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Developing an effective drug-screening assay for treating cardiac energetic deficiencies

Semester: Summer 2023

Presentation description

The mitochondrial calcium uniporter (MCU) may help compensate for energetic deficiencies characteristic of some heart diseases, via a mechanism dependent on breaking an interaction between its N-terminal domain (NTD) and complex I of the electron transport chain. Drugs that inhibit this interaction may offer new treatments for such heart diseases, but to identify such compounds we need a screening assay for MCU NTD interactions. The MCU NTDs also mediate dimerization of the MCU. To quantify the MCU NTD interactions, we used fluorescence resonance energy transfer (FRET), a highly distance-dependent process in which energy is transferred from a donor to an acceptor fluorescent protein. We constructed four cell lines to determine which construct would be the best indicator of drug effectiveness. Two cell lines take advantage of the dimerization of MCU to produce FRET; one has both fluorescent proteins on the N-terminal, while the other has them on opposite terminals. The third cell line uses the interaction between complex I and the MCU to produce FRET. The final cell line is used as a control for background levels of FRET. Each construct has a bidirectional promoter, activated by doxycycline, ensuring similar expression levels of each fluorescent protein. To prevent immature fluorescent proteins, which would lead to an underestimate of the FRET efficiency, we treated the cell lines with cycloheximide, a protein synthesis inhibitor, before measuring fluorescence with flow cytometry. Due to low MCU expression in the cells, we used fresh doxycycline and transfected the cells with Vpx, a viral protein that has allowed for the transcription of long coding mRNA in similar cells. Both were effective at increasing MCU expression. The cell constructs exhibiting the most FRET were the one with the fluorescent proteins on the N-terminal and the one involving complex I because of the closer proximity of the fluorescent proteins due to NTD interactions. These constructs will be used to quantify the efficacy of drugs in breaking the interaction between complex I and the MCU.

Presenter Name: Ashley Cluff

Presentation Type: Poster
Presentation Format: In Person
Presentation #6
College: Medicine
School / Department: Internal Medicine
Research Mentor: Dipayan Chaudhuri
Date | Time: Thursday, Aug 3rd | 10:30 AM