Modulation of Bax/Bak Dependent Apoptosis by Sirtuin 3 and Mitochondrial Permeability Transition by Sirtuin 4

Mitochondria are dynamic organelles that regulate a myriad of cellular functions, including energy production and metabolic regulation. Mitochondria are also a critical regulator of cell death signaling cascades modulating both apoptotic and necrotic cell death. However, what determines which cell death pathway is activated is still unclear. The mitochondrial/intrinsic pathway of apoptosis is dependent on the activation of pro-apoptotic proteins, Bax and Bak, which induce mitochondrial outer membrane permeabilization (MOMP). Once the integrity of outer mitochondrial membrane (OMM) is compromised, pro-apoptotic intermembrane space proteins like cytochrome c, Smac/Diablo, Omi/HtrA2 and AIF are released into the cytoplasm, which activates the post-mitochondrial phase of apoptosis. In humans, there are seven sirtuins (Sirtl-7), three of which are localized to the mitochondria (Sirt3-5). Sirt3 and Sirt5 have acetyltransferase activity, whereas Sirt4 has ADP-ribosyltransferase activity. Sirt3 is the major deacetylase in the mitochondrial matrix, and regulates various metabolic pathways by modulating the acetylation status of key metabolic enzymes. Although, metabolic regulation by Sirt3 has been studied extensively, the role of Sirt3 in cancer progression and regulation of the apoptotic pathway remains unclear. To sustain their rapid growth, cancer cells have a high glycolytic rate. This is achieved by overexpression of a key glycolytic enzyme, hexokinase II (HKII), which attaches to the OMM. HK.II binding to the OMM transmembrane voltage-dependent anion channel (VDAC) protein renders cancer cells resistant to a variety of cell death stimuli (e.g. DNA damage, oxidative stress and TNFa) by inhibiting activation of Bax/Bak. In this thesis, I present evidence that Sirt3 acts a mitochondrial localized tumor suppressor by modulating the binding of HKII on the OMM. Sirt3 dependent dissociation ofHKII from the OMM increases activation of pro apoptotic protein Bax/Bak in response to pro death stimuli, t-Bid and cisplatin. In contrast to apoptosis, opening of a multiprotein mitochondrial permeability transition pore (MPTP) by oxidative stress or mitochondrial calcium overload leads to necrotic cell death. The constituents of the MPTP, though controversial, consist of cyclophilin-D in the matrix, adenine nucleotide translocator (ANT) in the inner mitochondrial membrane, and VDAC on the OMM. Sirt4-mediated ribosylation of glutamate dehydrogenase-1 (GDH-1) inhibits its activity. In the second part of this thesis, I demonstrate that Sirt4 regulates the induction of the permeability transition by regulating the activity of GDH-1. Down-regulation of Sirt4 inhibits PIP induction by calcium overload and the thiol reactive agent phenylarsine oxide by increasing GDH-1 activity. Similarly, in intact cells, depletion of Sirt4 protected against PIP induction by cytotoxic drugs like INFa and doxorubicin. The evidence presented here demonstrates possible mechanisms by which mitochondria, more specifically, Sirt3 and Sirt4 modulate apoptotic and necrotic cell death, respectively