Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University, 3-1-1 Tsushimanaka, Okayama, 700-8530, Japan.
Department of Organ Anatomy and Nanomedicine, Yamaguchi University, 1-1-1 Minami-Kogushi, Ube, 755-8505, Japan.
Sci Rep. 2020 Nov 5;10(1):19087. doi: 10.1038/s41598-020-76100-7.
Investigation of the relevance between cell cycle status and the bioactivity of exogenously delivered biomacromolecules is hindered by their time-consuming cell internalization and the cytotoxicity of transfection methods. In this study, we addressed these problems by utilizing the photochemical internalization (PCI) method using a peptide/protein-photosensitizer conjugate, which enables immediate cytoplasmic internalization of the bioactive peptides/proteins in a light-dependent manner with low cytotoxicity. To identify the cell-cycle dependent apoptosis, a TatBim peptide-photosensitizer conjugate (TatBim-PS) with apoptotic activity was photo-dependently internalized into HeLa cells expressing a fluorescent ubiquitination-based cell cycle indicator (Fucci2). Upon irradiation, cytoplasmic TatBim-PS internalization exceeded 95% for all cells classified in the G, S, and G/M cell cycle phases with no significant differences between groups. TatBim-PS-mediated apoptosis was more efficiently triggered by photoirradiation in the G/S transition than in the G and S/G/M phases, suggesting high sensitivity of the former phase to Bim-induced apoptosis. Thus, the cell cycle dependence of Bim peptide-induced apoptosis was successfully investigated using Fucci2 indicator and the PCI method. Since PCI-mediated cytoplasmic internalization of peptides is rapid and does not span multiple cell cycle phases, the Fucci-PCI method constitutes a promising tool for analyzing the cell cycle dependence of peptides/protein functions.
研究细胞周期状态与外源性生物大分子生物活性之间的相关性受到其细胞内化耗时以及转染方法细胞毒性的阻碍。在本研究中，我们利用肽/蛋白质-光感受器缀合物的光化学内化（PCI）方法解决了这些问题，该方法能够以低细胞毒性的方式，以光依赖性的方式立即将生物活性肽/蛋白质内化到细胞质中。为了鉴定细胞周期依赖性凋亡，我们用光依赖性地将具有凋亡活性的 TatBim 肽-光感受器缀合物（TatBim-PS）内化到表达荧光泛素化细胞周期指示剂（Fucci2）的 HeLa 细胞中。照射后，G、S 和 G/M 细胞周期阶段的所有细胞中细胞质 TatBim-PS 的内化率均超过 95%，各组之间无显著差异。与 G 和 S/G/M 期相比，TatBim-PS 介导的凋亡在 G/S 转换期经光照射后更容易被触发，这表明前一阶段对 Bim 诱导的凋亡具有较高的敏感性。因此，我们成功地使用 Fucci2 指示剂和 PCI 方法研究了 Bim 肽诱导的凋亡的细胞周期依赖性。由于肽的 PCI 介导的细胞质内化是快速的，并且不跨越多个细胞周期阶段，因此 Fucci-PCI 方法构成了分析肽/蛋白质功能的细胞周期依赖性的有前途的工具。
