Analysis and comparison of response of the new probe PxIII-roGFP2 in different intracellular environments

• Grant number: 24/16691-7
• Support Opportunities: Scholarships abroad - Research Internship - Scientific Initiation
• Start date: March 10, 2025
• End date: July 09, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Flavia Carla Meotti
• Grantee: Isadora Medeiros
• Supervisor: Jose Pedro Friedmann Angeli
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: Julius-Maximilians-Universität Würzburg (JMU), Germany
• Associated to the scholarship: 23/08303-4 - Analysis of the production of organic hydroperoxides by human promyelocytic leukemia cells (HL-60) in real-time through the biosensor PxIII-roGFP2, BP.IC

Abstract

Lipid hydroperoxides are hallmarks of ferroptosis, a non-apoptotic cell death based on redox imbalance by phospholipid peroxidation, organic hydroperoxides production and dysfunction of iron metabolism. It is essential to thoroughly understand the role these molecules play in each stage of ferroptosis processes to better understand its development in different clinical conditions. However, assessing the real-time cellular redox status remains highly challenging. Based on this, we are focusing on characterizing a recently developed biosensor to monitor the production of organic hydroperoxides in mammalian cells. The probe PxIII-roGFP2 was obtained by binding the redox-sensitive Green Fluorescent Protein 2 (roGFP2) to the enzyme non-selenium glutathione peroxidase (PxIII) from Trypanosoma brucei. In our previous experiments, PxIII-roGFP2 showed itself to be more specific to react with organic hydroperoxides in vitro and had a more intense response in transfected cells when treated with this class of oxidants, showing to be a promising tool to detect organic hydroperoxides produced in intracellular context. Now we intend to deepen this characterization through the evaluation of the probe's response against oxidants produced internally. During this BEPE, we will first transfect HeLa cells to express PxIII-roGFP2 and then knockout glutathione peroxidase 4 (GPx4) gene, which codifies the enzyme responsible for phospholipids peroxides elimination. Next, cells will be treated with Erastin, a compound that can intensify lipid peroxidation by depleting intracellular synthesis of glutathione and dysfunction of iron metabolism. By this, inducing ferroptosis and creating an environment with prevalence of phospholipid peroxides, we can assess if the new probe can detect this type of oxidants produced internally. Next, we will transfect and treat peroxiredoxin 6 (PRDX6) knockout cells, to value probe's response in another lipid peroxides generating context, once this enzyme can eliminate lipid peroxides in a different mechanism from GPx4. We will also analyze PxIII-roGFP2 in front of ferroptosis caused exclusively by Erastin treatment. Manipulating these groups, we could generate intracellular environments with the prevalence of organic hydroperoxides, that are favorable to be detected by the new probe, and compare its functioning in these three different causing conditions. HeLa expressing PxIII-roGFP2 will be also treated with epidermal growth factor (EGF) to promote an intracellular production of hydrogen peroxide (H2O2), building a condition that is expected a less intense or slower probe's response. Then, the probe's response will be measured by its fluorescence variance in the two different redox contexts (one favorable to the probe's response and other that is not) and then compared. We will also perform these conditions in HeLa cells transfected to express the sensor Hyper7, which detects H2O2 in a specific way, to be used as a control of the H2O2 prevalence (when performing EGF treatment) and H2O2 reduction (when inducing ferroptosis). Moreover, with an internship at José Pedro Friedmann Angeli's Laboratory - a group with senior experience at manipulating, applying, and analyzing ferroptotic conditions - will be possible to acquire expertise in working with lipid peroxidation and non-apoptotic cellular death pathways that is beyond our current expertise, achieving an even higher level of academic production by our Laboratory.