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The Molecular Interaction of Apolipoprotein A-I and Lecithin: Cholesterol Acyl Transferase

Cooke, Allison L, B.A.
http://orcid.org/0000-0002-5888-2611
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1543580636448357

Abstract Details

2018, PhD, University of Cincinnati, Medicine: Pathobiology and Molecular Medicine.
Background: Atherosclerosis is a multifactorial inflammatory disease that begins with the accumulation of lipid in arterial endothelium. Atherosclerosis is the leading cause of deaths attributable to cardiovascular disease in the United States. Epidemiological studies showing high-density lipoprotein (HDL) cholesterol is inversely correlated with atherosclerosis has made it a pharmacological target for preventing cardiovascular disease. However, outcomes from clinical trials have raised questions about HDL’s protective properties. Investigating the molecular interactions of apolipoprotein (apo)A-I, which accounts for approximately 70% of total HDL protein, can help translate HDL structure to cardioprotective function. Lecithin: cholesterol acyl transferase (LCAT) is a critical HDL-modifying protein that performs a key function in reverse cholesterol transport by using apoA-I as a cofactor to esterify cholesterol. Data from our lab and others demonstrate that apoA-I molecules dimerize into an antiparallel stacked ring-structure that encapsulates lipid in reconstituted (r)HDL. Cross-linking analysis of rHDL implies that apoA-I molecules exist in at least two distinct organizations: one with helix 5 of an apoA-I molecule adjacent to helix 5 of its antiparallel partner (5/5 helical registry), and the other in a 5/2 registry. We hypothesized that the orientation of apoA-I molecules on rHDL modulates LCAT activity. Objective: Identify the mechanism by which apoA-I activates LCAT to determine how HDL interacts with its immense proteome. Linking HDL structure and function will allow for therapeutic development that targets HDL-associated inflammatory diseases. Major Findings: 1) Antiparallel apoA-I molecules adopt a thumbwheel mechanism to generate a discontinuous epitope for LCAT activation. Site-directed cysteine mutagenesis was used to “lock” two apoA-I molecules into an antiparallel 5/5, 5/2, and 5/1 helical registry on rHDL. The 5/5 mutant demonstrated higher LCAT activity than wildtype (WT) rHDL, while the 5/2 and 5/1 mutants showed dramatically lower LCAT activity (p<0.001). Surface plasmon resonance (SPR) analysis revealed no differences in the binding affinity of LCAT to rHDL mutants versus WT. Chemical cross-linking of LCAT and WT rHDL resulted in identification of intermolecular interaction sites between K240 of LCAT and helices (H)4-H7 of apoA-I. We propose that LCAT binds to rHDL particles of comparable size and lipid composition, but is activated by a discontinuous epitope comprised of two antiparallel apoA-I molecules operating in a thumbwheel mechanism on rHDL. Altering the 5/5 registry disrupts this epitope and prevents LCAT activation. These studies provide a basis for understanding how apoA-I structure may modulate the association and activity of HDL associated-proteins. 2) Helix-helix cooperation in antiparallel apoA-I molecules is essential for LCAT activation. Point-mutations in H3 and H4 of apoA-I inhibited LCAT activation to the same extent as point-mutations in H6 and H7 (p<0.001). Potential cholesterol binding motifs were identified in members of the apolipoprotein family (apoA-I, apoA-VI, and apoE); mutating these residues in apoA-I significantly decreases LCAT activation. Cooperation between H3 and H4 on one apoA-I molecule and H6 and H7 on its anti-parallel partner is essential for LCAT activation. Further, cholesterol binding motifs may provide a recurrent mechanism for cholesterol trafficking and interaction in the HDL proteome.
William Sean Davidson, Ph.D. (Committee Chair)
Christopher A. Crutchfield, Ph.D. (Committee Member)
Philip Howles, Ph.D. (Committee Member)
Francis McCormack, M.D. (Committee Member)
Thomas Thompson, Ph.D. (Committee Member)
Laura Woollett, Ph.D. (Committee Member)
217 p.
Pathology
apolipoprotein A-I; LCAT; high density lipoprotein; subspecies; cholesterol; atherosclerosis

Recommended Citations

Citations

  • Cooke, A. L. (2018). The Molecular Interaction of Apolipoprotein A-I and Lecithin: Cholesterol Acyl Transferase [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1543580636448357

    APA Style (7th edition)

  • Cooke, Allison. The Molecular Interaction of Apolipoprotein A-I and Lecithin: Cholesterol Acyl Transferase. 2018. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1543580636448357.

    MLA Style (8th edition)

  • Cooke, Allison. "The Molecular Interaction of Apolipoprotein A-I and Lecithin: Cholesterol Acyl Transferase." Doctoral dissertation, University of Cincinnati, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1543580636448357

    Chicago Manual of Style (17th edition)

ucin1543580636448357
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This open access ETD is published by University of Cincinnati and OhioLINK.