Structure-function and expression studies of mouse Cyp4f14 and Cyp4f39 and analysis of odor leakage from human remains containment units for use in space
Jayakody, Madushika Madri
MetadataShow full item record
The availability of both mouse and human genome sequences along with the fact that mouse is a good model organism to study mammals, especially humans, has prompted comparisons between the two species at multiple levels. Cytochrome P450 4F is a relatively new CYP subfamily that has gained a great deal of attention due to its involvement in inflammation and lung cancer. Mouse Cyp4f14 and Cyp4f39 show significant sequence similarity to human CYP4F2, CYP4F3 and CYP4F22 and are involved in limiting neuroinflammation and autosomal recessive congenital ichthyosis, respectively. Nevertheless, there are not many studies done on the structure and substrate binding by these enzymes. The first part of this thesis focuses on the mouse cytochrome P450 4F subfamily enzymes Cyp4f14 and Cyp4f39, their structure, substrate binding and expression by using a combination of experimental and computational chemistry approaches. Here we report initial expression studies of Cyp4f14 that were carried out by subcloning mouse Cyp4f14 (from Origene #MR208397) into pEx-N-His-GST vector and optimizing expression in E. coli as a function of OD600 at different isopropyl β-D-1-thiogalactopyranoside (IPTG) levels. Based on p-nitrocarboxylic acid (pNCA) oxidation assays and spectroscopy carried out with crude enzyme, induction at an OD600 of 0.6 with 0.5-0.8 mM of IPTG seems to be optimal for expression in the presence of cytochrome P450 reductase. Analysis of sequence alignments and preliminary work with homology models yielded insights into how mouse Cyp4f14 interacts with its substrate. Molecular docking studies revealed the possible binding sites and modes for substrates such as leukotriene B4, 8-pNCA, 11-pNCA and arachidonic acid. It is hoped that these results will provide insights into enzyme-substrate interactions and form a basis for future research. This study also reports a detailed structural analysis of mouse Cyp4f39 which is the ortholog of human CYP4F22. Our goal is to provide a strong basis to help future studies of these important enzymes. The second part of this thesis involves testing human remains containment units (HRCUs), or ‘body bags’. Volatile organic compounds emitted by decomposing cadavers were analyzed by polydimethylsiloxane/divinylbenzene solid phase microextraction (PDMS/DVB SPME) fibers and Gas Chromatography/Mass Spectrometry (GC/MS). These HRCUs will be used in the International Space Station (ISS) and potentially in colonies on the moon or Mars to store cadavers in the situation of a death (including return of the cadaver to Earth). The ability of bags containing non-refrigerated cadavers to contain volatiles was tested over time, with particular focus on volatiles causing human odor sensation. Different types of bags were found to have very different breakthrough times for odorous volatiles.