Open Access Thesis
Date of Award
Biochemistry and Molecular Biology
Cytochrome c is a 12.4kDa ubiquitously expressed protein known to be involved in many physiological processes of the cell such as respiration and apoptosis. The goal of our lab is to increase our knowledge of the regulation of cytochrome c in these opposite activities, and our working model posits that cytochrome c is decisively regulated by phosphorylation. When phosphorylated, cytochrome c leads to an "optimal" functioning in the electron transport chain by lowering electron flux, preventing harmful high mitochondrial membrane potentials and thus ROS production under healthy conditions. However, under cellular stress cytochrome c might be dephosphorylated favoring high mitochondrial membrane potentials and ROS and its participation in apoptosis. Our lab has previously published two phosphorylation sites in cow, namely Y48 in the liver and Y97 in the heart. The aim of my thesis was to identify phosphorylation site(s) on kidney cytochrome c and to perform functional characterization of the cow kidney protein. Kidney cytochrome c was found to be phosphorylated on S47 and based on this, suitable cytochrome c variants were over expressed in a prokaryotic system. These cytochrome c variants were used to study the effect of phosphorylation on the most common activities of cytochrome c protein i.e., cellular respiration and apoptosis. The results of the in vitro study revealed that the phosphomimetic mutant Ser47Glu has lower rates of respiration compared to wild type as well as S47A mutant which is in line with the working model of our lab. In addition, any mutation of the Ser47 residue resulted in almost fully diminished caspase activity when compared to wild type, suggesting that this residue might be key to the regulation of the apoptotic activity of cytochrome c.
Varughese, Ashwathy Mary, "In Vitro Characterization Of Serine 47 Phosphorylated Cytochrome C" (2014). Wayne State University Theses. 358.