Redox biology has been increasingly recognized as one of the key themes in cell signaling. A major obstacle in this field is the lack of capabilities to monitor clearly defined redox processes in live cells (including subcellular domains in live cells), tissues, or organisms. Consequently, molecular mechanisms of redox signaling and their connections to diseases remain largely uncharacterized. . The Ai laboratory has a focus on the development of genetically encoded, fluorescent-protein-based redox biosensors, with a long-term goal to develop intervention methods that may lead to novel therapeutics.
One direction of our work is to develop selective biosensors for individual reactive oxygen, nitrogen, and sulfur species (ROS/RNS/RSS). Our lab expanded the strategies for creating fluorescent redox probes, by introducing noncanonical amino acid (ncAAs) using a genetic code expansion technology into circularly permuted fluorescent proteins to derive novel biosensors. In particular, we developed the first, genetically encoded, selective fluorescent probes for hydrogen sulfide and peroxynitrite, both of which are important small molecules involved in intracellular signaling and oxidative stress.
Another direction is to engineer biosensors to directly sense thiol-based signaling components.We introduced disulfide bridges into a circularly permuted red fluorescent protein and created one of the first genetically encoded red fluorescent probes that can sense general redox changes in live cells. We further engineered this protein to derive a panel of redox-sensitive red fluorescent proteins (rxRFPs) showing different midpoint redox potential for imaging redox dynamics in various cellular compartments, such as mitochondria, ER, and the nucleus. Thioredoxins are highly conserved proteins playing essential roles in redox homeostasis and redox signaling, but there is a lack of method in monitoring thioredoxin redox dynamics in live cells. We developed the first genetically encoded fluorescent biosensor for thioredoxin redox by engineering a redox relay between human thioredoxin 1 (Trx1) and rxRFP. We utilized the resultant biosensor, TrxRFP1, to selectively monitor chemically induced perturbation of Trx redox in various mammalian cell lines. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells. We show that the glutathione and Trx redox systems can be individually perturbed without shifting the other. This finding corroborates the emerging perspective that various cellular redox couples are quasi-independent from each other and a thermodynamic equilibrium is not reached in live cells.
Currently, we are expanding genetically encoded fluorescent redox indicators for additional ROS/RNS/RSS and for further red-shifted excitation and emission. Moreover, we are combining redox biosensors with biosensors for Zn2+ and nucleotide sugars to explore the connections between redox signaling and Zn2+ or metabolism.
Y. Fan, M. Makar, M.X. Wang, and H-w. Ai*, Monitoring thioredoxin redox with a genetically encoded red fluorescent biosensor, Nat. Chem. Biol., 2017, 13: 1045-1052.
Z. Chen, and H-w. Ai*, Single Fluorescent Protein-Based Indicators for Zinc Ion (Zn2+), Anal. Chem., 2016, 88: 9029-9036.
Z. Chen, Z. Tian, K. Kallio, A.L. Oleson, A. Ji, D. Brochardt, D. Jiang, S.J. Remington*, and H-w. Ai*, The N-B Interaction through a Water Bridge: Understanding the Chemoselectivity of a Fluorescent Protein Based Probe for Peroxynitrite, J. Am. Chem. Soc., 2016, 138: 4900-4907.
Z. Chen, W. Ren, Q.E. Wright and H-w. Ai*, “Genetically Encoded Fluorescent Probe for the Selective Detection of Peroxynitrite“, J. Am. Chem. Soc., 2013, 135: 14940-14943.