EXPRESSION OF GLYCOLIPID ANCHORED PRIONS IN TRYPANOSOMES

布鲁氏锥虫引起一种动物和人类疾病，通常被称为非洲锥虫病、昏睡病或昏睡病。锥虫可以在被感染的宿主体内无限期地持续存在，通过抗原变异过程逃避免疫系统，抗原变异是由变异表面糖蛋白（VSGs）的顺序表达介导的，后者在锥虫表面形成一层坚若摧的“外衣”。每个锥虫表达1000万个单个糖基磷脂酰肌醇（GPI）锚定的VSG分子，占细胞总蛋白的10%。布氏体的表面积与红细胞的表面积大致相同，在大鼠或小鼠感染的高峰期，锥虫的产量约为红细胞数量的三分之一。对VSGs的研究提供了明确的化学证据，随后，GPI膜锚的第一个完整结构和GPI合成复杂途径的许多细节的阐明。随后，在真核细胞的数百种蛋白质中发现了糖脂锚点，它们具有共同的核心结构。布鲁氏锥虫是内源性gpi锚定蛋白表面表达的“工厂”：VSG和前环酸性重复蛋白（PARP）各占总锥虫蛋白的10%，分别存在于血液和昆虫（Glossina, Tesetse）的前环形式中。大多数哺乳动物的gpi锚定蛋白占细胞总蛋白的比例不到0.1%。尽管积累的知识表明朊病毒或突变朊病毒基因与几种脑病有关，但由于无法从天然来源或通过重组技术大量生产感染性PrPSc或天然PrPC，因此中心理论（即朊病毒传播涉及内源性细胞蛋白（PrPC）向感染性痒病朊病毒（PrPSc）的自催化转化）无法轻易得到验证。尽管最近发表的核磁共振研究必须对再生的非糖基化非gpi锚定重组蛋白进行，但关于PrPC结构的几个问题仍然存在。由锥虫产生的大量gpi锚定VSG，以及允许锥虫稳定转化表达异位基因的方法的发展，为生产高兴趣、高价值的gpi锚定哺乳动物蛋白，特别是PrP，提供了可靠的前景，可以快速评估。这一建议将侧重于生产不同形式的PrP在T.布鲁斯。

Trypanosoma brucei causes a disease of animals and humans that is commonly called African Trypanosomiasis, Nagana or Sleeping Sickness. Trypanosomes can persist indefinitely in the infected host, evading the immune system through a process of antigenic variation, mediated by the sequential expression of variant surface glycoproteins (VSGs) that form an impregnable 'coat' on the trypanosome surface. Each trypanosome expresses 10 million molecules of a single glycosylphosphatidylinositol- (GPI) anchored VSG, representing 10 percent of the total cell protein. The surface area of T. brucei is about the same as that of an erythrocyte and the yield of trypanosomes, at the peak of infection in a rat or mouse, is about one third of the number of erythrocytes. Studies of VSGs provided the definitive chemical evidence and, subsequently, the first complete structure of a GPI membrane anchor and the elucidation of many details of a complex pathway for GPI synthesis. The glycolipid anchors subsequently found in hundreds of proteins in a wide range of eukaryotic cells share a common core structure. T. brucei is a 'factory' for the surface expression of endogenous GPI-anchored proteins: VSG and the procyclic acidic repetitive protein (PARP) each comprise 10 percent of the total trypanosome protein, in the bloodstream and insect (Glossina, the Tesetse) 'procyclic' forms, respectively. Most mammalian GPI-anchored proteins comprise less than 0.1 percent of the total cellular protein. Despite the accumulated knowledge implicating prions or mutant prion genes in several encephalopathies, the central theory - that prion propagation involves a self-catalysed conversion of the endogenous cellular protein (PrPC) to the infectious scrapie prion (PrPSc) - cannot readily be tested because of the inability to produce infective PrPSc or native PrPC in large amounts from natural sources or by recombinant techniques. Several questions about the structure of PrPC remain, despite recently published NMR studies that were necessarily performed on renatured non-glycosylated non-GPI-anchored recombinant protein. The vast amount of GPI-anchored VSG that is made by trypanosomes and the development of methods that allow trypanosomes to be stably transformed to express ectopic genes, offer credible prospects, which can be rapidly evaluated, for producing high-interest high-value GPI-anchored mammalian proteins in general and PrP in particular. This proposal will focus on producing different forms of PrP in T. brucei.