PROJECT SUMMARY AND ABSTRACT Cytochrome P450 enzymes (P450, CYP) are essential for the metabolism of xenobiotics and endobiotics. The CYP4F family of fatty acid -hydroxylases consists of the isoforms CYP4F2, CYP4F3A, CYP4F3B, CYP4F11, and CYP4F22 which share amino acid sequence identities up to 93%. All of these isoforms assume different functions in the human body spanning vitamin K and drug metabolism, ceramide production, and the production of the important lipid mediator 20-hydroxteicosatetraenoic acid (20-HETE) from arachidonic acid. 20-HETE regulates the blood pressure but is also associated with human disease, such as hypertension, traumatic brain injury, and cancer. An inhibition of 20-HETE producing CYP4 isoforms ameliorates these diseases in animal models which demonstrated their tremendous potential as drug targets. Yet, the involvement of individual CYP4F isoforms in distinct diseases has not been examined yet and too little is known about their differential structure and function to target them individually. My research vision is to perform an in-depth functional and structural characterization of CYP4F -hydroxylases to unravel their impact on cellular processes and promote their use as drug targets. My lab will close three significant gaps in current CYP4F research. First, we will elucidate the function of CYP4F isoforms in physiology and disease. My lab has previously shown that the isoform CYP4F11 is significantly overexpressed in lung cancer. A transient knockdown of CYP4F11 in lung cancer cell lines leads to a significantly reduced cell proliferation and migration which is associated with reduced 20-HETE production. Using lung cancer as a model disease, we will perform transcriptomics and lipidomics studies to unravel metabolic pathways and differences in lipid composition which impact CYP4F11- dependent cell proliferation. We will extend our studies to additional CYP4F isoforms and their involvement in noncancerous diseases. Second, we will demonstrate that the function of CYP4F enzymes is regulated by the redox partner proteins cytochrome P450 reductase (CPR) and cytochrome b5 (CYB5). Both proteins show a significant allosteric effect on catalytic efficiency and ligand binding affinity for drug metabolizing and steroidogenic P450 enzymes. However, their impact on CYP4F -hydroxylases has never been investigated. Using recombinant human CPR and CYB5, my team will conduct a thorough evaluation of their impact on CYP4F catalysis and the binding of substrates and drugs. Third, there is currently no structural information available for any of the human CYP4 -hydroxylase which significantly hampers our understanding of enzyme function and isoform specific substrate specificities. We will solve the very first structure of a human CYP4F -hydroxylase using X-ray protein crystallography. My lab has successfully generated crystals of the isoform CYP4F11 in complex with the inhibitor HET0016 which will soon be screened for diffraction at the synchrotron. Our findings will promote our understanding of structure and function of human CYP4F -hydroxylases and pave the way for their use as drug targets exploiting significant differences in protein architecture.

NARRATIVE The cytochrome P450 4F (CYP4F) family are fatty acid -hydroxylases and involved in generating lipid mediators from arachidonic acid, the clearance of drugs, and the metabolism of vitamins and proinflammatory eicosanoids. They are associated with human diseases such as hypertension, cancer, and traumatic brain injury, yet little is known about their cellular function and their structural properties. The Brixius lab proposes in-depth functional and structural studies to unravel their role in cellular processes and their association with human disease using cell biology, biochemistry, and X-ray protein crystallography.