Understanding the role of apolipoproteinA-I in atherosclerosis. Post-translational modifications synergize dysfunction?
Graphical abstract
Introduction
An extensive research field supports a key role of human high density lipoproteins (HDL) and their major protein apolipoprotein A-I (apoA-I) in the protection against cardiovascular disease [1,2]. An overall agreement highlights their beneficial participation in the reverse cholesterol transport (RCT), which delivers excess of cholesterol from peripheral cells to the liver for its catabolism [3]. In addition, it has been recognized the crucial participation of HDL and apoA-I in pathways protecting endothelial functions: stimulation of nitric oxide-mediated vasodilatation [4], reduction of vascular cell adhesion molecule-1 (VCAM-I) expression [5,6], and inhibition of apoptosis promoting proliferation [7]. The multiple functions of apoA-I have been extensively related to its highly flexible structure. Also, a dynamic interconversion between lipid-free and lipid-bound states mediates its efficiency to interact with membranes and recruit phospholipids and cholesterol. Most of the protein circulates bound as complex HDL particles, and a minor fraction, about 5% is recycled as the lipoprotein is catabolized, yielding lipid-free or lipid-poor conformations which are more effective to interact with key proteins such as ATP-binding cassette proteins A1 and G1 (ABCA1 and ABCG1 receptors) and lecithin: cholesterol acyltransferase (LCAT) ([8] and references described there). Moreover, many other functions of apoA-I have been identified in vitro, suggesting that the protein mimics some of the pathways observed for HDL, as decreasing the respiratory burst induced by oxidized low density lipoproteins (oxLDL) [9] and binding to bacterial lipopolysaccharide (LPS) [10,11].
Despite the aforementioned information, the “HDL-cholesterol hypothesis” has been revised as it seemed to be oversimplified by setting an atherosclerosis-risk index from LDL/HDL ratio [8,12]. Beyond the usefulness of this ratio as a clinical parameter, atherosclerosis is a complex chronic pathological scenario, becoming a pro-oxidant environment with the release of reactive oxygen species (ROS) related to the failure of cholesterol homeostasis Thereby, quality of lipoproteins is considered to be a good predictor in addition to their quantity. Atherosclerotic plaques are characterized by dysfunctional apoA-I aggregated in the artery walls [13]. Protein distribution in human aorta is quite different from the circulating conformations. It is identified in high amounts deposited in chronic lesions predominantly lipid-poor, not associated with HDL, extensively oxidized and cross-linked, and functionally impaired [14]. Whether protein misfolding is a cause or a consequence of this microenvironment is not known. In this regard, it was shown the presence of diffuse deposits of apoA-I as amyloid patches in complicated atherosclerotic plaques [15]. This fact indicates that amyloidosis and atherosclerosis may be closely associated [16].
About twenty natural variants of apoA-I have been described inducing amyloidosis with deposit and failure of target organs such as heart, liver or kidney [17]. Interestingly, one naturally occurring deletion variant (K107del) was described inducing a unique pathologic pattern, as amyloidosis was associated with severe atherosclerosis [18]. The facts that determine this behavior are far to be known. The loss of function the increased tendency to misfold, or the ability to elicit a pro inflammatory environment were suggested to mediate its role in these diseases [19]. Taking advantage of the structural comparative study among K107del and the protein with the native sequence (Wt), we proposed here to understand the reasons that may affect apoA-I misfunction and aggregation in the atherosclerotic plaques. Deep structural knowledge of this variant may help clarify the participation of apoA-I (or its failure to fulfill the protective roles). As oxidation and crosslinking events are present in atherosclerotic plaques, we analyzed the variant's natural flexibility and function under native conditions and under controlled chemical modifications that could mimic those undergoing in a chronic pathological scenario.
Section snippets
Materials
Guanidine hydrochloride (GndHCl), thioflavin T (ThT), cholesterol (Chol), sodium cholate, ethylenediaminetetraacetic acid (EDTA), sodium chloride (NaCl), sodium dodecyl sulfate (SDS),Terbium -III chloride (Tb) and dipicolinic acid (DPA) were from Sigma-Aldrich (St Louis, MO); 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and dimyristoylphosphatidylcholine (DMPC) were purchased from Avanti Polar Lipids (Alabaster, AL); His-purifying resin was from Novagen (Darmstadt, Germany).
Proteins purification
ApoA-I variants (either Wt or K107del) were isolated and purified from bacterial strains in high yield and purity (Suppl Fig. 1). We have previously shown that Wt purified under this protocol behaves almost indistinguishable from plasma apoA-I [20].
Leakage
In order to characterize the influence of the deletion of the positive Lys residue in position 107 on protein function, we first compared the interaction of the variant with lipid bilayers, monitoring protein-induced leakage of SUVs. Energy transfer
Discussion and conclusions
In the present study, we set out to characterize local conditions that may shift apoA-I structure from the native folding, inducing its aggregation and dysfunction in the atherosclerotic plaques. From the analysis of this chronic pro inflammatory scenario, common factors are to be considered: 1) chemical modifications due to reactive oxygen species (ROS) freed by activated macrophages, among those oxidation and crosslinking [34]; 2) intrinsic modifications caused by hereditary mutations in the
Author contribution statement
RAG and IDL performed the experiments, did data analysis and designed the research; HAG did data analysis and searched for funding, MCG did data analysis and discussed results, NAR and MAT designed the research, wrote the manuscript and searched for funding.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
Authors acknowledge Mr. Mario Ramos for invaluable help with figure design, Mrs. Rosana del Cid for English assistance and Miss Letizia Bauzá for expert contributions with size exclusion chromatography assays. This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PUE 22920160100002 to HG); Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, PICT-2016-0849 to MAT and PICT-2016-0915 to HG); Universidad Nacional de La Plata (UNLP) (M187 and
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