PhD Oral Exam - Alan de Aguiar Lopes, Chemistry

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.

Abstract

Intracellular heme trafficking is important for cell respiration, antioxidant defence, and ergosterol biosynthesis in yeast, but information about this process is lacking. In Saccharomyces cerevisiae, heme production ends up with FeII insertion in the protoporphyrin IX by ferrochelatase (FECH), yet no heme acceptors from FECH have been identified. Cytochrome c peroxidase (Ccp1) is a candidate due to its heme-independent synthesis and involvement in heme trafficking. However, FECH and Ccp1 are separated by the inner mitochondrial membrane, so this thesis examines Pet9, an ADP/ATP carrier, as a candidate in delivering heme from FECH to Ccp1. In Chapter 2, potential tools for this study were characterized including hem15Δ S. cerevisiae cells, which were shown to be devoid of FECH activity, and cells homologously expressing N-terminally GST-tagged proteins. The expression of these proteins was examined to probe their applicability for GST pull-down assays, but protein cleavage impairs the use of these proteins. Chapter 3 reports on the LC-MS/MS analysis of GST pull-downs of soluble and membrane proteins from S. cerevisiae following addition to GST-apoCcp1 bait with different amount of cell lysates. Screening the pull-downs at a 1:5 bait-prey ratio from lysates containing 0.6% N-octylglucoside identified 81 mitochondrial proteins as Ccp1-specific interactors including FECH and Pet9. In Chapter 4, ternary complexes of Ccp1-Pet9-FECH were evaluated by protein docking analysis. Examination of the electrostatic potential surfaces reveals that complexation is driven by electrostatic complementarity. The outcomes of chapters 3 and 4 support FECH, Pet9, and Ccp1 form a functional complex for heme transfer. Heme is an essential coenzyme for the ergosterol pathway in S. cerevisiae. To probe links between heme and ergosterol biosynthesis, in Chapter 5 the S. cerevisiae proteome was analyzed by LC-MS/MS after cells were challenged with miconazole, an Erg11 inhibitor. Proteins of 20 biochemical pathways were altered, including ergosterol and heme biosynthesis. Overall, this thesis supports the role of heme as the last step for Ccp1 maturation and adaptive response to miconazole. These roles and mechanisms behind it may clarify how heme trafficking occurs between proteins and how heme production is a strategy to protect the cell from deleterious effects of antifungals.