The cathepsin L of Toxoplasma gondii (TgCPL) and its endogenous macromolecular inhibitor, toxostatin.
The cathepsin L of Toxoplasma gondii (TgCPL) and its endogenous macromolecular inhibitor, toxostatin.
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DOI：
10.1016/j.molbiopara.2008.11.012
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发表时间：
2009-03
影响因子：
1.5
通讯作者：
Reed, Sharon
中科院分区：
文献类型：
--
作者：
Huang, Robert;Que, Xuchu;Hirata, Ken;Brinen, Linda S.;Lee, Ji Hyun;Hansell, Elizabeth;Engel, Juan;Sajid, Mohammed;Reed, Sharon
T.弓形虫是所有脊椎动物包括人类的专性细胞内寄生虫。2成功的入侵和复制需要寄生虫蛋白的同步释放，其中许多蛋白需要蛋白水解加工。与大多数寄生虫不同，T.弓形虫具有有限数量的CA家族、C1家族半胱氨酸蛋白酶，其具有一个组织蛋白酶B（TgCP B）、一个组织蛋白酶L（TgCPL）和三个组织蛋白酶Cs（TgCPC 1、2、3）。在此之前，我们的特点是toxopain，唯一的组织蛋白酶B酶，其定位于棒状体细胞器。两个组织蛋白酶C被贩运通过致密颗粒的寄生虫液泡，在那里他们降解肽。我们现在报告的克隆，表达和建模的唯一组织蛋白酶L基因和两个新的内源性抑制剂的鉴定。TgCPL与人组织蛋白酶L不同，其最适pH为6.5，并且其底物在P2位置对亮氨酸（相对于苯丙氨酸）具有偏好性。这种独特的偏好解释了同源性建模，揭示了一个非典型的天冬氨酸（天冬氨酸216）在预测的活性位点S2口袋，这限制了基板访问的基础。为了进一步了解T.在弓形虫中，我们鉴定了两个编码内源性半胱氨酸蛋白酶抑制剂（ICP或toxostatins）的基因，其在纳摩尔范围内对TgCPB和TgCPL都有活性。过表达的toxostatin-1显着降低整体半胱氨酸蛋白酶活性的寄生虫裂解物，但没有检测到的影响，入侵或细胞内增殖。这些发现为T.弓形虫及其内源性控制。
T. gondii is an obligate intracellular parasite of all vertebrates, including man. Successful invasion and replication requires the synchronized release of parasite proteins, many of which require proteolytic processing. Unlike most parasites, T. gondii has a limited number of Clan CA, family C1 cysteine proteinases with one cathepsin B (TgCPB), one cathepsin L (TgCPL) and three cathepsin Cs (TgCPC1, 2, 3). Previously, we characterized toxopain, the only cathepsin B enzyme, which localizes to the rhoptry organelle. Two cathepsin Cs are trafficked through dense granules to the parasitophorous vacuole where they degrade peptides. We now report the cloning, expression, and modeling of the sole cathepsin L gene and the identification of two new endogenous inhibitors. TgCPL differs from human cathepsin L with a pH optimum of 6.5 and its substrate preference for leucine (vs. phenylalanine) in the P2 position. This distinct preference is explained by homology modeling, which reveals a non-canonical aspartic acid (Asp 216) at the base of the predicted active site S2 pocket, which limits substrate access. To further our understanding of the regulation of cathepsins in T. gondii, we identified two genes encoding endogenous cysteine proteinase inhibitors (ICPs or toxostatins), which are active against both TgCPB and TgCPL in the nanomolar range. Over expression of toxostatin-1 significantly decreased overall cysteine proteinase activity in parasite lysates, but had no detectable effect on invasion or intracellular multiplication. These findings provide important insights into the proteolytic cascades of T. gondii and their endogenous control.
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