Lysosomal biogenesis & function in African trypanosomes

溶酶体生物合成

• 批准号: 6830282
• 负责人: James D. Bangs
• 金额: $ 32.24万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6830282
• 关键词: Lysosomal; biogenesis; function; African; trypanosomes

EXCEED THE SPACE PROVIDED. African trypanosomes are parasitic protozoa that cause sleeping sickness in humans. Little is known about lysosomal biogenesis in these ancient eukaryotes, p67 is an essential membrane glycoprotein located primarily in the lysosome, but also in the flagellar pocket and endosomes. It trafficks to the lysosome in a stage-specific manner, and in bloodstream cells its N-glycans are modified by synthesis of poly-lactosamine (pNAL) chains. In trypanosomes secretion and endocytosis obligately intersect in the flagellar pocket; as an essential protein that exists at this intersection p67 is a unique tool for studying how these critical processes shape lysosomal biogenesis and function. We will study of the signals and machinery of p67 targeting and its role in lysosomal structure/function. Our underlying hypotheses are: i) that p67 targeting is mediated by specific signals in its cytoplasmic domain; and ii) that p67 is essential for lysosomal biogenesis and function. This work will expand our understanding of the novel aspects of trypanosomal secretory/endocytic trafficking, and of mechanisms of protein targeting conserved over many millions of years of eukaryotic evolution. In AIM #1 membrane protein reporters will be fused to the p67 cytoplasmic domain and mutagenesis will be used to identify the specific sequence motifs that mediate lysosomal targeting. In AIM #2 the cytoplasmic domain will be used as bait to identify interactive proteins that are part of the p67 trafficking machinery. The cytoplasmic domain will be expressed in trypanosomes as a TAP-tag fusion and in situ complexes will be isolated by affinity chromatography. The cytoplasmic domain will also be used as genetic bait in a yeast two-hybrid screen to identify genes for interactive proteins. In Aim #3 we will use RNAi silencing to examine the role of p67 in lysosomal function. Preliminary RNAi experiments indicate that p67 is an essential protein in bloodstream trypanosomes and our hypothesis is that p67 provides a protective glycocalyx against internal hydrolases and against potentially lytic host serum factors. In Aim #4 we will employ chemical mutagenesis to generate cell lines deficient in synthesis of the unusual pNAL N-glycans that are found on p67 and other membrane proteins of the flagellar pocket/endosomal system in bloodstream parasites. Mutants will be selected for failure to endocytose fluorescent tomato lectin and then characterized for general deficiencies in lysosomal/endocytic processes, and for specific defects in p67 targeting and function. PERFORMANCE SITE ========================================Section End===========================================

超出所提供的空间。 非洲锥虫是一种寄生原生动物，会引起人类昏睡病。在这些古老的真核生物中，对溶酶体的生物发生知之甚少，p67是一种重要的膜糖蛋白，主要位于溶酶体中，但也位于鞭毛袋和内体中。它以阶段特异性方式运输至溶酶体，并且在血流细胞中，其N-聚糖通过合成聚乳糖胺（pNAL）链而被修饰。在锥虫中，分泌和内吞作用在鞭毛口袋中必然相交;作为存在于这个相交处的必需蛋白，p67是研究这些关键过程如何塑造溶酶体生物发生和功能的独特工具。我们将研究p67靶向的信号和机制及其在溶酶体结构/功能中的作用。我们的基本假设是：i）p67靶向由其胞质结构域中的特异性信号介导;和ii）p67对于溶酶体生物发生和功能是必需的。这项工作将扩大我们对锥虫分泌/内吞运输的新方面的理解，以及在数百万年的真核生物进化中保守的蛋白质靶向机制。 在AIM#1中，膜蛋白报告基因将与p67胞质结构域融合，并且诱变将用于鉴定介导溶酶体靶向的特定序列基序。在AIM #2中，胞质结构域将用作诱饵以鉴定作为p67运输机制的一部分的相互作用蛋白。胞质结构域将在锥虫中表达为TAP-标签融合体，并通过亲和色谱分离原位复合物。胞质结构域也将在酵母双杂交筛选中用作遗传诱饵，以鉴定相互作用蛋白质的基因。在目标#3中，我们将使用RNAi沉默来检查p67在溶酶体功能中的作用。初步的RNAi实验表明，p67是一个重要的蛋白质在血流锥虫和我们的假设是，p67提供了一个保护性的糖萼对内部水解酶和对潜在的溶解宿主血清因子。在目标#4中，我们将采用化学诱变来产生在血流寄生虫中鞭毛袋/内体系统的p67和其他膜蛋白上发现的不寻常的pNAL N-聚糖的合成中缺陷的细胞系。将选择不能内吞荧光番茄凝集素的突变体，然后表征溶酶体/内吞过程中的一般缺陷，以及p67靶向和功能中的特定缺陷。性能现场=