Santos Camila C, Sant'anna Celso, Terres Amanda, Cunha-e-Silva Narcisa L, Scharfstein Julio, de A Lima Ana Paula C
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, C.C.S., Ilha do Fundão, Rio de Janeiro, 21949-900 RJ, Brazil.
J Cell Sci. 2005 Mar 1;118(Pt 5):901-15. doi: 10.1242/jcs.01677. Epub 2005 Feb 15.
Chagasin is a Trypanosoma cruzi protein that was recently characterized as a tight-binding inhibitor of papain-like cysteine proteases (CPs). Considering that parasite virulence and morphogenesis depend on the endogenous activity of lysosomal CPs of the cruzipain family, we sought to determine whether chagasin and cruzipain interact in the living cell. Ultrastructural studies showed that chagasin and cruzipain both localize to the Golgi complex and reservosomes (lysosome-like organelles), whereas free chagasin was found in small intracellular vesicles, suggesting that chagasin trafficking pathways might intersect with those of cruzipain. Taking advantage of the fact that sodium dodecyl sulphate and beta-mercaptoethanol prevent binding between the isolated proteins but do not dismantle preformed cruzipain-chagasin complexes, we obtained direct evidence that chagasin-cruzipain complexes are indeed formed in epimastigotes. Chagasin transfectants (fourfold increase in CP inhibitory activity) displayed low rates of differentiation (metacyclogenesis) and exhibited increased resistance to a synthetic CP inhibitor. These phenotypic changes were accompanied by a drastic reduction of soluble cruzipain activity and by upregulated secretion of cruzipain-chagasin molecular complexes. Analysis of six T. cruzi strains revealed that expression levels of cruzipain and chagasin are variable, but the molar ratios are fairly stable ( approximately 50:1) in most strains, with the exception of the G strain (5:1), which is poorly infective. On the same vein, we found that trypomastigotes overexpressing chagasin are less infective than wild-type parasites in vitro. The deficiency of chagasin overexpressers is caused by lower activity of membrane-associated CPs, because membranes recovered from wild-type trypomastigotes restored infectivity and this effect was nullified by the CP inhibitor E-64. In summary, our studies suggest that chagasin regulates the endogenous activity of CP, thus indirectly modulating proteolytic functions that are essential for parasite differentiation and invasion of mammalian cells.
查加辛是一种克氏锥虫蛋白，最近被鉴定为木瓜蛋白酶样半胱氨酸蛋白酶（CPs）的紧密结合抑制剂。鉴于寄生虫的毒力和形态发生依赖于克鲁兹蛋白酶家族溶酶体CPs的内源性活性，我们试图确定查加辛和克鲁兹蛋白酶在活细胞中是否相互作用。超微结构研究表明，查加辛和克鲁兹蛋白酶都定位于高尔基体复合体和贮存体（类溶酶体细胞器），而游离的查加辛存在于小的细胞内囊泡中，这表明查加辛的运输途径可能与克鲁兹蛋白酶的运输途径相交。利用十二烷基硫酸钠和β-巯基乙醇可阻止分离蛋白之间的结合但不会破坏预先形成的克鲁兹蛋白酶-查加辛复合物这一事实，我们获得了直接证据，证明查加辛-克鲁兹蛋白酶复合物确实在无鞭毛体中形成。查加辛转染子（CP抑制活性增加四倍）表现出低分化率（循环后期发育），并对合成CP抑制剂表现出增强的抗性。这些表型变化伴随着可溶性克鲁兹蛋白酶活性的急剧降低以及克鲁兹蛋白酶-查加辛分子复合物分泌的上调。对六种克氏锥虫菌株的分析表明，克鲁兹蛋白酶和查加辛的表达水平是可变的，但在大多数菌株中摩尔比相当稳定（约50:1），除了感染性较差的G菌株（5:1）。同样，我们发现体外过表达查加辛的锥鞭毛体比野生型寄生虫的感染性更低。查加辛过表达菌株的缺陷是由膜相关CPs的活性降低引起的，因为从野生型锥鞭毛体回收的膜恢复了感染性，并且这种作用被CP抑制剂E-64消除。总之，我们的研究表明，查加辛调节CP的内源性活性，从而间接调节对寄生虫分化和入侵哺乳动物细胞至关重要的蛋白水解功能。
