Chelsea Ohaka
Research Mentor(s): Alexandria Chabez
Mentor Department: Biological Chemistry
Authors: Chelsea Ohaka, Alexi Chabez, Dr. Emily Scott
Session: Session 1 (9:00am – 9:50am)
Presentation Type: Poster 8
Abstract
Our research focuses on understanding the functional characteristics of human cytochrome P450 7B1 (CYP7B1), a protein involved in the bile acid synthesis pathway in the liver. In this pathway, CYP7B1 hydroxylates substrates 25-hydroxycholesterol and 27-hydroxycholesterol to generate chenodeoxycholic acid. Mutations in CYP7B1 result in loss-of-function protein, which leads to the accumulation of both substrates in plasma and 27-hydroxycholesterol in cerebrospinal fluid. These mutations cause spastic paraplegia type 5 (SPG5), a neurodegenerative disease that causes progressive spasticity and weakness of the lower limbs. This has led our lab to focus on the significance of substrate binding to CYP7B1 mutations to further understand how these mutations affect protein function to cause SPG5. We are interested in the interaction between CYP7B1 and its substrates, as substrate binding at the enzyme’s active site is crucial in understanding disease mechanisms. Using a UV-visible spectrophotometer, we measure the absorbance of substrate binding by titrating in the substrate to interact with CYP7B1. The assays are conducted in triplicate and are analyzed to determine the binding affinity (Kd value) of the substrate. By plotting the difference in absorbance against substrate concentration, we can identify how tightly the substrate binds to the protein. Currently, we have observed lower Kd values for CYP7B1, indicating tight binding of 25-hydroxycholesterol. Although our results are still preliminary, they provide a general binding affinity value for comparing to the mutants in the future. This research could ultimately guide the development of genetic therapies for SPG5 patients, benefiting scientists, healthcare professionals, and pharmaceutical developers alike.
