细胞穿透肽通过互补亮氨酸拉链系统有效递送至细胞内的蛋白质。
Complementary leucine zippering system for effective intracellular delivery of proteins by cell-penetrating peptides.
机构信息
Department of Applied Chemistry, Faculty of Science and Engineering, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan.
Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, kita-ku, Okayama 700-8558, Japan.
出版信息
Bioorg Med Chem. 2021 Mar 1;33:116036. doi: 10.1016/j.bmc.2021.116036. Epub 2021 Jan 21.
A heterodimeric leucine zipper composed of a pair of leucine zipper peptides containing acidic or basic amino acid residues at appropriate positions in each peptide was used as a molecular glue to connect protein cargos to a cell-penetrating peptide (CPP) carrier. To investigate the hybridization properties by fluorescence experiments, we prepared an enhanced green fluorescent protein (EGFP) fused with an acidic leucine zipper (LzK), EGFP-LzK, and a basic leucine zipper (LzE) modified with a CPP, LzE-CPP. The LzK and LzE formed a 1:1 hybrid when EGFP-LzK and LzE-CPP were mixed in phosphate buffer saline, thereby conjugating the EGFP with the CPP. The formation of the 1:1 hybrid was confirmed by fluorescence spectra and fluorescence titration curves. Results from fluorescence microscopy experiments showed that EGFP was successfully delivered into cells by conjugating with the CPP via formation of the LzK/LzE hybrid. We also fused the apoptotic protein p53 with LzK (p53-LzK) and investigated the inhibition of cell proliferation of various cell lines by incubation with the p53-LzK/LzE-CPP hybrid. This hybrid was found to localize in nuclei and successfully inhibited cell-specific proliferation. The LzE/LzK zipper system inhibited cell proliferation more efficiently than the directly fused conjugate, p53-CPP. Our method will be a useful drug delivery system for delivering bioactive proteins to treat various diseases.
一对由两条亮氨酸拉链肽组成的杂二聚体，每条肽链在适当位置含有酸性或碱性氨基酸残基，可作为分子胶将蛋白 cargo 连接到穿膜肽（CPP）载体上。为了通过荧光实验研究杂交特性，我们制备了一种融合了酸性亮氨酸拉链（LzK）的增强型绿色荧光蛋白（EGFP），即 EGFP-LzK，以及一种带有 CPP 的碱性亮氨酸拉链（LzE），即 LzE-CPP。当在磷酸盐缓冲盐水中混合 EGFP-LzK 和 LzE-CPP 时，LzK 和 LzE 形成 1:1 杂合体，从而将 EGFP 与 CPP 连接起来。荧光光谱和荧光滴定曲线证实了 1:1 杂合体的形成。荧光显微镜实验结果表明，通过形成 LzK/LzE 杂合体，EGFP 成功地与 CPP 缀合并被递送到细胞内。我们还将凋亡蛋白 p53 与 LzK 融合（p53-LzK），并研究了与 p53-LzK/LzE-CPP 杂合体孵育对各种细胞系增殖的抑制作用。发现该杂合体定位于细胞核内，并成功抑制了细胞特异性增殖。与直接融合的缀合物 p53-CPP 相比，LzE/LzK 拉链系统更有效地抑制了细胞增殖。我们的方法将成为一种将生物活性蛋白递送到治疗各种疾病的有用的药物输送系统。
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