Trypanosoma brucei Metacaspase 4 Is a Pseudopeptidase and a Virulence Factor
Trypanosoma brucei Metacaspase 4 Is a Pseudopeptidase and a Virulence Factor
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DOI：
10.1074/jbc.m111.292334
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发表时间：
2011-11-18
影响因子：
4.8
通讯作者：
Mottram, Jeremy C.
中科院分区：
文献类型：
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作者：
Proto, William R.;Castanys-Munoz, Esther;Mottram, Jeremy C.
后半胱天冬酶是在植物、真菌和原生动物中发现的半胱天冬酶家族半胱氨酸肽酶，但在哺乳动物中不存在。布氏锥虫的不同寻常之处在于具有五个元半胱天冬酶 (MCA1-MCA5)，其中 MCA1 和 MCA4 具有活性位点替换，使它们可能成为非酶同源物。在这里，我们证明重组 MCA4 尽管保持了功能性肽酶结构，但缺乏可检测的肽酶活性。 MCA4 主要以寄生虫的血流形式表达，并通过双重肉豆蔻酰化/棕榈酰化与鞭毛膜结合。功能丧失表型揭示了 MCA4 的关键作用； RNAi 的快速消耗对寄生虫的细胞周期造成致命破坏，但 MCA4 无效突变寄生虫（Delta mca4）的产生是可能的。 Delta mca4 在无菌培养物中生长正常，但在小鼠中毒力显着降低。进一步的分析表明，MCA4 从寄生虫中释放出来，并由 MCA3 特异性加工，MCA3 是唯一一种棕榈酰化且具有酶活性的元半胱天冬酶。因此，我们已经确定布氏锥虫中的多个元半胱天冬酶形成膜相关的蛋白水解级联，从而产生假肽酶毒力因子。
Metacaspases are caspase family cysteine peptidases found in plants, fungi, and protozoa but not mammals. Trypanosoma brucei is unusual in having five metacaspases (MCA1-MCA5), of which MCA1 and MCA4 have active site substitutions, making them possible non-enzymatic homologues. Here we demonstrate that recombinant MCA4 lacks detectable peptidase activity despite maintaining a functional peptidase structure. MCA4 is expressed primarily in the bloodstream form of the parasite and associates with the flagellar membrane via dual myristoylation/palmitoylation. Loss of function phenotyping revealed critical roles for MCA4; rapid depletion by RNAi caused lethal disruption to the parasite's cell cycle, yet the generation of MCA4 null mutant parasites (Delta mca4) was possible. Delta mca4 had normal growth in axenic culture but markedly reduced virulence in mice. Further analysis revealed that MCA4 is released from the parasite and is specifically processed by MCA3, the only metacaspase that is both palmitoylated and enzymatically active. Accordingly, we have identified that the multiple metacaspases in T. brucei form a membrane-associated proteolytic cascade to generate a pseudopeptidase virulence factor.