Statins lower plasma cholesterol by as much as 50%, thus reducing future cardiovascular events. However, the physiological effects of statins are diverse and not all are related to low density lipoprotein cholesterol (LDL-C) lowering. We performed a small clinical pilot study to assess the impact of statins on lipoprotein-associated proteins in healthy individuals (n = 10) with normal LDL-C (<130 mg/ dL), who were treated with rosuvastatin (20 mg/day) for 28 days. Proteomic analysis of size-exclusion chromatography isolated LDL, large high density lipoprotein (HDL-L), and small HDL (HDL-S) fractions and spectral counting was used to compare relative protein detection before and after statin therapy. Significant protein changes were found in each lipoprotein pool and included both increases and decreases in several proteins involved in lipoprotein metabolism, complement regulation and acute phase response. The most dramatic effect of the rosuvastatin treatment was an increase in α-1-antirypsin (A1AT) spectral counts associated with HDL-L particles. Quantitative measurement by ELISA confirmed an average 5.7-fold increase in HDL-L associated A1AT. Molecular modeling predictions indicated that the hydrophobic reactive center loop of A1AT, the functional domain responsible for its protease inhibitor activity, is likely involved in lipid binding and association with HDL was found to protect A1AT against oxidative inactivation. Cell culture experiments, using J774 macrophages, demonstrated that the association of A1AT with HDL enhances its antiprotease activity, preventing elastase induced production of tumor necrosis factor α. In conclusion, we show that statins can significantly alter the protein composition of both LDL and HDL and our studies reveal a novel functional relationship between A1AT and HDL. The up-regulation of A1AT on

Apolipoprotein B (apoB) is a large amphipathic protein that is the structural scaffold for the formation of several classes of lipoproteins involved in lipid transport throughout the body. The goal of the present study was to identify specific domains in the apoB sequence that contribute to its lipid binding properties. A sequence analysis algorithm was developed to identify stretches of hydrophobic amino acids devoid of charged amino acids, which are referred to as hydrophobic cluster domains (HCDs). This analysis identified 78 HCDs in apoB with hydrophobic stretches ranging from 6 to 26 residues. Each HCD was analyzed in silico for secondary structure and lipid binding properties, and a subset was synthesized for experimental evaluation. One HCD peptide, B38, showed high affinity binding to both isolated HDL and LDL, and could exchange between lipoproteins. All-atom molecular dynamics simulations indicate that B38 inserts 3.7 Å below the phosphate plane of the bilayer. B38 forms an unusual α-helix with a broad hydrophobic face and polar serine and threonine residues on the opposite face. Based on this structure, we hypothesized that B38 could efflux cholesterol from cells. B38 showed a 12-fold greater activity than the 5A peptide, a bihelical Class A amphipathic helix (EC50 of 0.2658 vs. 3.188 µM; p<0.0001), in promoting cholesterol efflux from ABCA1 expressing BHK-1 cells. In conclusion, we have identified novel domains within apoB that contribute to its lipid biding properties. Additionally, we have discovered a unique amphipathic helix design for efficient ABCA1-specific cholesterol efflux.

Introduction: Statins, by inhibiting HMG-CoA reductase and up regulating hepatic LDL receptors, effectively lower plasma LDL-C by as much as 50%, thus reducing future CVD events. However, the physiological effects of statins are diverse and not all are related to lowering of LDL-C. Goal: The goal of this study was to test our hypothesis that some of these pleiotropic alternative effects from statins may be driven by compositional changes to lipoproteins distinct from their cholesterol content. We, therefore, performed a small clinical pilot study to assess the impact of statins on lipoprotein associated proteins in healthy individuals. Methods: Ten subjects with normal LDL-C (<130 mg/dL) were given rosuvastatin (20 mg/day) for 28 days. Plasma samples collected at baseline and after treatment were used for lipid measurement, nuclear magnetic resonance (NMR) lipoprotein profiling and lipoprotein proteomics. Results: The effects of rosuvastatin treatment on clinical lipid measures and NMR profile were consistent with established findings. Proteomic analysis of FPLC fractions representing LDL, HDL-1 (large) and HDL-2 (small) identified a total of 124 different proteins. Spectral counting was used to compare relative protein detection before and after statin therapy. Significant protein changes were found in each lipoprotein pool: LDL = 9, HDL-1 = 9 and HDL-2 = 4. These changes included both increases and decreases in proteins involved in lipoprotein metabolism, complement regulation and acute phase response. The most dramatic effect of the treatment was a profound increase in alpha-1-antirypsin (A1AT) spectral counts association with HDL-1 particles. Quantitative measurement by ELISA revealed an average 5.7 fold increase in HDL-1 associated A1AT. Preliminary in vitro studies indicate a potential functional role for A1AT enriched HDL in the formation of neutrophil extracellular traps (NETs), a pro-inflammatory component of vascular lesions. Summary: Based on these results, statins can significantly change the protein composition of both LDL and HDL. Some of these changes, such as the up regulation of A1AT on HDL, may convey anti-inflammatory functionality on lipoproteins and might contribute to some of the non-lipid lowering effects of statins.