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Dissertation Defense Announcement
To:  The George Mason University Community

Candidate: Rafique Islam
Program: PhD in Biosciences
 
Date:   Monday December 4, 2017
Time:   12:00 PM
Place:  George Mason University
             Science & Tech campus
             Bull Run Hall, Room 257            
  
Title: "Structure-Function Studies of Apolipoprotein Mimetic Peptides for Cholesterol Efflux Mediated by ABCA1 Transporter"

Dissertation Director: Dr. Alan Remaley, NIH/NHLBI
Committee Chair:
Dr. Ancha Baranova
Committee Members:
  Dr. Lance Liotta, Dr. Robert Lipsky

All are invited to attend the defense.

ABSTRACT:
The atherosclerotic plaque is the major pathologic hallmark of coronary syndrome and a primary driver of its formations is excessive blood cholesterol, particularly on Low Density Lipoproteins (LDL). In contrast, cholesterol on High density lipoprotein (HDL) is inversely related to cardiovascular disease, most likely because of its rule in removing excess cholesterol in peripheral tissues and delivering it to the liver for excretion by a pathway called Reverse Cholesterol Transport (RCT). However, for a significant part of the population, HDL levels are insufficient to maintain cholesterol homeostasis. Furthermore, our current cholesterol reducing agents, such as statins, are only partially effective in reducing cardiovascular events and do not act acutely in reducing events after a myocardial infarction. As an alternative, infusion of reconstituted HDL made with full-length apoA-I protein, the main protein on HDL, has been shown in animal studies and early stage clinical trials to be beneficial in rapidly reducing atherosclerotic plaques, but this strategy is limited by the gram quantities of either purified or recombinant apoA-I protein needed per dose for this therapy. This structure-function study was designed to test a series of apolipoprotein A-I mimetic peptides to determine the structural features that are necessary for the for the promotion of cholesterol efflux by the ABCA1 transporter, the main pathway for efflux of cellular cholesterol.  ApoA-I mimetic peptides that are effective in cholesterol efflux could potentially be used as low cost alternatives for HDL therapy and could be readily changed to enhance their biological properties, as well as improve their pharmacokinetics and pharmacodynamics features. To achieve these goals, we investigate the following questions: (1) what structural features of peptides accounts for their ability to bind to lipoproteins and HDL in particular, (2) what structural features are necessary for peptides to efflux cholesterol by the ABCA1 transporter and (3) can stabilization of helix formation by the incorporation of alpha-methyl residues protect the peptides from proteolysis and improve their cholesterol efflux ability. For the first study, we designed fluorescent amphipathic peptides with a wide range of sizes and charges, and hydrophobicity. This study revealed that those peptides with a neutral charge containing at least 18 residues and hydrophobic face of at least 189 degrees readily bound to HDL. For the second study, we designed 4 peptides with a variable number of glutamate (E), leucine (L), lysine (K) and alanine (A) residues arranged in an amphipathic helix and determined their ability to form HDL-like particle and promote ABCA1-dependent cholesterol efflux. Based on their unique physical properties these peptides were named: neu=ELK-neutral (EKLKELLEKLLEKLKELL), hyd=ELK-hydrophobic (EKLLELLKKLLELLKELL), pos=ELK-positive (EKLKALLEKLKAKLKELL), neg=ELK-negative (EELKEKLEELKEKLEEKL). CD-spectroscopy showed all peptides have greater than 40% helicity. However, hyd and pos were mostly helical in an aqueous buffer (52% and 22%, respectively), and in a TFE-containing solvent, mimicking a lipid environment. In DMPC vesicle solubilization assay, we observed following order: pos>neg>hyd>>neg.  By non-denaturing gel electrophoresis, neu, hyd, pos formed approximately 8, 8, and 18 nm size lipid particles, respectively when combined with DMPC, however, neg did not form any detectable particles. Using BHK-ABCA1 transfected cells, cholesterol efflux studies showed following results: hyd>neu>>pos, whereas neg peptide was inactive.  Using all-atom Molecular Dynamic Simulations, we showed that those peptides that were most active in cholesterol efflux were able to stabilize the nascent HDL-discoidal structures produced after cholesterol efflux by wrapping around the sides of the disc and covering up the hydrophobic acyl chains.  Using the Anton-2 supercomputer, we further showed for the neu peptide that the peptides align along the side of the disc in a picket fence-like orientation, which is in contrast to the full length apoA-I protein that wraps around the side of the disc in a belt-like configuration.  In the third study, we showed that incorporation of methyl groups in the alpha position of amino acids for the last natural helix of apoA-I increased peptide helicity. This was true for Leu and Lys substitutions and to a lesser degree when Asp was replaced with alpha-methyl aspartic acid.  This increased helicity was associated with an overall resistant to proteolysis to trypsin, chymotrypsin and endoproteinsase Asp-N due to increase helicity and site specific resistant to proteolysis at the site of substitution.   The native unsubstituted helix was inactive in ABCA1 cholesterol efflux, whereas the alpha-methyl amino acid substituted peptides, particularly those containing Leu and Lys substitutions were comparable to a hydrocarbon stapled variant of the peptide that sterically locks the peptide into a helical conformation and is very potent in inducing ABCA1-dependent cholesterol efflux. In summary, we have identified a number of structural features of apoA-I mimetic peptides that confers to them the ability to bind to HDL and promote cholesterol efflux by ABCA1, which can be used in the future design of therapeutic peptides for the treatment of cardiovascular disease.

 


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