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1.
J Lipid Res ; 54(12): 3293-302, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24123812

ABSTRACT

We studied the significance of four hydrophobic residues within the 225-230 region of apoA-I on its structure and functions and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of an apoA-I[F225A/V227A/F229A/L230A] mutant in apoA-I⁻/⁻ mice decreased plasma cholesterol, HDL cholesterol, and apoA-I levels. When expressed in apoA-I⁻/⁻ × apoE⁻/⁻ mice, approximately 40% of the mutant apoA-I as well as mouse apoA-IV and apoB-48 appeared in the VLDL/IDL/LDL. In both mouse models, the apoA-I mutant generated small spherical particles of pre-ß- and α4-HDL mobility. Coexpression of the apoA-I mutant and LCAT increased and shifted the-HDL cholesterol peak toward lower densities, created normal αHDL subpopulations, and generated spherical-HDL particles. Biophysical analyses suggested that the apoA-I[225-230] mutations led to a more compact folding that may limit the conformational flexibility of the protein. The mutations also reduced the ability of apoA-I to promote ABCA1-mediated cholesterol efflux and to activate LCAT to 31% and 66%, respectively, of the WT control. Overall, the apoA-I[225-230] mutations inhibited the biogenesis of-HDL and led to the accumulation of immature pre-ß- and α4-HDL particles, a phenotype that could be corrected by administration of LCAT.


Subject(s)
Apolipoprotein A-I/chemistry , Apolipoprotein A-I/metabolism , Hydrophobic and Hydrophilic Interactions , Lipoproteins, HDL/biosynthesis , Adenoviridae/genetics , Animals , Apolipoprotein A-I/blood , Apolipoprotein A-I/genetics , Chemical Phenomena , HEK293 Cells , Humans , Mice , Mutation
2.
J Lipid Res ; 54(12): 3281-92, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23990662

ABSTRACT

We investigated the significance of hydrophobic and charged residues 218-226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I⁻/⁻ mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-ß- and α4-HDL particles. In apoA-I⁻/⁻ × apoE⁻/⁻ mice, the same mutant formed few discoidal and pre-ß-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I⁻/⁻ mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218-222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218-222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-ß particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype.


Subject(s)
Apolipoprotein A-I/chemistry , Apolipoprotein A-I/metabolism , Hydrophobic and Hydrophilic Interactions , Lipoproteins, HDL/biosynthesis , Adenoviridae/genetics , Animals , Apolipoprotein A-I/blood , Apolipoprotein A-I/genetics , Cell Line , Humans , Lipoproteins, HDL/blood , Mice , Mutation , Protein Structure, Secondary , Protein Unfolding , Temperature , Transgenes/genetics
3.
J Lipid Res ; 52(7): 1363-72, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21504968

ABSTRACT

In this study, we investigated the role of positively and negatively charged amino acids within the 89-99 region of apolipoprotein A-I (apoA-I), which are highly conserved in mammals, on plasma lipid homeostasis and the biogenesis of HDL. We previously showed that deletion of the 89-99 region of apoA-I increased plasma cholesterol and phospholipids, but it did not affect plasma triglycerides. Functional studies using adenovirus-mediated gene transfer of two apoA-I mutants in apoA-I-deficient mice showed that apoA-I[D89A/E91A/E92A] increased plasma cholesterol and caused severe hypertriglyceridemia. HDL levels were reduced, and approximately 40% of the apoA-I was distributed in VLDL/IDL. The HDL consisted of mostly spherical and a few discoidal particles and contained preß1 and α4-HDL subpopulations. The lipid, lipoprotein, and HDL profiles generated by the apoA-I[K94A/K96A] mutant were similar to those of wild-type (WT) apoA-I. Coexpression of apoA-I[D89A/E91A/E92A] and human lipoprotein lipase abolished hypertriglyceridemia, restored in part the α1,2,3,4 HDL subpopulations, and redistributed apoA-I in the HDL2/HDL3 regions, but it did not prevent the formation of discoidal HDL particles. Physicochemical studies showed that the apoA-I[D89A/E91A/E92A] mutant had reduced α-helical content and effective enthalpy of thermal denaturation, increased exposure of hydrophobic surfaces, and increased affinity for triglyceride-rich emulsions. We conclude that residues D89, E91, and E92 of apoA-I are important for plasma cholesterol and triglyceride homeostasis as well as for the maturation of HDL.


Subject(s)
Apolipoprotein A-I/chemistry , Apolipoprotein A-I/metabolism , Cholesterol/metabolism , Homeostasis , Lipoproteins, HDL/biosynthesis , Triglycerides/metabolism , Anilino Naphthalenesulfonates/metabolism , Animals , Apolipoprotein A-I/genetics , Cholesterol/blood , Conserved Sequence , Dimyristoylphosphatidylcholine/metabolism , Emulsions , Gene Expression Regulation , Homeostasis/genetics , Humans , Kinetics , Lipoproteins, HDL/metabolism , Male , Mice , Mice, Inbred C57BL , Mutagenesis , Mutation , Protein Stability , Protein Structure, Secondary , Protein Structure, Tertiary , Temperature , Triglycerides/blood
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