While historically regarded as an unfortunate consequence of aerobic metabolism, hydrogen peroxide and other related reactive species play vital roles in many physiological processes, ranging from cell signaling to the oxidative burst in the immune response. Hydrogen peroxide levels are most often perturbed by use of bolus addition, in which peroxide is added to cell culture and must diffuse through the membrane to exert intracellular effects. Although this technique has helped elucidate a qualitative relationship between peroxide levels and the resulting phenotype, its limitations prevents development of a mechanistic, quantitative relationship that resembles actual physiology. In my thesis, I have developed novel protein engineering methodologies to screen for next-generation enzymatic generators of peroxide to advance the field towards an ideal generator, one capable of tuning peroxide levels inside cells with minimal other perturbations. I have also coupled this work with development of a transport model, to predict changes in peroxide levels upon use of bolus addition or an enzymatic generator and thus initiate feedback between experiment and theory, refining the criteria for an ideal generator. The development of a peroxide generator will allow us to mechanistically link peroxide levels with relevant biological events and advance our understanding of redox biology.
Troy F. Langford, Beijing K. Huang, Joseph B. Lim, Sun Jin Moon, Hadley D. Sikes. “Monitoring the action of redox-directed cancer therapeutics using a human peroxiredoxin-2-based probe,” Nature Communications, 2018, in press.
Joseph B. Lim, Troy F. Langford, Beijing K. Huang, William M. Deen and Hadley D. Sikes. “A reaction diffusion model of cytosolic hydrogen peroxide,” Free Radical Biology and Medicine, 2016, 89: 85-90. DOI:10.1016/j.freeadbiomed. 2015.11.005.
B. Lim and H. D. Sikes. “Use of a genetically encoded sensor for high-throughput screening of enzymatic activity,” Protein Engineering, Design and Selection, 2015, 28 (3): 79-83. DOI:10.1093/protein/gzv003.
B. Lim, Kimberly A. Barker, Linda Jiang, Veronica Molina, Jessica Saifee, Hadley D. Sikes. “Insights into electron leakage in the reaction cycle of cytochrome P450 BM3 revealed by kinetic modeling and mutagenesis,” Protein Science, 2015, 24 (11): 1874-1883. . (Cover Story)
Joseph B. Lim, Beijing K. Huang, William M. Deen and Hadley D. Sikes. “Analysis of the lifetime and spatial localization of hydrogen peroxide generated in the cytosol using a reduced kinetic model,” Free Radical Biology and Medicine, 2015, 89: 47-53. DOI:10.1016/ j.freeradbiomed.2015.07.009.
B. Lim, K. A. Barker, B. K. Huang, and H. D. Sikes. “In-depth characterization of the fluorescent signal of HyPer, a probe for hydrogen peroxide, in bacteria exposed to external oxidative stress,” Journal of Microbiological Methods, 2014, 106: 33-39. DOI:10.1016/ j.mimet.2014.07.038.
Consultant, Bertha Center for Social Innovation and Entrepreneurship