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| Identifier |
000358521 |
| Title |
Functional characterization of naturally occuring APOA-I mutations found in the Copenhagen city heart study : a substitution L144R in APOA-I results in abnormal HDL phenotype that can be corrected with treatment with LCAT |
| Alternative Title |
Χαρακτηρισμός δύο φυσικών μεταλλάξεων της ΑΠΟΑ-I που εντοπίστηκαν κατά την επιδημιολογική μελέτη του πληθυσμού της Κοπενχάγης : |
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Author
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Κατεϊφίδης, Ανδρέας
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Thesis advisor
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Ζαννής, Βασίλειος
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| Abstract |
ApoA-I is an essential protein for the formation and the physiological functions of HDL particles. Epidemiological and genetic data have shown that low or high levels of HDL or apoA-I are associated with increased or decreased risk of developing atherosclerosis, respectively, indicating the importance of apoA-I in cellular cholesterol homeostasis. Two mutants were identified by screening the population of the Copenhagen City Heart Study (CCHS). I have generated recombinant adenoviruses expressing these two naturally occurring apoA-I mutants, apoA-I(L144R) and apoA-I(A164S). Cell culture studies showed that when HTB-13 cells were infected with adenoviruses expressing the wild type or the mutant apoA-I forms in all cases apoA-I was secreted efficiently in to the culture medium. Adenovirus mediated gene transfer in apoA-I-deficient mice showed that administration of the adenovirus expressing ApoA-I(L144R) resulted in low total cholesterol levels and decreased cholesterol esters to total cholesterol ratio (CE/TC) as compared to mice that received the adenovirus expressing the wild type apo A-I or the apoA-I(A164S). The difference in total cholesterol and CE/TC ratio between WT and apoA-I(L144R) persisted even when the level of expression of the mutant, as determined by the steady state hepatic apoA-I mRNA levels, were increased. FPLC fractionation of plasma showed that the HDL cholesterol peak of mice expressing the apoA-I(L144R) was very small and resembled the HDL fraction cholesterol peak of mice expressing the control protein (GFP). Fractionation of plasma by density gradient ultracentrifugation
showed that apoA-I in mice expressing apoA-I(L144R) was greatly reduced and was distributed mainly in the HDL-3 region as compared to mice expressing the wild type apoA-I or apoA-I(A164S). Electron microscopy of the HDL fraction showed that apoA-I(L144R) promoted the formation of few discoidal particles as well as small size particles similar to those observed in mice expressing the control protein (GFP), in contrast wild type apoA-I and apoA-I(A164S) promoted the formation of spherical particles. Two-dimensional gel electrophoresis showed that the mutant apoA-I(L144R) promoted the formation of preβ- and α4-HDL particles, whereas WT apoA-I and the mutant apoA-I(A164S) promoted the formation of normal preβ and α HDL subpopulations of different sizes (α1, α2, α3 and α4). Simultaneous treatment of the mice with the virus expressing the apoA-I(L144R) and human LCAT normalized the total cholesterol level and the CE/TC ratio. The LCAT treatment restored the HDL cholesterol peak, promoted the formation of spherical HDL and restored normal preβ- and α-HDL subpopulations. The findings establish that apoA-I(L144R) has an aberrant and apoA-I(A164S) has a normal HDL phenotype. The present study also suggest that the low HDL levels of mice expressing the apoA-I(L144R) results from LCAT insufficiency that inhibits the efficient conversion of the lipidated apoA-I to discoidal and subsequently spherical HDL particles. The correction of the aberrant HDL phenotypes by treatment with LCAT suggest a potential therapeutic intervention for HDL abnormalities that result from specific mutation in apoA-I.
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| Physical description |
107 σ. : πιν. ; 30 εκ. |
| Language |
Greek, English |
| Subject |
Apolipoproteins genetics |
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Απολιποπρωτεϊνες Γενετική |
| Issue date |
2008-04-03 |
| 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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| Notes |
Πρόγραμμα μεταπτυχιακών σπουδών: "Κυτταρική και γενετική αιτιολογία, διαγνωστική και θεραπευτική των ασθενειών του ανθρώπου". |
| Views |
368 |
Functional characterization of naturally occuring APOA-I mutations found in the Copenhagen city heart study : a substitution L144R in APOA-I results in abnormal HDL phenotype that can be corrected with treatment with LCAT
