| Abstract |
In order to investigate how structural mutations in apoA-I affect the biogenesis and the
plasma levels of high density lipoprotein (HDL) and explain the etiology of genetically
determined low levels of HDL, I have generated recombinant adenoviruses expressing
naturally occurring and bioengineered mutations of apolipoprotein A-I and studied their
properties in vitro and in vivo.
The naturally occurring mutants apoA-I(Leu141Arg)Pisa, apoA-I(Leu159Arg)FIN, apoAI(
Arg151Cys)Paris and apoA-I(Arg160Leu)Oslo as well as the bioengineered mutant apoAI(
Arg149Ala) were secreted efficiently from cells in culture. The capacity of the mutant
proteins to activate LCAT in vitro was greatly reduced to 0.41%, 2.07%, 28.4%, 2.7%
and 5.2% of that of the WT apoΑ-Ι, respectively, in vitro and their ability to promote
ABCA1-mediated cholesterol efflux from macorphages was similar to that of the WT
apoA-I protein.
Gene transfer of the mutants in apoA-I deficient (apoA-I-/-) mice generated aberrant HDL
phenotypes. Mice expressing the apoΑ-Ι(Leu141Arg)Pisa or apoΑ-Ι(Leu159Arg)FIN
mutants had almost 90% decrease in plasma HDL-cholesterol and apoA-I levels (85%
and 91% for apoΑ-Ι(Leu141Arg)Pisa, and 89% and 87% for apoΑ-Ι(Leu159Arg)FIN,
respectively), compared to those of mice expressing the WT αποΑ-Ι. apoA-/- mice
expressing apoA-I(Arg160Leu)Oslo, apoA-I(Arg149Ala) and apoA-I(Arg151Cys)Paris
mutants had a reduction in HDL levels by 78%, 59% and 61% while the apoA-I levels
were reduced by 68%, 64% and 55%, respectively, as compared to mice expressing the
WT apoA-I.
The cholesteryl ester to total cholesterol (CE/TC) ratio of HDL from mice expressing any
of the five mutants was significantly decreased and the apoA-I was distributed in the
HDL3 region. Similar experiments for the mutant apoA-I(Leu178Pro) showed no
difference in the phenotype produced by the mutant compared to the one produced by the
WT apoA-I.
The expression of mutants apoΑ-Ι(Leu141Arg)Pisa or apoΑ-Ι(Leu159Arg)FIN in apoA-I-/-
mice caused accumulation of preβ1-HDL and small size α4-HDL particles in the plasma
and generated only few spherical HDL particles, similar in number to those formed by
the control protein GFP. The mutants apoA-I(Arg160Leu)Oslo and apoA-I(Arg149Ala)
promoted the formation of preβ1 and mainly α4-HDL subpopulations and gave a mixture
of discoidal and spherical particles. Interestingly, apoA-I(Arg151Cys)Paris generated
subpopulations of different sizes that migrate between preβ and α-HDL and formed
mostly spherical and a few discoidal particles.
Simultaneous treatment of mice with adenoviruses expressing any of the five mutants and
human LCAT normalized plasma apoA-I, HDL cholesterol levels and the CE/TC ratio. It
also led to the formation of spherical HDL particles consisting mostly of α-HDL
subpopulations with slower electrophoretic mobility.
The study establishes that apoA-I(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN inhibit an
early step in the biogenesis of HDL due to inefficient esterification of the cholesterol of
the preβ1-HDL particles by the endogenous LCAT. On the other hand, the mutants apoAI(
Arg160Leu)Oslo, apoA-I(Arg149Ala) and apoA-I(Arg151Cys)Paris inhibit a later step in
the biogenesis of HDL due to inefficient esterification of the cholesterol of the discoidal
particles.
The correction of the aberrant HDL phenotypes by treatment with LCAT suggests a
potential therapeutic intervention for HDL abnormalities that result from specific
mutations in apoA-I.
Adenovirus-mediated gene transfer of C-terminal apoA-I mutants in apoA-I-/- mice,
showed that the plasma HDL levels were greatly reduced in mice expressing the carboxyterminal
deletion mutants apoA-I[Δ(185-243)] and apoA-I[Δ(220-243)], shown
previously to deminsh the ABCA1-mediated lipid efflux. The HDL levels were normal in
mice expressing the WT apoA-I, the apoA-I[Δ(232-243)] deletion mutant or the apoAI[
E191A/H193A/K195A] point mutant, which promote normal ABCA1-mediated lipid
efflux. EM and 2D gel electrophoresis analyses showed that the apoA-I[Δ(185-243)] and
apoA-I[Δ(220-243)] mutants formed mainly preβ-HDL particles and few spherical
particles enriched in apoE, while WT apoA-I, apoA-I[Δ(232-243)] and apoAI[
E191A/H193A/K195A] formed spherical α-HDL particles. The findings establish that
(a) deletions that eliminate the 220-231 region of apoA-I prevent the synthesis of α-HDL
but allow the synthesis of preβ-HDL particles in vivo, (b) the amino-terminal segment 1-
184 of apoA-I can promote synthesis of preβ-HDL-type particles in an ABCA1-
independent process, and (c) the charged residues in the 191-195 region of apoA-I do not
influence the biogenesis of HDL.
Finally, in order to elucidate the contribution of different transcription factors to the
regulation of apoΑ-Ι and apoCIII genes we used a series of recombinant adenoviruses
expressing nuclear receptors and members of the TGFβ signaling pathway. Preliminary
results indicated that human apoA-I gene expression is negatively regulated, in vivo, by
the TGFβ signaling pathway and specifically through SMAD3 protein.
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Σχέση δομής-λειτουργίας και μεταγραφική ρύθμιση της απολιποπρωτεϊνης Α-Ι του ανθρώπου
