Doctoral theses
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| Identifier |
uch.biology.phd//2000liadaki |
| Title |
Σχέση Δομής - Λειτουργίας της Απολιποπρωτεΐνης Α-Ι του ανθρώπου |
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Author
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Λιαδάκη, Καλλιόπη Ν
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| Abstract |
Apolipoprotein A-I is the major protein constituent of the high density lipoprotein (HDL) particles and plays a crucial role in lipid transport and metabolism. The subject of this study was the structural and functional analysis of human apoA-I. The main focus was to understand how specific domains and residues of apoA-I contribute to its physiological functions, that is its ability to bind to phospholipids and form high density lipoprotein (ΗDL) particles, to activate lecithin: cholesterol acyltransferase (LCAT) and to bind to scavenger receptor, class B, type I (SR-BI). We mutated the human apoA-I gene and generated by transfection and selection stable cells lines expressing variant forms of apoA-I. The mutant proteins were purified from the culture media and were used in a variety of functional and structural assays. Dimyristoyl phosphatidyl choline (DMPC) binding assays showed that the carboxy-terminal region of apoA-I is critical for the interaction of apoA-I with multilamellar phospholipid vesicles. The deletion of regions in the carboxy-terminal domain (residues 167-243) (helices 7-10) inhibits the mutant proteins from solubilizing DMPC vesicles. In contrast the substitution of glycines at positions 185 and 186 with prolines did not affect the ability of the mutant protein to interact and solubilize phospholipid vesicles. LCAT analysis, using reconstituted HDL particles containing the mutant apoA-I forms, showed that the variants (Δ165-175) and (G185>P, G186>P) reduced LCAT activation (60% compared to the wild type apoA-I). Mutants (Δ187-197), (Δ185-243), (Δ198-243), (Δ220-243) and (Δ232-243) reduced even more the activation to 37-48 % of the wild type apoA-I. These findings indicate that helix 7 and the carboxy-terminal helices 8-10 are important for LCAT activation. Cross-linking experiments showed that the carboxy-terminal domain of apoA-I participates in the self association of the protein in solution. The deletion of carboxy-terminal domains of the protein results in formation of lower quantities of dimers, and inhibits the formation of higher order oligomers (aggregates) in solution. Pulse chase experiments showed a delay in the kinetics of secretion of the mutant forms (Δ165-243) and (Δ175-243), as demonstrated by the double increase in the half time of the intracellular forms of the mutant proteins compared to that of the wild type apoA-I. The deletion of the carboxy terminal amino acids 165-243 or 175-243 probably destabilizes the protein inside the cell and delays its secretion. In the context of studying the binding of apoA-I to the HDL receptor SR-BI, a new technique, called immunoreceptor assay, was developed and was used as a simple and powerful alternative to evaluate receptor-binding parameters of a variety of apoA-I-containing ligands, without the need for iodination. In this assay the unlabeled receptor-associated ligand is detected by quantitative immunoblotting. The analysis of different ligands showed that discoidal reconstituted HDL particles containing apoA-I bind with high affinity to SR-BI, while the lipid-free apoA-I and the pre-β-1 HDL do not exhibit SR-BI-dependent binding. Moreover the increase in the density of spherical HDL resulted in decrease in the binding affinity of HDL to SR-BI. Studies of the binding of reconstituted HDL particles containing the variant apoA-I froms showed that optimal binding of discoidal particles to SR-BI can be achieved by the central domain of apoA-I (residues 60-184) combined with either the amino or carboxy terminal domain. These findings demonstrate the important role of apoA-I in HDL binding to SR-BI and indicate that the amino (1-59) and carboxy (185-243) termini of apoA-I independently influence binding to SR-BI, either directly or through changes in the conformation of the core of apoA-I (residues 60-184). In addition to their use in the present study, the variant apoA-I forms generated will be valuable in the future for studies of selective uptake of cholesterol, mediated by SR-BI, for promoting cholesterol efflux from cells, for binding to other cell surface receptors, and for in vivo studies in transgenic animals.
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| Language |
Greek |
| Issue date |
2000-11-02 |
| Collection
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School/Department--School of Sciences and Engineering--Department of Biology--Doctoral theses
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Type of Work--Doctoral theses
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| Permanent Link |
https://elocus.lib.uoc.gr//dlib/8/d/c/metadata-dlib-2000liadaki.tkl
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| Views |
269 |
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