Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 04763, Korea.
Research Institute for Natural Sciences, Hanyang University, Seoul 04763, Korea.
Int J Mol Sci. 2018 Mar 8;19(3):779. doi: 10.3390/ijms19030779.
Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates cellular responses such as proteasomal degradation and DNA repair upon interaction with its substrate. We identified a highly cationic region within the PAR-binding motif of Iduna; the region was similar among various species and showed amino acid sequence similarity with that of known cell-penetrating peptides (CPPs). We hypothesized that this Iduna-derived cationic sequence-rich peptide (Iduna) could penetrate the cell membrane and deliver macromolecules into cells. To test this hypothesis, we generated recombinant Iduna-conjugated enhanced green fluorescent protein (Iduna-EGFP) and its tandem-repeat form (d-Iduna-EGFP). Both Iduna-EGFP and d-Iduna-EGFP efficiently penetrated Jurkat cells, with the fluorescence signals increasing dose- and time-dependently. Tandem-repeats of Iduna and other CPPs enhanced intracellular protein delivery efficiency. The delivery mechanism involves lipid-raft-mediated endocytosis following heparan sulfate interaction; d-Iduna-EGFP was localized in the nucleus as well as the cytoplasm, and its residence time was much longer than that of other controls such as TAT and Hph-1. Moreover, following intravenous administration to C57/BL6 mice, d-Iduna-EGFP was efficiently taken up by various tissues, including the liver, spleen, and intestine suggesting that the cell-penetrating function of the human Iduna-derived peptide can be utilized for experimental and therapeutic delivery of macromolecules.
依度那是一种依赖多聚 ADP-核糖（PAR）的 E3 泛素连接酶，在与底物相互作用时，它可以调节细胞反应，如蛋白酶体降解和 DNA 修复。我们在依度那的 PAR 结合基序内发现了一个高度正电荷区域；该区域在各种物种中相似，并且与已知的细胞穿透肽（CPP）具有氨基酸序列相似性。我们假设这个源自依度那的富含正电荷的序列肽（Iduna）可以穿透细胞膜并将大分子递送到细胞内。为了验证这一假设，我们生成了重组的依度那缀合的增强型绿色荧光蛋白（Iduna-EGFP）及其串联重复形式（d-Iduna-EGFP）。Iduna-EGFP 和 d-Iduna-EGFP 都能有效地穿透 Jurkat 细胞，荧光信号随着剂量和时间的增加而增加。依度那和其他 CPP 的串联重复增强了细胞内蛋白质的递送效率。递药机制涉及与硫酸乙酰肝素相互作用后的脂筏介导内吞作用；d-Iduna-EGFP 定位于细胞核和细胞质中，其停留时间比 TAT 和 Hph-1 等其他对照物长得多。此外，在向 C57/BL6 小鼠静脉给药后，d-Iduna-EGFP 被各种组织（包括肝脏、脾脏和肠道）有效摄取，表明人源依度那衍生肽的细胞穿透功能可用于大分子的实验和治疗性递药。
