In silico identification and experimental validation of cellular uptake and intracellular labeling by a new cell penetrating peptide derived from CDN1

Abstract

Bioactive therapeutic molecules are generally impermeable to the cell membrane, hindering their utility and efficacy. A group of peptides called cell-penetrating peptides (CPPs) were found to have the capability of transporting different types of cargo molecules across the cell membrane. Here, we identified a short peptide named P2, which has a higher proportion of basic residues than the CDN1 (cyclin-dependent kinase inhibitor 1) protein it is derived from, and we used bioinformatic analysis and experimental validation to confirm the penetration property of peptide P2. We found that peptide P2 can efficiently enter different cell lines in a concentration-dependent manner. The endocytosis pathway, especially receptor-related endocytosis, may be involved in the process of P2 penetration. Our data also showed that peptide P2 is safe in cultured cell lines and red blood cells. Lastly, peptide P2 can efficiently deliver self-labeling protein HaloTag into cells for imaging. Our study illustrates that peptide P2 is a promising imaging agent delivery vehicle for future applications.

Cell-Permeable PROTAC Degraders against KEAP1 Efficiently Suppress Hepatic Stellate Cell Activation through the Antioxidant and Anti-Inflammatory Pathway

“…FITC-labeled KKP1 in the cell has the strongest fluorescence intensity during the initial 1 and 2 h of treatment and gradually decreased after 4, 8, 16, and 24 h (Figures D,E and S3). Our previous publications suggest that a penetration enhancer − can significantly enhance the penetration efficiency of the well-known different CPPs including hPP3, hPP10, , MT23, Scp01-b, Dot1l, P2, and P1 . We wanted to address whether the penetration efficiency of KKP can further be enhanced.…”

Improved transfer efficiency of supercharged 36 + GFP protein mediate nucleic acid delivery

“…Our studies also suggested although 36 + GFP can significantly enter cells and tissues compared with GFP protein, the penetration efficiency of 36 + GFP is still limited. Following our previous studies (Ma et al., 2015 ; Wang et al., 2016a , 2016b , 2016c ; Zhou et al., 2017 ; Ding et al., 2019 ; Zhang et al., 2019 ; Chen et al., 2021 ; Guo et al., 2021 ), we conducted 5% DMSO treatment in cultured cells, and found that the efficiency of 36 + GFP mediated transduction itself and its mediated transfection were significantly increase thus these results are consistent with our previous published studies. These data indicated that penetration enhancer application is another alternative approach to enhance the penetration and relative delivery efficiency of peptide or supercharged proteins- based delivery vectors.…”

Emerging Landscape of Cell-Penetrating Peptide-Mediated Organelle Restoration and Replacement

“…CPPs are a diverse group of long peptides of 4–30 amino acids capable of delivering bioactive cargos of proteins, − peptides, , nucleic acids, − nanoparticles, and pharmacologic agents into the cellular cytoplasm through different internalization mechanisms mainly including energy-dependent endocytosis and energy-independent direct penetration. ,− Since the discovery of the first qualitative CPP-TAT, − increasing research activities and preclinical evaluations of CPP-derived therapies have provided us with promising results in various disease models. , Thus, these breakthroughs bring new prospects for the development of CPP-derived therapeutics to treat human diseases related to intracellular organelles.…”