The Lysosome of Trypanosoma brucei: A Proteomic Analysis

布氏锥虫的溶酶体：蛋白质组学分析

• 批准号: 8197811
• 负责人: James D. Bangs
• 金额: $ 16.59万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8197811
• 关键词: Affinity; Africa; Africa South of the Sahara; African; African Trypanosomiasis; Antigen-Antibody Complex; Area; Attention; Biochemical; Biogenesis; Biology; Bite; Blood Circulation; Candidate Disease Gene; Cathepsin L; Cathepsins B; Cattle; Cell surface; Cells; Country; Disease; Drops; Eflornithine; Enzymes; Epitopes; Equilibrium; Event; Fractionation; Frequencies; Genes; Genetic Recombination; Genome; Genomics; Goals; Human; Human Genetics; Hybrids; Immunofluorescence Microscopy; Immunoprecipitation; Infection; Intervention; Isopycnic Centrifugation; Life Cycle Stages; Livestock; Lysosomal Storage Diseases; Lysosomes; Lytic; Mass Spectrum Analysis; Melarsoprol; Membrane Glycoproteins; Metabolism; Neuraxis; Nutritional; Orphan; Parasites; Penetration; Peptide Hydrolases; Pharmaceutical Preparations; Physiologic pulse; Physiology; Proteins; Proteome; Proteomics; Protozoa; Pump; Radiolabeled; Regimen; Resistance; Risk; Sampling; Scheme; Sedimentation process; Serum; Staging; Therapeutic; Tropical Disease; Trypanosoma; Trypanosoma brucei brucei; Tsetse Flies; Vaccination; Validation; base; drug development; gel electrophoresis; human disease; killings; lysosomal proteins; nagana; neglect; nutrition; pathogen; public health relevance; radiotracer; repository; reversed phase chromatography; trafficking; trait; transmission process; vector

DESCRIPTION (provided by applicant): African trypanosomes (Trypanosoma brucei ssp.) are parasitic protozoa that cause human African trypanosomiasis (HAT, sleeping sickness) and nagana in livestock. These diseases have devastating impact throughout Africa where the tsetse fly vector is found. ~60 million people in 36 countries are at risk of tsetse bite and transmission, consequently HAT is considered to be a great neglected tropical disease. Few drugs are available, the best of which (eflornithine) is costly and requires a prolonged regimen, and the worst (melarsoprol) kills up to 10% of recipients. Infection is inevitably fatal without treatment, and since vaccination is not an option there is a critical need for new drug development. Thus a better understanding of the basic parasite biology is essential, particularly of aspects amenable to therapeutics. One such area is the lysosome because it impacts the host-pathogen balance in multiple ways. Expression of lysosomal activities is differentially regulated during the life cycle [1], and there are stage specific differences in the trafficking of essential lysosomal components [2]. The lysosome is the final repository of endocytic cargo acquired from the host for nutrition [3], as well as for lytic immune complexes removed from the cell surface [4]. Release of lysosomal proteases is a factor in the signature event of human infection, penetration of the central nervous system [5]. Lysosomal physiology is also critical to the activity of an innate human serum resistance trait, trypanolytic factor, which limits the host range of Trypanosoma species [6]. And finally, lysosomal hydrolytic activities have drawn considerable attention as potential chemotherapeutic targets [7]. However, only three lysosomal components have been characterized in T. brucei, p67 an essential membrane glycoprotein [6, 8], the a cathepsin L orthologue, TbCatL [1, 9], and a cathepsin B orthologue, TbCatB [10]. Mammalian lysosomes contain ~75 proteins that have been validated by biochemical and/or proteomic analyses, and of these ~half are associated with human genetic in-born errors in metabolism (also known as lysosomal storage diseases) [11]. Of the mammalian total only 11 obvious orthologues (~1/7th) can be identified in the T. brucei genome (treating the multi-subunit vacuolar protein pump as a single entity), despite the fact that biochemical analyses indicate many of these activities should be present. One would predict based on the percent of all genes in the genome for which function is known or can predicted by homology (~1/3rd) that this number would be much higher. The overarching goal of this proposal is to rectify this situation by direct proteomic analysis. Given the circumstances this is the only approach to filling the gaps that cannot be filled by standard genomic approaches. The uber-rationale is that these proteins will provide ample opportunities for subsequent drug development, and for further basic studies of lysosomal biogenesis. This rationale is amply supported by the critical functions of the lysosome in trypanosome biology, and the frequency that human disease is associated with lysosomal deficiencies. PUBLIC HEALTH RELEVANCE: African trypanosomes (Trypanosoma brucei ssp.) are parasitic protozoa that cause human African trypanosomiasis (HAT, sleeping sickness), as well as nagana in cattle and other livestock. Current drugs are toxic and vaccination is not an option, consequently new chemotherapeutic targets are needed. We propose a protoeomic analysis of purified lysosomes with the rationale that identification of critical parasite-specific enzymes will provide unique opportunities for subsequent parasite-specific drug development.

描述（由申请人提供）：非洲锥虫（Trypanosoma brucei ssp.）是一种寄生原生动物，可引起人类非洲锥虫病（HAT，昏睡病）和牲畜的那加那病。这些疾病对发现采采蝇媒介的整个非洲产生了毁灭性影响。 36 个国家约 6000 万人面临采采蝇叮咬和传播的风险，因此 HAT 被认为是一种严重被忽视的热带疾病。可用的药物很少，其中最好的药物（依氟鸟氨酸）价格昂贵且需要长期治疗，而最差的药物（美拉丙醇）会导致多达 10% 的接受者死亡。如果不进行治疗，感染将不可避免地致命，并且由于无法选择疫苗接种，因此迫切需要开发新药。因此，更好地了解寄生虫的基本生物学至关重要，特别是适合治疗的方面。其中一个区域是溶酶体，因为它以多种方式影响宿主-病原体平衡。溶酶体活性的表达在生命周期中受到差异性调节[1]，并且重要溶酶体成分的运输存在阶段特异性差异[2]。溶酶体是从宿主获得的内吞物质的最终储存库，用于营养 [3] 以及从细胞表面去除的裂解免疫复合物 [4]。溶酶体蛋白酶的释放是人类感染、渗透中枢神经系统的标志性事件的一个因素[5]。溶酶体生理学对于先天人类血清抗性特征（锥虫分解因子）的活性也至关重要，它限制了锥虫物种的宿主范围[6]。最后，溶酶体水解活性作为潜在的化疗靶点引起了相当大的关注[7]。然而，T. brucei 中仅对三种溶酶体成分进行了表征，p67 是一种必需的膜糖蛋白 [6, 8]，组织蛋白酶 L 直向同源物 TbCatL [1, 9] 和组织蛋白酶 B 直向同源物 TbCatB [10]。哺乳动物溶酶体含有约 75 种蛋白质，这些蛋白质已通过生化和/或蛋白质组分析验证，其中约一半与人类先天性代谢缺陷有关（也称为溶酶体贮积病）[11]。在哺乳动物总数中，仅 11 个明显的直向同源物（约 1/7）可以在 T. brucei 基因组中被识别（将多亚基液泡蛋白泵视为单个实体），尽管生化分析表明其中许多活性应该存在。人们可以根据基因组中功能已知或可以通过同源性预测的所有基因的百分比（~1/3）来预测，这个数字会高得多。该提案的总体目标是通过直接蛋白质组分析来纠正这种情况。鉴于这种情况，这是填补标准基因组方法无法填补的空白的唯一方法。最重要的理由是这些蛋白质将为后续药物开发以及溶酶体生物合成的进一步基础研究提供充足的机会。溶酶体在锥虫生物学中的关键功能以及人类疾病与溶酶体缺陷相关的频率充分支持了这一基本原理。 公共卫生相关性：非洲锥虫（布氏锥虫亚种）是一种寄生原生动物，可导致人类非洲锥虫病（HAT，昏睡病）以及牛和其他牲畜的那加那病。目前的药物有毒且无法接种疫苗，因此需要新的化疗靶点。我们提出对纯化的溶酶体进行蛋白质组学分析，其基本原理是鉴定关键的寄生虫特异性酶将为随后的寄生虫特异性药物开发提供独特的机会。