Cell-Penetrating Function of the Poly(ADP-Ribose) (PAR)-Binding Motif Derived from the PAR-Dependent E3 Ubiquitin Ligase Iduna

Abstract

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.

Intracellular delivery of nucleic acid by cell‐permeable hPP10 peptide

“…To employ hPP10 as a pDNA delivery carrier, the cytotoxicity of hPP10/pDNA complexes was evaluated by the MTT assay ( Figure 1b). Consistent with other groups and our published data (Chen and Wu, 2018;Koo, Yoon, Kim, Cha, & Choi, 2018;Tsai, Lin, Chang, Chen, & Hu, 2018), we observed no significant cytotoxicity of TAT/pDNA or hPP10/pDNA at different N/P ratios in Caski and B16 cell lines for 24 or 48 hr (Figure 1b). These results suggested that hPP10 was able to bind with pDNA and did not induce cytotoxicity to different cell lines.…”

Cell penetrating peptides: the potent multi-cargo intracellular carriers

“…Moreover, Pep-1 peptide was used to determine the antitumor effects of antisense PNAs targeting cyclin B1 as well as to evaluate early embryonic development using the delivery of antibodies and proteins (e.g. p53) into immature bovine and mouse oocytes [188][189][190][191].…”

The Cysteine-Containing Cell-Penetrating Peptide AP Enables Efficient Macromolecule Delivery to T Cells and Controls Autoimmune Encephalomyelitis

“…As shown in oligoarginine, transduction efficiency increases with increasing arginine counts up to R15 [ 32 , 33 ]. Similarly, tandem-repeat forms of CPP, such as dNP2 [ 18 ], 2pIL-1αNLS [ 34 ], d-Iduna [ 35 ], HHph-1 [ 36 ], and Pas2r12 [ 37 ], have improved delivery efficiency over single forms. Interestingly, cellular uptake was significantly improved by simply repeating the CPP sequence, but the degree differed greatly depending on the sequence ( Figure 2 e) or cell lines [ 35 ].…”

“…Similarly, tandem-repeat forms of CPP, such as dNP2 [ 18 ], 2pIL-1αNLS [ 34 ], d-Iduna [ 35 ], HHph-1 [ 36 ], and Pas2r12 [ 37 ], have improved delivery efficiency over single forms. Interestingly, cellular uptake was significantly improved by simply repeating the CPP sequence, but the degree differed greatly depending on the sequence ( Figure 2 e) or cell lines [ 35 ]. In the case of AP, the tandem repeat form of AP, dAP, showed improved delivery efficiency, but not as much as twice the cutoff value.…”