Novel cell-penetrating peptide targeting mitochondria
Novel cell-penetrating peptide targeting mitochondria
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DOI：
10.1096/fj.14-269225
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发表时间：
2015-11-01
期刊：
影响因子：
4.8
通讯作者：
Langel, Ulo
中科院分区：
文献类型：
--
作者：
Cerrato, Carmine Pasquale;Pirisinu, Marco;Langel, Ulo
细胞穿透肽（CPPs）是一种短而无毒的肽，具有阳离子和/或两性特性，能够穿过细胞膜。CPPs用于递送各种各样的货物，如蛋白质、寡核苷酸和治疗分子。本研究的目的是基于Szeto-Schiller肽（SS-31）合成针对线粒体的异常小的新型CPPs，以影响线粒体内过程并改善生物学效应。所用肽均采用9-氟酰甲基羰基化学手工合成。在研究的第一部分，HeLa 705， U87和bEnd。取3个细胞作为体外分娩模型。细胞在37℃和5% CO2下与不同浓度的我们的肽孵育24小时。细胞增殖法测定细胞活力。对线粒体膜电位和抗氧化活性等生物效应进行了评价。以H2O2为阳性对照。使用5(6)-羧基荧光素（FAM）偶联的肽进行摄取研究。荧光显微镜用于确定多肽在细胞中的存在和定位。用预处理细胞分离的线粒体和分离后处理的线粒体来证实肽的靶向能力。单独摄取FAM作为阴性对照。显微镜研究证实了多肽穿透细胞的能力。定位分析表明，与SS-31相比，摄取增加了35%。线粒体CPP-1 （mtCPP-1）对线粒体膜电位无影响，并能阻止活性氧的形成。3个细胞比SS-31大2倍。即使高浓度（100 μ M）也未见细胞毒性。这些数据表明mtCPP-1是线粒体CPP，由于其自身的抗氧化活性而保护线粒体免受氧化损伤。
Cell-penetrating peptides (CPPs) are short, nontoxic peptides with cationic and/or amphipathic properties able to cross the cellular membrane. CPPs are used for the delivery of awide variety of cargoes, such as proteins, oligonucleotides, and therapeuticmolecules. The aim of the present study was to synthesize unusually small novel CPPs targeting mitochondria based on the Szeto-Schiller peptide (SS-31) to influence intramitochondrial processes and to improve the biologic effects. All the peptides used were synthesized manually using 9-fluorenylmethyloxycarbonyl chemistry. In the first part of the study, HeLa 705, U87, and bEnd. 3 cells were used as in vitro delivery model. Cells were incubated for 24 h at 37 degrees C and 5% CO2 with different concentrations of our peptides. Cell proliferation assay was performed to evaluate cell viability. Biologic effects such as mitochondrial membrane potential and antioxidant activity were evaluated. H2O2 was used as positive control. Uptake studies were performed using peptides conjugated with 5(6)-carboxyfluorescein (FAM). Fluorescent microscopy was used to determine presence and localization of peptides into the cells. Isolated mitochondria from pretreated cells and mitochondria treated after isolation were used to confirmthe targeting ability of the peptide. Uptake of FAM alone was used as negative control. Microscopy studies confirmed the ability of peptides to penetrate cell. Localization analysis showed increase in uptake by35% compared with SS-31. Mitochondrial CPP 1 (mtCPP-1) had no effect on mitochondrial membrane potential and prevented reactive oxygen species formation in bEnd. 3 cells by 2-fold compared with SS-31. No cytotoxicity was observed even at high concentration (100 mu M). These data suggest that mtCPP-1 is a mitochondrial CPP and protect mitochondria from oxidative damage due to its own antioxidant activities.