What is known: Disulfide reduction-fueled enzymes s!upport homeostasis and combat oxidative damage that contributes to neurodegeneration, inflammatory diseases, and cancer. NADPH provides the reducing power for most anabolic and cytoprotective reduction reactions, yet only two enzymes can use NADPH to reduce cytosolic disulfides: thioredoxin reductase-1 (TrxR1) and glutathione reductase (Gsr) 1. Both TrxR1 and Gsr have active sites that are dominantly inhibited by electrophilic toxins and oxidants 2, 3. In Co-PI Schmidt?s lab, mice with TrxR1/Gsr-null livers uncovered unexpected robustness in the disulfide reductase systems, including an NADPH-independent pathway that uses catabolism of methionine (Met) to sustain redox homeostasis 4. Importantly, this pathway is also thought to sustain normal cells under oxidative or electrophilic stress 5. Met and Cys are the 2 sulfur (S)-amino acids found in proteins, but S-containing molecules synthesized from Met or Cys, including S-adenosyl-Met (SAM), glutathione (GSH), CoA, and others, are also important in redox, detox, energetics, biosynthesis, regulation, and other processes. Co-PI DeNicola has been studying the roles of altered S-amino acid metabolism in sustaining some cancers6. These studies are revealing how some cancers use altered S-amino acid redox metabolism, which could uncover targetable cancer-specific susceptibilities. Unresolved questions: It remains unknown how other metabolic activities, including those that directly utilize Met or Cys, as well as more peripheral systems that either (i) supply resources to these pathways; (ii) depend upon these pathways; or (iii) might, in some conditions, compete with these pathways for substrates, are realigned to help cells survive stress. We hypothesize that conversion to Met-dependence involves realignment of diverse metabolic pathways. This work is significant because a better understanding of these processes will uncover processes that can be therapeutically targeted to either specifically increase the robustness of critical cells under oxidative or toxic stress, or specifically increase the vulnerability of pathogenic cells in cancer or inflammatory diseases. New preliminary investigations in this resubmission demonstrate our ability to perform stable isotope flux labeling studies in whole mice and in mouse-derived hepatic organoids. What is proposed: In this revised multi-institution collaborative project, we will define the metabolic pathway realignments that occur when hepatocytes switch from NADPH-dependent to -independent disulfide reduction. We propose 3 Specific Aims:
Aim 1, Define how NADPH- versus Met-fueled disulfide reductase homeostasis influences S-metabolism prioritization.
Aim 2, Define how re-wiring of serine metabolism supports Met-fueled disulfide reductase homeostasis.
Aim 3, Test whether Met-dependent survival increases the activity and dependence on liver methyltransferases. Anticipated outcomes, value: This project will help us understand how global shifts in hepatic metabolism occurs in response to severe oxidative or electrophilic stress in liver, and how this helps sustain health.
! Recent investigations have revealed that mammalian cells, but no microbes including bacterial or lower eukaryotic pathogens, have a backup system for maintaining redox- and metabolic-homeostasis when the canonical thioredoxin reductase- or glutathione reductase-driven systems are impeded. Unlike the canonical systems, which are fueled by oxidation of NADPH, this system has been shown to use catabolism of the essential sulfur amino acid methionine via a pathway that has been rigorously defined, yet little is known about how ancillary metabolic pathways must be reprioritized to support this pathway. Here two teams, one with expertise in mouse models of redox biology and one with expertise on sulfur amino acid metabolism-based cell strengths and vulnerabilities, use cutting-edge approaches to understand how these pathways work, with a vision toward future clinical applications that exploit this knowledge to support beneficial cells and ablate pathogenic cells in patients.
