Modulation of Orai1 by cationic peptides triggers their direct cytosolic uptake.

At concentrations exceeding 10 µM, arginine-rich cell-penetrating peptides (CPPs) trigger a rapid cytoplasmic import that involves activation of acid sphingomyelinase (ASMase). ASMase activation occurs through a variety of stress signals and has also been related to the reorganization of membrane microdomains during entry of pathogens. However, in none of these cases has the initial trigger for ASMase activation been established on a molecular level. We here show that rapid cytosolic CPP import depends upon an increase in intracellular calcium, likely caused by modulation of the Orai1 calcium channel. At low peptide concentration, cytoplasmic import could be induced by thapsigargin, a known activator of Orai1. Compounds known to block Orai1 inhibited rapid uptake. Peptide-mediated modulation of Orai1 involved cell surface sialic acids as inhibition of sialylation as well as chemical blocking of sialic acids reduced rapid cytoplasmic uptake, which could be reconstituted by thapsigargin. These results establish a link between the known propensity of arginine-rich CPPs to interact with the glycocalyx and calcium influx as the initial step triggering direct cytosolic peptide uptake.

Effective Design of Multifunctional Peptides by Combining Compatible Functions.

Multifunctionality is a common trait of many natural proteins and peptides, yet the rules to generate such multifunctionality remain unclear. We propose that the rules defining some protein/peptide functions are compatible. To explore this hypothesis, we trained a computational method to predict cell-penetrating peptides at the sequence level and learned that antimicrobial peptides and DNA-binding proteins are compatible with the rules of our predictor. Based on this finding, we expected that designing peptides for CPP activity may render AMP and DNA-binding activities. To test this prediction, we designed peptides that embedded two independent functional domains (nuclear localization and yeast pheromone activity), linked by optimizing their composition to fit the rules characterizing cell-penetrating peptides. These peptides presented effective cell penetration, DNA-binding, pheromone and antimicrobial activities, thus confirming the effectiveness of our computational approach to design multifunctional peptides with potential therapeutic uses. Our computational implementation is available at http://bis.ifc.unam.mx/en/software/dcf.

Is the peptide-mediated gene delivery a feasible and reliable technology for efficient transgenesis?

Background: Cell penetrating peptides (CPPs) are natural or synthetic, cationic or amphipathic short peptide sequences with high affinity for biological membranes, capable of crossing the lipid bilayer systems and mediating the delivery of biomolecules, drugs or nanoparticles into the cells. Review: In this work, I presented a brief overview of some significant contributions from several groups around the world dealing with the theme. Our own groups succeeded in characterizing natural and synthetic peptides with cell penetration properties. These peptides and derived sequences were found in natural proteins, like animal toxins, and improved by rational design and chemical synthesis. For instance, peptide derivatization of crotamine ­ a cationic and defensin-like peptide found in the venom of South American rattlesnake (Crotalus durissus terrificus) ­ allowed us to produce shorter derived and analogs with improved nuclear homing property and intrinsic nucleolar targeting capabilities. Conclusion: The potential of biotechnological and biomedical applications of natural or synthetic CPPs, for the field of cell biology could include: the fluorescent image analysis of subcellular and organellar structures; the delivery of hydrophilic drugs and antiproliferative agents to treat cancer and degenerative diseases; the delivery of biomolecules into the mammalian cells for cancer treatment, cell and gene therapy, gene silencing and transgenesis.