title: Inhibitory nitrosylation of mammalian thioredoxin reductase 1: Molecular characterization and evidence for its functional role in cellular nitroso-redox imbalance.
abstract: Mammalian thioredoxin 1 (Trx1) and the selenoprotein Trx reductase 1 (TrxR1) are key cellular enzymes that function coordinately in thiol-based redox regulation and signaling. Recent studies have revealed that the Trx1/TrxR1 system has an S-nitrosothiol reductase (denitrosylase) activity through which it can regulate nitric oxide-related cellular processes. In this study we revealed that TrxR1 is itself susceptible to nitrosylation, characterized the underlying mechanism, and explored its functional significance. We found that nitrosothiol or nitric oxide donating agents rapidly and effectively inhibited the activity of recombinant or endogenous TrxR1. In particular, the NADPH-reduced TrxR1 was partially and reversibly inhibited upon exposure to low concentrations (<10μM) of S-nitrosocysteine (CysNO) and markedly and continuously inhibited at higher doses. Concurrently, TrxR1 very efficiently reduced low, but not high, levels of CysNO. Biochemical and mass spectrometric analyses indicated that its active site selenocysteine residue renders TrxR1 highly susceptible to nitrosylation-mediated inhibition, and revealed both thiol and selenol modifications at the two redox active centers of the enzyme. Studies in HeLa cancer cells demonstrated that endogenous TrxR1 is sensitive to nitrosylation-dependent inactivation and pointed to an important role for glutathione in reversing or preventing this process. Notably, depletion of cellular glutathione with l-buthionine-sulfoximine synergized with nitrosating agents in promoting sustained nitrosylation and inactivation of TrxR1, events that were accompanied by significant oxidation of Trx1 and extensive cell death. Collectively, these findings expand our knowledge of the role and regulation of the mammalian Trx system in relation to cellular nitroso-redox imbalance. The observations raise the possibility of exploiting the nitrosylation susceptibility of TrxR1 for killing tumor cells.
authors.affiliation: Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Rotem
authors.initials: R
authors.lastname: Engelman
authors.affiliation: Smoler Proteomics Center and Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Tamar
authors.initials: T
authors.lastname: Ziv
authors.affiliation: Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Elias S J
authors.initials: ESJ
authors.lastname: Arnér
authors.affiliation: Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel. Electronic address: benhar@tx.technion.ac.il.
authors.forename: Moran
authors.initials: M
authors.lastname: Benhar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D003545:Cysteine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009249:NADP / Q000737:chemistry; D020030:Nitric Oxide Donors / Q000737:chemistry / Q000494:pharmacology; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D051381:Rats; D026403:S-Nitrosothiols / Q000737:chemistry / Q000494:pharmacology; D017279:Selenocysteine / Q000737:chemistry; D054481:Thioredoxin Reductase 1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D020030:Nitric Oxide Donors; D026403:S-Nitrosothiols; D017279:Selenocysteine; D009249:NADP; C021315:S-nitrosocysteine; C516124:TXNRD1 protein, human; D054481:Thioredoxin Reductase 1; C516126:Txnrd1 protein, rat; D005978:Glutathione; D003545:Cysteine
keywords: Cell death; Nitrosylation; Redox regulation; Thioredoxin reductase