A Parasite Cysteine Protease Is Key to Host Protein Degradation and Iron Acquisition
A Parasite Cysteine Protease Is Key to Host Protein Degradation and Iron Acquisition
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DOI：
10.1074/jbc.m805824200
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发表时间：
2008-10-24
影响因子：
4.8
通讯作者：
McKerrow, James H.
中科院分区：
文献类型：
--
作者：
O'Brien, Theresa C.;Mackey, Zachary B.;McKerrow, James H.
Can CA(木瓜蛋白酶)家族的半胱氨酸蛋白酶是原始无脊椎动物的主要水解酶。半胱氨酸蛋白酶抑制剂可以阻止原生动物布鲁氏锥虫的感染。RNA干扰研究表明，组织蛋白酶B样蛋白酶tbcatB是关键的抑制物靶标。利用tbcatB的两个等位基因中的一个缺失的寄生虫，描述了该酶在降解内吞的宿主蛋白中的关键作用。TbcatB缺乏导致鞭毛袋和邻近内细胞室的生长速度下降和变形。Western印迹和显微镜分析表明，tbcatB的缺失会导致寄主和寄生虫蛋白的积累，包括溶酶体标记p67。寄生的一个关键功能是宿主转铁蛋白的降解，这是获得铁所必需的。重组tbcatB的底物专一性分析揭示了该酶的最佳多肽切割序列，并用基于FRET的底物进行了实验验证。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和N-末端测序鉴定转铁蛋白的降解情况。因为即使是轻微的tbcatB缺乏对寄生虫也是致命的，tbcatB是开发新的抗锥虫化疗的合乎逻辑的靶点。
Cysteine proteases of the Clan CA (papain) family are the predominant protease group in primitive invertebrates. Cysteine protease inhibitors arrest infection by the protozoan parasite, Trypanosoma brucei. RNA interference studies implicated a cathepsin B-like protease, tbcatB, as a key inhibitor target. Utilizing parasites in which one of the two alleles of tbcatb has been deleted, the key role of this protease in degradation of endocytosed host proteins is delineated. TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment. Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67. A critical function for parasitism is the degradation of host transferrin, which is necessary for iron acquisition. Substrate specificity analysis of recombinant tbcatB revealed the optimal peptide cleavage sequences for the enzyme and these were confirmed experimentally using FRET-based substrates. Degradation of transferrin was validated by SDS-PAGE and the specific cleavage sites identified by N-terminal sequencing. Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy.