Access Type

Open Access Thesis

Date of Award

January 2017

Degree Type

Thesis

Degree Name

M.S.

Department

Biochemistry and Molecular Biology

First Advisor

Maik Hüttemann

Abstract

Prostate cancer is the second leading cause of cancer death among men in America. The progression of cancer goes along with the Warburg effect, a metabolic switch from depending primarily on mitochondrial respiration to glycolysis. In addition, cancer cells manage to evade apoptosis. Cell signaling, via posttranslational modifications (PTMs), is one of the most important means of regulation, and most commonly dysregulated in cancer. In prostate cancer, androgen signaling plays a crucial role in driving cell proliferation.

Mammalian Cytochrome c (Cytc) is a multifunctional protein involved in cellular life and death decision. It is an essential component of the electron transport chain (ETC), where it shuttles electrons to cytochrome c oxidase (COX) to eventually generate ATP. Cytc also functions as a trigger of apoptosis when released into the cytosol. However, its regulation is not well understood. Previously, it was shown that Cytc can be posttranslationally modified and regulated by phosphorylation through cell signaling pathways, controlling the protein functions.

In this study, we demonstrated that development of prostate cancer causes changes in Cytc posttranslational modifications which in turn, alter the main protein functions, including respiration and apoptosis. The preliminary data demonstrated that human Cytc is acetylated on lysine 53 in eight independent castrate-resistant and -sensitive human tumor xenografts, suggesting that this is a cancer-specific modification. To characterize the functional effects, Lys-53 was mutated to the acetylmimetic glutamine, a non-acetylated arginine that carries a positive charge, and to the nonpolar isoleucine as an additional control. Cytc variants were overexpressed in bacteria and purified to homogeneity. COX activity, analyzed with purified Cytc variants, demonstrated that the acetylmimetic Lys53Gln Cytc showed reduced respiration compared to the nonacetylated WT. Remarkably, compared to WT, acetylmimetic Lys53Gln mutant was unable to trigger caspase-3 activity and hence, to induce apoptosis. This data suggests that the Lys-53 epitope is directly involved in the interaction between Cytc and Apaf-1. Our study shows that acetylmimetic Lys53Gln causes a reduction of peroxidase activity of Cytc compared to WT control, proving the results that Lys53Gln is incapable of triggering apoptosis. We observed that the redox potentials of the Cytc variants were in a range between the redox potential of complex III and IV, suggesting the ability of proteins to function properly, which was also confirmed by spectrophotometric analysis. We also showed that Lys53Gln Cytc mutant has a higher ability to degrade H2O2 and act as a stronger ROS scavenger compared to WT. Additionally, Lys53Gln Cytc mutant demonstrated a better capacity to accept electrons further supporting its role as ROS scavenger. In summary, our data suggest that cell signaling regulates cellular respiration and apoptosis via PTMs of Cytc, and suggest distinct regulation of Cytc in cancer.

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