Role of lysosomes in oxidative stress induced regulated cell death
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Date
2020
Authors
Pandian, Nagakannan
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Abstract
Oxidative stress caused either due to overproduction of reactive oxygen species (ROS) or failure of endogenous antioxidants leads to damage of cellular macromolecules and intracellular organelles, which if remain uncontrolled may lead to cell death. Lysosomes play a vital role in maintaining cellular homeostasis by acting as the terminal degradative organelles in autophagic process. Autophagy is a highly regulated pro-survival pathway that delivers the toxic cellular debris and damaged organelles to the highly acidic lysosomes for digestion. Any impairment in lysosomal function can lead to inhibition of autophagic process and protein accumulation. Importantly, due to the presence of potent hydrolyzing enzymes within its lumen, any compromise in lysosomal membrane integrity can lead to severe cellular damage and cell death. Several factors including ROS has been shown to contribute to lysosomal membrane permeabilization (LMP); however, the molecular mechanisms involved in the induction of cell death by LMP during uncontrolled oxidative stress is poorly understood.
In this thesis, I demonstrate that excessive oxidative stress induced by inactivation of endogenous thiol antioxidants such as thioredoxin (Trx) and glutathione (GSH) systems promote oxidative damage of lysosomes and results in activation of apoptotic and non-apoptotic cell death mechanisms. Inactivation of Trx system led to blockade of autophagic process and protein accumulation, which was attributed to loss of lysosomal proteolytic ability and acidity. On further exploration, I discovered that inhibition of Trx system caused oxidative inactivation of cathepsin L, a pro-autophagic lysosomal protease and LMP resulting in the induction of apoptotic pathway. Using small molecule inhibitor screening, cathepsin B was found to be the executioner of apoptotic cell death in this model.
Complementary studies in another model of a recently identified non-apoptotic cell death mechanism known as ferroptosis further confirms cathepsin B as a major executioner of cell death under oxidative stress conditions. Iron mediated lipid peroxidation is a major player in ferroptosis. GSH and glutathione peroxidase 4 (GPX4) are required for proper scavenging of lipid peroxides and hence depletion of GSH or deletion of GPX4 results in ferroptosis. Using pharmacologic and gene-knockout approaches, cathepsin B was identified as the cell death executioner in ferroptosis which was confirmed in primary fibroblasts derived from cathepsin B knockout (Ctsb-/-) mice. Accordingly, inhibition of cathepsin B also attenuated several markers of cell death and preserved the integrity of mitochondrial and lysosomal membranes. Using inhibitor screening and knockout cell lines, cathepsin B was found to act as a histone H3 protease during ferroptosis that was not previously recognized in any physiological or pathological settings.
Taken together, this thesis establishes that cathepsin B acts as an executioner of cell death induced by LMP following failure of intracellular redox balance. These findings can have implications in several disease conditions including traumatic injuries, neurodegenerative diseases and cancers where oxidative stress plays a key role in occurrence and development of pathologies.
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Keywords
oxidative stress, lysosomes, cathepsins, autophagy, ferroptosis, apoptosis