The Lysosome of Trypanosoma brucei: A Proteomic Analysis

性状（由申请方提供）：非洲锥虫（布氏锥虫亚种）是寄生原生动物，引起人类非洲锥虫病（HAT，昏睡病）和牲畜的长角线虫病。这些疾病在发现采采蝇媒介的整个非洲具有破坏性影响。36个国家的约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（依氟鸟氨酸）价格昂贵，需要长期治疗，最差的（美拉胂醇）可导致10%的接受者死亡。如果不进行治疗，感染不可避免地是致命的，由于疫苗接种不是一种选择，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是必不可少的，特别是在治疗方面。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]，并且在必需溶酶体组分的运输中存在阶段特异性差异[2]。溶酶体是从宿主获得的用于营养的内吞货物的最终储存库[3]，以及从细胞表面去除的溶解性免疫复合物[4]。溶酶体蛋白酶的释放是人类感染的标志性事件，即中枢神经系统渗透的一个因素[5]。溶酶体生理学对先天性人血清抗性性状（锥虫溶解因子）的活性也至关重要，锥虫溶解因子限制了锥虫属物种的宿主范围[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，在T.布氏杆菌，p67，必需膜糖蛋白[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种已通过生物化学和/或蛋白质组学分析验证的蛋白质，其中约一半与人类遗传性先天代谢缺陷（也称为溶酶体贮积病）相关[11]。在哺乳动物中，只有11个明显的直系同源物（约1/7）可以在T。布鲁氏菌基因组（将多亚基液泡蛋白泵作为单一实体处理），尽管事实上生化分析表明这些活性中的许多应该存在。基于基因组中已知功能或可通过同源性预测的所有基因的百分比（约1/3），可以预测该数字会高得多。该提案的总体目标是通过直接蛋白质组学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。最基本的原理是，这些蛋白质将为随后的药物开发和溶酶体生物发生的进一步基础研究提供充足的机会。这种原理得到了锥虫生物学中溶酶体的关键功能以及人类疾病与溶酶体缺陷相关的频率的充分支持。 公共卫生相关性：非洲锥虫（布氏锥虫亚种）非洲锥虫是一种寄生原生动物，可引起人类非洲锥虫病（HAT，昏睡病），以及牛和其他牲畜的长角线虫病。目前的药物是有毒的，疫苗接种不是一种选择，因此需要新的化疗靶点。我们提出了一个纯化的溶酶体的protoeomic分析的基本原理，识别关键的寄生虫特异性酶将提供独特的机会，为后续的寄生虫特异性药物开发。

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.