The Lysosome of Trypanosoma brucei: A Proteomic Analysis

描述(申请人提供)：非洲锥虫(Trypanosoma brucei SSP.)是导致人类非洲锥虫病(HAT、昏睡病)和家畜Nagana的寄生原虫。这些疾病在发现采采蝇媒介的整个非洲都造成了毁灭性的影响。36个国家的约6000万人面临采采症咬伤和传播的风险，因此，采采症被认为是一种被严重忽视的热带疾病。可用的药物很少，其中最好的(依氟鸟氨酸)价格昂贵，需要长时间的治疗，最差的(美拉索洛尔)可导致高达10%的接受者死亡。感染在不治疗的情况下不可避免地是致命的，而且由于接种疫苗不是一种选择，因此迫切需要开发新药。因此，更好地了解基本的寄生虫生物学，特别是对可用于治疗的方面的了解是至关重要的。其中一个区域是溶酶体，因为它以多种方式影响宿主-病原体的平衡。溶酶体活性的表达在生命周期中受到不同的调节[1]，并且在必需的溶酶体成分的运输上存在阶段特异性的差异[2]。溶酶体是从宿主获取的内吞货物的最终储存库[3]，以及从细胞表面移除的裂解免疫复合体[4]。溶酶体蛋白水解酶的释放是人类感染中枢神经系统的标志性事件中的一个因素[5]。溶酶体生理学对于一种天生的人类血清抵抗特性--胰酶溶解因子的活性也是至关重要的，它限制了锥虫物种的宿主范围[6]。最后，溶酶体的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，在布鲁氏毛滴虫中，只有三种溶酶体成分被鉴定：必需的膜糖蛋白p67[6，8]、组织蛋白酶L同源物TbCatL[1，9]和组织蛋白酶B同源物TbCatB[10]。哺乳动物溶酶体含有约75种蛋白质，已被生化和/或蛋白质组学分析证实，其中约一半与人类先天代谢缺陷(也称为溶酶体储存疾病)有关[11]。在哺乳动物的全部基因组中，只有11个明显的同源基因(~1/7)可以在布氏毛滴虫基因组中识别(将多亚单位液泡蛋白泵视为一个单一实体)，尽管生化分析表明其中许多活动应该存在。人们会根据同源性(~1/3)已知或可以预测的基因组中所有功能已知的基因的百分比来预测，这个数字将会高得多。这项建议的首要目标是通过直接的蛋白质组学分析来纠正这种情况。在这种情况下，这是填补标准基因组方法无法填补的空白的唯一方法。其基本原理是，这些蛋白质将为后续的药物开发和进一步的溶酶体生物发生的基础研究提供充足的机会。溶酶体在锥体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，充分支持了这一理论。 公共卫生相关性：非洲锥虫(锥虫SSP.)是导致人类非洲锥虫病(HAT、昏睡病)的寄生原虫，以及牛和其他牲畜的Nagana。目前的药物是有毒的，接种疫苗不是一个选择，因此需要新的化疗靶点。我们建议对纯化的溶酶体进行原基因组学分析，其基本原理是鉴定关键的寄生虫特异性酶将为后续的寄生虫特异性药物开发提供独特的机会。

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.