Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.

We report a systematic and detailed analysis of recombinant neurolysin (EC 3.4.24.16) specificity in parallel with thimet oligopeptidase (TOP, EC 3.4.24.15) using Bk sequence and its C- and N-terminal extensions as in human kininogen as motif for synthesis of internally quenched fluorescent substrates. The influence of the substrate size was investigated, and the longest peptide susceptible to TOP and neurolysin contains 17 amino acids. The specificities of both oligopeptidases to substrate sites P(4) to P(3)' were also characterized in great detail using seven series of peptides based on Abz-GFSPFRQ-EDDnp taken as reference substrate. Most of the peptides were hydrolyzed at the bond corresponding to P(4)-F(5) in the reference substrate and some of them were hydrolyzed at this bond or at F(2)-S(3) bond. No restricted specificity was found for P(1)' as found in thermolysin as well for P(1) substrate position, however the modifications at this position (P(1)) showed to have large influence on the catalytic constant and the best substrates for TOP contained at P(1), Phe, Ala, or Arg and for neurolysin Asn or Arg. Some amino acid residues have large influence on the K(m) constants independently of its position. On the basis of these results, we are hypothesizing that some amino acids of the substrates can bind to different sub-sites of the enzyme fitting P-F or F-S bond, which requires rapid interchange for the different forms of interaction and convenient conformations of the substrate in order to expose and fit the cleavage bonds in correct position for an efficient hydrolysis. Finally, this plasticity of interaction with the substrates can be an essential property for a class of cytosolic oligopeptidases that are candidates to participate in the selection of the peptides to be presented by the MHC class I.

No abstract