The Interaction of Thioredoxin with Txnip

The thioredoxin system plays an important role in maintaining a reducing environment in the cell. Recently, several thioredoxin binding partners have been identified and proposed to mediate aspects of redox signaling, but the significance of these interactions is unclear in part due to incomplete understanding of the mechanism for thioredoxin binding. Thioredoxin-interacting protein (Txnip) is critical for regulation of glucose metabolism, the only currently known function of which is to bind and inhibit thioredoxin. We explored the mechanism of the Txnip-thioredoxin interaction and present evidence that Txnip and thioredoxin form a stable disulfide-linked complex. We identified two Txnip cysteines that are important for thioredoxin binding and showed that this interaction is consistent with a disulfide exchange reaction between oxidized Txnip and reduced thioredoxin. These cysteines are not conserved in the broader family of arrestin domain-containing proteins, and we demonstrate that the thioredoxin-binding property of Txnip is unique. These data suggest that Txnip is a target of reduced thioredoxin and provide insight into the potential role of Txnip as a redox-sensitive signaling protein. The thioredoxin system plays an important role in maintaining a reducing environment in the cell. Recently, several thioredoxin binding partners have been identified and proposed to mediate aspects of redox signaling, but the significance of these interactions is unclear in part due to incomplete understanding of the mechanism for thioredoxin binding. Thioredoxin-interacting protein (Txnip) is critical for regulation of glucose metabolism, the only currently known function of which is to bind and inhibit thioredoxin. We explored the mechanism of the Txnip-thioredoxin interaction and present evidence that Txnip and thioredoxin form a stable disulfide-linked complex. We identified two Txnip cysteines that are important for thioredoxin binding and showed that this interaction is consistent with a disulfide exchange reaction between oxidized Txnip and reduced thioredoxin. These cysteines are not conserved in the broader family of arrestin domain-containing proteins, and we demonstrate that the thioredoxin-binding property of Txnip is unique. These data suggest that Txnip is a target of reduced thioredoxin and provide insight into the potential role of Txnip as a redox-sensitive signaling protein. Thioredoxin is a ubiquitous disulfide oxidoreductase that, along with the glutathione system, plays a major role in maintaining the cytoplasm in a reducing environment. Thioredoxin activity is mediated by a pair of cysteine thiols at its active site (human thioredoxins C32 and C35) that are oxidized during reduction of the substrate. By maintaining this reducing environment, thioredoxin is a critical defense against excess concentrations of reactive oxygen species, which are deleterious to cells and implicated in the pathophysiology of diseases such as atherosclerosis (1Griendling K.K. FitzGerald G.A. Circulation. 2003; 108: 1912-1916Crossref PubMed Scopus (774) Google Scholar, 2Ozcan U. Cao Q. Yilmaz E. Lee A.H. Iwakoshi N.N. Ozdelen E. Tuncman G. Gorgun C. Glimcher L.H. Hotamisligil G.S. Science. 2004; 306: 457-461Crossref PubMed Scopus (2972) Google Scholar), diabetes (3Furukawa S. Fujita T. Shimabukuro M. Iwaki M. Yamada Y. Nakajima Y. Nakayama O. Makishima M. Matsuda M. Shimomura I. J. Clin. Invest. 2004; 114: 1752-1761Crossref PubMed Scopus (4000) Google Scholar), and arthritis (4Hitchon C.A. El-Gabalawy H.S. Arthritis Res. Ther. 2004; 6: 265-278Crossref PubMed Scopus (492) Google Scholar, 5Henrotin Y. Kurz B. Aigner T. Osteoarthritis Cartilage. 2005; 13: 643-654Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). The reducing environment of the cell is also important for keeping protein thiols reduced, so that under normal conditions proteins contain many free sulfhydryl groups and relatively rare accessible disulfides (6Gilbert H.F. Adv. Enzymol. Relat. Areas Mol. Biol. 1990; 63: 69-172PubMed Google Scholar). In addition to the classic reducing activity of thioredoxin, recent evidence suggests that the redox state of thioredoxin may itself be an important component of redox signaling pathways. Thioredoxin reportedly binds a number of transcription factors and signaling molecules, including NF-κB p50 subunit (7Qin J. Clore G.M. Kennedy W.M. Huth J.R. Gronenborn A.M. Structure. 1995; 3: 289-297Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar), Ref-1 (8Qin J. Clore G.M. 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One of the better characterized interactions is that of reduced thioredoxin with apoptosis signal-regulating kinase 1 (ASK1), 2The abbreviations used are: ASK1, apoptosis signal-regulating kinase 1; GST, glutathione S-transferase; DTT, dithiothreitol; GFP, green fluorescent protein; Bis-Tris, 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)propane-1,3-diol.2The abbreviations used are: ASK1, apoptosis signal-regulating kinase 1; GST, glutathione S-transferase; DTT, dithiothreitol; GFP, green fluorescent protein; Bis-Tris, 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)propane-1,3-diol. which plays a key role in promoting stress-induced apoptosis (12Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2075) Google Scholar). Because only reduced thioredoxin is thought to bind ASK1, the interaction can be controlled by the redox state of the cell: during conditions of oxidative stress, ASK1 is released from thioredoxin and promotes apoptosis. The identification of thioredoxin and ASK1 cysteines required for the interaction (13Zhang R. Al-Lamki R. Bai L. Streb J.W. Miano J.M. Bradley J. Min W. Circ. Res. 2004; 94: 1483-1491Crossref PubMed Scopus (213) Google Scholar) supports the hypothesis that thioredoxin forms a mixed disulfide complex with ASK1. However, the implications of this interaction are not clear in part due to (i) the lack of direct evidence that thioredoxin can form a stable mixed disulfide and (ii) the lack of a plausible mechanism for formation of this disulfide in the normal reducing environment of the cell. Txnip (thioredoxin-interacting protein, also called “vitamin-D3-up-regulated protein-1” (14Chen K.S. DeLuca H.F. Biochim. Biophys. Acta. 1994; 1219: 26-32Crossref PubMed Scopus (269) Google Scholar) and “thioredoxin-binding protein 2”), is a 50-kDa protein with structural homology to the arrestins. Despite the arrestin homology, currently the only known function of Txnip is to bind thioredoxin and inhibit thioredoxin-reducing activity (15Yamanaka H. Maehira F. Oshiro M. Asato T. Yanagawa Y. Takei H. Nakashima Y. Biochem. Biophys. Res. Commun. 2000; 271: 796-800Crossref PubMed Scopus (102) Google Scholar, 16Junn E. Han S.H. Im J.Y. Yang Y. Cho E.W. Um H.D. Kim D.K. Lee K.W. Han P.L. Rhee S.G. Choi I. J. Immunol. 2000; 164: 6287-6295Crossref PubMed Scopus (399) Google Scholar, 17Nishiyama A. Matsui M. Iwata S. Hirota K. Masutani H. Nakamura H. Takagi Y. Sono H. Gon Y. Yodoi J. J. Biol. Chem. 1999; 274: 21645-21650Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). Txnip, like ASK1, appears to form a disulfide bond with thioredoxin, because the interaction requires the presence of the thioredoxin active-site cysteines (17Nishiyama A. Matsui M. Iwata S. Hirota K. Masutani H. Nakamura H. Takagi Y. Sono H. Gon Y. Yodoi J. J. Biol. Chem. 1999; 274: 21645-21650Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). Consistent with this role, overexpression of Txnip decreases thioredoxin-reducing activity, increases redox stress, inhibits growth and hypertrophy, and causes increased apoptosis (16Junn E. Han S.H. Im J.Y. Yang Y. Cho E.W. Um H.D. Kim D.K. Lee K.W. Han P.L. Rhee S.G. Choi I. J. Immunol. 2000; 164: 6287-6295Crossref PubMed Scopus (399) Google Scholar, 18Schulze P.C. De Keulenaer G.W. Yoshioka J. Kassik K.A. Lee R.T. Circ. Res. 2002; 91: 689-695Crossref PubMed Scopus (130) Google Scholar, 19Yoshioka J. Schulze P.C. Cupesi M. Sylvan J.D. MacGillivray C. Gannon J. Huang H. Lee R.T. Circulation. 2004; 109: 2581-2586Crossref PubMed Scopus (91) Google Scholar), whereas loss of Txnip expression is associated with tumor growth and metastasis (20Goldberg S.F. Miele M.E. Hatta N. Takata M. Paquette-Straub C. Freedman L.P. Welch D.R. Cancer Res. 2003; 63: 432-440PubMed Google Scholar, 21Han S.H. Jeon J.H. Ju H.R. Jung U. Kim K.Y. Yoo H.S. Lee Y.H. Song K.S. Hwang H.M. Na Y.S. Yang Y. Lee K.N. Choi I. Oncogene. 2003; 22: 4035-4046Crossref PubMed Scopus (227) Google Scholar, 22Nishinaka Y. Nishiyama A. Masutani H. Oka S. Ahsan K.M. Nakayama Y. Ishii Y. Nakamura H. Maeda M. Yodoi J. Cancer Res. 2004; 64: 1287-1292Crossref PubMed Scopus (112) Google Scholar). In addition, increasing evidence suggests major physiological roles for Txnip in glucose metabolism and cell differentiation. Both a naturally occurring mouse strain “hyplip1” with a truncation in Txnip (23Bodnar J.S. Chatterjee A. Castellani L.W. Ross D.A. Ohmen J. Cavalcoli J. Wu C. Dains K.M. Catanese J. Chu M. Sheth S.S. Charugundla K. Demant P. West D.B. de Jong P. Lusis A.J. Nat. Genet. 2002; 30: 110-116Crossref PubMed Scopus (179) Google Scholar, 24Hui T.Y. Sheth S.S. Diffley J.M. Potter D.W. Lusis A.J. Attie A.D. Davis R.A. J. Biol. Chem. 2004; 279: 24387-24393Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 25Sheth S.S. Castellani L.W. Chari S. Wagg C. Thipphavong C.K. Bodnar J.S. Tontonoz P. Attie A.D. Lopaschuk G.D. Lusis A.J. J. Lipid Res. 2005; 46: 123-134Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and mice with targeted deletion of Txnip (26Oka S.I. Liu W. Masutani H. Hirata H. Shinkai Y. Yamada S.I. Yoshida T. Nakamura H. Yodoi J. FASEB J. 2005; 20: 121-123Crossref PubMed Scopus (101) Google Scholar) have low blood glucose, hyperlipidemia, and a severely dysregulated response to fasting. It is possible that these roles for Txnip are not mediated by inhibition of thioredoxin function alone. Understanding Txnip function will require that the mechanism for the interaction of Txnip and thioredoxin be better defined. In the present work we explored the mechanism of the Txnip-thioredoxin interaction and present evidence that Txnip and thioredoxin form a stable disulfide-liked complex. We identified two Txnip cysteines that are important for thioredoxin binding and showed that this interaction is consistent with a disulfide exchange reaction between oxidized Txnip and reduced thioredoxin. These two critical cysteines are not conserved in the broader family of arrestin domain-containing proteins, suggesting that Txnip is a unique redox-sensitive signaling protein. 293 Cell Culture and Transfection—HEK 293 cells were maintained in Dulbecco's modified Eagle's medium with high glucose, no sodium pyruvate, 10% fetal bovine serum, and antibiotics (100 units/ml penicillin and streptomycin). Plasmid-encoded proteins were expressed by transfection with FuGENE 6 (Roche Applied Science) using 3% FuGENE and a 1:3 ratio of micrograms of DNA to microliters of FuGENE. Three days after transfection, cells were washed in ice-cold phosphate-buffered saline, harvested in lysis buffer (Tris-buffered saline with 0.5% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma)), and clarified at 16,000 × g for 10 min. Adipocyte Culture—3T3-L1 mouse fibroblasts (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium with high glucose (25 mm), penicillin, streptomycin, and 10% newborn calf serum (Invitrogen). Differentiation was induced by adding 5 μg/ml insulin, 0.25 μm dexamethasone, and 0.5 mm 3-isobutyl-1-methylxanthine to the media according to a standard E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). By days after of cells were and Adipocyte Txnip were in by with a was into the and the was by were by transfection of an into cells green fluorescent protein was with in a The of of the was by the number of cells after days after cells were cultured in low glucose mm), with a of of and to proteins for and a overexpression of Txnip and Txnip in cells not of with a of was from by with in the presence of and mm and into (Invitrogen). The were and and the the was from and into (Invitrogen). were and and Txnip was from into using and and were into and by with by of DNA with were by was from cell and between the and of to the glutathione proteins were induced in cells by 0.5 mm for Cell were in and units/ml and the were with for at by with 0.5% Triton in 293 cell were with of protein. with at by with lysis proteins were released by in were at and were to were 10% (Invitrogen). of Txnip was with a from to the and were from Thioredoxin activity in cell was by the of A. M. Enzymol. 1995; PubMed Scopus Google Scholar) with cell were in excess insulin, thioredoxin and at for min. The reaction was by the addition of and of to was by at was used as a between groups were by with were for using 1 and number of Txnip and Thioredoxin a have that Txnip not with the thioredoxin active-site (15Yamanaka H. Maehira F. Oshiro M. Asato T. Yanagawa Y. Takei H. Nakashima Y. Biochem. Biophys. Res. Commun. 2000; 271: 796-800Crossref PubMed Scopus (102) Google Scholar, 17Nishiyama A. Matsui M. Iwata S. Hirota K. Masutani H. Nakamura H. Takagi Y. Sono H. Gon Y. Yodoi J. J. Biol. Chem. 1999; 274: 21645-21650Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar), suggesting that Txnip and thioredoxin by formation of a disulfide bond with of the thioredoxin active-site However, is direct evidence that thioredoxin forms stable mixed disulfides with its binding partners under physiological thioredoxin is thought to be an active disulfide in part due to the of the mixed disulfide R. 1995; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have identified interactions of and Ref-1 with thioredoxin, but because the were to a thioredoxin not provide evidence the physiological of the mixed disulfide complex (7Qin J. Clore G.M. Kennedy W.M. Huth J.R. Gronenborn A.M. Structure. 1995; 3: 289-297Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, J. Clore G.M. Kennedy W.P. Kuszewski J. Gronenborn A.M. 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We we a stable disulfide complex of Txnip and thioredoxin by a binding Txnip was in 293 which have of Txnip, and the cell were with to proteins were released by in in a of and to with a against Txnip Txnip at was The of this increased with increasing concentrations of DTT, consistent with of the Txnip from disulfide-linked by were also at the of the complex this was with mm but in a from to 10 mm The was not in the The identification of an that is to is consistent with the identification of a stable Txnip-thioredoxin disulfide-linked complex. We also that the Txnip showed a with increasing of suggesting that Txnip is to the for the disulfide of PTEN J. Lee Yang K.S. Y. Kim Rhee S.G. U. S. A. 2004; PubMed Scopus Google Scholar). However, this evidence was by the of the to the Txnip that be for and disulfide forms of Txnip in that Txnip a disulfide bond with thioredoxin, at Txnip cysteine be required for the thioredoxin interaction were conserved Txnip this cysteine be of the PubMed mouse and data proteins to Txnip of function Txnip and these contain structural homology to the which are proteins that have important roles in and signaling S.K. Science. 2005; PubMed Scopus Google Scholar). The have been arrestin domain-containing 1 Three have so been and as in 1 Y. M. Fujii G. K. M. S. Clin. Cancer Res. Google Scholar), as by E. J. 2004; PubMed Scopus Google Scholar), and as protein S.I. Masutani H. Liu W. H. D. S. Yodoi J. 2005; PubMed Google Scholar). The protein, is for its The of these proteins and the significance of the arrestin of the proteins with the to Txnip a conserved cysteine in cysteine is also conserved in arrestin domain-containing to and that the Txnip cysteine be required for interaction with thioredoxin and that the arrestin domain-containing proteins a family of Txnip for the (15Yamanaka H. Maehira F. Oshiro M. Asato T. Yanagawa Y. Takei H. Nakashima Y. Biochem. Biophys. Res. Commun. 2000; 271: 796-800Crossref PubMed Scopus (102) Google Scholar) identified that deletion of the arrestin not binding to thioredoxin. We a Txnip for of the cysteine in the of the protein and in 293 Txnip and of the including the to bind a of Txnip cysteine Txnip not the conserved is critical for the interaction with thioredoxin. Txnip is conserved Txnip was not present in of the arrestin domain-containing proteins Txnip and Txnip was not present in the and suggests that the family of proteins is not by interaction with thioredoxin. to the hypothesis that the proteins not with thioredoxin, we the mouse with homology to Txnip and as as mouse Txnip, into expression The were in 293 and of the proteins was in cell to an interaction with under conditions that Txnip Consistent with these Oka S.I. Masutani H. Liu W. H. D. S. Yodoi J. 2005; PubMed Google Scholar) have that also not bind thioredoxin. These that the arrestin domain-containing protein family is not by thioredoxin binding. The structural homology with the suggests a role as proteins for interaction and signaling, as for the S.K. Science. 2005; PubMed Scopus Google Scholar). It may be that for interaction with and that of binding partners will be important for In addition, because the of Txnip to bind thioredoxin is Txnip may conserved signaling of the Txnip evidence that Txnip forms a complex with thioredoxin, with the identification of a Txnip cysteine critical for the that the interaction is mediated by formation of a disulfide However, in the reducing environment of the normal by de formation of protein disulfides is in the of an to the redox reaction C.A. Nat. Mol. Biol. 2002; 3: PubMed Scopus Google Scholar). protein disulfides are in the in a that requires protein disulfide and to to oxygen J.S. J. Cell Biol. 2004; 164: PubMed Scopus Google Scholar, S. J. M. C. R. D. A. A.M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). We that Txnip a disulfide bond with thioredoxin, a disulfide exchange mechanism be two Txnip with oxidized thioredoxin, Txnip a disulfide bond and with reduced thioredoxin. Nishiyama (17Nishiyama A. Matsui M. Iwata S. Hirota K. Masutani H. Nakamura H. Takagi Y. Sono H. Gon Y. Yodoi J. J. Biol. Chem. 1999; 274: 21645-21650Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar) have that the interaction of Txnip with thioredoxin is by of thioredoxin, suggesting that Txnip with reduced thioredoxin. However, of thioredoxin in can of thioredoxin by at cysteine binding to Txnip M. B. A. PubMed Scopus Google Scholar, D.A. J.R. A. Powis G. PubMed Scopus Google Scholar). this we the of redox state the interaction of with were in lysis buffer and reduced by addition of μm DTT, oxidized by addition of μm The was by to with cell a not bind Txnip of Txnip was not by reduction of with In of binding to Txnip, that thioredoxin be reduced for interaction with We also the redox conditions during binding the Txnip-thioredoxin were with 293 cell in the presence of DTT, no Txnip as a and no Txnip binding was However, redox during binding the from redox of thioredoxin addition of μm no addition of μm binding The of these two are consistent with a disulfide exchange mechanism and suggest that Txnip is present as an Because Txnip with reduced thioredoxin, the disulfide not from thioredoxin. addition of during binding inhibits the suggesting that the Txnip disulfide and disulfide We evidence that Txnip have an for Txnip in from 293 cells Txnip Txnip from and cell showed evidence for at the under The of evidence for an with the redox-sensitive of Txnip was consistent with the hypothesis that Txnip Txnip Thioredoxin an Txnip disulfide were required for interaction with thioredoxin, the hypothesis be that the disulfide cysteine and that its Txnip cysteine also be required for interaction with thioredoxin. We of the cysteines in the Txnip arrestin and for interaction with The Txnip binding by a Txnip cysteine that is important for thioredoxin supports the hypothesis that Txnip an disulfide between and that interaction with reduced thioredoxin by disulfide the that, binding of the Txnip to thioredoxin was no binding at of the Txnip was We that Txnip is critical for thioredoxin because forms the disulfide with thioredoxin, whereas Txnip is required for reaction by disulfide but of this cysteine not the interaction hypothesis is also consistent with the that a Txnip truncation with deletion of the thioredoxin (15Yamanaka H. Maehira F. Oshiro M. Asato T. Yanagawa Y. Takei H. Nakashima Y. Biochem. Biophys. Res. Commun. 2000; 271: 796-800Crossref PubMed Scopus (102) Google Scholar). Txnip of Thioredoxin in the Txnip mixed disulfide is important for the inhibition of thioredoxin activity by Txnip, we and Txnip in by Because these cells are of thioredoxin activity is days after activity in the cell was to protein and to the activity of the cells with an In cells with Txnip, thioredoxin activity was reduced to of In in cells with Txnip thioredoxin activity was of for with not for with These data provide evidence that Txnip-thioredoxin mixed disulfide formation is important for the inhibition of the reducing activity of thioredoxin by However, because these may physiological of Txnip the of the Txnip-thioredoxin interaction in may be the overexpression was only In addition, of Txnip and thioredoxin may not be by mixed disulfide The evidence for a stable mixed disulfide between Txnip and thioredoxin also the hypothesis that the role of thioredoxin in redox signaling is not to in reduction but may also interactions that are by the state of its In several aspects of the Txnip-thioredoxin interaction are to the interaction of ASK1 with thioredoxin. Txnip, ASK1 not bind the thioredoxin active-site and binds to reduced thioredoxin, suggesting the thioredoxin active-site cysteines are of a thioredoxin active-site cysteine appears to the consistent with the of the mixed disulfide complex by (7Qin J. Clore G.M. Kennedy W.M. Huth J.R. Gronenborn A.M. Structure. 1995; 3: 289-297Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). of ASK1 identified a that is critical for the interaction with thioredoxin. the we that ASK1 may also bind thioredoxin by a disulfide exchange However, for this hypothesis require evidence to demonstrate an ASK1 In the provide evidence that Txnip and thioredoxin form a stable mixed disulfide by disulfide We that oxidized Txnip a disulfide between cysteines and and the oxidized Txnip with thioredoxin to form a disulfide between Txnip cysteine and thioredoxin cysteine In this reduction of Txnip of thioredoxin the We that identification of the mechanism and of that not bind thioredoxin may identification of of Txnip, its arrestin These also the hypothesis that thioredoxin, in addition to its reducing activity, can in redox signaling the formation of mixed disulfide with signaling proteins in a with