Interleukin 4 is inactivated via selective disulfide-bond reduction by extracellular thioredoxin.
Proceedings of the National Academy of Sciences of the United States of America
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 muM-1min-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.
View details for DOI 10.1073/pnas.1805288115
View details for PubMedID 30104382
Cystamine and Disulfiram Inhibit Human Transglutaminase 2 via an Oxidative Mechanism
2018; 57 (24): 3359–63
The catalytic activity of transglutaminase 2 (TG2), a ubiquitously expressed mammalian enzyme, is regulated by multiple post-translational mechanisms. Because elevated activity of TG2 in the extracellular matrix is associated with organ-specific diseases such as celiac disease and renal fibrosis, there is growing therapeutic interest in inhibitors of this enzyme. Cystamine, a symmetric disulfide compound, is one of the earliest reported TG2 inhibitors. Despite its widespread use as a tool compound to block TG2 activity in vitro and in vivo, its mechanism of action has remained unclear. Here, we demonstrate that cystamine irreversibly inhibits human TG2 ( kinh/ Ki = 1.2 mM-1 min-1) via a mechanism fundamentally distinct from those proposed previously. Through mass spectrometric disulfide mapping and site-directed mutagenesis, we show that cystamine promotes the formation of a physiologically relevant disulfide bond between Cys370 and Cys371 that allosterically abrogates the catalytic activity of human TG2. This discovery led us to evaluate clinically useful thiol → disulfide oxidants for TG2 inhibitory activity. It is demonstrated that disulfiram, a relatively safe oral thiuram disulfide, is a fairly potent TG2 inhibitor ( kinh/ Ki = 8.3 mM-1 min-1) and may therefore provide a practical tool for clinically validating this emerging therapeutic target in intestinal disorders such as celiac disease.
View details for DOI 10.1021/acs.biochem.8b00204
View details for Web of Science ID 000436026000006
View details for PubMedID 29570977
View details for PubMedCentralID PMC6008213
Pharmacologic inhibition of the enzymatic effects of tissue transglutaminase reduces cardiac fibrosis and attenuates cardiomyocyte hypertrophy following pressure overload
JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY
2018; 117: 36–48
Tissue transglutaminase (tTG) is a multifunctional protein with a wide range of enzymatic and non-enzymatic functions. We have recently demonstrated that tTG expression is upregulated in the pressure-overloaded myocardium and exerts fibrogenic actions promoting diastolic dysfunction, while preventing chamber dilation. Our current investigation dissects the in vivo and in vitro roles of the enzymatic effects of tTG on fibrotic remodeling in pressure-overloaded myocardium. Using a mouse model of transverse aortic constriction, we demonstrated perivascular and interstitial tTG activation in the remodeling pressure-overloaded heart. tTG inhibition through administration of the selective small molecule tTG inhibitor ERW1041E attenuated left ventricular diastolic dysfunction and reduced cardiomyocyte hypertrophy and interstitial fibrosis in the pressure-overloaded heart, without affecting chamber dimensions and ejection fraction. In vivo, tTG inhibition markedly reduced myocardial collagen mRNA and protein levels and attenuated transcription of fibrosis-associated genes. In contrast, addition of exogenous recombinant tTG to fibroblast-populated collagen pads had no significant effects on collagen transcription, and instead increased synthesis of matrix metalloproteinase (MMP)3 and tissue inhibitor of metalloproteinases (TIMP)1 through transamidase-independent actions. However, enzymatic effects of matrix-bound tTG increased the thickness of pericellular collagen in fibroblast-populated pads. tTG exerts distinct enzymatic and non-enzymatic functions in the remodeling pressure-overloaded heart. The enzymatic effects of tTG are fibrogenic and promote diastolic dysfunction, but do not directly modulate the pro-fibrotic transcriptional program of fibroblasts. Targeting transamidase-dependent actions of tTG may be a promising therapeutic strategy in patients with heart failure and fibrosis-associated diastolic dysfunction.
View details for DOI 10.1016/j.yjmcc.2018.02.016
View details for Web of Science ID 000430035200004
View details for PubMedID 29481819
View details for PubMedCentralID PMC5892840
Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease
2017; 356 (6333): 44-?
Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.
View details for DOI 10.1126/science.aah5298
View details for Web of Science ID 000398689100033
View details for PubMedID 28386004
Thioredoxin-1 Selectively Activates Transglutaminase 2 in the Extracellular Matrix of the Small Intestine: IMPLICATIONS FOR CELIAC DISEASE.
journal of biological chemistry
2017; 292 (5): 2000-2008
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 Cys(370)-Cys(371) disulfide bond of TG2 as one of clearest examples of an allosteric disulfide bond in mammals.
View details for DOI 10.1074/jbc.M116.767988
View details for PubMedID 28003361
View details for PubMedCentralID PMC5290969
- An unprecedented dual antagonist and agonist of human Transglutaminase 2. Bioorganic & medicinal chemistry letters 2015; 25 (21): 4922-4926
- Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 2014; 98 (15): 6689-6700