Interleukin 4 is inactivated via selective disulfide-bond reduction by extracellular thioredoxin.

Thioredoxin 1 (TRX), an essential intracellular redox regulator, is also secreted by mammalian cells. Recently, we showed that TRX activates extracellular transglutaminase 2 via reduction of an allosteric disulfide bond. In an effort to identify other extracellular substrates of TRX, macrophages derived from THP-1 cells were treated with NP161, a small-molecule inhibitor of secreted TRX. NP161 enhanced cytokine outputs of alternatively activated macrophages, suggesting that extracellular TRX regulated the activity of interleukin 4 (IL-4) and/or interleukin 13 (IL-13). To test this hypothesis, the C35S mutant of human TRX was shown to form a mixed disulfide bond with recombinant IL-4 but not IL-13. Kinetic analysis revealed a kcat/KM value of 8.1 µM-1⋅min-1 for TRX-mediated recognition of IL-4, which established this cytokine as the most selective partner of extracellular TRX to date. Mass spectrometry identified the C46-C99 bond of IL-4 as the target of TRX, consistent with the essential role of this disulfide bond in IL-4 activity. To demonstrate the physiological relevance of our biochemical findings, recombinant TRX was shown to attenuate IL-4-dependent proliferation of cultured TF-1 erythroleukemia cells and also to inhibit the progression of chronic pancreatitis in an IL-4-driven mouse model of this disease. By establishing that IL-4 is posttranslationally regulated by TRX-promoted reduction of a disulfide bond, our findings highlight a novel regulatory mechanism of the type 2 immune response that is specific to IL-4 over IL-13.

Thioredoxin-1 Selectively Activates Transglutaminase 2 in the Extracellular Matrix of the Small Intestine: IMPLICATIONS FOR CELIAC DISEASE.

Transglutaminase 2 (TG2) catalyzes transamidation or deamidation of its substrates and is ordinarily maintained in a catalytically inactive state in the intestine and other organs. Aberrant TG2 activity is thought to play a role in celiac disease, suggesting that a better understanding of TG2 regulation could help to elucidate the mechanistic basis of this malady. Structural and biochemical analysis has led to the hypothesis that extracellular TG2 activation involves reduction of an allosteric disulfide bond by thioredoxin-1 (TRX), but cellular and in vivo evidence for this proposal is lacking. To test the physiological relevance of this hypothesis, we first showed that macrophages exposed to pro-inflammatory stimuli released TRX in sufficient quantities to activate their extracellular pools of TG2. By using the C35S mutant of TRX, which formed a metastable mixed disulfide bond with TG2, we demonstrated that these proteins specifically recognized each other in the extracellular matrix of fibroblasts. When injected into mice and visualized with antibodies, we observed the C35S TRX mutant bound to endogenous TG2 as its principal protein partner in the small intestine. Control experiments showed no labeling of TG2 knock-out mice. Intravenous administration of recombinant TRX in wild-type mice, but not TG2 knock-out mice, led to a rapid rise in intestinal transglutaminase activity in a manner that could be inhibited by small molecules targeting TG2 or TRX. Our findings support the potential pathophysiological relevance of TRX in celiac disease and establish the Cys370-Cys371 disulfide bond of TG2 as one of clearest examples of an allosteric disulfide bond in mammals.

An unprecedented dual antagonist and agonist of human Transglutaminase 2.

Transglutaminase 2 (TG2) is a ubiquitously expressed, Ca(2+)-activated extracellular enzyme in mammals that is maintained in a catalytically dormant state by multiple mechanisms. Although its precise physiological role in the extracellular matrix remains unclear, aberrantly up-regulated TG2 activity is a hallmark of several maladies, including celiac disease. Previously, we reported the discovery of a class of acylideneoxoindoles as potent, reversible inhibitors of human TG2. Detailed analysis of one of those inhibitors (CK-IV-55) led to an unprecedented and striking observation. Whereas this compound was a non-competitive inhibitor (3.3±0.9 µM) of human TG2 at saturating Ca(2+) concentrations, it activated TG2 in the presence of sub-saturating but physiologically relevant Ca(2+) concentrations (0.5-0.7 mM). This finding was validated in a cellular model of TG2 activation and inhibition. Mutant TG2 analysis suggested that CK-IV-55 and its analogs bound to a low-affinity Ca(2+) binding site on the catalytic core of TG2. A mechanistic model for the dual agonistic/antagonistic action of CK-IV-55 on TG2 is presented, and the pathophysiological implications of basal activation of intestinal TG2 by small molecules are discussed.

Electronic and geometric structures of the organophosphate-degrading enzyme from Agrobacterium radiobacter (OpdA).

The organophosphate-degrading enzyme from Agrobacterium radiobacter (OpdA) is a highly efficient catalyst for the degradation of pesticides and some nerve agents such as sarin. OpdA requires two metal ions for catalytic activity, and hydrolysis is initiated by a nucleophilic hydroxide that is bound to one of these metal ions. The precise location of this nucleophile has been contentious, with both a terminal and a metal-ion-bridging hydroxide as likely candidates. Here, we employed magnetic circular dichroism to probe the electronic and geometric structures of the Co(II)-reconstituted dinuclear metal center in OpdA. In the resting state the metal ion in the more secluded α site is five-coordinate, whereas the Co(II) in the solvent-exposed ß site is predominantly six-coordinate with two terminal water ligands. Addition of the slow substrate diethyl 4-methoxyphenyl phosphate does not affect the α site greatly but lowers the coordination number of the ß site to five. A reduction in the exchange coupling constant indicates that substrate binding also triggers a shift of the µ-hydroxide into a pseudoterminal position in the coordination sphere of either the α or the ß metal ion. Mechanistic implications of these observations are discussed.

Cobalt(II) "Scorpionate" complexes as electronic ground state models for cobalt-substituted zinc enzymes: Structure investigation by magnetic circular dichroism.

Zinc centers in pseudo-tetrahedral geometry are widely found in biology, often with three histidine ligands from protein. The trispyrazolylborate "scorpionate" ligand is used as a model for this tris(histidine) motif, and spectroscopically active CoII is often used as a substitute for spectroscopically silent ZnII. In this work, four pseudo-tetrahedral scorpionate complexes with the formula (Tpt-Bu,Tn)CoL, where Tpt-Bu,Tn = hydrotris(3-tert-butyl, 5-2'-thienyl-pyrazol-1-yl)borate anion and L = Cl-, N3-, NCO-, or NCS-, were studied using variable-temperature, variable-field magnetic circular dichroism (VTVH MCD) spectroscopy. The major goal was to determine the axial and rhombic zero field splitting (ZFS) parameters (D and E, respectively) of these S = 3/2 systems and compare these ZFS parameters to those determined previously by high-frequency and -field electron paramagnetic resonance (HFEPR) spectroscopy on the same (L = Cl- and NCS-) or closely related complexes. Additionally, HFEPR studies were undertaken here on the complexes with L = N3-, NCO-. Crystal structures for these two complexes are also first reported here. The values of D determined by VTVH MCD were + 12.8 and + 3.6 cm-1 for the L = Cl- and NCS- complexes, respectively. These values are in close agreement with those for the same complexes as previously determined by HFEPR. The values of D determined by VTVH MCD were + 3.0 and + 6.6 cm-1 for the L = N3- and NCO- complexes, respectively. These values were not as close to those determined by HFEPR in the present study, which are 4.2 cm-1 ≤ |D| ≤ 5.6 cm-1 in Tpt-Bu,TnCoN3, and 8.3 cm-1 ≤ |D| ≤ 11.0 cm-1 in Tpt-Bu,TnCoNCO. The bands in MCD spectra of these complexes were assigned in C3v symmetry and a complete ligand-field analysis of the MCD data was made using the Angular Overlap Model (AOM), which is compared to previous results.

Therapeutic approaches for celiac disease.

Celiac disease is a common, lifelong autoimmune disorder for which dietary control is the only accepted form of therapy. A strict gluten-free diet is burdensome to patients and can be limited in efficacy, indicating there is an unmet need for novel therapeutic approaches to supplement or supplant dietary therapy. Many molecular events required for disease pathogenesis have been recently characterized and inspire most current and emerging drug-discovery efforts. Genome-wide association studies (GWAS) confirm the importance of human leukocyte antigen genes in our pathogenic model and identify a number of new risk loci in this complex disease. Here, we review the status of both emerging and potential therapeutic strategies in the context of disease pathophysiology. We conclude with a discussion of how genes identified during GWAS and follow-up studies that enhance susceptibility may offer insight into developing novel therapies.

Selective inhibition of extracellular thioredoxin by asymmetric disulfides.

Whereas the role of mammalian thioredoxin (Trx) as an intracellular protein cofactor is widely appreciated, its function in the extracellular environment is not well-understood. Only few extracellular targets of Trx-mediated thiol-disulfide exchange are known. For example, Trx activates extracellular transglutaminase 2 (TG2) via reduction of an intramolecular disulfide bond. Because hyperactive TG2 is thought to play a role in various diseases, understanding the biological role of extracellular Trx may provide critical insight into the pathogenesis of these disorders. Starting from a clinical-stage asymmetric disulfide lead, we have identified analogs with >100-fold specificity for Trx. Structure-activity relationship and computational docking model analyses have provided insights into the features important for enhancing potency and specificity. The most active compound identified had an IC(50) below 0.1 µM in cell culture and may be appropriate for in vivo use to interrogate the role of extracellular Trx in health and disease.