Authored by Sabrina Lazar - Cell Biology and Professional Writing '20
Using enzymes as tools
What if you could directly visualize the location and amount of both toxic and essential molecules in your body? Dr. Marie Heffern’s lab in the Department of Chemistry is developing methods to do just that. Dr. Heffern trains and mentors several undergraduates in her lab, many of whom also help work on these projects – this is a snapshot of just one of them.
The team developed a system to observe copper (Cu+) mineral pools in the body using a mouse model. Our bodies cannot create or destroy copper, so its baseline level has to be tightly regulated. Both an excess and deficiency in copper are harmful. Incorrect copper regulation can lead to several maladies, such as anemia, non-alcoholic fatty liver disease, bone loss, and cardiovascular diseases.
The team had to find a way to first locate copper ions, and then to somehow create a visual marker to show where it was concentrated within the body. To accomplish this, they first synthesized a probe called Copper-Caged Luciferin-1 (CCL-1) which would circulate and bind to Cu+ ions. The binding would create a substance called D-Luciferin, which is a luminescent molecule from fireflies, and is the substrate for an enzyme called Luciferase. Luciferase binding its substrate creates a bioluminescent signal, allowing Cu+ to be detected using bioluminescence imaging on the whole animal.
Fig. 1: A simplified diagram showing the bioluminescent reporter pathway. Courtesy of Marie Heffern, Ph. D., and permission obtained from PNAS.
Since the liver is a key organ for copper storage, the team also altered the DNA sequence in one mouse line to include a liver-specific luciferase enzyme. This allowed them to more precisely monitor the levels of copper ion in live mice livers.
Fig. 2: A: CCL-L in mice reporting copper concentrations. B: More photons are emitted when CCL-1 and copper are together. Courtesy of Marie Heffern, Ph. D., and permission obtained from PNAS.
Utilizing the remarkable ability of specific enzymes in this creative way allows the monitoring of free-moving ions in a living system. Imagine the future medical applications of such experiments! (If you want to read more: In vivo bioluminescence imaging reveals copper deficiency in a murine model of nonalcoholic fatty liver disease).
The Heffern lab continues to make similar advances in other projects, working on a variety of different molecules and diseases. Faculty members, graduate students, undergraduate students and staff work together towards these common goals.
Despite all working in the same lab, every undergraduate has their own motivations and unique paths that led them to this research. Fourth-year undergraduate Ian F. is working on a project about the internalization of a certain peptide, and how metals affect the process. He “likes uncovering things [he] doesn’t understand and seeing the connection to topics in biology”. Third-year undergraduate Joey A. is interested in metalloendocrinology – the study of the interaction of metals and hormone systems, and developing molecular probes, a curiosity which stems from his own medical history. His advice to undergraduates looking to narrow down their research focus is “speak to TAs of your favorite classes, and explore. Make sure to choose an area that you genuinely enjoy and are fascinated by.” Third-year undergraduate Quang P. shared that even though he was relatively new to the lab, he was adjusting to the demanding life and workload of a researcher. To his fellow undergrads and anyone looking to acquiring research experience, he said “Don’t be afraid to step out of your comfort zone. Look for an opportunity wherever you can, and don’t worry about knowing everything- it’s an opportunity where you’re going to learn”.
"Luciferase Reporter Assay" - Bitesize Bio. https://bitesizebio.com/10774/the-luciferase-reporter-assay-how-it-works/
"In vivo bioluminescence imaging" Heffern et al. Bioluminescence imaging of copper in liver disease. Marie C. Heffern, Hyo Min Park, Ho Yu Au-Yeung, Genevieve C. Van de Bittner, Cheri M. Ackerman, Andreas Stahl, Christopher J. Chang. Proceedings of the National Academy of Sciences Dec 2016, 113 (50) 14219-14224; DOI:10.1073/pnas.1613628113
"Metalloendocrinology" - UC Davis Department of Chemistry. https://chemistry.ucdavis.edu/news/metals-and-hormones