CHARACTERIZATION OF THE ROLE OF THE MITOCHONDRIAL AIF IN THE REGULATION OF PROGRAMMED CELL DEATH INDUCED BY CYTOTOXIC MOLECULES

• Main Author: LINA WIRASWATI ( NIM: 30511015 ), HESTI
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/22443
• Institution: Institut Teknologi Bandung

<p align="justify">While in healthy cells, through the activity of its respiratory chain, the mitochondrion acts as an energy factory and is necessary for cell survival, it is established that in various conditions of stress stimuli, this organelle plays an important role in the cellular response to death decision. Cytotoxic molecules can affect cell survival by not only provoking the disruption of vital mitochondrial functions but also by causing the release of mitochondrial proteins into the cytoplasm. The lethal action of those proteins can take the cell to the irreversible phase of death. One of these proteins is AIF (Apoptosis-Inducing Factor) that was initially as caspase-independent death. In cells induced to die, upon mitochondrial outer membrane permeabilization, AIF is translocated to the nucleus, where it fulfills its lethal action by participating to the chromatine condensation and degradation. AIF plays also an indispensible role in the mitochondrion of the healthy cell by regulating the activity of the respiratory chain. Recently, it was proposed that in addition to the regulation of the respiratory chain machinery, the mitochondrial activity of AIF could also be implicated in the reduction of molecules belonging to the quinones family by acting as a NADPH:quinone reductase (such as menadione, 2-methyl-1,4-naphtoquinone), facilitated the formation of semiquinone radical or hydroquinone that associated with rapid redox cycling to lead oxidative stress in the cell. Menadione cytotoxicity also associated with its arylation capacity with an important thiol proteins to form menadione-S-conjugate compound that toxic to the cell. As the cellular reduction and metabolization of quinones is established to be an important prerequisite for their cytotoxicity, understanding their cellular metabolization is of great importance in the field of oncology because members of this family have been successfully explored for their anti-tumor activity. Moreover, study the role of mitochondrial AIF is important, especially related to the development of cancer resistance and could contribute for finding a new cancer therapies aimed and targeting AIF. The research program was dedicated to the characterization of the impact of the mitochondrial activity of AIF on the cytotoxicity of menadione, a compound that belongs to the quinone family. The U2OS cell line used as model for the study was derived from a human osteosarcoma. Mammalian cell culture, drug treatment, and transfection experiments with siRNA (small interference Ribonucleic acid) were employed in order to study the cell respons to drug. Flow cytometry-based approaches were applied in order to study the cytotoxic effects of menadione on U2OS cells. Computational approaches were applied to evaluate and analysis the interaction of functional groups of menadione to AIF residues. Structural alignment was employed using FATCAT software. Molecular docking studies were performed by AutoDock Vina software in order to evaluate and analyze the role of mitochondrial AIF to menadione metabolism. The result showed the contribution of AIF to cell death induced by menadione, in condition this drug does not cause the loss of mitochondrial membrane permeabilization (MMP). It indicates that AIF stays in mitochondria to act its lethal activity and does not use its nuclease apoptotic function. Type of death by menadione induction is apoptosis in caspase-independent manner. Inhibition of Complex I of respiratory chain revealed that the cytoprotective effect of AIF depletion is independent-respiratory chain activity. It also suggests that the lethal action of AIF is not related to NADH:quinone reductase activity. Flow cytometry analysis showed the appearance of tiodione formation that also early event of cell death induced by menadione. siRNA technique also showed that AIF depletion reduced thiodione formation. This cytoprotective effect was accompanied by the maintenance of high levels of reduced gluthatione (GSH), which are normally depleted by menadione. Altogether, these results revealed that AIF facilitate menadione toxicity, thereby precipitating protein arylation and gluthatione depletion. Finally, computational study confirmed the involvement of AIF in menadione-induced death that showed AIF-menadione has interaction stability 20% higher than NQO1-menadione (enzyme that responsible to complete reduction of quinone). Structural alignment between AIF and Gluthatione-S-transferase (GST) showed that AIF does not have transferase catalytic residues. Binding energy analysis showed that thiodione has better affinity to AIF compare to GSH or menadione. Although the number of contact residues that surrounds thiodione is 45% higher compare to menadione or GSH, more than half of thiodione structure is not in frame of FAD position, even being outside the AIF. Meanwhile the structure of menadione and GSH is in frame of FAD position and were buried inside FAD domain of AIF. It indicated that AIF modulates conjugation process by means of stabilizing thiodione formation rather than interact with menadione or GSH directly.<p align="justify"> <br />