Cytochrome P450 Research

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.

CYP4 phylogeny

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.

Spectrum of expressed zebrafish CYP

So how do we test function of novel enzymes?  The first step is typically to express the gene to provide a pool of enzyme to work with.  In our case, we typically express CYPs of interest in bacteria with the human co-enzyme, cytochrome P450 NADPH reductase.  We have expressed several CYP genes from zebrafish from the CYP1 and CYP3 family.

NAPH and NADP+ detected by CE

The next major step is to perform some biochemistry: enzymatic reactions containing our expressed protein, NADPH cofactor, and possible substrates of interest.  We have used a number of fluorogenic probe substrates (e.g. resorufin based compounds) to asses metabolism to a fluorogenic metabolite but because most of these probe substrates were developed for mammals,  they are less informative for non-mammalian species.  While most enzymatic assays monitor the loss of substrate or generation of metabolite, these assays are very specific for  a single substrate or a single metabolite.  The measurement of NADPH consumption is an indirect measure of CYP catalytic activity but provides a generic assay for CYP activity which allows for screening of CYP function with any compound of interest. When we couple this indirect assay with the power of robotics, we can undertake high throughput screening of chemical libraries.

Our current research is aimed at using high throughput screening approaches to better understand CYP function.  For example, the zebrafish CYP1A and CYP3C1 enzymes have been screened with thousands of compounds, using NADPH consumption as the endpoint.  These screens have identified ~100 potential substrates. Follow up tests on the hits provide additional data on the CYP-substrate interaction such as kinetic parameters and reactive oxygen species production. This research is completed in McMaster’s High Throughput Screening Laboratory using libraries of chemicals derived from natural products and off-label drugs and environmental chemicals.

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),  Ontario Innovation Trust (OIT), and US Environmental Protection Agency.

Recent Posts

Research Leave is where?

Research leave has rolled around and again, it seems to follow family catastrophe.  I *thought* we would be in Bergen, Norway right now but new elder care duties seriously cancelled that plan. So now I am local.  Research Leave in Hamilton!!!  At the end, it looks like COVID might have cancelled things anyways because I am not sure relocating in the middle of the largest wave of the pandemic would have been so attractive.  Still, it was Norway and I am missing the sea.

My priority for research leave has always been to do something significant for my research program.  For my first, I spent 13 months in Sweden and this was where I fist started doing behavioural research with fish.  I also learned to experiment with primary hepatocytes, although this hasn’t been incorporated in my lab in the same way.  That is mostly because the results of the experiments were a bit blah and unexciting for the compounds we were testing in hepatocyte culture.  But the behavioural research is a different story.  Now we have added in a suite of behavioural assays in different life stages.  For juvenile and adults, we have used courtship, aggression, behavioural choice experiments with odorants and thermal preference tests. For larval fish, we now do a whole range of swimming (general swimming, light:dark response, thigmotaxis), startle responses and feeding behaviour.  My year away pushed us in a new direction that has been rewarding.

So what is on the table this year?  Planning is still an active process but one thing is for sure is that I have some training and planning to do. First, is training to work with human subjects as we have some projects that will cross into social sciences.  The other major training need is to brush up on transcriptomics analyses and R so that we are better equipped for some of the new data we will be generating in the next few years.  Second, is planning for both the lab and field for our perch embryo experiments. We learned so much from last year’s experiments and have to adjust. We will adjust the lab for better rearing protocols and really minimize some of the labour issues we encountered last year. We are adding  new lights to help with feed training the larval fish too. We are also adjusting our field sites and I need to get new permits in place. This is really exciting and I already have new Windermere traps under construction with the engineering lab.  I can’t wait for perch spawning.

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