Complementary leucine zippering system for effective intracellular delivery of proteins by cell-penetrating peptides
Complementary leucine zippering system for effective intracellular delivery of proteins by cell-penetrating peptides
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DOI：
10.1016/j.bmc.2021.116036
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发表时间：
2021-01-23
影响因子：
3.5
通讯作者：
Michiue, Hiroyuki
中科院分区：
文献类型：
--
作者：
Kitamatsu, Mizuki;Yuasa, Hiroki;Michiue, Hiroyuki
一种异二聚体亮氨酸拉链由一对亮氨酸拉链肽组成，这些亮氨酸拉链肽在每个肽的适当位置含有酸性或碱性氨基酸残基，用作分子胶将蛋白质货物连接到细胞穿透肽（CPP）载体上。为了通过荧光实验研究其杂交特性，我们制备了与酸性亮氨酸拉链（LzK）融合的增强型绿色荧光蛋白(EGFP) -LzK，以及与CPP修饰的碱性亮氨酸拉链(LzE) - LzE。将EGFPLzK与LzE-CPP混合在磷酸盐缓冲盐水中，使EGFP与CPP偶联，LzK与LzE形成1:1的杂种。荧光光谱和荧光滴定曲线证实了1:1杂化产物的形成。荧光显微镜实验结果表明，EGFP通过与CPP结合形成LzK/LzE杂交体成功进入细胞。我们还将凋亡蛋白p53与LzK （p53-LzK）融合，并通过p53-LzK/LzE-CPP杂交培养，研究了对多种细胞系细胞增殖的抑制作用。这种杂交被发现在细胞核中定位，并成功地抑制了细胞特异性增殖。LzE/LzK拉链体系比直接融合的p53-CPP更有效地抑制细胞增殖。我们的方法将成为一种有效的药物输送系统，用于输送生物活性蛋白来治疗各种疾病。
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 EGFPLzK 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.