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When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.
Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.
Cytochrome c peroxidase (Ccp1), independently of its peroxidase activity, functions H2O2 sensing and signaling to regulate another heme enzyme such as catalase A (Cta1) in vivo. The maturation of Cta1, a key H2O2 detoxifying heme enzyme, involves the recruitment of heme from cytochrome c peroxidase (Ccp1) in respiring yeast mitochondria. An interest of this research was to track the heme status of Ccp1 in live cells spatiotemporally. To date, there is no tool to investigate when and where an endogenous heme-binding protein is heme-loaded or heme-lost in live cells. The first goal of my research was thus to develop a tool to track the heme of Ccp1 in live cells. Heme is known as efficiently quenching green fluorescent protein (GFP). The Ccp1-GFP fusion that is chromosomally expressed by yeast, along with the native promotor of Ccp1, is hence selected to track heme-bound and heme-free form of Ccp1. The analysis of GFP fluorescence lifetimes using fluorescence lifetime imaging microscopy (FLIM) reveals that Ccp1-GFP is fully heme-loaded and resides in the mitochondria of 2-day respiring cells, compared to the partly heme-free form of extra-mitochondrial Ccp1-GFP in 7-day respiring cells. Notably, the FLIM approach presents a method for tracking the heme of endogenous heme-binding protein in various subcellular compartments. Mitochondrial heme-loaded Ccp1 is also identified as the first heme-based H2O2 sensing protein in yeast cells and its W191F variant is known as amplified H2O2 sensing. The second goal of the research was thus to identify the H2O2 responsive proteins, regulated by Ccp1 and its W191F variant in live cells using label-free proteomics. The findings revealed that the global response to exogenous H2O2 shows a switch of cellular activity from biosynthesis to defense and a redirection of carbohydrate metabolism to NADPH production in both strains. Nevertheless, the amplified H2O2 sensing Ccp1W191F generates a significantly more sustained response. The label-free proteomics approach further offers a way to fully identify the stress responsive proteins in live cells. Overall, my research findings provide insights into the heme-mediated physiological role of Ccp1 as a heme donor protein and the H2O2 stimulon regulator using FLIM and label-free proteomics.