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.
Highlights
Cell-penetrating peptide (CPP) is a short amino acid sequence that enables macromolecules such as proteins, DNA, and RNA to be transported into cells across the plasma membrane [1,2,3]
The PAR-binding motif (F144 to R167) [27] of Iduna-derived cationic sequence-rich peptide (Iduna) contains a highly cationic sequence, and in this study, we investigated the cell-penetrating ability of this sequence by generating recombinant proteins comprising the CPP conjugated with a fluorescent protein
We found the highly cationic sequence (RRRKIKR) in the PAR-binding motif (PBM) of Iduna
Summary
Cell-penetrating peptide (CPP) is a short amino acid sequence that enables macromolecules such as proteins, DNA, and RNA to be transported into cells across the plasma membrane [1,2,3]. The most widely used CPPs were TAT [4,5,6], Antp [7,8], VP22 [9], transportan [10], and poly-arginine [11,12]. Most CPPs, including the ones mentioned here, are derived from non-human sources, such as viruses or drosophila, or are synthesized artificially. We have previously reported novel human protein-derived CPPs including LPIN [18], 2pIL-1aNLS [19], dNP2 [20], and AP [21]. Iduna ( known as RNF146) is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that can degrade
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