Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.
Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.
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DOI：
10.1038/cddis.2010.51
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发表时间：
2010-09-02
影响因子：
9
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中科院分区：
文献类型：
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在几项报告低等真核生物和人类原生动物寄生虫利什曼原虫中细胞死亡（CD）的研究中，使用泛半胱天冬酶底物或抑制剂（如苄氧羰基-缬氨酰-丙氨酰-乙酰基-[O-甲基]-氟甲基酮（Z-VAD-FMK））揭示了蛋白水解活性。然而，大多数低等真核生物不编码半胱天冬酶，而是编码MCA，其在底物特异性上不同于半胱天冬酶，并且不能解释Z-VAD-FMK的识别。在本研究中，我们感兴趣的是确定哪种酶捕获Z-VAD底物。我们发现，热休克（HS）诱导利什曼原虫CD和导致Z-VAD-FMK的细胞内结合。我们排除了Z-VAD-FMK与主要利什曼原虫后半胱天冬酶（LmjMCA）的结合和抑制，并鉴定了半胱氨酸蛋白酶C（LmjCPC），一种组织蛋白酶B样（CPC）酶，作为Z-VAD-FMK结合酶。我们证实了Z-VAD-FMK与CPC的特异性相互作用，表明Z-VAD结合在CPC基因缺失的墨西哥利什曼原虫菌株中不存在。我们还表明，寄生虫暴露于各种压力条件下释放CPC成可溶性部分。最后，我们证实了CPC在利什曼原虫CD中的作用，当暴露于氧化剂过氧化氢（H2 O2）时，CPC敲除寄生虫比野生型寄生虫（WT）存活得更好。总之，这项研究确定了CPC作为利什曼原虫Z-VAD-FMK的底物，并作为利什曼原虫CD级联反应中潜在的额外执行蛋白酶，可能在其他低等真核生物中。
In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H2O2), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.
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影响因子：
6.4
作者：
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