Design and application of a genetically-encoded probe for peroxiredoxin-2 oxidation in human cells

Abstract

Hydrogen peroxide (H₂O₂) is a well-known oxidant species commonly produced in eukaryotic organisms as a result of cellular metabolism that plays a central role in numerous processes in cells, and dysregulation of this species can result in a number of different disease states in human cells. In the case of cancer, elevated metabolism is believed to result in higher rates of H₂O₂ production in these cells, as well as more susceptibility to H₂O₂-induced apoptosis than normal cells. To this end, researchers have identified several therapeutic compounds that are believed to kill cancer cells via the intracellular elevation of one or more oxidants. However, due to the limitations of current tools for detection of these species, little is known about which therapeutic compounds induce toxicity via elevation of specific oxidants, which would aid in the identification of susceptible tumors to these treatments.

Currently, the main limitation of genetically-encoded tools for detection of H₂O₂ in these applications is the low sensitivity to H₂O₂ . Most genetically-encoded probes for this species used in human cells utilize H₂O₂-responsive domains with reaction rate coefficients nearly two orders of magnitude lower than other, more reactive peroxidases in the cell, such as peroxiredoxins (Prxs). In this regard, several studies have demonstrated that Prxs should react with the majority of intracellular H₂O₂ on the basis of a high reaction rate coefficient with H₂O₂ and intracellular abundance. In light of these studies, research in the field of redox biology has shifted to focus more on Prxs' role as natural sensors of H₂O₂ fluctuations in human cells. To this end, the first part of my thesis project focuses on the development of a genetically-encoded probe for H₂O₂-mediated human Prx2 oxidation in human cells.

The second part of my thesis focuses on the application of this probe in a high-throughput screen to identify small-molecule cancer therapeutics that act through H₂O₂-mediated mechanisms. Together, this thesis lays the foundation for a new class of genetically-encoded sensors that enable specific, sensitive measurement of H₂O₂ perturbations in human cells in response to redox-based therapeutics, which will facilitate the advancement of these therapeutic compounds in the future.