TY - JOUR
T1 - Inhibitory nitrosylation of mammalian thioredoxin reductase 1
T2 - Molecular characterization and evidence for its functional role in cellular nitroso-redox imbalance
AU - Engelman, R
AU - Ziv, T
AU - Arner, ESJ
AU - Benhar, M
AU - Arnér, Elias S.J.
N1 - Funding Information: M.B. acknowledges funding from the Israel Science Foundation (ISF 1336/10 and 1574/14 ) and the Israel Cancer Research Fund (Project Grant). E.S.J.A. acknowledges funding support from Swedish Research Council (Medicine and Natural Sciences), Swedish Cancer Society and Karolinska Institutet . Publisher Copyright: © 2016 Elsevier Inc.
PY - 2016/8/1
Y1 - 2016/8/1
N2 - 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 (<10 μM) 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.
AB - 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 (<10 μM) 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.
KW - Cell death
KW - Nitrosylation
KW - Redox regulation
KW - Thioredoxin reductase
UR - https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=technion_pure2wos&SrcAuth=WosAPI&KeyUT=WOS:000381924100034&DestLinkType=FullRecord&DestApp=WOS
UR - http://www.scopus.com/inward/record.url?scp=84978419555&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.freeradbiomed.2016.06.032
DO - https://doi.org/10.1016/j.freeradbiomed.2016.06.032
M3 - Article
C2 - 27377780
SN - 0891-5849
VL - 97
SP - 375
EP - 385
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
ER -