Subramanian Shoba, Hardt Markus, Choe Youngchool, Niles Richard K, Johansen Eric B, Legac Jennifer, Gut Jiri, Kerr Iain D, Craik Charles S, Rosenthal Philip J
Department of Medicine, Division of Infectious Diseases, University of California San Francisco, San Francisco, California, United States of America.
PLoS One. 2009;4(4):e5156. doi: 10.1371/journal.pone.0005156. Epub 2009 Apr 9.
The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P(1) - P(4) amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P(2) position. Second, with overlapping peptides spanning alpha and beta globin and proteolysis-dependent (18)O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P(2) Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents.
恶性疟原虫半胱氨酸蛋白酶恶性疟原虫蛋白酶-2和恶性疟原虫蛋白酶-3降解宿主血红蛋白，为寄生虫蛋白质合成提供游离氨基酸。血红蛋白水解被描述为由天冬氨酸蛋白酶启动的有序过程，但半胱氨酸蛋白酶抑制剂完全阻断该过程，这表明半胱氨酸蛋白酶也可启动血红蛋白水解。为了表征恶性疟原虫蛋白酶的具体作用，我们采用了三种方法。首先，使用随机的P(1)-P(4)氨基酸底物文库，恶性疟原虫蛋白酶-2和恶性疟原虫蛋白酶-3对P(2)位置为亮氨酸的切割位点表现出强烈偏好。其次，对于跨越α和β珠蛋白的重叠肽以及蛋白水解依赖性(18)O标记，在许多切割位点观察到水解。第三，对于完整的血红蛋白，鉴定出了许多切割产物。我们的结果表明，疟原虫对血红蛋白的水解不是一个高度有序的过程，而是由恶性疟原虫蛋白酶在多个位点快速切割进行的。然而，恶性疟原虫蛋白酶-2和恶性疟原虫蛋白酶-3对小肽底物中的P(2)亮氨酸表现出很强的特异性，这与先前鉴定的优化小分子抑制剂中的特异性一致。这些结果与恶性疟原虫蛋白酶-2和恶性疟原虫蛋白酶-3在恶性疟原虫血红蛋白水解中的主要作用以及开发具有优化特异性的小分子抑制剂作为抗疟剂的可能性是一致的。
