CPP-Ts: a new intracellular calcium channel modulator and a promising tool for drug delivery in cancer cells.

Scorpion sting envenoming impacts millions of people worldwide, with cardiac effects being one of the main causes of death on victims. Here we describe the first Ca2+ channel toxin present in Tityus serrulatus (Ts) venom, a cell penetrating peptide (CPP) named CPP-Ts. We show that CPP-Ts increases intracellular Ca2+ release through the activation of nuclear InsP3R of cardiomyocytes, thereby causing an increase in the contraction frequency of these cells. Besides proposing a novel subfamily of Ca2+ active toxins, we investigated its potential use as a drug delivery system targeting cancer cell nucleus using CPP-Ts's nuclear-targeting property. To this end, we prepared a synthetic CPP-Ts sub peptide14-39 lacking pharmacological activity which was directed to the nucleus of specific cancer cell lines. This research identifies a novel subfamily of Ca2+ active toxins and provides new insights into biotechnological applications of animal venoms.

Membrane-permeable Rab27A is a regulator of the acrosome reaction: Role of geranylgeranylation and guanine nucleotides.

The acrosome reaction is the regulated exocytosis of mammalian sperm's single secretory granule, essential for fertilization. It relies on small GTPases, the cAMP binding protein Epac, and the SNARE complex, among other components. Here, we describe a novel tool to investigate Rab27-related signaling pathways: a hybrid recombinant protein consisting of human Rab27A fused to TAT, a cell penetrating peptide. With this tool, we aimed to unravel the connection between Rab3, Rab27 and Rap1 in sperm exocytosis and to deepen our understanding about how isoprenylation and guanine nucleotides influence the behaviour of Rab27 in exocytosis. Our results show that TAT-Rab27A-GTP-γ-S permeated into live sperm and triggered acrosomal exocytosis per se when geraylgeranylated but inhibited it when not lipid-modified. Likewise, an impermeant version of Rab27A elicited exocytosis in streptolysin O-permeabilized - but not in non-permeabilized - cells when geranylgeranylated and active. When GDP-ß-S substituted for GTP-γ-S, isoprenylated TAT-Rab27A inhibited the acrosome reaction triggered by progesterone and an Epac-selective cAMP analogue, whereas the non-isoprenylated protein did not. Geranylgeranylated TAT-Rab27A-GTP-γ-S promoted the exchange of GDP for GTP on Rab3 and Rap1 detected by far-immunofluorescence with Rab3-GTP and Rap1-GTP binding cassettes. In contrast, TAT-Rab27A lacking isoprenylation or loaded with GDP-ß-S prevented the activation of Rab3 and Rap1 elicited by progesterone. Challenging streptolysin O-permeabilized human sperm with calcium increased the population of sperm with Rap1-GTP, Rab3-GTP and Rab27-GTP in the acrosomal region; pretreatment with anti-Rab27 antibodies prevented the activation of all three. The novel findings reported here include: the description of membrane permeant TAT-Rab27A as a trustworthy tool to unveil the regulation of the human sperm acrosome reaction by Rab27 under physiological conditions; that the activation of endogenous Rab27 is required for that of Rab3 and Rap1; and the connection between Epac and Rab27 and between Rab27 and the configuration of the SNARE complex. Moreover, we present direct evidence that Rab27A's lipid modification, and activation/inactivation status correlate with its stimulatory or inhibitory roles in exocytosis.

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.

Design and implementation of a high yield production system for recombinant expression of peptides.

BACKGROUND: Making peptide pharmaceuticals involves challenging processes where many barriers, which include production and manufacture, need to be overcome. A non common but interesting research area is related to peptides with intracellular targets, which opens up new possibilities, allowing the modulation of processes occurring within the cell or interference with signaling pathways. However, if the bioactive sequence requires fusion to a carrier peptide to allow access into the cell, the resulting peptide could be such a length that traditional production could be difficult. The goal of the present study was the development of a flexible recombinant expression and purification system for peptides, as a contribution to the discovery and development of these potentially new drugs. RESULTS: In this work, a high throughput recombinant expression and purification system for production of cell penetrating peptides in Escherichia coli has been designed and implemented. The system designed produces target peptides in an insoluble form by fusion to a hexahistidine tagged ketosteroid isomerase which is then separated by a highly efficient thrombin cleavage reaction procedure. The expression system was tested on the anticancer peptides p53pAnt and PNC27. These peptides comprise the C-terminal region and the N-terminal region of the protein p53, respectively, fused by its carboxyl terminal extreme to the cell penetrating peptide Penetratin. High yields of purified recombinant fused peptides were obtained in both cases; nevertheless, thrombin cleavage reaction was successful only for p53pAnt peptide release. The features of the system, together with the procedure developed, allow achievement of high production yields of over 30 mg of highly pure p53pAnt peptide per g of dry cell mass. It is proposed that the system could be used for production of other peptides at a similar yield. CONCLUSIONS: This study provides a system suitable for recombinant production of peptides for scientific research, including biological assays.

Crotamine: a novel cell-penetrating polypeptide nanocarrier with potential anti-cancer and biotechnological applications.

Crotamine is a basic, 42-residue polypeptide derived from snake venom that has been shown to possess cell-penetrating properties. Crotamine forms nanoparticles with a variety of DNA and RNA molecules, and crotamine-plasmid DNA nanoparticles are selectively delivered into actively proliferating cells in culture or in mice. As such, these nanoparticles could form the basis for a nucleic acid drug-delivery system. Here we describe the preparation, purification, and biochemical and biophysical analysis of venom-derived, recombinant, chemically synthesized, and fluorescent-labeled crotamine; the formation and characterization of crotamine-DNA and -RNA nanoparticles; and the delivery of these nanoparticles into cells and animals.