Engel J C, Doyle P S, Palmer J, Hsieh I, Bainton D F, McKerrow J H
Department of Pathology, University of California, San Francisco, California 94143, USA.
J Cell Sci. 1998 Mar;111 ( Pt 5):597-606. doi: 10.1242/jcs.111.5.597.
Cruzain, the major cysteine protease of the protozoan parasite Trypanosoma cruzi, is a target of rational drug design for chemotherapy of Chagas' disease. The precise biological role of cruzain in the parasite life cycle and the mechanism involved in the trypanocidal effect of cysteine protease inhibitors are still unclear. Here we report biological and ultrastructural alterations caused by cysteine protease inhibitors in T. cruzi epimastigotes. Cruzain, a glycoprotein that transits the Golgi-endosomal pathway, localized to pre-lysosomes/lysosomes in the posterior end of untreated epimastigotes by fluorescent microscopy utilizing either a biotinylated cysteine protease inhibitor to tag the active site, or a specific anti-cruzain antibody. Radiolabeled or biotinylated cysteine protease inhibitors bound exclusively to cruzain in intact epimastigotes confirming that cruzain is accessible to, and is targeted by the inhibitors. Treatment of T. cruzi epimastigotes with specific cysteine protease inhibitors arrested growth, altered the intracellular localization of cruzain, and induced major alterations in the Golgi complex. Following treatment, cruzain accumulated in peripheral dilations of Golgi cisternae. There was a concomitant 70% reduction in gold-labeled cruzain transported to lysosomes. Cisternae abnormalities in the Golgi compartment were followed by distention of ER and nuclear membranes. Brefeldin A increased the number and size of cisternae in epimastigotes. Pre-treatment of epimastigotes with cysteine protease inhibitors followed by exposure to brefeldin A induced a more rapid appearance of the cysteine protease inhibitor-induced Golgi alterations. Our results suggest that cysteine protease inhibitors prevent the normal autocatalytic processing and trafficking of cruzain within the Golgi apparatus. Accumulation of cruzain may decrease mobility of Golgi membranes and result in peripheral distention of cisternae. These major alterations of the Golgi complex parallel the death of T. cruzi epimastigotes.
克鲁兹蛋白酶是原生动物寄生虫克氏锥虫的主要半胱氨酸蛋白酶，是恰加斯病化疗合理药物设计的靶点。克鲁兹蛋白酶在寄生虫生命周期中的精确生物学作用以及半胱氨酸蛋白酶抑制剂杀锥虫作用所涉及的机制仍不清楚。在此，我们报告了半胱氨酸蛋白酶抑制剂对克氏锥虫前鞭毛体造成的生物学和超微结构改变。克鲁兹蛋白酶是一种通过高尔基体 - 内体途径转运的糖蛋白，利用生物素化的半胱氨酸蛋白酶抑制剂标记活性位点或特异性抗克鲁兹蛋白酶抗体，通过荧光显微镜观察，在未处理的前鞭毛体后端定位于前溶酶体/溶酶体。放射性标记或生物素化的半胱氨酸蛋白酶抑制剂在完整的前鞭毛体中仅与克鲁兹蛋白酶结合，证实克鲁兹蛋白酶可被抑制剂作用并成为其作用靶点。用特异性半胱氨酸蛋白酶抑制剂处理克氏锥虫前鞭毛体可使生长停滞，改变克鲁兹蛋白酶的细胞内定位，并诱导高尔基体复合体发生重大改变。处理后，克鲁兹蛋白酶积聚在高尔基体扁平囊的外周扩张处。同时，转运至溶酶体的金标记克鲁兹蛋白酶减少了70%。高尔基体区室的扁平囊异常之后是内质网和核膜的扩张。布雷菲德菌素A增加了前鞭毛体中扁平囊的数量和大小。用半胱氨酸蛋白酶抑制剂预处理前鞭毛体，然后暴露于布雷菲德菌素A，可使半胱氨酸蛋白酶抑制剂诱导的高尔基体改变更快出现。我们的结果表明，半胱氨酸蛋白酶抑制剂可阻止克鲁兹蛋白酶在高尔基体中正常的自催化加工和运输。克鲁兹蛋白酶的积聚可能会降低高尔基体膜的流动性，并导致扁平囊外周扩张。高尔基体复合体的这些重大改变与克氏锥虫前鞭毛体的死亡平行。
