Exploration of the function of a non-prolyl cis-peptide bond in human plasma platelet-activating factor acetylhydrolase
| Author(s) | Xie, Shilin | |
| Date Accessioned | 2018-02-06T13:31:53Z | |
| Date Available | 2018-02-06T13:31:53Z | |
| Publication Date | 2017 | |
| SWORD Update | 2017-11-10T14:19:38Z | |
| Abstract | Plasma platelet-activating factor acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A2, is an extracellular 45 KDa monomer that circulated in plasma and associates with lipoproteins. As a group-VIIA PLA2 enzyme, it catalyzes the hydrolysis of the acetyl group at the sn-2 position of platelet-activating factor (PAF) to form the products lyso-PAF and acetate. Additionally, PAF-AH can hydrolase a wide range of substrates with a short acetyl chain at the sn-2 position like oxidized phospholipids from LDL oxidation. In order to keep PAF-AH constitutively active without undermining cellular integrity, the enzyme only targets oxidatively fragmented phospholipids. A majority of PAF-AH is bound to low-density lipoprotein (LDL), which might be correlated with inflammatory atherogenesis. Studies show that an increase of PAF-AH concentration or activity could possibly lead to an increase in risk of atherogenesis, which plays a significant role in heart diseases. Many reversible PAF-AH inhibitors were developed in the pharmaceutical industry for the use as an extra treatment for patients with heart diseases. ☐ According to the solved structure via x-ray crystallization, there is a rare non-prolyl cis-peptide bond located between Phe-72 and Asp-73, which is far away from the active site catalytic triad formed by Ser-273, Asp-296 and His-351. Due to the high energy barrier of cis-trans isomerization, this conformational change is thought to be the rate-limit step in the protein folding process. Therefore, the existence of a cis-peptide bond usually is thought to play a great role in protein function or enhance structural stability in some way. The purpose of the existence of this non-prolyl cis-peptide in PAF-AH is still unsolved. Based on the previous work of plasma PAF-AH’s cis-peptide bond, a hypothesis was formed that this non-prolyl cis-peptide bond plays a role in maintaining the binding between LDL and PAF-AH. ☐ Work presented in this thesis aims to test this hypothesis. An E. coli expression construct of a truncated form of PAF-AH (ΔPAF-AH) was obtained from former lab member from the Bahnson lab. In an effort to explore the function role of a cis-peptide bond at this position, two site-directed mutants (D73G and D73P) were created at the position of this cis-peptide bond. D73G mutant aims to mimic the mouse PAF-AH that is reported only bound to high density lipoprotein (HDL) and D73P mutant aims to mimic the common prolyl cis-peptide bond. Expression and purification of ΔPAF-AH, as well as cis-peptide bond mutants, were performed using E. coli expression construct. Enzyme activity assays using a general substrate were carried out and proved that there were no functional d ifferences between wild type and cis-peptide bond mutants, indicating the cis-peptide bond does not play a role in enzyme activity. Moreover, there are no significant differences of circular dichroism (CD) secondary structure test between wild type and mutants, eliminating the possibility that this cis-peptide bond may have a influence on protein folding. Through CD thermodynamics test, there was a significant decrease of stability of D73G mutant while D73P mutant only show a little decline of stability. Possible explanation is that D73G might have changed this position to a trans configuration, which leads to the instability of protein. In order to test this explanation, high-purity mutant protein samples were produced for further crystallization trials. | en_US |
| Advisor | Bahnson, Brian J. | |
| Degree | M.S. | |
| Department | University of Delaware, Department of Chemistry and Biochemistry | |
| Unique Identifier | 1021882766 | |
| URL | http://udspace.udel.edu/handle/19716/22657 | |
| Language | en | |
| Publisher | University of Delaware | en_US |
| URI | https://search.proquest.com/docview/1972023406?accountid=10457 | |
| Keywords | Pure sciences | |
| Keywords | CD thermodynamics | |
| Keywords | Cis-peptide bond | |
| Keywords | PAF-AH | |
| Title | Exploration of the function of a non-prolyl cis-peptide bond in human plasma platelet-activating factor acetylhydrolase | en_US |
| Type | Thesis | en_US |