Molecular and biological mechanism studies of protein photooxidation to control cell fate and their therapeutic applications

Abstract

Oxidation in biological systems has long been studied in terms of longevity. The oxidation generally occurs via labile molecules such as reactive oxygen species (ROS). Current studies have recognized oxidation as an important endogenous signaling agent where its subcellular location, amount and temporal resolution determine cell fate. This notion offered a plethora of research focusing on oxidative modulation of biological processes, but the complex mechanism and the dynamic nature of ROS hindered its application beyond plain apoptosis. This dissertation focuses on the therapeutic potency and biological mechanism of protein photooxidation with respect to molecular engineering and molecular biology. The thesis covers molecular design strategies to benefit from ROS and ROS-related pathways, structural biological studies of a protein photooxidation route, plausible mode-of-actions (MoAs) regarding oxidized proteins based on proteomics, and potential clinical advantages of controlled protein oxidation. In Chapter 2, therapeutic implication of photooxidative autophagy inhibition in drug resistant cancer is discuessed using Ir(III) complexes. A molecular design utilizing biocompatible neutral Ir(III) photosensitizers to regulate reactive oxygen species (ROS) generation at lysosomes and to inhibit autophagy is presented. The molecular mechanisms governing the biocompatibility and lysosome specificity of these Ir(III) complexes, the biological mechanisms underlying autophagy inhibition through lysosomal oxidation, and the potency of this strategy in drug resistant cells are investigated in vitro and in vivo with a compound optimized for therapeutic window of photodynamic therapy. In Chapter 3, a molecular design to overcome hypoxia and induce trigger immunogenic cell death is presented based on photoelectrochemical and protein-based analyses. An amphiphilic photocatalyst oxidizes water to supply H2O2 which can be converted into a hydroxyl radical (•OH) after accepting an electron. The catalytic process can be accelerated under hypoxia, making the strategy advantageous for future clinical use. The photocatalyst localizes to intracellular membranes, oxidizing proteins and inducing non-canonical pyroptosis. This process destabilizes protein folding, exacerbates mitochondrial and ER stress, and activates inflammasome caspases, leading to gasdermin D-mediated pyroptosis, revealing membrane protein oxidation as a trigger for non-canonical pyroptosis. Chapter 4 investigates an unexpected oxidation occurring at core region of a folded protein. A novel photooxidation mechanism of a protein is suggested as O2-confinement oxidation pathway. The oxidation conveys trapping of dissolved O2 within protein cavities, where it interacts with photosensitizing tryptophan residues, generating ROS under visible light exposure. It occurs via spin- flip based electron transfer and accelerate oxidative damage to core residues, collapsing protein integrity. A broad influence of this exceptional photooxidation pathway was further confirmed based on whole- cell proteomics.