Distant Homology Modeling of LCAT and Its Validation through <i>In Silico</i> Targeting and <i>In Vitro</i> and <i>In Vivo</i> Assays

<div><p>LCAT (lecithin:cholesterol acyltransferase) catalyzes the transacylation of a fatty acid of lecithin to cholesterol, generating a cholesteryl ester and lysolecithin. The knowledge of LCAT atomic structure and the identification of the amino acids relevant in controlling its structure and function are expected to be very helpful to understand the enzyme catalytic mechanism, as involved in HDL cholesterol metabolism. However - after an early report in the late ‘90 s - no recent advance has been made about LCAT three-dimensional structure. In this paper, we propose an LCAT atomistic model, built following the most up-to-date molecular modeling approaches, and exploiting newly solved crystallographic structures. LCAT shows the typical folding of the α/β hydrolase superfamily, and its topology is characterized by a combination of α-helices covering a central 7-strand β-sheet. LCAT presents a Ser/Asp/His catalytic triad with a peculiar geometry, which is shared with such other enzyme classes as lipases, proteases and esterases. Our proposed model was validated through different approaches. We evaluated the impact on LCAT structure of some point mutations close to the enzyme active site (Lys218Asn, Thr274Ala, Thr274Ile) and explained, at a molecular level, their phenotypic effects. Furthermore, we devised some LCAT modulators either designed through a de novo strategy or identified through a virtual high-throughput screening pipeline. The tested compounds were proven to be potent inhibitors of the enzyme activity.</p></div

<i>In vitro</i> inhibitory assays on LCAT of the heptadecylcholesteryl (R, S) phosphonyl chloridate and the two top-scoring compounds.

<p><i>In vitro</i> inhibitory assays on LCAT of the heptadecylcholesteryl (R, S) phosphonyl chloridate and the two top-scoring compounds.</p

Stability score of WT and T274 mutant LCAT.

<p>*Stability is the absolute thermostability of the mutation and, for the generated ensemble, it is equal to the Boltzmann average of the stabilities of the ensemble.</p><p>**dStability is the relative thermostability of the mutation in comparison with the wild-type protein, and it is equal to the Boltzmann average of the relative stabilities of the ensemble.</p

Superposition of the top 10 conformations obtained by LowMode MD for wild-type LCAT and T274[A/I] mutants.

<p>Protein backbone is rendered in ribbons, whereas Phe103, Ser181 and Thr274[Ala/Ile] side chains are rendered as sticks. Color code: wild-type LCAT = orange, T274A = blue, T274I = green.</p

Mass spectrometry data of human recombinant LCAT and of the covalent adduct between LCAT and its irreversible inhibitor.

<p>*Molecular weight calculated from the aminoacid sequence. An increase of 643 Da is expected in the presence of one molecule of inhibitor bound to the protein. The higher MW experimentally observed for LCAT in comparison with the value calculated from the sequence can be ascribed to the glycosylation of the protein.</p

Molecular docking results: a) lowest energy pose in LCAT binding site for heptadecylcholesteryl R phosphonyl chloridate b) compound #1 and c) compound #2 and its chemical structures.

<p>The surface of the protein binding site is colored according to lipophilicity (hydrophilic area in blue in grey, lipophilic in gold and neutral in white).</p

Effects of HDL isolated from carriers of CETP mutations and controls on eNOS activation in HUVEC.

<p>Cells were incubated for 10-sex matches controls (n = 8), at the concentration of 1.0 mg of protein/ml. Western blot analysis of the phosphorylated and total forms of eNOS was performed, and the phosphorylated/total eNOS ratios were calculated by densitometric analysis and expressed as fold of increase in treated vs. untreated cells. Data points for each study participant are shown. cells. Results are mean±SEM of 3 separate experiments performed with 1 preparation of homozygote HDL2, 3 preparations of control HDL2, and 3 batches of cells. *<i>P</i><0.05 vs. untreated homozygote HDL2.</p

GGE analysis of purified HDL2 and HDL3.

<p>HDL fractions isolated from the homozygote, and a representative heterozygote and control were analyzed by GGE.</p

2D electrophoresis analysis of purified HDL.

<p>HDL isolated from the homozygote, and a representative heterozygote and control were separated by 2D electrophoresis and immunodetected with anti apoA-I and anti apoE antibodies.</p

Effects of HDL isolated from carriers of CETP mutations and controls on VCAM-1 expression in TNFα-stimulated HUVEC.

<p>Cells were incubated overnight with HDL, HDL2, or HDL3 isolated from 7 heterozygous carriers of CETP mutations and age-sex matched controls (n = 8), at the concentration of 1.0 mg of protein/ml, before stimulation with TNFα for 8 hours. Results are expressed as percentage of VCAM-1 concentration in conditioned media of untreated TNFα-stimulated cells. Data points for each study participant are shown.</p