Δομικές και λειτουργικές μελέτες πεπτιδικών μοντέλων της απολιποπρωτεΐνης Α-Ι. Ρόλος στην αθηρωμάτωση

Abstract

Atherosclerosis is a chronic inflammatory disease of the artery wall which may lead to heart disease and stroke. Numerous epidemiological studies over the past few decades have inversely correlated high plasma HDL levels with atherosclerosis and high risk of cardiovascular disease. Apolipoprotein A-I (apoA-I), the main protein constituent of HDL, contributes significantly to its antiatherogenic properties and has been under close investigation. Human apoA-I consists of 243 amino acids which are organised in 10 amphipathic )-helices. The distinct distribution in each helix of positively and negatively charged residues, as well as the hydrophobic residues, classifies apoA-I helices into 3 main distinct classes; class A, Y and G. The protein is remarkably flexible and assumes numerous lipid-dependent conformations. It plays a key role in lipid and cholesterol metabolism and has been found to exert significant antioxidant and antiinflammatory properties. However, despite the continuous and extensive research efforts to elucidate its exact structural and biological properties, many details still remain unanswered. A useful approach to better understanding its atheroprotective abilities is the use of peptide analogues of apoA-I. A new series of K-class amphipathic )-helical peptide analogues of human apoA-I was designed and synthesized in solid phase, according to the Fmoc-strategy: 1. Ac-RMSDSAERAVDRL-NH2 helix 6, region 147-159 (1) 2. Ac-RASDSAERAVDRL-NH2 helix 6, region 147-159 (2) 3. Ac-AEKAVDKL(NVa)KALD-NH2 helix 6, region 152-164 (3) 4. Ac-PALE(NVa)LREGLKPVLESFKVSF-NH2 helix 9 and 10, region 209-229 (4) 5. palm-RMSDSAERAVDRL-NH2 helix 6, region 147-159 (5) 6. palm-RASDSAERAVDRL-NH2 helix 6, region 147-159 (6) 7. palm-AEKAVDKL(NVa)KALD-NH2 helix 6, region 152-164 (7) All peptides were synthesized in Rink Amide AM resin, identified by Electrospray Ionization Mass Spectrometry (ESI-MS) and purified by Semi-Preparative Reverse Phase high performance liquid chromatography (RP-HPLC). The purified peptides were confirmed by ESI-MS and analytical RPHPLC. Their structure was assessed by Circular Dichroism Spectroscopy (CD) in the following aqueous peptide solutions; sodium dodecylsulphate (SDS=8mM and molar ratio peptide:SDS 1:80), in trifluoroethanol (TFE/H2O 50:50 v/v and Cpep=100μM), in tetradecylphosphocholine (C14PC=5mM and molar ratio peptide:C14PC 1:50) and in H2O (Cpep=100μM). 190 The biological activity of the peptides containing Met – peptide (1) and its palmitoylated analogue (5) – as well as their analogues where Met has been substituted by Ala –peptides (2) and (6) – was tested in order to elucidate the role of Met148 of apoA-I against LDL oxidation in vitro. Peptide (3) and its palmitoylated analogue (7), corresponding to helix 6, region 152-164 of human apoA-I were synthesized in order to examine the role of this region in lecithin:cholesterol acyltransferase (LCAT) activity (in progress). Finally, K-class peptide model (4), corresponding to helices 9 and 10 of human apoA-I (region 209-229), was selected in order to test this region’s ability to promote ABCA1-mediated phospholipid efflux from cells, in vitro (in progress).