Access Type

Open Access Embargo

Date of Award

January 2021

Degree Type

Thesis

Degree Name

M.S.

Department

Biochemistry and Molecular Biology

First Advisor

Maik Hüttemann

Abstract

Cytochrome c (Cytc) is a pivotal multifunctional mitochondrial protein that serves as a single electron carrier between complexes III and IV of the electron transport chain. It has important roles in both cellular respiration and apoptosis. The novel Thr49 (T49) phosphorylation of Cytc likely affects mitochondrial respiration, membrane potential, ROS production, ATP production, and apoptosis. Based on the functional characterization of previously mapped phosphorylation sites (Tyr97, Tyr48, Thr28, Ser47, Thr58) of the lab, we hypothesize that T49 phosphorylation will lead to controlled respiration, optimal intermediate mitochondrial membrane potential, lower ROS production, and inhibition of apoptosis compared to unphosphorylated Cytc. Here we report some of the functional studies that aid in the functional characterization of phosphorylation of T49. Characterization of this specific post-translational modification (PTM) can demonstrate overall regulation of Cytc in human tissue types that contain the modification, specifically in the context of respiration and apoptosis. To complete this objective, we used an in vitro model with recombinant protein and a cell culture model.In the recombinant protein model, T49A and T49E reduction and oxidation rate demonstrated that Cytc may exhibit a slower electron transfer rate and increased ROS scavenging capabilities. The significantly higher reduction rate suggests that phosphorylation at T49 may play an essential role in the ROS scavenging capabilities of Cytc. Additionally, the phosphomimetic variant demonstrated an increased susceptibility of heme degradation by H2O2 demonstrating oxidative stress is able to alter the function of the protein. T49A and T49E Cytc both demonstrated a drastically lower reduction rate by superoxide compared to WT Cytc, suggesting that modifying T49 alters Cytc’s superoxide scavenging ability. Further studies demonstrated Cytc’s role in apoptosis. Cardiolipin peroxidase activity demonstrated that T49A and T49E results in lower peroxidase activity compared to WT suggesting that the amino acid substitution at this residue may affect the conformation of the protein. Additionally, the phosphomimetic Cytc variant demonstrated a decrease in caspase-3 activation suggesting lower levels of apoptosome formation in the presence of the mutants. Further experiments must be done to elucidate the functional effects of T49 phosphorylation.

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