Muscular Dystrophy Association
Neuromuscular Disease Research Grant
01/01/07-12/31/09
PI: Patrice Hamel
Unraveling the mitochondrial redox pathway in cytochrome c maturation.

Lay summary:
This proposal aims to understand how crucial molecules such as c-type cytochromes that function in the production of energy but also in programmed cellular death, are made in the context of a cell. The energy status of the cell is critical for survival, when compromised cardiac and other myopathies result.C-type cytochromes are essential players in this status and their assembly process which is complex and needs to be well understood. Only, two c-type cytochrome assembly factors, CCHLs and Cyc2p are known. We propose to study how these proteins work to synthesize cytochromes c and also discover new molecules that operate alongside CCHL and Cyc2p through biochemical and molecular genetics approaches.

Abstract:
It is well established that mitochondrial dysfunctions can be the direct cause of myopathies and neurodevelopmental diseases. Besides the textbook function of respiration and ATP generation, the emerging role of mitochondria in metal and redox metabolism, oxidative stress, apoptosis, aging and communication with the rest of the cell has also become widely recognized. Therefore, there is increasing attention on mitochondrial contribution to disease. In order to develop therapies for appropriate cure of mitochondrial diseases, it is critical that we understand the basics of mitochondria structure and biogenesis. The c-type cytochromes are universal heme proteins in which the heme cofactor is covalently bound. Cytochromes c function, not only in transducing energy, but also in signaling in the cell death pathways (apoptosis). Their distinctive feature is the covalent binding of the heme cofactor to the cysteines of the apocytochromes, an assembly step which requires catalysis in vivo and can occur via one of the three possible pathways, referred to as Systems I, II and III. Therefore, the study of their biogenesis is fundamental to the understanding of mitochondrial function. Common to systems I and II is the operation of a cytochrome c assembly machinery with heme delivery systems and multiple redox components. In contrast, system III  which operates in animal and fungal mitochondria is only defined by two components, the cytochrome c and c1 heme lyases (CCHL and CC1HL) specific for their respective substrates cytochrome c and cytochrome c1. The biogenesis of mitochondrial c-type cytochromes is poorly understood at a mechanistic level and needs to be reinvestigated in the context of the situation where loss of human CCHL was found to cause a neurodevelopmental disease with cardiomyopathic manifestations. The complexity of systems I and II suggests that additional players are implicated in the biogenesis of mitochondrial c-type cytochromes as well. We have recently discovered Cyc2p, a unique mitochondrial flavoprotein with a yet-to-be defined redox activity in the assembly process. We will exploit yeast as model experimental system to further dissect the biochemistry and molecular genetics of mitochondrial c-type cytochromes biogenesis, with the goal of detailing its fundamental mechanism. We proposed a broad experimental plan involving 1) the exploration of the participation of Cyc2p in redox chemistry by the comparison of the thiol redox status of apocytochrome c1 in mitochondria, 2) the biochemical identification of CCHL and Cyc2p interacting partners via the resolution of the components of a CCHL-containing complex, and 3) the discovery of new assembly factors acting alongside Cyc2p through molecular genetics using the synthetic genetic array methodology.