Toward a Comprehensive Framework for Investigating Copper Dyshomeostasis: The Role of His-Tag Modularity and Alternative Expression Systems in Evaluating Dopamine β-Hydroxylase

zoe semersky

Name: Zoë Semersky
Majors: Biochemistry, Molecular Biology
Advisors: Annastassia Gallo, Sara Martin

Research Supported by the Copeland Fund

Dopamine β-Hydroxylase (DBH) is a copper-containing monooxygenase responsible for the benzylic hydroxylation of dopamine to produce norepinephrine, a neurotransmitter vital to the control of nervous system functions including learning and memory.1 Norepinephrine depletion is associated with neurodegenerative diseases like Alzheimer’s and Parkinson’s, where hallmarks include plaque formation and neural death in brain regions where DBH is localized.2 Extensive data demonstrates irregular metal levels in these diseased brain regions compared to healthy brains, leading to the prominent view that impaired metal homeostasis is involved in the pathology of neurodegeneration;3 however, the mechanism behind how decreased DBH activity impacts this has yet to be understood.

Since DBH is bound to copper, investigating the impact of variable copper levels on its activity can offer a practical explanation of the loss observed clinically. The preliminary work herein approach establishing a DBH expression system and elucidating the role of copper ion dyshomeostasis on its structure and function while instituting a new lab. Through expression optimization, it is revealed that temperature plays a role in the folding of the soluble enzyme. High-pressure liquid chromatography and steady-state kinetics demonstrate that a recombinant bacterial expression system yields soluble but enzymatically inactive DBH, suggesting that glycosylation is a critical post-translational modification for its function. Finally, the presence of a His-tag in glycosylated DBH inhibits steady-state behavior.

Posted in Comments Enabled, Independent Study, Symposium 2023 on April 13, 2023.

11 responses to “Toward a Comprehensive Framework for Investigating Copper Dyshomeostasis: The Role of His-Tag Modularity and Alternative Expression Systems in Evaluating Dopamine β-Hydroxylase”

  1. Torence says:

    Great work!! What was the hardest part of the research process?

  2. Meagen Pollock says:

    Your 3D model is awesome! Nice work!

  3. Ann Agurkis says:

    This is amazing, though I’m not totally comprehending it all. Has your study lead you to believe a hypothesis regarding how to potentially control for copper or DBH, or temperature regulation to prevent memory loss? Or maybe that’s just way down the line.
    Very impressive and important work! Congratulations!

  4. Shelley Judge says:

    Great work, Zoe. I’m such a visual person, so I loved the software you used to model your research. You should be so proud.

  5. Zoë Semersky says:

    Hi Torence,

    One of the most challenging parts was troubleshooting experiments that didn’t go according to plan. Although that was the case, I’d say it was also the most fun and rewarding piece! It forced me to consider my approach from different angles creatively.

  6. Zoë Semersky says:

    Thank you, Dr. Pollock!

  7. Zoë Semersky says:

    I appreciate it, Dr. Judge! Glad the visual was useful.

  8. Zoë Semersky says:

    Thank you, Mrs. Agurkis! I like your thinking. Those are definitely areas to consider down the line. I’d say this is still in the very preliminary stages, but exciting nonetheless!

  9. Isabelle Dwyer says:

    Do you know why temperature was seen to play such a big role on the expression of DBH-His? If temperature was able to increase the expression of DBH, couldn’t we theoretically translate that to some sort of potential treatment options for AD and PD if they are seen to have a lower DBH concentrations? Also, this is incredibly cool research and so so impressive!!!

  10. Prof. Claire Eager says:

    This is such a compelling poster, Zoë! How did you balance the work of doing the research and representing it in so many visual and written ways? Congratulations!

  11. Zoë Semersky says:

    Hi Isabelle, the temperature played a role in this expression system, but it’s also important to consider that it was in bacteria. The temperature of the human body is about 37C, and altering that in a person would likely have serious negative effects. The decreased norepinephrine production clinically could suggest that DBH concentrations are lower or malfunctioning in some other way (which is where this work is hopefully headed in uncovering). Great thinking!