Apolipoprotein A-IV Structural Models and Functional Implications
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University of Cincinnati / OhioLINK
2008
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ndltd-OhioLink-oai-etd.ohiolink.edu-ucin12188260622021-08-03T06:12:52Z Apolipoprotein A-IV Structural Models and Functional Implications TUBB, MATTHEW ROBERT Biochemistry apolipoprotein cholesterol mass spectrometry cross-linking protein structure amphipathic Human apolipoprotein (apo)A-IV is a 46 kDa exchangeable plasma protein produced mainly in the small intestine in response to lipid meals with numerous proposed functions ranging from forward and reverse cholesterol transport to anti-inflammation, anti-oxidation, and satiation. Unfortunately, there is very little structural basis for these functions largely due to the lack of a three-dimensional model of the protein. The following aims were constructed in order to answer specific questions about apoA-IV structure and function.AIM 1: <i>Determine the specific sequences in apoA-IV that modulate its ability to bind to lipid</i>. We have used site-directed mutagenesis to introduce point mutations into apoA-IV in order to discover what regions influence lipid binding. Once these regions were identified we performed experiments to conclusively demonstrate that the two regions, distant in terms of the primary sequence, were quite near each other in the native lipid-free conformation. We propose that there is a direct intramolecular interaction within apoA-IV that inhibits its inherent lipid binding ability.AIM 2: <i>Create a three dimensional model of apoA-IV, both lipid-free and lipid-bound, using mass spectrometry and computer modeling</i>. Since both lipid-free and lipid-bound apoA-IV likely play distinct physiological roles, we set out to develop models of apoA-IV structure in both states. First, we developed a method to produce highly homogeneous apoA-IV recombinant HDL particles. We then used cross-linking chemistry and high resolution mass spectrometry to create "proximity maps" of lipid-free and lipid-bound apoA-IV. This was followed by sequence threading and computer modeling to generate the first ever all-atom molecular models of apoA-IV.AIM 3: <i>Identify an apoA-IV binding protein or receptor</i>. A novel apoA-IV receptor has yet to be identified and, although many groups have attempted, discovering it would provide tremendous information about the way in which apoA-IV is able to carry out its diverse functions <i>in vivo</i>. We employed various experimental methods to attempt to identify a novel protein partner for apoA-IV. Among the techniques used were affinity chromatography, far western blotting, yeast two hybrid and label transfer. Certain results remain promising, yet identification of a specific protein has eluded us. 2008-09-26 English text University of Cincinnati / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ucin1218826062 http://rave.ohiolink.edu/etdc/view?acc_num=ucin1218826062 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |
| collection |
NDLTD |
| language |
English |
| sources |
NDLTD |
| topic |
Biochemistry apolipoprotein cholesterol mass spectrometry cross-linking protein structure amphipathic |
| spellingShingle |
Biochemistry apolipoprotein cholesterol mass spectrometry cross-linking protein structure amphipathic TUBB, MATTHEW ROBERT Apolipoprotein A-IV Structural Models and Functional Implications |
| author |
TUBB, MATTHEW ROBERT |
| author_facet |
TUBB, MATTHEW ROBERT |
| author_sort |
TUBB, MATTHEW ROBERT |
| title |
Apolipoprotein A-IV Structural Models and Functional Implications |
| title_short |
Apolipoprotein A-IV Structural Models and Functional Implications |
| title_full |
Apolipoprotein A-IV Structural Models and Functional Implications |
| title_fullStr |
Apolipoprotein A-IV Structural Models and Functional Implications |
| title_full_unstemmed |
Apolipoprotein A-IV Structural Models and Functional Implications |
| title_sort |
apolipoprotein a-iv structural models and functional implications |
| publisher |
University of Cincinnati / OhioLINK |
| publishDate |
2008 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1218826062 |
| work_keys_str_mv |
AT tubbmatthewrobert apolipoproteinaivstructuralmodelsandfunctionalimplications |
| _version_ |
1719432867542466560 |