Modulation of cytochrome c release by mitochondrial redox status and caspase-2

Abstract

The release of cytochrome c is an important event during apoptosis, induced by diverse stimuli. Our laboratory has previously proposed that cytochrome c release occurs via a two-step process, involving the detachment of the hemoprotein from its binding to the inner mitochondrial membrane, followed by its release into the cytosol through pores in the outer mitochondrial membrane - an event that is usually triggered by proapoptotic Bcl-2 family proteins, such as Bid, Bax and Bak. Cytochrome c specifically and stoichiometrically binds to cardiolipin, thus anchoring the hemoprotein to the inner mitochondrial membrane to participate in electron transport. Mitochondria are the main intracellular source of reactive oxygen species (ROS), and it has been shown that cardiolipin might become oxidized and lose its interaction with cytochrome c as a result of increased ROS production, or deficient ROS scavenging, within the mitochondria.

This thesis investigates the mechanism of cytochrome c release from mitochondria, and how this may be modulated by caspase-2 or the mitochondrial redox status. Caspase-2 is one of the best conserved caspases among species, and it is unique in the sense that it shares features of both initiator and executioner caspases.

We have demonstrated a new role for caspase-2 in apoptosis signaling, and propose a novel mechanism for cytochrome c release, mediated by caspase-2 and possibly involving pore formation in the mitochondrial membrane by this protease (papers I and II). Caspase-2 seemingly plays a role in apoptosis induction by exerting a direct effect on mitochondria, thereby releasing cytochrome c. Interestingly, this effect seems to also involve an interaction between caspase-2 and cardiolipin. On the contrary, we have shown that cardiolipin is not a pre-requisite for Bax-mediated cytochrome c release (Paper III). However, cardiolipin must be affected by protein binding or oxidation in order for solubilization of cytochrome c to occur, allowing release of the hemoprotein through the Bax-pores.

One typical dissociation factor for cytochrome c is oxidation of cardiolipin. The glutathione (GSH; gamma-glu-cys-gly) system is one of the most important intracellular redox systems. This abundant tripeptide protects from ROS and has been linked to apoptosis by several observations. In paper IV, apart from describing a new method for GSH visualization in the cell, we also demonstrated the capability of mitochondria to scavenge GSH during oxidative stress.

Moreover, papers V and VI indicated that mitochondrial Grx2 is a possible inhibitor of apoptosis, since knocking down the protein by siRNA (paper V) or overexpressing Grx2 (paper VI) influence cell death signaling, probably by preventing oxidation or degradation of cardiolipin (paper VI). It is clear that the mitochondrial redox environment is crucial for keeping cardiolipin reduced and preventing cytochrome c release. Lowering the level of Grx2, or other mitochondrial redox enzymes, may thus have a lethal effect on the cell.

In conclusion, it is clear that the release of cytochrome c may occur by different mechanisms, depending on the apoptotic inducer and on the type of cell. While caspase-2 is able to form pores in the mitochondrial membrane, as well as promote dissociation of cytochrome c from cardiolipin, we cannot exclude that this protease also may work in concert with other pore forming agents, such as Bax. However, cardiolipin is not required for Bax pore-formation of the mitochondrial membrane. In addition, we have shown that mitochondria require a unctional redox system for protection from apoptosis.