One of the functions of apolipoproteins is to promote cholesterol and phospholipid release from cells. Apolipoprotein A-I (apoA-I) is a foremost facilitator of this process as it extracts lipids from peripheral cells and generates high density lipoproteins (HDL), which transport excess cholesterol from the periphery to the liver for excretion. Lipid release from cholesterol-laden macrophages mediated by apoA-I is thought to be one of the mechanisms that reduce foam cell formation and atherosclerosis progression. High plasma levels of apoA-I have been shown to correlate with lower incidence of cardiovascular disease.
What would normally be a beneficial apolipoprotein, however, can be harmful when its levels in parts of the body are very high. Amyloids are insoluble deposits that consist of different types of proteins. An increasing number of clinical studies indicate that amyloid deposits are a significant component of the atherosclerotic plaque and that apoA-I is a major precursor of these amyloids. ApoA-I is a very abundant protein in plasma (about 1 mg/ml in normal conditions), where it is mostly associated with HDL. Local levels of apoA-I and other HDL-associated apolipoproteins in the subendothelial space of atherosclerotic arteries can be several folds higher. Mechanisms promoting protein misfolding, such as oxidation and glycosylation, can lead to apolipoprotein aggregation due to the high local concentration of these proteins in the proximity of the atherosclerotic plaque; thus stability of apoA-I and other HDL-associated apolipoproteins is an important factor in atherosclerosis.
1. Impact of self-association on exchangeable apolipoproteins' structure and function
Exchangeable apolipoproteins are dynamic molecules that can transition between their lipid-free and lipid-associated forms and transfer between different lipoprotein particles. Lipid binding of apolipoproteins is mediated by their most characteristic structural motif: the amphipathic a-helix. Hydrophobic and hydrophilic surfaces are segregated on opposite faces of amphipathic a-helical domains. Lipids bind to the hydrophobic regions and shield them from the aqueous environment. In the absence of lipids, apolipoproteins fold in tertiary and quaternary structures, which sequester the extensive hydrophobic surfaces of amphipathic a-helices in hydrophobic pockets that exclude water. Apolipoprotein stability and solubility in the polar medium are thus maintained. Intermolecular apolar interactions between amphipathic a-helices aid in shielding the hydrophobic surfaces from solvent. The tendency for lipid-free exchangeable apolipoproteins to self-associate is a result of these interactions. Apolipoprotein A-I, A-II, A-IV, E, and Cs all form soluble self-associated species in solution. The Cavigiolio lab is currently investigating how exchangeable apolipoproteins’ self-association impacts their function.
Impact of self-association on exchangeable apolipoproteins' structure and function
2. Development of a clinically-viable measure of HDL functionality
Large population studies have shown that low plasma HDL-cholesterol levels (HDL-C) are associated with high risk of developing cardiovascular disease (CVD). Nonetheless, there are numerous cases of severe CVD in patients with high HDL-C. A recent genetic study challenges the existing paradigm and proposes that a better biomarker of CVD risk could be obtained by evaluating the functional quality of HDL, rather than merely quantifying HDL cholesterol content. Reliable routine analytical methods are needed to quantify HDL functionality in clinical plasma samples in order to produce such a biomarker. We are using the inherent ability of apoA-I to transition between the lipid-bound and lipid-free states as a functional parameter to assess HDL quality.
We have previously designed a fluorescent variant of apoA-I that exhibits distinct emission spectra, depending on whether it is lipid-free or HDL-associated. Using this fluorescent apoA-I as a probe of apoA-I lipidation state, we showed that the pools of HDL-associated and lipid-free apoA-I participate in a spontaneous ‘exchange’ reaction. The kinetics of the exchange reaction, which we named “apoA-I exchangeability”, were significantly reduced by apoA-I crosslinking or oxidation by myeloperoxidase, which are apolipoprotein modifying events similar to those occurring in atherosclerosis. The Cavigiolio lab is building on this previous research by exploring whether “exchangeability” of apoA-I can be used clinically as a measure of HDL functionality and as a predictor of a patient’s risk of developing CVD.
Development of a clinically-viable measure of HDL functionality
3. Role of apolipoprotein A-II in HDL structure-function
Apolipoprotein A-II (apoAII) is an exchangeable apolipoprotein produced in the liver with higher affinity for lipids than apoA-I. HDL particles in human plasma contain either apoA-I (HDL-AI) or both apoA-I and A-II (HDL-AI-AII). Different HDL subclasses contain a variable amount of apoA-I molecules but, strikingly, the molar ratio of apoA-I vs. apoA-II is roughly constant; it is equal to 2:1 in all HDL-AI-AII particle sizes.
Despite thirty years of research on apoA-II, both the structure of apoA-II-containing HDL and the metabolic role of this protein are poorly understood. For example, the mechanism and the timing of apoA-II insertion into HDL are not fully explained. Furthermore, a correlation between apoA-II levels and incidence of CVD has not been completely established. The primary obstacle in understanding apoA-II function has been the unavailability of bacterially expressed recombinant apoA-II. To overcome this technical limitation, the Cavigiolio lab, in collaboration with the laboratory of Prof. Sean Davidson (University of Cincinnati, Ohio, USA), has developed an intein-based bacterial expression system that produces highly pure mature human apoA-II at substantial yields. The Cavigiolio lab is now using this expression system to generate variants of apoA-II and investigate the structure and function of apoA-II.
Role of apolipoprotein A-II in HDL structure-function
Wednesday, February 24, 2016 9:18 AM