The genetically encoded biosensor (PxIII-roGFP) as a tool to monitor the production and role of organic peroxides in redox signaling in mammalian inflammatory cells

• Grant number: 21/00349-0
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: February 01, 2022
• End date: February 01, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Flavia Carla Meotti
• Grantee: Mikaela Peglow Pinz
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 18/14898-2 - Investigations of the redox processes in inflammatory response and associated pathologies, AP.JP2
• Associated scholarship(s): 23/14552-7 - Use of a new biosensor (PxIII-roGFP2) to study the redox signaling in mammalian cells in an atherosclerosis cenarium., BE.EP.PD

Abstract

Inflammation is a physiological response to injury in order to repair damaged tissue and restore homeostasis. However, the perpetuation of inflammation is associated to an increase in oxidative species and redox imbalance. Organic peroxides formed in oxidative processes act as signaling molecules that stimulate gene expression and cell survival, but can also act as cytotoxic mediators. Recently, the development of biosensors based on fluorescent proteins such as GFP (green fluorescent protein) has received great prominence in the scientific community. These probes allow non-invasive and real-time monitoring of the cell oxidant production. In addition, they can be genetically directed to different cell compartments and promote dynamic studies in a sensitive and specific way. The sensitive redox variants of GFP (roGFP) can be coupled with enzymes sensitive to certain oxidants, for their specific monitoring. So far, there are no biosensors capable of detecting organic hydroperoxides. Since these hydroperoxides are important mediators of the inflammatory response, the first goal of this project is to perform the expression of a new roGFP2 biosensor fused to glutathione-like peroxidase from Trypanosoma brucei (PxIII-roGFP2) in Escherichia coli (E. coli) followed by the purification and characterization of the reaction kinetics with organic hydroperoxides of biological relevance. The second goal is to transfect this probe into different cell lines: human kidney embryonic cells (HEK293T), human promyelocytic leukemic cells (HL-60), human monocytic leukemic cells (THP-1) and Bone Marrow-derived Macrophage (BMDM) from murine to monitor the production of organic hydroperoxides under different stimuli. The third goal detect organic hydroperoxides in HL-60, THP-1, BMDM and HEK293T cells by mass spectrometry and compare the efficiency of the biosensor with the technique currently used. The fourth goal to perform a dynamic and spatio-temporal analysis of the levels of organic hydroperoxides. For this, the PxIII-roGFP2 biosensor will be genetically directed to the cytosol and mitochondria of HL-60 and THP-1 cells. The fifth goal to evaluate how the expression of genes involved in redox and inflammatory signaling can be affected in HL-60, THP-1 and BMDM cells (expressing the PxIII-roGFP2 biosensor) under different stimuli. The sixth goal is monitoring the redox status of activated HL-60, THP-1 and BMDM cells and correlate the expression of markers from the apoptotic process with the levels of organic hydroperoxides. In general, the present project seeks a better understanding of the role played by oxidants (more specifically organic hydroperoxides) in inflammatory processes. The use of the PxIII-roGFP2 biosensor is a very innovative biological instrument and, based on the characterization and application studies of this project, we will generate unprecedented opportunities for additional and more detailed studies in redox biology in different processes.