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8.A.39 The Homeobox; Penetratin (Penetratin) Family

Calcium-activated chloride currents (CaCCs) are required for epithelial electrolyte and fluid secretion, fertilization, sensory transduction and excitability of neurons and smooth muscle. Defolliculated Xenopus oocytes express a robust CaCC formed by a heterologous group of proteins including transmembrane protein 16A (TMEM16A) and bestrophins. Penetratin, a 16-amino acid peptide, potentiates endogenous oocyte CaCCs by ~50-fold at 10 μM. CaCC potentiation was rapid and dose-dependent (EC50=3.2 μM). Penetratin-potentiated currents reversed at -18 mV and were dependent on the extracellular divalent cations present, showing positive regulation by Ca2+ and Mg2+ but effective block by Zn2+ (IC50=5.9 μM). Extracellular Cd2+, Cu2+ and Ba2+ resulted in bimodal responses: CaCC inhibition at low but potentiation at high concentrations. Intracellular BAPTA injection, which prevents activation of CaCCs, and the Cl- channel blockers niflumic acid and DIDS, significantly reduced potentiation. In contrast, the K+ channel blockers Cs+, TEA, tertiapin-Q and halothane had no effect. This pharmacological profile is consistent with penetratin potentiation of zinc-sensitive CaCCs that are activated by influx of extracellular Ca2+ (Kanjhan and Bellingham, 2011; Durzyńska et al. 2015).

Cell penetrating peptides (CPPs) are small peptides that are able to penetrate the plasma membranes of mammalian cells. Because these peptides can also carry large hydrophilic cargos such as proteins, they could potentially be used to transport biologically active drugs across cell membranes. One characteristic feature of the CPPs is that they typically have a net positive charge. Therefore, a key issue associated with the transport mechanism is the role of the transmembrane electrochemical potential in driving the peptides across the membrane. Björklund et al. 2006 reconstituted bacteriorhodopsin (bR) in large unilamellar vesicles (LUVs) with fluorescein-labeled CPP penetratin enclosed within the LUVs under conditions when the fluorescence is quenched. Illumination of the bacteriorhodopsin-containing LUVs resulted in creation of a transmembrane proton electrochemical gradient (positive on the inside). Upon generation of this gradient, an increase in fluorescence was observed, showing that the proton gradient drives the translocation of penetratin (Björklund et al. 2006).

Arginine-rich CPPs, including Tat, Penetratin and oligoarginine peptides, are short peptides that can be efficiently internalized into cells and have been widely used as carriers for intracellular delivery of bioactive molecules (Takeuchi and Futaki 2016). CPPs can transport into the cell a wide variety of biologically active conjugates (or cargoes), and are, therefore, promising in the treatment and in the diagnosis of several types of cancer (Vale et al. 2020). Some notable examples are TAT and Penetratin, capable of penetrating the central nervous system (CNS) (Vale et al. 2020). Cationic and amphipathic CPPs, transactivator of transcription (TAT), and penetratin may be  the best among all the identified penetration enhancers for drug delivery to the fundus oculi via topical eye drop instillation (Thareja et al. 2021). 

CPPs colocalized with lysosomes and macropinosomes (Zhu et al., 2022; PMID 35128758). Chlorpromazine, wortmannin, cytochalasin D, and the ATPase inhibitor oligomycin had dose-dependent endocytosis-inhibitory effects on CC12. Oligomycin had the most significant inhibitory effect on CC12 uptake; CC12 was co-located with the lysosome, but not with the macropinosome. Penetratin, cytochalasin D and oligomycin had obvious inhibitory effects with oligomycin having the most significant inhibitory effect on penetratin uptake; the co-localization of penetratin with the lysosome was higher than that with the macropinosome. Cation-independent CC12 and cationic penetratin may be internalized into cells primarily through caveolae and clathrin-mediated endocytosis, typically dependent on ATP. The transport of penetratin could be partly achieved through a direct transmembrane pathway, as the positive charge of penetratin interacts with the negative charge of the cell membrane, and partly through the endocytic pathway (Zhu et al., 2022; PMID 35128758).

References associated with 8.A.39 family:

Batta, G., L. Kárpáti, G.F. Henrique, G. Tóth, S. Tarapcsák, T. Kovács, F. Zákány, I.M. Mándity, and P. Nagy. (2021). Statin-boosted cellular uptake and endosomal escape of penetratin due to reduced membrane dipole potential. Br J Pharmacol. [Epub: Ahead of Print] 33908640
Björklund, J., H. Biverståhl, A. Gräslund, L. Mäler, and P. Brzezinski. (2006). Real-time transmembrane translocation of penetratin driven by light-generated proton pumping. Biophys. J. 91: L29-31. 16782795
Durzyńska, J., &.#.3.2.1.;. Przysiecka, R. Nawrot, J. Barylski, G. Nowicki, A. Warowicka, O. Musidlak, and A. Goździcka-Józefiak. (2015). Viral and other cell-penetrating peptides as vectors of therapeutic agents in medicine. J Pharmacol Exp Ther 354: 32-42. 25922342
Kanjhan, R. and M.C. Bellingham. (2011). Penetratin peptide potentiates endogenous calcium-activated chloride currents in Xenopus oocytes. J. Membr. Biol. 241: 21-29. 21442407
Pescina, S., C. Ostacolo, I.M. Gomez-Monterrey, M. Sala, A. Bertamino, F. Sonvico, C. Padula, P. Santi, A. Bianchera, and S. Nicoli. (2018). Cell penetrating peptides in ocular drug delivery: State of the art. J Control Release 284: 84-102. 29913221
Takeuchi, T. and S. Futaki. (2016). Current Understanding of Direct Translocation of Arginine-Rich Cell-Penetrating Peptides and Its Internalization Mechanisms. Chem Pharm Bull (Tokyo) 64: 1431-1437. 27725497
Thareja, A., H. Hughes, C. Alvarez-Lorenzo, J.J. Hakkarainen, and Z. Ahmed. (2021). Penetration Enhancers for Topical Drug Delivery to the Ocular Posterior Segment-A Systematic Review. Pharmaceutics 13:. 33670762
Vale, N., D. Duarte, S. Silva, A.S. Correia, B. Costa, M.J. Gouveia, and A. Ferreira. (2020). Cell-penetrating peptides in oncologic pharmacotherapy: A review. Pharmacol Res 162: 105231. 33027717