Post-graduate theses
Current Record: 838 of 1236
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| Identifier |
000381980 |
| Title |
The role of amino acids 218-222 of apolipoprotein A-I in the biogenesis of HDL |
| Alternative Title |
Ο ρόλος των αμινοξέων 218-222 της απολιποπρωτεϊνης Α-Ι στη βιογέννεση της HDL |
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Author
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Beck, Melissa Ashley
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Thesis advisor
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Ζαννής, Β.
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Reviewer
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Καρδάσης, Δ.
Παπακωνσταντή Ε.
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| Abstract |
Apolipoprotein A-I (apoA-I) is the predominant protein component of the high density
lipoprotein (HDL) particle, and deficiency of apoA-I prevents the formation of HDL. ApoA-I
activates the enzyme lecithin:cholesterol acyltransferase (LCAT), which is necessary for the
esterification of cholesterol in HDL, a process associated with the conversion of nascent discoidal
to mature spherical HDL particles. Previous studies showed that the hydrophobic amino acids in the
220-231 region of apoA-I are required for cholesterol efflux in vitro, and amino acid substitution of
apoA-I within the 220-231 region diminishes the ability of the mutant protein to create mature
spherical HDL particles in vivo.
Adenovirus-mediated gene transfer of WT apoA-I, mutant apoAI[
L218A/L219A/V221A/L222A], or mutant apoA-I plus LCAT in apoA-I-/- x apoE-/- double
deficient mice was used to elucidate the role of hydrophobic residues in the 218-222 region of
apoA-I in the biogenesis of HDL. Plasma obtained four days post gene transfer was analyzed by
various assays to monitor the formation and maturation of HDL. Fast protein liquid
chromatography (FPLC) analysis of plasma showed that in mice expressing the mutant protein, the
HDL peak was greatly diminished as compared to the mice expressing the WT apoA-I. Density
gradient ultracentrifugation showed that, compared to WT apoA-I, expression of the mutant protein
was associated with low levels of apoA-I that floated mainly in the HDL3 region. Two dimensional
gel electrophoresis of plasma and electromicroscopy of the HDL fraction showed that mice
expressing the mutant protein generated pre-β HDL particles and a small number of discoidal HDL
particles. In contrast, mice expressing WT apoA-I generated predominantly α1, α2, and α3 spherical
HDL particles. Mice co-expressing the apoA-I mutant and LCAT generated a pronounced
cholesterol shoulder in the VLDL/IDL/LDL region, shifted the apoA-I toward the lower densities,
and promoted the formation of small sized HDL particles.
The findings suggest that substitution of the hydrophobic residues in the 218-222 region of
the apoA-I by alanine disrupts the biogenesis of HDL. The disruption appears to result from
diminished interactions of the mutant apoA-I with ABCA1, combined with inefficient conversion
of nascent discoidal to mature spherical HDL particles. The observed phenotype generated by the
apoA-I[L218A/L219A/V221A/L222A] mutant can be partially restored when the apoA-I mutant is
co-expressed with LCAT.
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| Language |
English |
| Subject |
APOA-I |
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Adenovirus -mediated gene transfer |
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HDL Biogenesis |
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HDL Βιογένεση |
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LCAT |
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Απολιποπρωτίνη Α-Ι |
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Γονιδιακή μεταφορά μέσω αδενοιών |
| Issue date |
2012-07-24 |
| Collection
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School/Department--School of Medicine--Department of Medicine--Post-graduate theses
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Type of Work--Post-graduate theses
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| Permanent Link |
https://elocus.lib.uoc.gr//dlib/7/d/6/metadata-dlib-1390204317-423906-28034.tkl
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| Views |
147 |
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