Botulinum neurotoxins (BoNTs) represent a revolution in cosmetic science because of their remarkable and long-lasting antiwrinkle activity. However, their high neurotoxicity seriously limits their use. Thus, there is a need to design and validate non-toxic molecules that mimic the action of BoNTs. The hexapeptide Ac-EEMQRR-NH(2) (coined Argireline) was identified as a result of a rational design programme. Noteworthy, skin topography analysis of an oil/water (O/W) emulsion containing 10% of the hexapeptide on healthy women volunteers reduced wrinkle depth up to 30% upon 30 days treatment. Analysis of the mechanism of action showed that Argireline significantly inhibited neurotransmitter release with a potency similar to that of BoNT A, although as expected, it displayed much lower efficacy than the neurotoxin. Inhibition of neurotransmitter release was due to the interference of the hexapeptide with the formation and/or stability of the protein complex that is required to drive Ca(2+)-dependent exocytosis, namely the vesicular fusion (known as SNARE) complex. Notably, this peptide did not exhibit in vivo oral toxicity nor primary irritation at high doses. Taken together, these findings demonstrate that Argireline is a non-toxic, antiwrinkle peptide that emulates the action of currently used BoNTs. Therefore, this hexapetide represents a biosafe alternative to BoNTs in cosmetics.

Amperometry in chromaffin cells expressing green f luorescent protein (GFP) fused to synaptosomeassociated protein of 25 kDa (SNAP-25) have been used to test the involvement of single amino acids in exocytotic function, overcoming some of the limitations of studies based on Botulinum neurotoxin cleavage, as this occurs at defined sites of the protein. Constructs containing either the whole SNAP-25 polypeptide or several deleted forms lacking its C-terminal domain were heavily overexpressed in transfected cells. All GFP-fusions were located in both the cytoplasm and the plasma membrane. Although a construct containing complete SNAP-25 sustained normal secretion, removal of four or more amino acids of its C terminus greatly altered the overall rate and extent of exocytosis. Further mutational analysis proved that Leu 203 , the fourth residue from the C terminus, is critical for secretion. Kinetics of single granule fusions from cells expressing truncated forms showed slow onset and decay times when compared with control cells expressing full SNAP-25. Thus, these data provide direct evidence for the involvement of a specific residue of SNAP-25 in exocytosis and show that overexpression of GFP-SNAP contructs combined with single vesicle fusion measurements constitutes a powerful approach to dissect the structural elements playing a role in individual steps of the exocytotic cascade.In the nervous system, information is sent across synapses between individual cells by exocytosis of chemical neurotransmitters. For this reason, the elucidation of the molecular basis of exocytosis is essential to understanding neurotransmission. In this sense, the characterization of a variety of proteins [soluble N-ethylmaleimide-sensitive fusion protein, attachment protein receptors (SNAREs)], which constitute the complex involved in specific docking and fusion of neurotransmitter-containing vesicles, has stimulated an unprecedented progress in this matter (1-5). Assembly of the initial core complex between plasma membrane proteins (t-SNAREs) such as syntaxin (6) and synaptosome-associated protein of 25 kDa (SNAP-25) (7) and vesicle associated proteins (vSNAREs) such as synaptobrevin (8) provides the specificity required for vesicle docking and, probably, the minimal machinery for membrane fusion (9) whereas the interaction between these proteins and other membrane-attached (synaptotagmin, munc-18, etc.) or cytosolic (SNAPs, ATP, etc) factors ensures the necessary control for the process. Cleavage of SNAREs by Clostridial neurotoxins has been established as an essential tool to investigate the role of these proteins in neurotransmission (10). More specifically, botulinum neurotoxins A and E (BoNT A and E) proteolize the C-terminal domain of 12), causing partial inhibition of secretion in both neuronal (11) and endocrine systems (13,14). Given that the neurotoxins are unable to cleave SNAP-25 when this protein is forming part of mature trimeric complexes, as demonstrated in in vitro assays (15), it has been proposed that the in...

Bovine adrenomedullary cells in culture have been used to study the role of myosin in vesicle transport during exocytosis. Amperometric determination of calcium-dependent catecholamine release from individual digitonin-permeabilized cells treated with 3 microM wortmannin or 20 mM 2,3-butanedione monoxime (BDM) and stimulated by continuous as well as repetitive calcium pulses showed alteration of slow phases of secretion when compared with control untreated cells. The specificity of these drugs for myosin inhibition was further supported by the use of peptide-18, a potent peptide affecting myosin light-chain kinase activity. These results were supported also by studying the impact of these myosin inhibitors on chromaffin granule mobility using direct visualization by dynamic confocal microscopy. Wortmannin and BDM affect drastically vesicle transport throughout the cell cytoplasm, including the region beneath the plasma membrane. Immunocytochemical studies demonstrate the presence of myosin types II and V in the cell periphery. The capability of antibodies to myosin II in abrogating the secretory response from populations of digitonin-permeabilized cells compared with the modest effect caused by anti-myosin V suggests that myosin II plays a fundamental role in the active transport of vesicles occurring in the sub-plasmalemmal area during chromaffin cell secretory activity.

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Fusion proteins made of green fluorescent protein coupled to SNAP-25 or synaptobrevin were overexpressed in bovine chromaffin cells in order to study the role of critical protein domains in exocytosis. Point mutations in the C-terminal domain of SNAP-25 (K201E and L203E) produced a marked inhibition of secretion, whereas single (Q174K, Q53K) and double mutants (Q174K/Q53K) of amino acids from the so-called zero layer only produced a moderate alteration in secretion. The importance of the SNAP-25 C-terminal domain in exocytosis was also confirmed by the similar effect on secretion of mutations in analogous residues of synaptobrevin (A82D, L84E). The effects on the initial rate and magnitude of secretion correlated with the alteration of single vesicle fusion kinetics since the amperometric spikes from cells expressing SNAP-25 L203E and K201E and synaptobrevin A82D and L84E mutants had lower amplitudes and larger half-width values than the ones from controls, suggesting slower neurotransmitter release kinetics than that found in cells expressing the wild-type proteins or zero layer mutants of SNAP-25. We conclude that a small domain of the SNAP-25 C terminus and its counterpart in synaptobrevin play an essential role in the final membrane fusion step of exocytosis.The SNAP 1 (soluble NSF attachment protein) receptor (SNARE) hypothesis (1) has been crucial to our understanding of the molecular machinery responsible for exocytosis. The molecular events taking place during the exocytotic fusion of cellular and vesicular membranes should provide fundamental information about the mechanism of membrane fusion common to different membrane trafficking processes (2). The assembly of a ternary complex formed by the plasma membrane proteins syntaxin (3) and SNAP-25 (4) and the vesicle-associated protein synaptobrevin (5) is considered to be one of the molecular events driving vesicle priming, involving maturation steps needed to promote the apposition and final fusion of membranes (6). A heptad repeat structural motif typical of coiledcoils forming proteins is present and could be the basis for the formation of the core complex. This was confirmed by the observed increase in ␣-helix content following assembly of the complex (7,8). In recent years, precise structural studies on the nature of the complex (9, 10) have shown that single fragments of 60 -70 residues of syntaxin and synaptobrevin together with two segments of SNAP-25 form helices in a four-stranded coil. Although most of the interactions between these helices are hydrophobic, there is a polar layer embedded in the middle of this rod-shaped structure formed by three glutamines and one arginine (zero layer), which is believed to be critical for SNARE complex formation. In addition, functional studies using specific neurotoxins (11), antibodies against critical domains (12), peptides imitating regions of SNAREs (13), and overexpression of altered forms of these proteins (14, 15) have revealed important details about the participation of SNAREs in the exocytotic proce...