Cell membrane penetrating function of the nuclear localization sequence in human cytokine IL-1α

Cytokines are released from the cell, bind to their receptors, and affect cellular responses. The precursor form of interleukin 1 alpha (pIL-1 alpha) has a nuclear localization sequence (NLS) that causes it to be localized to the nucleus and regulate specific gene expression. The amino acids of the NLS are basic amino acid-rich sequences, as is the cell penetrating peptide (CPP), which has been widely studied as a way to deliver macromolecules into cells. Here, we hypothesized that the NLS in pIL-1 alpha (pIL-1 alpha NLS) can penetrate the cell membrane and it could deliver macromolecules such as protein in vivo. We characterized cell membrane penetration ability of pIL-1 alpha NLS or its tandem repeated form (2pIL-1 alpha NLS) to enhance its intracellular delivery efficiency. 2pIL-1 alpha NLS showed comparable protein delivery efficiency to TAT-CPP and it mediates endocytosis following heparan sulfate interaction. 2pIL-1 alpha NLS conjugated enhanced green fluorescence protein was localized to the nucleus and the cytoplasm. Intra-peritoneal administration of 2pIL-1 alpha NLS conjugated dTomato protein showed remarkable in vivo intracellular delivery efficiency in various tissues including spleen, liver, and intestine in mice. Moreover, cytotoxicity of 2pIL-1 alpha NLS was not observed even at 100 mu M. Our results demonstrate cell membrane-penetrating function of NLS in pIL-1 alpha, which can be used as a safe therapeutic macromolecular delivery peptide.