Abstract
In addition to the role of the mitochondria in energy metabolism, these organelles play a key role in cell death signaling. In particular, mitochondrial alterations, such as stimulation of reactive oxygen species (ROS) formation, decreased ATP synthesis, loss of membrane potential and the release of pro-apoptotic proteins from the mitochondrial intermembrane space (IMS), have been shown to be involved in, and often responsible for, various manifestations of cell death. Among the pro-apoptotic proteins to be released are cytochrome c and Apoptosis-Inducing Factor (AIF). While cytochrome c initiates caspase-dependent cell death, AIF leads to caspase-independent cell death. The release of pro-apoptotic proteins from the IMS is considered as a “point of no return” in apoptotic signaling, although the mechanisms regulating permeabilization of the outer mitochondrial membrane remain controversial. Two main mechanisms have been elaborated including the induction of mitochondrial permeability transition (MPT) and selective pore formation by the Bcl-2 (B-cell lymphoma 2) family proteins, Bax and Bak. Although, distinct cell death pathways can be triggered by various signals, they often merge at a common regulator of this multistep process. Currently, it is widely accepted that the mitochondria serve as such a regulator. Therefore, the main goal of this project was to investigate the involvement of mitochondria in cell death.
While cytosolic Bax is known to target the mitochondria to induce mitochondrial outer membrane permeabilization (MOMP) during apoptosis, less is known about its mitochondria-specific target. We addressed this issue and could show that a functional translocase of the outer mitochondrial membrane (TOM complex) is required for Bax to release cytochrome c from the mitochondria.
Previous work in our group has established that AIF-mediated, and caspase- independent apoptosis is the main cell death mechanism in non-small cell lung carcinomas treated with anticancer drugs. However, the molecular mechanisms underlying AIF release remained obscure. We addressed this issue and could show that Ca2+ was imported into cells upon treatment of cells with protein kinase C (PKC) inhibitors. The imported Ca2+ had two critical functions; a) the activation of a mitochondrial calpain that could cleave AIF, a step essential for its release and b) to stimulate mitochondrial ROS production. This, in turn, led to a selective posttranslational modification, carbonylation of AIF, which significantly sensitized AIF to calpain-mediated processing. Recently, we were also able to define the pathway by which Ca2+ entered cells exposed to the PKC inhibitors. We found that the hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) mediated a sustained Ca2+ import, which was triggered by dephosphorylation of a critical residue within the conserved C-terminus of the channel in cells treated with PKC-inhibitors. Our findings reveal a novel role for the HCN2 channel in cell death signaling and uncover a new potential therapeutic drug target.