Driving forces in the delivery of penetratin conjugated G protein fragment.

A42 is a chimera peptide consisting of Galphas(374-394)C379A--the 21-mer C terminus of the Galphas protein, able of adenosine inhibitory activity--and penetratin--the 16 residue fragment, derived from the homeodomain of the Drosophila transcription factor Antennapedia. A42 is able to cross cell membranes and to inhibit A2A and A2B adenosine and beta-adrenergic receptor stimulated camps (D'Ursi et al. Mol. Pharmacol. 2006, 69, 727-36). Here we present an extensive biophysical study of A42 in different membrane mimetics, with the objective to evaluate the molecular mechanisms which promote the membrane permeation. Fluorescence, CD, and NMR data were acquired in the presence of negatively charged and zwitterionic sodium dodecyl sulfate and dodecylphosphocholine surfactants. To validate the spectroscopic results in a larger scale, fluorescence microscopy experiments were performed on negatively charged and zwitterionic dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine vesicles. Our results show that the internalization of A42 is mainly driven by electrostatic interactions, hydrophobic interactions playing only a secondary, sinergistic role. The distribution of the charges along the molecule has an important role, highlighting that internalization is a process which requires a specific matching of peptide and membrane properties.

A membrane-permeable peptide containing the last 21 residues of the G alpha(s) carboxyl terminus inhibits G(s)-coupled receptor signaling in intact cells: correlations between peptide structure and biological activity.

Cell-penetrating peptides are able to transport covalently attached cargoes such as peptide or polypeptide fragments of endogenous proteins across cell membranes. Taking advantage of the cell-penetrating properties of the 16-residue fragment penetratin, we synthesized a chimeric peptide that possesses an N-terminal sequence with membrane-penetrating activity and a C-terminal sequence corresponding to the last 21 residues of G alpha(s). This G alpha(s) peptide was an effective inhibitor of 5'-N-ethylcarboxamidoadenosine (NECA) and isoproterenol-stimulated production of cAMP in rat PC12 and human microvascular endothelial (HMEC-1) cells, whereas the carrier peptide had no effect. The maximal efficacy of NECA was substantially reduced when PC12 cells were treated with the chimeric peptide, suggesting that it competes with G alpha(s) for interaction with receptors. The peptide inhibited neither G(q)- nor G(i)-coupled receptor signaling. The use of a carboxy-fluorescein derivative of the peptide proved its ability to cross the plasma membrane of live cells. NMR analysis of the chimeric peptide structure in a membrane-mimicking environment showed that the G alpha(s) fragment assumed an amphipathic alpha-helical conformation tailored to make contact with key residues on the intracellular side of the receptor. The N-terminal penetratin portion of the molecule also showed an alpha-helical structure, but hydrophobic and hydrophilic residues formed clustered surfaces at the N terminus and center of the fragment, suggesting their involvement in the mechanism of penetratin internalization by endocytosis. Our biological data supported by NMR analysis indicate that the membrane-permeable G alpha(s) peptide is a valuable, nontoxic research tool to modulate G(s)-coupled receptor signal transduction in cell culture models.

Galphas protein C-terminal alpha-helix at the interface: does the plasma membrane play a critical role in the Galphas protein functionality?

The heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins, Galphabetagamma) mediate the signalling process of a large number of receptors, known as G protein-coupled receptors. The C-terminal domain of the heterotrimeric G protein alpha-subunit plays a key role in the selective activation of G proteins by their cognate receptors. The interaction of this domain can take place at the end of a cascade including several successive conformational modifications. Galpha(s)(350-394) is the 45-mer peptide corresponding to the C-terminal region of the Galpha(s) subunit. In the crystal structure of the Galpha(s) subunit it encompasses the alpha4/beta6 loop, the beta6 beta-sheet segment and the alpha5 helix region. Following a previous study based on the synthesis, biological activity and conformational analysis of shorter peptides belonging to the same Galpha(s) region, Galpha(s)(350-394) was synthesized and investigated. The present study outlines the central role played by the residues involved in the alpha4/beta6 loop and beta6/alpha5 loops in the stabilization of the C-terminal Galpha(s)alpha-helix. H(2)O/(2)H(2)O exchange experiments, and NMR diffusion experiments show interesting evidence concerning the interaction between the SDS micelles and the polypeptide. These data prompt intriguing speculations on the role of the intracellular environment/cellular membrane interface in the stabilization and functionality of the C-terminal Galpha(s) region.