Cytochrome P450 Enzymes; CYPs
My basic research program focuses on the cytochrome P450 superfamily of proteins with an emphasis on their evolution and function in aquatic species such as fish. Cytochrome P450 enzymes, or CYPs, are heme proteins critical for generation of major biological signaling molecules (e.g. steroid hormones) and for the detoxification of xenobiotics (e.g. drugs, environmental contaminants). CYPs are a key component of the defensome – the genes that aid in protection and defense from toxic compounds. Vertebrate species have 50-100 CYP genes in their genome but the function of many of these genes in non-mammalian species is unclear. We have a primary interest in understanding the evolution and function of CYPs in aquatic organisms.
Our projects involve genome annotation of CYP sequences, phylogenetic studies of CYP families, protein expression and functional testing of CYPs. This research raises fundamental questions about CYP protein function and attracts students with strong interests in protein evolution, bioinformatics, molecular biology and biochemistry.
Bioinformatics approaches are used in genome annotations of CYP genes and phylogenetic studies that raise functional hypotheses regarding novel CYP sequences. With each new genome completed, an array of CYP sequences are identified for which functional knowledge is lacking. Our basic science research is directly aimed at uncovering the function of these novel genes and understanding the capacity of CYP systems in aquatic species.
Much of the tools used to examine CYP function were designed for mammalian systems. My lab has undertaken experiments meant to directly compare and contrast the function of CYP systems in mammalian and piscine liver; the major organ for xenobiotic metabolism. We have tested the capacity of fish hepatic CYP mediated metabolism using fluorogenic substrates and examined CYP enzyme inhibition with typical mammalian CYP inhibitors. Since the liver expresses multiple CYP isoforms, we have expressed several CYP enzymes including those in the CYP1 and CYP3 family to investigate the function of specific CYPs important for drug, xenobiotic, and estradiol metabolism.
Our ability to study CYP systems is limited by the available tools. In collaboration with the lab of Dr. Philip Britz-McKibbin (Department of Chemistry, McMaster University), we have developed a capillary electrophoresis assay for CYP function. CYPs require the donation of electrons from NADPH to complete their catalytic cycle, typically hydroxylation reactions. While most assays monitor the loss of substrate or generation of metabolite, these assays are very specific for single compounds. The measurement of NADPH consumption would provide a generic assay for CYP activity that would allow for screening of CYP function with low cost and higher throughput. Our current research is aimed at using high throughput screening approaches to build structure-activity relationships and uncover function of our expressed CYP proteins. This research is completed in McMaster’s High Throughput Screening Laboratory using libraries of chemicals derived from natural products and off-label drugs.
This research has been funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery and Accelerator Programs, the Canadian Foundation of Innovation (CFI), and Ontario Innovation Trust (OIT).