Thioredoxin Cross-Linking by Nitrogen Mustard in Lung Epithelial Cells: Formation of Multimeric Thioredoxin/Thioredoxin Reductase Complexes and Inhibition of Disulfide Reduction.

The thioredoxin (Trx) system, which consists of Trx and thioredoxin reductase (TrxR), is a major cellular disulfide reduction system important in antioxidant defense. TrxR is a target of mechlorethamine (methylbis(2-chloroethyl)amine; HN2), a bifunctional alkylating agent that covalently binds to selenocysteine/cysteine residues in the redox centers of the enzyme, leading to inactivation and toxicity. Mammalian Trx contains two catalytic cysteines; herein, we determined if HN2 also targets Trx. HN2 caused a time- and concentration-dependent inhibition of purified Trx and Trx in A549 lung epithelial cells. Three Trx cross-linked protein complexes were identified in both cytosolic and nuclear fractions of HN2-treated cells. LC-MS/MS of these complexes identified both Trx and TrxR, indicating that HN2 cross-linked TrxR and Trx. This is supported by our findings of a significant decrease of Trx/TrxR complexes in cytosolic TrxR knockdown cells after HN2 treatment. Using purified recombinant enzymes, the formation of protein cross-links and enzyme inhibition were found to be redox status-dependent; reduced Trx was more sensitive to HN2 inactivation than the oxidized enzyme, and Trx/TrxR cross-links were only observed using reduced enzyme. These data suggest that HN2 directly targets catalytic cysteine residues in Trx resulting in enzyme inactivation and protein complex formation. LC-MS/MS confirmed that HN2 directly alkylated cysteine residues on Trx, including Cys32 and Cys35 in the redox center of the enzyme. Inhibition of the Trx system by HN2 can disrupt cellular thiol-disulfide balance, contributing to vesicant-induced lung toxicity.

[1]  E. Weerapana,et al.  Covalent protein modification: the current landscape of residue-specific electrophiles. , 2015, Current opinion in chemical biology.

[2]  A. Miele,et al.  Thioredoxin Reductase and its Inhibitors , 2014, Current protein & peptide science.

[3]  D. Laskin,et al.  Cross-linking of thioredoxin reductase by the sulfur mustard analogue mechlorethamine (methylbis(2-chloroethyl)amine) in human lung epithelial cells and rat lung: selective inhibition of disulfide reduction but not redox cycling. , 2014, Chemical research in toxicology.

[4]  A. Holmgren,et al.  The thioredoxin antioxidant system. , 2014, Free radical biology & medicine.

[5]  C. Lillig,et al.  Thioredoxins, glutaredoxins, and peroxiredoxins--molecular mechanisms and health significance: from cofactors to antioxidants to redox signaling. , 2013, Antioxidants & redox signaling.

[6]  A. DeCaprio,et al.  Covalent adduction of nitrogen mustards to model protein nucleophiles. , 2013, Chemical research in toxicology.

[7]  Francisco J. Garcia,et al.  Bifunctional electrophiles cross-link thioredoxins with redox relay partners in cells. , 2013, Chemical research in toxicology.

[8]  A. Gow,et al.  Attenuation of acute nitrogen mustard-induced lung injury, inflammation and fibrogenesis by a nitric oxide synthase inhibitor. , 2012, Toxicology and applied pharmacology.

[9]  R. Begum,et al.  Sulfur, oxygen, and nitrogen mustards: stability and reactivity. , 2012, Organic & biomolecular chemistry.

[10]  A. Holmgren,et al.  Thioredoxin system in cell death progression. , 2012, Antioxidants & redox signaling.

[11]  W. Loke,et al.  Sulfur mustard and respiratory diseases , 2012, Critical reviews in toxicology.

[12]  A. Holmgren,et al.  The role of thioredoxin in the regulation of cellular processes by S-nitrosylation. , 2012, Biochimica et biophysica acta.

[13]  K. Fritz-Wolf,et al.  Crystal structure of the human thioredoxin reductase-thioredoxin complex. , 2011, Nature communications.

[14]  D. Laskin,et al.  Sulfur mustard-induced pulmonary injury: therapeutic approaches to mitigating toxicity. , 2011, Pulmonary Pharmacology & Therapeutics.

[15]  A. Gow,et al.  Functional and inflammatory alterations in the lung following exposure of rats to nitrogen mustard. , 2011, Toxicology and applied pharmacology.

[16]  D. Laskin,et al.  Selective targeting of selenocysteine in thioredoxin reductase by the half mustard 2-chloroethyl ethyl sulfide in lung epithelial cells. , 2010, Chemical research in toxicology.

[17]  N. Tretyakova,et al.  Proteomic analysis of DNA-protein cross-linking by antitumor nitrogen mustards. , 2009, Chemical research in toxicology.

[18]  R. Hartmann-Petersen,et al.  Thioredoxin Txnl1/TRP32 Is a Redox-active Cofactor of the 26 S Proteasome* , 2009, Journal of Biological Chemistry.

[19]  J. Yodoi,et al.  Thioredoxin 1 delivery as new therapeutics. , 2009, Advanced drug delivery reviews.

[20]  G. Di Trapani,et al.  Thioredoxin system inhibitors as mediators of apoptosis for cancer therapy. , 2009, Molecular nutrition & food research.

[21]  A. Holmgren,et al.  Regulation of the Catalytic Activity and Structure of Human Thioredoxin 1 via Oxidation and S-Nitrosylation of Cysteine Residues* , 2008, Journal of Biological Chemistry.

[22]  A. Holmgren,et al.  Inhibition of the Human Thioredoxin System , 2008, Journal of Biological Chemistry.

[23]  S. Muro,et al.  Thioredoxin-1 Ameliorates Cigarette Smoke-Induced Lung Inflammation and Emphysema in Mice , 2008, Journal of Pharmacology and Experimental Therapeutics.

[24]  Elias S. J. Arnér,et al.  Cell Death by SecTRAPs: Thioredoxin Reductase as a Prooxidant Killer of Cells , 2008, PloS one.

[25]  A. Holmgren,et al.  Targeting thioredoxin reductase is a basis for cancer therapy by arsenic trioxide , 2007, Proceedings of the National Academy of Sciences.

[26]  K. Fritz-Wolf,et al.  The structure of human thioredoxin reductase 1 provides insights into C-terminal rearrangements during catalysis. , 2007, Journal of molecular biology.

[27]  M. Ghanei,et al.  Long Term Consequences from Exposure to Sulfur Mustard: A Review , 2007, Inhalation toxicology.

[28]  Pedro A Fernandes,et al.  Similarities and differences in the thioredoxin superfamily. , 2006, Progress in biophysics and molecular biology.

[29]  Elias S. J. Arnér,et al.  Selenocysteine in proteins-properties and biotechnological use. , 2005, Biochimica et biophysica acta.

[30]  T. Sandström,et al.  The nitrogen mustard melphalan activates mitogen‐activated phosphorylated kinases (MAPK), nuclear factor‐κB and inflammatory response in lung epithelial cells , 2005, Journal of applied toxicology : JAT.

[31]  K. Becker,et al.  The thioredoxin system—From science to clinic , 2004, Medicinal research reviews.

[32]  J. Yodoi,et al.  Redox-active protein thioredoxin prevents proinflammatory cytokine- or bleomycin-induced lung injury. , 2003, American journal of respiratory and critical care medicine.

[33]  L. Herzenberg,et al.  Circulating thioredoxin suppresses lipopolysaccharide-induced neutrophil chemotaxis , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Elias S. J. Arnér,et al.  Physiological functions of thioredoxin and thioredoxin reductase. , 2000, European journal of biochemistry.

[35]  A. Holmgren,et al.  Essential Role of Selenium in the Catalytic Activities of Mammalian Thioredoxin Reductase Revealed by Characterization of Recombinant Enzymes with Selenocysteine Mutations* , 2000, The Journal of Biological Chemistry.

[36]  R. Glockshuber,et al.  Importance of Redox Potential for the in VivoFunction of the Cytoplasmic Disulfide Reductant Thioredoxin fromEscherichia coli * , 1999, The Journal of Biological Chemistry.

[37]  C. Fenselau,et al.  Covalent sequestration of the nitrogen mustard mechlorethamine by metallothionein. , 1998, Drug metabolism and disposition: the biological fate of chemicals.

[38]  M. Kunkel,et al.  Mechanisms of inhibition of the thioredoxin growth factor system by antitumor 2-imidazolyl disulfides. , 1998, Biochemical pharmacology.

[39]  N. Isowa,et al.  Amelioration of ischemia-reperfusion injury by human thioredoxin in rabbit lung. , 1997, The Journal of thoracic and cardiovascular surgery.

[40]  G. Powis,et al.  Crystal structures of reduced, oxidized, and mutated human thioredoxins: evidence for a regulatory homodimer. , 1996, Structure.

[41]  J. Dacre,et al.  Toxicology and pharmacology of the chemical warfare agent sulfur mustard. , 1995, Pharmacological reviews.

[42]  S. Rink,et al.  A mechlorethamine-induced DNA interstrand cross-link bends duplex DNA. , 1995, Biochemistry.

[43]  A. Holmgren,et al.  Determination of the reduction-oxidation potential of the thioredoxin-like domains of protein disulfide-isomerase from the equilibrium with glutathione and thioredoxin. , 1993, Biochemistry.

[44]  A. Holmgren,et al.  Rat liver thioredoxin and thioredoxin reductase: purification and characterization. , 1982, Biochemistry.