AbstractsBiology & Animal Science

According to the current WHO statistic, cardiovascular diseases are the leading cause of deaths all over the world. Over 1.7 million people have died due to cardiovascular disorders like myocardial infarction, coronary artery diseases and stroke. A common condition underlying almost all of these disorders is an excessive accumulation of lipids- more specifically cholesterol. Thus, managing the levels of cholesterol can prove to be a very effective strategy in preventing or treating several cardiovascular disorders. ApoA-I mimetic peptides are promising candidates in this respect. L4F is a synthetic apoA-I mimetic peptide containing 18 L- amino acid residues of which four are phenylalanine and hence the name. It has been found to be capable of reducing lesions due plaque formation in atherosclerosis in mice. Animal models and cell studies have demonstrated the capability of L4F to reduce the expression of inflammatory cytokines IL-1, IL-6 and TNFa. L4F is also found to be capable of binding oxidized lipids. Although a lot is known of the biological activity of L4F, very little is known about its structure and how that relates to its varied functions. It is unknown how or if L4F is capable of binding cholesterol in solution state. Thus, our research is a step forward in that direction.We have successfully determined that L4F can bind cholesterol in solution state. The binding was detected using an enzyme assay. The principle of the assay is such that it can detect cholesterol that is in solution and available for the enzyme to detect. Thus L4F is capable of solubilizing cholesterol and making it available in solution. L4F showed detergent-like action where it solubilized cholesterol from a pellet of cholesterol and made it detectable by the enzyme. We were also successful in determining the time course of binding. The binding or complex formation starts almost instantaneously but L4F-cholesterol bound complex starts to fall apart over a period of time. The maximum binding was detected at 3hrs after which it gradually decreases and by 12hrs when barely any could be detected. This property of L4F is favorable for developing it as a drug molecule.We made efforts to express the peptide in bacterial cells to obtain isotopically labeled peptide. This isotopically labeled peptide would eventually be used for structural analysis by Hydrogen-Deuterium Exchange using NMR or Mass spectrometry. The gene construct (oligo) for the peptide was designed and primers were designed for PCR amplification of the double stranded oligos for insertion into vector for expression. PCR products were obtained successfully. The expression of isotopically labeled peptide and structural analysis of L4F are the ultimate goals of this research and efforts will be continued in that direction by other lab members.