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

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.

NAPH and NADP+ detected by CE

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).

Recent Posts

Recruitment begins

I’m not sure I can adequately describe the size or scale of the impact in our lab over the last year+ but this pandemic was dishing things out left, right and centre.  I feel like we are at a nexus of where the pandemic really delayed and harmed our research program.  The personal impacts for many in my group have likewise been large and the largest goal was to try to keep all of us upright, in science, and keeping our heads above the water.  In that regard, we need to reassess and redefine success over the last year.  We have survived (!!) and this was largely from the resilience of our group, as much as I think that is an overused term.  But we did get through this year together, pivoting efforts, helping each other, focusing on what we could do, taking things one step at a time.  Now we are starting to see progress in projects again and productivity is starting to leap back up.  That provides some space to look forward and plan for the future.  Let’s start with some lab success stories.  I’ll add in where we will be recruiting in the upcoming year in these projects.  A detailed outline of our open positions in 2021-2022 will be posted, so please check them out and get in touch if you are interested in our research program.

When the pandemic shut down McMaster, yellow perch were spawning and we were about to launch into working with a brand new species.  Waiting a full 12 months to restart was very hard but spring 2021 saw us undertaking the experiments we canceled the year before (and then some!) with very good outcomes.  The lab (especially Shamaila Fraz) came roaring back with ambitious plans that were very fruitful and lots of experiences in testing new protocols to help move research plans forward.  I am quite excited to see the data collection from our new samples and the analyses from the data already collected.  It will shape the direction of our research into developmental plasticity and impacts of temperature on fish development.  Much of our work this year focused on embryogenesis and immediate post-hatching periods while we work on the juvenile rearing stages. Perch are definitely a bit tough to rear post-hatching, compared to the other species we have worked on.  No surprise but we certainly learned some things not to do to keep them happy.  We are looking to recruit for this project in the upcoming months so we are ready to take on spring 2022 spawn.

While we graduated two graduate students from the lab in 2021, clear success stories, these were students who were done data collection prior to university shut down.  Other graduate students in the lab faced much bigger challenges. With major lost experiments and one totally new project later, we forged a new plan to get back on track. I’m really excited about these new directions.  Andrea Murillo has a freezer full of samples taken from our culture of the marine polychaete worm, Capitella teleta, and she is now extracting those samples to get geared up for gene expression and steroid hormone analyses.  Shemar Williams has completed a suite of experiments in zebrafish embryos and on track to finish his MSc.  Its great to see these project humming along.  This also means it is time to think about new graduate students in 2022 interested in the function of cytochrome P450 enzymes using either zebrafish or Capitella as a primary species of interest.

Success, of course, has also been obvious in more traditional ways. We have been so happy to celebrate Oana Birceanu’s success this year as she starts her new role at Western’s Physiology and Pharmacology department as an Assistant Professor. I look forward to continued collaboration and engagement with her research group in the years to come.

**  If you are interested in our lab, please check back under “Recruitment Opportunities” for more details in the upcoming days. I will be posting specific information there.  The much needed refresh of the lab website is just beginning.

 

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