Disulfide linked hetero dimeric peptide arrays for screening functional peptides inside cells
Disulfide linked hetero dimeric peptide arrays for screening functional peptides inside cells
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DOI：
10.1016/j.jbiosc.2019.11.012
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发表时间：
2020-05-01
影响因子：
2.8
通讯作者：
Honda, Hiroyuki
中科院分区：
文献类型：
--
作者：
Kozaki, Ikko;Shimizu, Kazunori;Honda, Hiroyuki
在细胞中具有重要作用的细胞内功能肽作为细胞活性的调节剂已被广泛研究。此前，我们提出了一种用于鉴定细胞内功能肽的筛选系统，该系统可用于合成各种细胞穿透肽（CPP）-功能肽复合物，然后可以使用各种基于细胞的测定来确定肽的细胞内功能。我们证明，通过氨基酸取代可以增加细胞死亡诱导肽的活性。然而，CPP 与功能性肽的共价缀合可能会破坏功能性肽的生物学特性。在本研究中，我们开发了一种新型细胞内功能肽筛选系统，并在细胞内递送后结合解离系统，以消除 CPP 的影响。为了构建该系统，我们开发了一种合成含有二硫键的 CPP 功能肽异二聚体的方法，该二硫键可被细胞内还原环境裂解。该系统使用Fmoc-Lys(ivDde)-OH在一个分子中合成CPP和功能性肽，选择性地形成异源二聚肽的二硫键。此外，我们评估了细胞死亡诱导肽（WELVVLGKL）的单个氨基酸取代文库。我们获得了6种比原始肽活性更高的肽。我们的结果表明，我们的新筛选系统可以消除 CPP 的影响，可用于细胞内的肽筛选。 (C) 2019 年，日本生物技术协会。版权所有。
Intracellular functional peptides with important roles in cells have been widely studied as regulators of cellular activity. Previously, we proposed a screening system for identifying intracellular functional peptides, which can be utilized to synthesize various cell-penetrating peptide (CPP)-functional peptide complexes, and the intracellular functions of the peptides can then be determined using various cell based assays. We demonstrated that the activity of a cell death-inducing peptide was increased by an amino acid substitution. However, covalent conjugation of CPPs with functional peptides may disrupt the biological characteristics of the functional peptides. In the present study, we developed a novel screening system for intracellular functional peptides combined with a dissociation system after delivery inside cells to eliminate the influence of CPPs. To construct this system, we developed a method for synthesizing CPP-functional peptide hetero-dimers containing a disulfide bond that is cleaved by the intracellular reducing environment. The system used Fmoc-Lys(ivDde)-OH to synthesize CPP and functional peptides in one molecule to selectively form a disulfide bond of hetero-dimeric peptides. Furthermore, we evaluated a single amino acid substitution library of the cell death inducing peptide (WELVVLGKL). We obtained 6 peptides with higher activity than the original peptide. Our results suggest that our new screening system can be eliminate the influence of CPPs and is useful for peptide screening inside cells. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.