.