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Structure–function analysis of naturally occurring apolipoprotein A-I L144R, A164S and L178P mutants provides insight on their role on HDL levels and cardiovascular risk

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Abstract

Naturally occurring point mutations in apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), may affect plasma HDL-cholesterol levels and cardiovascular risk. Here, we evaluated the effect of human apoA-I mutations L144R (associated with low HDL-cholesterol), L178P (associated with low HDL-cholesterol and increased cardiovascular risk) and A164S (associated with increased cardiovascular risk and mortality without low HDL-cholesterol) on the structural integrity and functions of lipid-free and lipoprotein-associated apoA-I in an effort to explain the phenotypes of subjects carrying these mutations. All three mutants, in lipid-free form, presented structural and thermodynamic aberrations, with apoA-I[L178P] presenting the greatest thermodynamic destabilization. Additionally, apoA-I[L178P] displayed reduced ABCA1-mediated cholesterol efflux capacity. When in reconstituted HDL (rHDL), apoA-I[L144R] and apoA-I[L178P] were more thermodynamically destabilized compared to wild-type apoA-I, both displayed reduced SR-BI-mediated cholesterol efflux capacity and apoA-I[L144R] showed severe LCAT activation defect. ApoA-I[A164S] was thermodynamically unaffected when in rHDL, but exhibited a series of functional defects. Specifically, it had reduced ABCG1-mediated cholesterol and 7-ketocholesterol efflux capacity, failed to reduce ROS formation in endothelial cells and had reduced capacity to induce endothelial cell migration. Mechanistically, the latter was due to decreased capacity of rHDL-apoA-I[A164S] to activate Akt kinase possibly by interacting with endothelial LOX-1 receptor. The impaired capacity of rHDL-apoA-I[A164S] to preserve endothelial function may be related to the increased cardiovascular risk for this mutation. Overall, our structure–function analysis of L144R, A164S and L178P apoA-I mutants provides insights on how HDL-cholesterol levels and/or atheroprotective properties of apoA-I/HDL are impaired in carriers of these mutations.

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Abbreviations

ABCA1:

ATP-binding cassette A1

ABCG1:

ATP-binding cassette G1

ANS:

8-anilino-1-naphthalenesulfonic acid

apoA-I:

Apolipoprotein A-I

C:

Cholesterol

CD:

Circular dichroism

cpt-cAMP:

8-(4-Chlorophenylthio)-cAMP

CVD:

Cardiovascular disease

DCF:

2′,7′-Dichlorofluorescein

DCFH-DA:

2′,7′-Dichlorodihydrofluorescein diacetate

DPBS:

Dulbecco’s phosphate-buffered saline

GdnHCl:

Guanidine hydrochloride

HCAEC:

Human coronary artery endothelial cells

HDL:

High-density lipoprotein

HDL-C:

HDL-cholesterol

IHD:

Ischemic heart disease

IMT:

Intima–media thickness

LCAT:

Lecithin:cholesterol acyltransferase

LOX-1:

Lectin-type oxidized low-density lipoprotein receptor 1

MDA:

Malondialdehyde

Ni-NTA:

Ni2+-nitrilotriacetic acid

oxPAPC:

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine

POPC:

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

rHDL:

Reconstituted HDL

ROS:

Reactive oxygen species

SR-BI:

Scavenger receptor class B type I

TBARS:

Thiobarbituric acid reactive substances

Trx:

Thioredoxin

WT:

Wild type

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Acknowledgements

We thank Dr. Dimitris Kardassis (Medical School of the University of Crete, Greece) and members of his lab for providing valuable help with the endothelial cell migration assay. We acknowledge support of this work by the projects: (a) “An Open-Access Research Infrastructure of Chemical Biology and Target-Based Screening Technologies for Human and Animal Health, Agriculture and the Environment (OPENSCREEN-GR)” (MIS 5002691) which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund), (b) “The National Research Infrastructure on Integrated Structural Biology, Drug Screening Efforts and Drug target functional characterization (INSPIRED)” (MIS 5002550) which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund), (c) “A Greek Research Infrastructure for Visualizing and Monitoring Fundamental Biological Processes (BioImaging-GR)” (MIS 5002755) which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund) and (d) “CURE-PLaN” a grant from the Leducq Foundation for Cardiovascular Research.

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Authors and Affiliations

  1. Institute of Biosciences and Applications, National Center for Scientific Research “Demokritos”, Agia Paraskevi, 15341, Athens, Greece

    Christina Gkolfinopoulou, Faye Soukou, Ioannis Dafnis & Angeliki Chroni

  2. Laboratory of Organic Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, Panepistimioupolis Zografou, Athens, Greece

    Tahsin F. Kellici & Thomas Mavromoustakos

  3. 4th Department of Internal Medicine, Clinical Genomics and Pharmacogenomics Unit, ‘Attikon’ Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece

    Despina Sanoudou

  4. Molecular Biology Division, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

    Despina Sanoudou

  5. Center for New Biotechnologies and Precision Medicine, Medical School, National and Kapodistrian University of Athens, Athens, Greece

    Despina Sanoudou

  6. Protein Chemistry Laboratory, Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, National Center for Scientific Research “Demokritos”, Agia Paraskevi, Athens, Greece

    Efstratios Stratikos

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  1. Christina Gkolfinopoulou
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Gkolfinopoulou, C., Soukou, F., Dafnis, I. et al. Structure–function analysis of naturally occurring apolipoprotein A-I L144R, A164S and L178P mutants provides insight on their role on HDL levels and cardiovascular risk. Cell. Mol. Life Sci. 78, 1523–1544 (2021). https://doi.org/10.1007/s00018-020-03583-y

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