Objective 1: To Investigate the role of mitochondrion in the molecular mechanisms of artemisinin toxicity
Does loss of respiratory activity result in decreased artemisinin toxicity?
If respiratory activity is required for artemisinin to exert its toxicity, yeast cells lacking functional electron transport chain (ETC) should display increased tolerance to artemisinin. If our results are consistent with the role of mitochondrial respiration on artemisinin toxicity, we will further refine the subunits of electron transport chain participate in artemisinin toxicity.
Which complexes of the electron transport chain contribute to artemisinin-induced cell death?
Previous study has shown that yeast cells lacking the alternative NADH dehydrogenase enzymes Nde1 and Ndi1, functioning to deliver reducing equivalents from the cytosol to ETC similar to complex I, are resistant to artemisinin and its derivatives. However, the exact site of ROS generation by artemisinin during oxidative phosphorylation remains elusive. This question can be addressed by screening mutant strains in each complex for sensitivity to artemisinin.
Does mitochondrial iron play an important role in artemisinin-induced cell death?
Free iron is important for bioactivation of artemisinin, although the involvement of mitochondrial iron for artemisinin toxicity has never been examined. It is possible that an extra-mitochondrial iron pool activates artemisinin prior to the drug exerting its toxicity toward mitochondria. Alternatively the drug may be transported into mitochondria in an inactive form before being activated by the free iron inside the mitochondria. We will therefore monitor if disruption in mitochondrial iron metabolism alters the cellular toxicity of artemisinin.
Does artemisinin exposure lead to elevated mitochondrial DNA damage?
The production of mitochondrial ROS can result in oxidative damage to mitochondrial proteins, membranes, and DNA. We will therefore monitor for mutations in mtDNA upon cellular exposure to toxic levels of artemisinin using an erythromycin resistance assay.
Is mitophagy involved in elimination of damaged mitochondria from artemisinin toxicity?
We predict that if mitophagy is involved in reducing cellular damage due to artemisinin toxicity cells deficient in mitophagy will exhibit a significant increased sensitivity to artemisinin. If this were the case, drugs that are able to inhibit mitophagy may have potential therapeutic use in combination with artemisinin.
Objective 2: To investigate the inhibition of ER to Golgi trafficking as a mechanism to sensitize cells to artemisinin
Our analysis using the S. cerevisiae models suggests a possible role for impaired ER to Golgi trafficking in the strong artemisinin sensitivity of cells lacking the lysine deacetylase RPD3. Retention of proteins in the ER leads to induction of the unfolded protein response (UPR) to clear the accumulated proteins, maintaining ER homeostasis [74, 75]. It is possible that the combined effect of ER stress, due to rpd3? mutations, with toxicity from artemisinin exposure may overwhelm the capacity of the cellular stress responses, leading to cell death.
