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Title:From macromolecular crowding to transcriptional regulation: Exploring the complexities of cytochrome P450s, cannabinoids, and inflammation
Author(s):Huff, Hannah Cates
Director of Research:Das, Aditi
Doctoral Committee Chair(s):Gennis, Robert B
Doctoral Committee Member(s):Leckband, Deborah E; Sinha, Saurabh; Sweedler, Jonathan V
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):cytochrome P450
macromolecular crowding
lipids
cannabinoids
inflammation
arachidonic acid
CYP2J2
CYP2D6
interleukin-6
Abstract:Cytochrome P450s are an extremely versatile group on enzymes capable of metabolizing fatty acids, eicosanoids, sterols, vitamins, and xenobiotics. Those that metabolize fatty acids are of particular interest since some of the eicosanoid products are anti-inflammatory and have been shown to interact with the endogenous cannabinoid system. The determination that eicosanoids also function as endocannabinoids has opened the door to comparisons with phytocannabinoids and questions about how the presence This work is aimed at untangling the complex web of P450 interactions with the environment and cannabinoids, the regulation of inflammation, and the intersection of these two topics. Described in Chapter 2 is the investigation of lipid composition and macromolecular crowding and their effects on the stability and protein-protein interactions of the Cytochrome P450 2J2 (CYP2J2) and its redox partner Cytochrome P450 Reductase (CPR). CYP2J2 is a membrane-bound epoxygenase expressed in cardiac tissue that is known to metabolize fatty acids as well as several xenobiotics. Previous studies on the effects of macromolecular crowding had all been performed on soluble enzymes, thus, we set out to determine the effects of macromolecular crowding on a membrane protein system. It was determined that crowding effects in a lipid-solubilized system are size-dependent and that crowding effects in nanodiscs depend on both size and crowding agent identity. Additionally, we thought to determine the effects of membrane lipids on protein activity as CYP2J2 is an integral membrane protein. By varying the percentage of anionic lipids, sphingolipids, and cholesterol, we were able to determine an optimal percentage range for anionic lipid composition. We were also able to determine that the presence of sphingolipids results in increased secondary metabolite, likely due to processive metabolism. Described in Chapter 3 is an exploration of the interactions of Cytochrome P450 2D6 (CYP2D6) and several of its polymorphisms with an array of phytocannabinoids (pCBs). CYP2D6 is a highly polymorphic CYP which has been shown previously to be inhibited by cannabidiol (CBD). In our current study, we examined the binding of eight pCBs to four common polymorphisms of CYP2D6 that are representative of different ethnic groups. We found that CYP2D6*17 has much larger spin-shifts than the other mutants tested, and that THC binds all four mutants more tightly than other pCBs. It was also determined that the inhibition of CYP2D6 by pCBs is dependent on the substrate molecules, as DXM and AEA metabolism were not inhibited by the same group of pCBs. Lastly, we were able use MD simulations to show differences in binding affinity and distance from the heme that are polymorphism dependent. Described in Chapter 4 is an examination of the interactions between different branches of the Arachidonase super-pathway. Arachidonic acid is an ω-6 polyunsaturated fatty acid that can be metabolized by COX, LOX, and CYP enzymes to produce pro- and anti-inflammatory metabolites. Each pathway metabolizes arachidonic acid into different products, which can either feed into each other, or work at cross-purposes. Described in Chapter 5 is the deconvolution of the murine IL-6 enhancer. Interleukin-6 (IL-6) is a known proinflammatory cytokine and participant in the acute inflammatory response. Although it is well studied, there is still much to be learned about the intricacies of how its expression is regulated. Gene expression is directly controlled through regulatory elements known as enhancers, which are regions of DNA containing transcription factor (TF) binding sites. Every TF has a unique motif to which it binds, which can in turn vary in strength according to sequence. Described within is the development of an IL-6 enhancer library which test multiple aspects of transcriptional regulation including binding site strength, distance, number, and order. Using MPRA, we are able to analyze our entire library simultaneously, affording us a high-throughput and experimentally equal view on how different aspects of the IL-6 enhancer affect regulation.
Issue Date:2020-05-01
Type:Thesis
URI:http://hdl.handle.net/2142/108272
Rights Information:Copyright 2020 Hannah Huff
Date Available in IDEALS:2020-08-27
Date Deposited:2020-05


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