| Abstract |
The protein components of lipoproteins are apolipoproteins. Αn important trait of most of the apolipoproteins is the presence of amphipathic α-helices in their secondary structure. The amphipathic α-helices contribute to the lipid-binding properties of the apolipoproteins. In addition to their lipid-binding functions, the apolipoproteins participate to the lipoprotein pathways due to several other characteristics they possess.
In order to investigate the different properties of the apolipoproteins, in vitro and in vivo studies should be performed. Apolipoprotein A-I is the major protein constituent of high density lipoproteins (ΗDL) and has been extensively studied in vitro and in vivo as it plays a crucial role in the HDL biogenesis pathway. ApoA-II and apoA-IV are also important protein constituents of HDL and also participate in the HDL biogenesis pathway. However, the physiological role and the functionality of these two apolipoproteins has not yet been completely understood and more in vivo studies should be designed and performed for this purpose.
In the first part of this study, we created two recombinant adenoviruses expressing the apolipoproteins A-II and A-IV respectively. These recombinant adenoviruses are going to be used for studying the properties of apoA-II and apoA-IV both in vitro and in vivo. We expect that these studies will enlighten us about the functions of these apolipoproteins.
In the second part of this study, we tried to create a form of apoA-I that could be biotinylated. This way HDL particles will be easily isolated from the blood during in vivo studies. In order to successfully biotinylate a secreted protein, apoA-I in this case, we created a form of apoA-I fused with the Bio-epitope and a secreted form of the biotin ligase BirA that successfully biotinylates proteins tagged to the Bio-epitope. The cotransfection of the secreted BirA construct along with the bio-apoA-I construct into HEK 293 T cells enabled the biotinylation of the secreted apolipoprotein A-I. In the future, recombinant adenoviruses expressing this particular form of apoA-I and the secreted form of BirA are going to be constructed. We expect that the infection of mice with these adenoviruses will lead to the formation of HDL particles that will contain biotinylated apoA-I.
This way, HDL particles will be easily isolated from the blood plasma of infected mice using high affinity chromatography and more especially Streptavidin-Sepharose beads as these beads bind biotin with very high affinity. This method will contribute to the extensive study of the structure and the composition of HDL particles that are formed when mutated forms of the A-I, A-II, A-IV and E apolipoproteins are involed in the HDL biogenesis pathway.
The results of multiple epidemiological studies show that the low HDL levels in blood plasma are considered as a strong independent risk factor for atherosclerotic vascular disease. The study of different proteins that are involed in the HDL biogenesis pathway will enlighten us about the relationship between the structure and the function of HDL and will hopefully lead us to the development of new strategies concerning the gene therapy of different dyslipidemias.
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Δημιουργία ανασυνδυασμένων αδενοϊών που εκφράζουν τις απολιποπρωτεiνες A-IV και A-II
