The Interaction of Thioredoxin with Txnip

Abstract

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 (1, 2), diabetes (3), and arthritis (4, 5). 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 (6).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-B p50 subunit (7), Ref-1 (8), Jab-1 (9), Oct-4 (10), and PTEN (11). One of the better characterized interactions is that of reduced thioredoxin with apoptosis signal-regulating kinase 1 (ASK1), 2 which plays a key role in promoting stress-induced apoptosis (12). 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 (13) 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) ...

Thioredoxin-interacting Protein (Txnip) Is a Critical Regulator of Hepatic Glucose Production

“…To increase the sensitivity of the assay to unavailable thioredoxin (through formation of either mixed disulfides at the active site or noncovalent protein complexes), we omitted the incubation at 70°C and the incubation with dithiothreitol (33). Livers were homogenized in a Tris buffer with 0.5% Triton X-100 and clarified at 16,000 ϫ g. Portions of the fresh clarified homogenate containing 50 g of total protein were incubated with insulin, rat liver thioredoxin reductase (Sigma), and NADPH for 15 min at 37°C.…”

“…Lentiviral Production and Tail Vein Injection-Lentiviral constructs for Txnip and Txnip C247S were made as described previously (33). Viral multiplicity of infection for liver infectivity was estimated based on in vitro primary hepatocyte transduction efficiency: 0.5 ml of undiluted viral stocks supplemented with Polybrene (7.5 g/ml) was added to 10 5 primary hepatocytes cultured in 12-well plates, and GFP-positive cells were counted 96 h after transduction.…”

“…Interestingly, the gene encoding Arrdc3 has recently been linked to human obesity in males (34). This suggests the hypothesis that the Arrdc family of Txnip-like proteins is a family of metabolic regulators whose functions are not limited to thioredoxin binding (33).…”

“…We recently reported that mutation of a single Txnip cysteine (Cys-247) abolishes the ability of Txnip to bind thioredoxin and to inhibit thioredoxin activity when overexpressed in 3T3-L1 adipocytes (33). We have now generated total and hepatocyte-specific Txnip gene deletion in mice to assess the functions of Txnip in vivo.…”

“…Txnip has distant sequence homology to the arrestins, intracellular proteins that bind to phosphorylated receptors and then direct further signaling, and/or endocytosis (32). In addition to Txnip, mammals have three Txnip-homologous proteins of unknown function, named Arrestin domain containing 2-4 (Arrdc2-4), which do not bind thioredoxin (33). Interestingly, the gene encoding Arrdc3 has recently been linked to human obesity in males (34).…”