Characterization of apolipoprotein A-I functional domains.

dc.contributor.advisorMarcel, Y.,
dc.contributor.authorFrank, Philippe Guy.
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 60-08, Section: B, page: 3739.
dc.description.abstractThe plasma concentration of high-density lipoprotein (HDL) is inversely correlated to the development of coronary heart disease. This inverse relationship has been attributed to the role that HDL and its major constituent, apolipoprotein A-I (apoA-I), play in reverse cholesterol transport. This pathway allows HDL to acquire excess cellular cholesterol from peripheral cells and mediate its transport to the liver for degradation and removal from the body. ApoA-I has three major functions in this process. In order to identify the different domains of human apoA-I involved in these functions, a bacterial expression system was designed. This expression system was used to express apoA-I mutants and to identify the specific domains responsible of its functions. Three apoA-I mutants, corresponding to deletions of apoA-I residues 100--143, 122--165, 144--186, have been characterized for their ability to bind phospholipids; and to form reconstituted apoA-I-containing lipoproteins, as well as for their structural and physical properties. The results show that the amphipathic alpha-helices within residues 100--186 are directly involved in the interactions with phospholipids and determine the lipid binding capacity of the protein. We have also examined the contribution of these amphipathic alpha-helices to the ability of apoA-I to promote cholesterol efflux from human skin fibroblasts. All central deletion mutants in reconstituted HDL could acquire cellular cholesterol from normal fibroblasts with similar affinity and binding capacity for cholesterol. The contribution of amphipathic: alpha-helices (central and C-terminal helices) to the ability of lipid-free apoA-I to promote cellular cholesterol efflux was examined with cholesterol-loaded fibroblasts and macrophages. ApoA-I interaction and cholesterol efflux from cells such as fibroblasts, which accumulate moderate amounts of cholesterol, do not require any specific sequence between residues 100 and 243. We have examined the ability of the central domain deletion mutants in reconstituted lipoproteins to activate the reaction mediated by LCAT. Deletion of residues 122--165 or 144--186 results in an inability of the mutant apoproteins to promote cholesterol esterification. This study and others suggest that apoA-I contains three functionally distinct domains: the N-terminal domain plays an important role in the stabilization of the lipid-free protein; the C-terminal domain is involved in the initial binding of lipids; the central domain mediates the maturation of high density lipoproteins. (Abstract shortened by UMI.)
dc.format.extent219 p.
dc.publisherUniversity of Ottawa (Canada)
dc.subject.classificationBiology, Molecular.
dc.titleCharacterization of apolipoprotein A-I functional domains.
CollectionTh├Ęses, 1910 - 2010 // Theses, 1910 - 2010

NQ38784.PDF8.7 MBAdobe PDFOpen