Farias Shirley L, Gazarini Marcos L, Melo Robson L, Hirata Izaura Y, Juliano Maria A, Juliano Luiz, Garcia Célia R S
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, SP, Brazil.
Mol Biochem Parasitol. 2005 May;141(1):71-9. doi: 10.1016/j.molbiopara.2005.01.015.
Bloodstage malaria parasites require proteolytic activity for key processes as invasion, hemoglobin degradation and merozoite escape from red blood cells (RBCs). We investigated by confocal microscopy the presence of cysteine-protease activity elicited by calcium stimulus in Plasmodium chabaudi and Plasmodium falciparum in free trophozoites or for the later parasite within RBC using fluorescence resonance energy transfer (FRET) peptides. Peptide probes access, to either free or intraerythrocytic parasites, was also tested by selecting a range of fluorescent peptides (653-3146 Da molecular mass) labeled with Abz or FITC. In the present work we show that Ca2+ stimulus elicited by treatment with either melatonin, thapsigargin, ionomicin or nigericin, promotes an increase of substrate hydrolysis, which was blocked by the specific cysteine-protease inhibitor E-64 and the intracellular Ca2+ chelator, BAPTA. When parasites were treated with cytoplasmic Ca2+ releasing compounds, a cysteine-protease was labeled in the parasite cytoplasm by the fluorescent specific irreversible inhibitor, Ethyl-Eps-Leu-Tyr-Cap-Lys(Abz)-NH2, where Ethyl-Eps is Ethyl-(2S,3S)-oxirane-2,3-dicarboxylate. In summary, we demonstrate that P. chabaudi and P. falciparum have a cytoplasmic dependent cysteine-protease activity elicited by Ca2+.
血液期疟原虫在入侵、血红蛋白降解以及裂殖子从红细胞（RBC）逸出等关键过程中需要蛋白水解活性。我们通过共聚焦显微镜，使用荧光共振能量转移（FRET）肽，研究了钙刺激在游离滋养体阶段的查巴迪疟原虫和恶性疟原虫中，以及在红细胞内后期疟原虫中引发的半胱氨酸蛋白酶活性的存在情况。还通过选择一系列用Abz或FITC标记的荧光肽（分子量653 - 3146 Da），测试了肽探针进入游离或红细胞内疟原虫的情况。在本研究中，我们发现用褪黑素、毒胡萝卜素、离子霉素或尼日利亚菌素处理引发的Ca2 +刺激，会促进底物水解增加，这被特异性半胱氨酸蛋白酶抑制剂E - 64和细胞内Ca2 +螯合剂BAPTA所阻断。当用细胞质Ca2 +释放化合物处理疟原虫时，荧光特异性不可逆抑制剂Ethyl - Eps - Leu - Tyr - Cap - Lys(Abz)-NH2（其中Ethyl - Eps是乙基-(2S,3S)-环氧乙烷-2,3 - 二羧酸盐）在疟原虫细胞质中标记了一种半胱氨酸蛋白酶。总之，我们证明查巴迪疟原虫和恶性疟原虫具有由Ca2 +引发的细胞质依赖性半胱氨酸蛋白酶活性。
