Analysis of the production of organic hydroperoxides by human promyelocytic leukemia cells (HL-60) in real-time through the biosensor PxIII-roGFP2

• Grant number: 23/08303-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: August 01, 2023
• End date: November 30, 2025
• Field of knowledge: Biological Sciences - Biochemistry
• Principal Investigator: Flavia Carla Meotti
• Grantee: Isadora Medeiros
• 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): 24/16691-7 - Analysis and comparison of response of the new probe PxIII-roGFP2 in different intracellular environments, BE.EP.IC

Abstract

The inflammatory response consists of the mobilization of several cellular and molecular mechanisms in the face of infection or tissue injury to prevent damage to the organism. The cells involved in this process are accountable for synthesizing large concentrations of oxidant molecules, mainly for combating microorganisms and cellular signaling. It is essential to thoroughly understand the role these molecules play in each stage of inflammation to better understand this process in different clinical conditions. The presence of oxidants and the cellular processes involved in their production can be analyzed using genetically encoded biosensors, such as those derived from the redox-sensitive Green Fluorescent Protein 2 (roGFP2) , whose use is promising. These tools have structural characteristics that allow real-time detection of oxidants produced in cells and have been widely used for the detection of hydrogen peroxide. However, none of these tools have been explored for the detection of organic peroxides, which also trigger significant inflammatory response. In this work, we will characterize the biosensor PxIII-roGFP2, obtained by fusing the roGFP2 protein with the non-selenium glutathione peroxidase enzyme (PxIII) from Trypanosoma brucei. This enzyme has a similar action to glutathione peroxidase, capable of mediating the reduction of phospholipid hydroperoxides. In this sense, this new biosensor may be a tool capable of detecting organic hydroperoxides involved in the inflammatory context at the intracellular level. Therefore, our first objective is to express and purify the biosensor PxIII-roGFP2, followed by characterizing kinetics with linoleic acid hydroperoxide. As a second objective, we aim to purify the pcDNA3.1 plasmid (vector that carries PxIII-roGFP2) and then, transfect human promyelocytic leukemia cells (HL-60) to express the biosensor PxIII-roGFP2 in their cytoplasm. This will be applied in the detection of oxidants produced by cells when differentiated and activated. Finally, as a third objective, we intend to evaluate the oxidative burst in HL-60 promyelocytic cells differentiated to neutrophil-like cells (dHL-60) and activated in groups transfected and non-transfected with PxIII-roGFP2, to ensure that the biosensor does not interfere with the cellular processes involved in the inflammatory response.