Understanding the role of apolipoproteinA-I in atherosclerosis. Post-translational modifications synergize dysfunction?

Highlights

• Oxidation is clue to induce protein misfolding.

• Natural mutation does not seem critical as a sole reason to determine pathogenicity.

• Atherosclerosis and amyloidosis are closely related.

• Intramolecular crosslinking restrains protein flexibility and function.

Abstract

Background

The identification of dysfunctional human apolipoprotein A-I (apoA-I) in atherosclerotic plaques suggests that protein structure and function may be hampered under a chronic pro inflammatory scenario. Moreover, the fact that natural mutants of this protein elicit severe cardiovascular diseases (CVD) strongly indicates that the native folding could shift due to the mutation, yielding a structure more prone to misfold or misfunction. To understand the events that determine the failure of apoA-I structural flexibility to fulfill its protective role, we took advantage of the study of a natural variant with a deletion of the residue lysine 107 (K107del) associated with atherosclerosis.

Methods

Biophysical approaches, such as electrophoresis, fluorescence and spectroscopy were used to characterize proteins structure and function, either in native conformation or under oxidation or intramolecular crosslinking.

Results

K107del structure was more flexible than the protein with the native sequence (Wt) but interactions with artificial membranes were preserved. Instead, structural restrictions by intramolecular crosslinking impaired the Wt and K107del lipid solubilization function. In addition, controlled oxidation decreased the yield of the native dimer conformation for both variants.

Conclusions

We conclude that even though mutations may alter protein structure and spatial arrangement, the highly flexible conformation compensates the mild shift from the native folding. Instead, post translational apoA-I modifications (probably chronic and progressive) are required to raise a protein conformation with significant loss of function and increased aggregation tendency.

General significance

The results learnt from this variant strength a close association between amyloidosis and atherosclerosis.

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.