Secretory cargo selection by p24 transmembrane proteins in African Trypanosomes

非洲锥虫中 p24 跨膜蛋白的分泌货物选择

• 批准号: 8717799
• 负责人: Emilia K. Kruzel
• 金额: $ 5.33万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-20 至 2017-09-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8717799
• 关键词: Africa South of the Sahara; African; African Trypanosomiasis; Anabolism; Antigen-Antibody Complex; Antigenic Variation; Binding; Biochemical; Biogenesis; Bioinformatics; Biology; Bite; Blood Circulation; Capsid Proteins; Cell Line; Cell membrane; Cell surface; Cells; Cellular biology; Cessation of life; Co-Immunoprecipitations; Coated vesicle; Complex; Country; Defect; Destinations; Development; Disease; Endocytosis; Endoplasmic Reticulum; Engineering; Epitopes; Eukaryota; Exhibits; Face; Family; GPI Membrane Anchors; Genes; Genome; Germ Cells; Glycosylphosphatidylinositols; Goals; Golgi Apparatus; Growth; Host Defense; Human; Immune; Immune system; Immunofluorescence Microscopy; In Vitro; Infection; Integral Membrane Protein; Intervention; Left; Length; Life; Locales; Lysosomes; Lytic; Mammalian Cell; Mammals; Mediating; Membrane; Membrane Glycoproteins; Nature; Nutritional; Organelles; Organism; Orthologous Gene; Parasites; Pathogenesis; Phenotype; Process; Protein Family; Proteins; RNA Interference; Role; Serum; Site; Specificity; Surface; System; Treatment Protocols; Tropical Disease; Trypanosoma; Trypanosoma brucei brucei; Tsetse Flies; Vaccines; Variant; Vesicle; Virulence; Virulence Factors; Wheat; Yeasts; base; in vivo; insight; killings; lysosomal proteins; member; neglect; novel; protein complex; protein function; public health relevance; receptor; receptor function; research study; secretory protein; stoichiometry; success; trafficking

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.

描述（由申请人提供）：本提案的目标是评估负责原生动物寄生虫布氏锥虫中毒力因子正确定位的内源性分泌机制。布氏锥虫引起非洲人类非洲锥虫病 (HAT)，该病在撒哈拉以南非洲 36 个国家流行，每年导致数万人死亡。如果不进行治疗，这种疾病都是致命的，现有的治疗方案毒性很大且难以施用，并且不存在疫苗。布氏锥虫通过受感染采采蝇的叮咬传播到宿主血液中，寄生虫在感染过程中的成功依赖于两种重要的毒力成分，这两种成分都是布氏锥虫分泌系统的产物。第一个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）涂层。第二个是寄生虫溶酶体，这是一种消化细胞器，可以处理内吞的营养因子并降解裂解宿主免疫复合物。 VSG 正确靶向细胞表面和细胞器成分靶向溶酶体是布氏锥虫发病机制的核心。尽管布氏锥虫在感染过程中发挥着至关重要的作用，但其分泌和内膜运输机制仍然广泛未被表征。 所有分泌货物（与其最终目的地无关）遇到的第一个运输步骤是从 COPII（外壳蛋白 II）包被的囊泡中的 ER 排出。在其他真核生物中，跨膜蛋白 p24 家族的成员形成特征不明的异聚复合物，在 COPII 囊泡出芽过程中介导货物选择。特别是，所描述的两种明确的 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。生物信息学分析确定了 T. brucei 基因组中 p24 家族的 8 个成员 (TbERP1-8)，我们最初假设它们可能参与 GPI-VSG 贩运。通过 RNAi 沉默单独评估 TbERP1-8。似乎没有一个是必需的，只有 TbERP2 沉默会导致中度生长缺陷。此外，TbERP1-8 沉默的细胞系没有表现出 VSG 运输或大量分泌的延迟。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 的前向运输发生显着的前高尔基体延迟。此外，TbERP1、TbERP2 或 TbERP8 的沉默导致 ER 驻留蛋白的分泌增加，表明在蛋白质靶向中具有次级调节作用。在这些敲低实验中观察到的平行表型表明，Erp1、Erp2 和/或 Erp8 在 ER 退出期间发挥作用，并且可能包含具有新货物特异性的 p24 复合物。本提案的目标是充分表征这些候选者，并描述 T. brucei 中功能性 TbErp 复合体的亚基身份和货物特异性。