Chemical biology approaches to investigate the role of extracellular thioredoxin-1 in pathophysiology [electronic resource]
Keywords: Chemistry.

Celiac disease is a lifelong autoimmune disease of the small intestine that affects 1% of most populations. The autoimmune features of the disease require the presence of dietary gluten and HLA-DQ2- or HLA-DQ8-restricted gluten-specific CD4+ T cells. Extracellular transglutaminase 2 (TG2), the principal autoantigen of the disease, facilitates a robust T cell (Th1) response to gluten. Proteolytically resistant, proline- and glutamine-rich peptides derived from gluten accumulate in the gut lumen. These peptides are weak antigens by themselves; their affinity for HLA-DQ2 or DQ8 is markedly enhanced by TG2-catalyzed deamidation of selected glutamine residues. However, extracellular TG2 in the small intestinal mucosa is maintained in an inactive state via an intramolecular disulfide bond, and thus must be activated before having its catalytic function. An improved understanding of the physiological regulation mechanisms of extracellular TG2 could enable novel therapeutic strategies for treating this lifelong condition. Thioredoxin-1 (TRX), an essential intracellular redox regulator, is also secreted by mammalian cells. Although its export mechanism and extracellular catalytic cycle are unclear, its serum concentration is correlated with various inflammatory conditions. Previous work from our lab demonstrated that TRX activated extracellular transglutaminase 2 (TG2) via reduction of an unusual disulfide bond. We determined that because hyperactive TG2 is thought to play a role in a variety of disease states including celiac disease, 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 identified analogs with > 100-fold for TRX over other dithiols. An advantage of disulfide inhibitors is that they can irreversibly oxidize extracellular TRX without affecting the function of its intracellular counterpart. Structure-activity relationship analysis provided insight into the features important for enhancing potency and specificity. A computational docking model was developed to rationalize the ability of these asymmetric disulfides to selectively recognize the superficial redox active site of TRX. The most active compound in this series, NP161, had an IC50 value below 0.1µM in cell culture, suggesting it may be appropriate for in vivo use to interrogate the role of extracellular TRX in health and disease. To test the physiological relevance of this hypothesis that extracellular TG2 activation involves reduction of an intramolecular disulfide bond by TRX, we first showed that macrophages exposed to pro-inflammatory stimuli released TRX in sufficient quantities to activate TG2 in their extracellular environment. By using the C35S mutant of human TRX, which was capable of forming a metastable mixed disulfide bond with TG2, we demonstrated that the two proteins could recognize each other with high specificity in the extracellular matrix of cultured fibroblasts. When injected into mice, the labeled C35S TRX mutant identified endogenous TG2 as its principal protein partner in small intestinal lamina propria. Control experiments showed no labeling of the small intestine of TG2-knockout mice. Intravenous administration of recombinant human TRX in wild-type, but not TG2-knockout mice, led to a rapid rise in intestinal transglutaminase activity in a manner that could be inhibited by small molecules targeting TG2 as well as TRX. In addition to underscoring the potential pathophysiological relevance of TG2 recognition by TRX in celiac disease, our data establishes the C370-C371 disulfide bond in TG2 as one of clearest examples of an allosteric disulfide bond in mammals. These results motivated us to harness the same tools, namely NP161 and C35S TRX, to search for other biologically relevant protein substrates of extracellular TRX. In an effort to identify other extracellular substrates of TRX, macrophages derived from THP-1 and human peripheral blood mononuclear cells were treated with a pharmacological inhibitor of secreted TRX. Not only did TRX inhibition attenuate macrophage polarization upon lipopolysaccharide and interferon-γ exposure, but it also promoted polarization toward an alternative state. From this data we hypothesized that extracellular TRX influenced macrophage function by redox regulation of either interleukin-4 (IL-4) or -13 (IL-13). In support of this hypothesis, the C35S mutant of human TRX formed a mixed disulfide bond with recombinant IL-4 but not IL-13. Quantitative analysis yielded kcat/KM values of 8.1 and 1.6 M-1min-1 for TRX-mediated recognition of IL-4 and IL-13, respectively. Mass spectrometry revealed that the C46-C99 bond in IL-4 was the primary target of TRX, consistent with the critical role of this disulfide bond in IL-4 activity. Exogenous TRX attenuated IL-4 dependent proliferation of the TF-1 erythroleukemia line and CD206 expression in THP-1-derived macrophages. By demonstrating that IL-4 is post-translationally regulated by TRX-promoted reduction of a disulfide bond, our findings highlight a novel, pharmacologically promising immunomodulatory mechanism. Collectively, the work presented here underscores the relevance of extracellular TRX in human physiology.