Formation, metabolism and cell signaling actions of tryptophan catabolism-derived electrophiles

Under steady-state conditions, >90% of tryptophan (Trp) catabolism occurs in the liver via the kynurenine (Kyn) pathway. However, this process has important pathophysiological roles in the brain, kidneys and heart, as well as in modulating immunity, tumor microenvironments, and pregnancy. Altered Kyn metabolism is observed in

aging, cardiovascular disease, organ injury, cancer, and transplantation, where it often is a strong predictor of outcome. Despite their relevance, the mechanisms behind the effects resulting from changes in systemic and local Kyn metabolism are poorly defined. Using untargeted and targeted mass spectrometry together with

isotopically labeled standards, we discovered the endogenous formation of the electrophilic mediator kynurenine- carboxyketoalkene (Kyn-CKA) secondary to in vivo Kyn deamination in humans and mice. Kyn-CKA reacts covalently with cysteine residues in transcription factors, transcriptional regulatory proteins and enzymes to

regulate protein and cellular function. We recently published that Kyn-CKA modulates TLR/NF-κB- and Keap1/Nrf2-dependent signaling and our preliminary data shows that Kyn-CKA also affects bioenergetic metabolism and impacts seemingly cysteine-independent processes. Furthermore, we now know that N-formyl-

kynurenine (NFK) and 3-hydroxy-kynurenine (OH-Kyn) also generate electrophiles in vivo. We hypothesize that Trp catabolism via the Kyn pathway results in the formation of novel bioactive products that adaptively modulate cellular responses via redox-dependent mechanisms. We propose to characterize the mechanisms that regulate

the formation of Trp-derived electrophiles via modulation of individual enzymes in the Kyn pathway and the identification of potential enzymes capable of catalyzing NFK, Kyn and OH-Kyn deamination. Trp-derived electrophiles undergo conjugation to glutathione and reductive inactivation, thus we will identify and kinetically

characterize the enzymes involved in these processes. Unbiased high resolution mass spectrometry and isotopically labeled precursors will be used to discover pathways for inactivation and biotransformation of Trp- derived electrophiles. Furthermore, we will characterize the effects of Trp-derived electrophiles on cellular and

tissue bioenergetic metabolism, first using hepatocytes and then expanding to other cell types as informed by in vivo formation data. In this regard, we will establish the impact of dietary Trp intake regulation on systemic and tissue Trp-derived electrophile levels, as well as assess a potential contribution of the microbiome to this process.

Finally, we will use spatial transcriptomics in conjunction with click chemistry-dependent labeling strategies to elucidate the sites of azido-Kyn deamination in vivo and define the impact of this process on electrophile-targeted and untargeted regions of interest to differentiate between proximal and paracrine signaling effects. Overall, our

discovery that the Kyn pathway is a source of bioactive electrophiles has the potential to transform our understanding of Trp biology. We expect our research provide a solid biochemical foundation that will enable the development of novel clinical approaches for conditions in which Trp metabolism is dysregulated.