Mechanisms of Differential Activation in H2O2-Mediated Cellular Responses

University Of Arizona, Tucson AZ

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT My lab investigates how human cells detect and respond to stress, how these mechanisms fail in disease, and how they can be targeted therapeutically. We develop fluorescent reporters to monitor key stress response proteins and employ live-cell imaging and quantitative image analysis to capture their spatial and temporal dynamics. By integrating mathematical modeling and 'omics methods, we uncover how dynamic responses influence cell fate decisions. Our research strategically focuses on stresses and pathways relevant to human disease, ultimately aiming to translate these findings into effective treatments. Recently, we discovered a dose-dependent switch where transcription factors and kinases respond differently to varying levels of H2O2. At low H2O2, 'Group 1' proteins like p53, NRF2, and AKT are activated, while 'Group 2' proteins such as FOXO1, NF-κB, and GCN2 remain inactive. However, at higher concentrations, Group 2 proteins are activated while activation of Group 1 proteins is delayed until Group 2 proteins switch off. Prolonged activation of Group 2 is linked to cell death, underscoring the concept that oxidative stress can be categorized as eustress (mild and beneficial) or distress (severe, causing damage and disease). Our findings reveal the shift between oxidative eustress to distress is not gradual but dramatic, resembling a phase transition. H2O2 activates proteins by oxidizing reactive cysteine residues to sulfenic acid (SOH), which is then further modified to more stable post-translational modifications. Thousands of cysteines on diverse sets of proteins are known to be oxidized by H2O2, highlighting one of the key knowledge gaps in redox signaling: given the overwhelming number of reactive cysteines, how does H2O2 selectively oxidize the proper target? The underlying hypothesis of our research plan is that the dose-dependent activation of the two groups of proteins offers a window into how specificity is achieved in redox signaling. We build on our prior research with four separate projects to address this goal. In the first project, we take a targeted approach to identify the proteins directly oxidized under high H2O2 that activate group 2 proteins, and those that block group 1 activation. In the second project, we take an unbiased proteomics approach to identify key cysteine residues that are oxidized under different levels of H2O2 exposure. By coupling these data with phosphoproteomics, we will dissect how cysteine oxidation affects kinase activity. In the third project we test three different models on how PRDX proteins selectively control protein oxidation. Finally in the fourth project we make use of a human colonic monolayer system we developed to understand how redox signaling impacts gut homeostasis. In sum, our research plan aims to uncover the molecular mechanisms behind H2O2-mediated protein regulation, providing critical insights into how cells differentiate between eustress and distress. By elucidating the dose- dependent switches controlling transcription factors and kinases, we will advance understanding of redox signaling specificity and identify new therapeutic targets for oxidative stress-related diseases.