Lysosomal biogenesis & function in African trypanosomes

溶酶体生物合成

基本信息

批准号：
7146720
负责人：
James D. Bangs
金额：
$ 30.54万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-07-01 至 2008-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7146720
关键词：
Affinity Chromatography African African Trypanosomiasis Behavior Binding Proteins Biochemistry Biogenesis Biological Biological Assay Blood Circulation C-terminal Calmodulin Cattle Cell Death Cell Line Cell membrane Cells Chemicals Complex Cytoplasmic Tail Data Defect Endocytosis Endosomes Environment Epitopes Eukaryota Eukaryotic Cell Event Evolution Failure Genes Genetic Glycocalyx Golgi Apparatus Green Fluorescent Proteins Hand Human Hydrolase In Situ Insecta Kinetics Leucine Life Cycle Stages Light Liquid substance Lysosomes Lytic Mediating Membrane Glycoproteins Membrane Proteins Modification Morphology Mutagenesis Organism Parasites Pathway interactions Phase Phenotype Polysaccharides Positioning Attribute Post-Translational Protein Processing Predisposition Process Production Proteins Protozoa RNA RNA Interference Rate Reporter Role Serum Shapes Signal Transduction Site-Directed Mutagenesis Specificity Staging Structure System TNF gene Technology Trypanosoma Trypanosoma brucei brucei Variant Vesicle Work Yeasts cell growth gene cloning in vivo interest lactosamine mutant nagana novel poly-N-acetyllactosamine receptor receptor mediated endocytosis research study stem tomato lectin tool trafficking trans-sialidase uptake yeast two hybrid system

项目摘要

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.

非洲锥虫是寄生原生动物，引起人类睡眠的疾病。鲜为人知关于这些古代真核生物中的溶酶体生物发生，p67是一种必不可少的膜糖蛋白主要是在溶酶体中，但也位于鞭毛口袋和内体中。它在一个阶段特异性的方式，在血液细胞中，其N-聚糖通过多聚乳糖胺的合成而修饰（Pnal）连锁。在锥虫分泌和内吞作用中，在鞭毛袋中有规则相交；作为在此交叉点p67中存在的必需蛋白质是研究这些关键过程的独特工具塑造溶酶体生物发生和功能。我们将研究p67靶向的信号和机械以及它在溶酶体结构/功能中的作用。我们的基本假设是：i）p67靶向是由其细胞质结构域中的特定信号； ii）p67对于溶酶体生物发生和功能至关重要。这项工作将扩大我们对锥虫分泌/内吞贩运的新颖方面的理解，以及在数百万年的真核进化中保存蛋白质靶向的机制。在AIM＃1中，膜蛋白报道器将融合到p67细胞质结构域，诱变将会用于识别介导溶酶体靶向的特定序列基序。在AIM＃2中细胞质域将被用作诱饵，以识别p67运输机制一部分的交互式蛋白质。这细胞质结构域将在锥虫中表示为Tap-tag融合，原位络合物将是通过亲和色谱分离。细胞质结构域也将用作酵母中的遗传诱饵两个杂交筛选以鉴定相互作用蛋白的基因。在AIM＃3中，我们将使用RNAi沉默检查 p67在溶酶体功能中的作用。初步RNAi实验表明p67是必不可少的蛋白在血液锥虫中，我们的假设是p67提供了针对内部的保护性糖脂水解酶和潜在的溶解宿主血清因子。在AIM＃4中，我们将采用化学诱变为了产生缺乏在p67和其他上发现的异常pnal n-glycans合成的细胞系血液寄生虫中鞭毛袋/内体系统的膜蛋白。突变体会选择因未能内吞荧光番茄凝集素而被选择，然后以一般缺陷为特征溶酶体/内吞过程，以及p67靶向和功能中的特定缺陷。