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Item Drug Inhibition of Redox Factor-1 Restores Hypoxia-Driven Changes in Tuberous Sclerosis Complex 2 Deficient Cells(MDPI, 2022-12-15) Champion, Jesse D.; Dodd, Kayleigh M.; Lam, Hilaire C.; Alzahrani, Mohammad A. M.; Seifan, Sara; Rad, Ellie; Scourfield, David Oliver; Fishel, Melissa L.; Calver, Brian L.; Ager, Ann; Henske, Elizabeth P.; Davies, David Mark; Kelley, Mark R.; Tee, Andrew R.; Pediatrics, School of MedicineTherapies with the mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for tuberous sclerosis complex (TSC) patients. Here, we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses a redox signaling activity that stimulates the transcriptional activity of STAT3, NF-kB, and HIF-1α, which are involved in inflammation, proliferation, angiogenesis, and hypoxia, respectively. Here, we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor APX3330 was effective at blocking the hyperactivity of STAT3, NF-kB, and HIF-1α. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors such as STAT3, NF-kB, and HIF-1α as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits compared to using mTORC1 inhibitors alone.Item In vitro and In vivo evidence demonstrating chronic absence of Ref-1 Cysteine 65 impacts Ref-1 folding configuration, redox signaling, proliferation and metastasis in pancreatic cancer(Elsevier, 2024) Mijit, M.; Kpenu, E.; Chowdhury, N. N.; Gampala, S.; Wireman, R.; Liu, S.; Babb, O.; Georgiadis, M. M.; Wan, J.; Fishel, M. L.; Kelley, M. R.; Pediatrics, School of MedicineRef-1/APE1 (Redox Effector/Apurinic Endonuclease 1) is a multifunctional enzyme that serves as a redox factor for several transcription factors (TFs), e.g., NF-kB, HIF-1α, which in an oxidized state fail to bind DNA. Conversion of these TFs to a reduced state serves to regulate various biological responses such as cell growth, inflammation, and cellular metabolism. The redox activity involves a thiol exchange reaction for which Cys65 (C65) serves as the nucleophile. Using CRISPR editing in human pancreatic ductal adenocarcinoma (PDAC) cells, we changed C65 to Ala (C65A) in Ref-1 to evaluate alteration of Ref-1 redox dynamics as well as chronic loss of Ref-1 redox activity on cell signaling pathways, specifically those regulated by NF-kB and HIF-1α. The redox activity of Ref-1 requires partial unfolding to expose C65, which is buried in the folded structure. Labeling of Ref-1 with polyethylene glycol-maleimide (PEGm) provides a readout of reduced Cys residues in Ref-1 and thereby an assessment of partial unfolding in Ref-1. In comparing Ref-1WT vs Ref-1C65A cell lines, we found an altered distribution of oxidized versus reduced states of Ref-1. Accordingly, activation of NF-kB and HIF-1α in Ref-1C65A lines was significantly lower compared to Ref-1WT lines. The bioinformatic data revealed significant downregulation of metabolic pathways including OXPHOS in Ref-1C65A expressing clones compared to Ref-1WT line. Ref-1C65A also demonstrated reduced cell proliferation and use of tricarboxylic acid (TCA) substrates compared to Ref-1WT lines. A subcutaneous as well as PDAC orthotopic in vivo model demonstrated a significant reduction in tumor size, weight, and growth in the Ref-1C65A lines compared to the Ref-1WT lines. Moreover, mice implanted with Ref-1C65A redox deficient cells demonstrate significantly reduced metastatic burden to liver and lung compared to mice implanted with Ref-1 redox proficient cells. These results from the current study provide direct evidence that the chronic absence of Cys65 in Ref-1 results in redox inactivity of the protein in human PDAC cells, and subsequent biological results confirm a critical involvement of Ref-1 redox signaling and tumorigenic phenotype.Item Metabolism of hydrogen sulfide (H2S) and Production of Reactive Sulfur Species (RSS) by superoxide dismutase(Elsevier, 2017-11-20) Olson, Kenneth R.; Gao, Yan; Arif, Faihaan; Arora, Kanika; Patel, Shivali; DeLeon, Eric. R.; Sutton, Thomas R.; Feelisch, Martin; Cortese-Krott, Miriam M.; Straub, Karl D.; Cellular and Integrative Physiology, School of MedicineReactive sulfur species (RSS) such as H2S, HS•, H2Sn, (n = 2–7) and HS2•- are chemically similar to H2O and the reactive oxygen species (ROS) HO•, H2O2, O2•- and act on common biological effectors. RSS were present in evolution long before ROS, and because both are metabolized by catalase it has been suggested that “antioxidant” enzymes originally evolved to regulate RSS and may continue to do so today. Here we examined RSS metabolism by Cu/Zn superoxide dismutase (SOD) using amperometric electrodes for dissolved H2S, a polysulfide-specific fluorescent probe (SSP4), and mass spectrometry to identify specific polysulfides (H2S2-H2S5). H2S was concentration- and oxygen-dependently oxidized by 1 μM SOD to polysulfides (mainly H2S2, and to a lesser extent H2S3 and H2S5) with an EC50 of approximately 380 μM H2S. H2S concentrations > 750 μM inhibited SOD oxidation (IC50 = 1.25 mM) with complete inhibition when H2S > 1.75 mM. Polysulfides were not metabolized by SOD. SOD oxidation preferred dissolved H2S over hydrosulfide anion (HS-), whereas HS- inhibited polysulfide production. In hypoxia, other possible electron donors such as nitrate, nitrite, sulfite, sulfate, thiosulfate and metabisulfite were ineffective. Manganese SOD also catalyzed H2S oxidation to form polysulfides, but did not metabolize polysulfides indicating common attributes of these SODs. These experiments suggest that, unlike the well-known SOD-mediated dismutation of two O2•- to form H2O2 and O2, SOD catalyzes a reaction using H2S and O2 to form persulfide. These can then combine in various ways to form polysulfides and sulfur oxides. It is also possible that H2S (or polysulfides) interact/react with SOD cysteines to affect catalytic activity or to directly contribute to sulfide metabolism. Our studies suggest that H2S metabolism by SOD may have been an ancient mechanism to detoxify sulfide or to regulate RSS and along with catalase may continue to do so in contemporary organisms., • Polysulfides are reactive sulfide species (RSS) and are similar to reactive oxygen species (ROS). • RSS may be the antecedent of redox regulatory and stress-related modalities. • RSS likely persist in modern-day organisms and are regulated by SOD.Item The protease activity of human ATG4B is regulated by reversible oxidative modification(Taylor & Francis, 2020-10) Zheng, Xueping; Yang, Zuolong; Gu, Qianqian; Xia, Fan; Fu, Yuanyuan; Liu, Peiqing; Yin, Xiao-Ming; Li, Min; Pathology and Laboratory Medicine, School of MedicineMacroautophagy/autophagy plays a pivotal role in cytoplasmic material recycling and metabolic turnover, in which ATG4B functions as a "scissor" for processing pro-LC3 and lipidated LC3 to drive the autophagy progress. Mounting evidence has demonstrated the tight connection between ROS and autophagy during various pathological situations. Coincidentally, several studies have shown that ATG4B is potentially regulated by redox modification, but the underlying molecular mechanism and its relationship with autophagy is ambiguous. In this study, we verified that ATG4B activity was definitely regulated in a reversible redox manner. We also determined that Cys292 and Cys361 are essential sites of ATG4B to form reversible intramolecular disulfide bonds that respond to oxidative stress. Interestingly, we unraveled a new phenomenon that ATG4B concurrently formed disulfide-linked oligomers at Cys292 and Cys361, and that both sites underwent redox modifications thereby modulating ATG4B activity. Finally, increased autophagic flux and decreased oxidation sensitivity were observed in Cys292 and Cys361 double site-mutated cells under normal growth conditions. In conclusion, our research reveals a novel molecular mechanism that oxidative modification at Cys292 and Cys361 sites regulates ATG4B function, which modulates autophagy.Item Synthesis, Redox and Spectroscopic Properties of Pterin of Molybdenum Cofactors(MDPI, 2022-05-22) Colston, Kyle J.; Basu, Partha; Chemistry and Chemical Biology, School of SciencePterins are bicyclic heterocycles that are found widely across Nature and are involved in a variety of biological functions. Notably, pterins are found at the core of molybdenum cofactor (Moco) containing enzymes in the molybdopterin (MPT) ligand that coordinates molybdenum and facilitates cofactor activity. Pterins are diverse and can be widely functionalized to tune their properties. Herein, the general methods of synthesis, redox and spectroscopic properties of pterin are discussed to provide more insight into pterin chemistry and their importance to biological systems.