Structure-guided engineering of a fast genetically encoded sensor for real-time H2O2 monitoring.

Lee, J. D.; Won, W.; Kimball, K.; Wang, Y.; Yeboah, F.; Evitts, K.; Neiswanger, C.; Schattauer, S.; Rappleye, M.; Bremner, S. B.; Chun, C.; Smith, N.; Mack, D. L.; Young, J. E.; Lee, C. J.; Chavkin, C.; Berndt, A.

doi:10.1101/2024.01.31.578117

bioRxiv · 10.1101/2024.01.31.578117

Structure-guided engineering of a fast genetically encoded sensor for real-time H2O2 monitoring.

Lee, J. D.·Won, W.·Kimball, K.·Wang, Y.·Yeboah, F.·Evitts, K.·Neiswanger, C.·Schattauer, S.·Rappleye, M.·Bremner, S. B.·Chun, C.·Smith, N.·Mack, D. L.·Young, J. E.·Lee, C. J.·Chavkin, C.·Berndt, A.

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Abstract

Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H2O2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS), leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity or response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP), we created a novel, green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics, ideal for monitoring intracellular H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse neurons and astrocytes in ex vivo brain slices. These applications demonstrate oROSs capabilities to monitor H2O2 as a secondary response to pharmacologically induced oxidative stress, G-protein coupled receptor (GPCR)-induced cell signaling, and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via A{beta}-putriscine-MAOB axis, highlighting the sensors relevance in validating neurodegenerative disease models. oROS is a versatile tool, offering a window into the dynamic landscape of H2O2 signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for diseases associated with oxidative stress, such as cancer, neurodegenerative disorders, and cardiovascular diseases.

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Lee, J. D., Won, W., Kimball, K., Wang, Y., Yeboah, F., Evitts, K., Neiswanger, C., Schattauer, S., Rappleye, M., Bremner, S. B., Chun, C., Smith, N., Mack, D. L., Young, J. E., Lee, C. J., Chavkin, C., Berndt, A.. 2024-02-04. Structure-guided engineering of a fast genetically encoded sensor for real-time H2O2 monitoring.. https://doi.org/10.1101/2024.01.31.578117

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