Synthetic peptides able to modulate the interaction of AKAP121 with mitochondria

摘要

Protein phosphorylation and dephosphorylation play a central role in the regulation of cellular functions in response to change in external stimuli.Phosphorilation is mediated by different kinases that are ubiquitous in cells.One of the best characterized protein kinase is the c-AMP-dependent kinase A(PKA).The subcellular localization of this enzyme is maintained through its association with A-kinase anchoring proteins(AKAPs).AKAPs represent a group of functionally related proteins,classified by their ability to interact with PKA inside cells.AKAP proteins are able to bring PKA to different membrane and cellular organelles and thus can mediate cAMP signalling events such as development,differentiation,cell survival and cell progression.In particular AKAP 121 localizes PKA on mitochondria and endoplasmic reticulum and mediate the protective effect of cAMP on cell survival.It was demonstrated that the targeting of AKAP 121 to the outer membrane of mitochondria both in male germ cells and in transfected heterologous cells is mediated by the first 30 N-terminal residues(MT)that are predict to form a highly hydrophobic alpha-helical wheel.Starting from the data available in the literature we chose to further investigate the conformational properties in solution of the 10-30 sequence of the MT domain in order to confirm the structural prevision.Thus,we designed a peptide containing this sequence and the two Lys and one b-Ala residues at N-terminus to improve solubility and enzymatic stability,respectively.Moreover,the peptide was acetylated and amidated to eliminate the charges at N-and C-termini.The final sequence was:Ac-~1XKKPLALPGMLALLGWWWFFSRKKX~(25)-NH_2(X=beta-Ala)(AKAPwt).CD and NMR studies were used to elucidate the structural features of the peptide in TFE aqueous solution.