Structural systems biology of microenvironmental oxidative stress and synthetic biology intervention

ABSTRACT I seek to characterize proteomic and fundamental molecular properties of bacteria and human cells under oxidative stress as a means to understand mechanistic underpinnings of sensitivity phenotypes. 1) Oxidative stress broadly impacts protein function, but it is very challenging to experimentally determine which protein malfunctions lead to cellular stress phenotypes. I propose a structural systems biology approach to answering these questions for induced stress in E. coli and human cells. Genome-scale metabolic network reconstruction will be integrated with solved and modeled protein structures to enable detailed models of proteomic oxidative damage and its impact on cellular metabolism, permitting stress simulations and prediction of metabolic bottlenecks. Predicted stress phenotypes will be validated by proteomics, metabolomics, and targeted in vitro enzyme activity assays under oxidative stress. This approach will reveal protein targets to inform future efforts in diagnosing and treating oxidative-stress-associated conditions including radiation toxicity, metabolic dysfunction, and aging. 2) I will develop a theoretical model of molecular sensitivity to oxidative damage of generic proteins of interest and serve for design and engineering more robust variants. Redox proteomics can identify oxidation sites at residue resolution on specific proteins or proteome-wide. Analysis of this data in the context of 3D protein structures will uncover molecular properties rendering some sites and proteins more vulnerable than others. I will validate the model in the context of mammalian glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which aggregates on the mitochondrial membrane causing dysfunction under oxidative stress. I will combine the model for molecular vulnerability to oxidation with evolutionary sequence conservation analysis to design oxidation-robust GAPDH variants. These designs will be experimentally characterized through recombinantly expressed proteins and cell-based assays for enzyme activity, oxidation states, and phenotypic outcomes under stress. Results will have implications for human diseases related to GAPDH dysfunction and will serve as a foundation for rational design of stress-resistant proteins, a significant technological advance. 3) I will investigate the functionality of specialized intrinsically disordered proteins (IDPs) for cellular protection against oxidative stress. Exploiting the model case of GAPDH oxidation again, here I will not alter GAPDH itself but introduce synthetic IDPs engineered to target the mitochondrial outer membrane or GAPDH directly through molecular interactions. Some IDPs are known to form protective barriers to reactive oxygen species (ROS) or disaggregate proteins, and I will investigate whether these can serve to protect GAPDH under stress. Designs will be tested on purified GAPDH in enzymatic activity assays and in cell-based assays for mitochondrial dysfunction and protein oxidation. This work would further the fundamental understanding of IDP function and lay groundwork for therapeutic development.