SUMOylation of Tissue Transglutaminase as Link between Oxidative Stress and Inflammation

Abstract

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. CF is characterized by chronic bacterial lung infections and inflammation, and we have previously reported that tissue transglutaminase (TG2), a multifunctional enzyme critical to several diseases, is constitutively up-regulated in CF airways and drives chronic inflammation. Here, we demonstrate that the generation of an oxidative stress induced by CFTR-defective function leads to protein inhibitor of activated STAT (PIAS)y-mediated TG2 SUMOylation and inhibits TG2 ubiquitination and proteasome degradation, leading to sustained TG2 activation. This prevents peroxisome proliferator-activated receptor (PPAR)γ and IkBα SUMOylation, leading to NF-κB activation and to an uncontrolled inflammatory response. Cellular homeostasis can be restored by small ubiquitin-like modifier (SUMO)-1 or PIASy gene silencing, which induce TG2 ubiquitination and proteasome degradation, restore PPARγ SUMOylation, and prevent IkBα cross-linking and degradation, thus switching off inflammation. Manganese superoxide dismutase overexpression as well as the treatment with the synthetic superoxide dismutase mimetic EUK-134 control PIASy-TG2 interaction and TG2 SUMOylation. TG2 inhibition switches off inflammation in vitro as well as in vivo in a homozygous F508del-CFTR mouse model. Thus, TG2 may function as a link between oxidative stress and inflammation by driving the decision as to whether a protein should undergo SUMO-mediated regulation or degradation. Targeting TG2-SUMO interactions might represent a new option to control disease evolution in CF patients as well as in other chronic inflammatory diseases, neurodegenerative pathologies, and cancer.

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator

“…We previously reported that in CF epithelial cells, cystamine induces an increase in peroxisome proliferator activated receptor-γ (PPARγ) levels and a decrease in p42-44 MAPK phosphorylation, TNFA secretion and TGM2 SUMOylation. 7,24,25 Here, we report that these effects persisted beyond cystamine washout for 24 h (Fig. S7A), unless either BECN1 (data not shown) or CFTR were depleted by siRNAs during cystamine washout (Fig.…”

“…3D). Sqstm1 shRNA significantly decreased the expression of validated markers of lung inflammation, 7,25,48 as it reduced the mRNA expression of the inflammatory cytokine Tnfa, the levels of Tnfa and CXCL2 protein, and macrophage infiltration in lungs (p < 0.001), as compared with control shRNA treated Cftr F508del mice ( Fig. 3E; Fig.…”

“…22,23 We previously reported that defective CFTR leads to the inhibition of autophagy in the epithelial cells of human and mouse CF airways. 7,8 Increased levels of reactive oxygen species (ROS), induced by defective CFTR function, lead to tissue transglutaminase-2 (TGM2) activation 24,25 driving cross-linking and aggresome accumulation of several TGM2-substrate proteins, 24 including the sequestration of the essential autophagy protein BECN1. 7 The functional sequestration of BECN1 dislodges the PtdIns3K complex III away from the endoplasmic reticulum (ER), thus inhibiting autophagosome formation.…”

“…To translate these findings to human CF airways, we used an ex-vivo model of cultured nasal polyp biopsies belonging to CF patients, a useful tool to test potential strategies of modulation of mucosal response to environmental triggers within their natural environment. 7,24,25,49 Nasal polyp biopsies from five ΔF508/ΔF508 patients ( Table 1A, Patients #1-5) were treated with cystamine for 18 h, washed and then cultured for further 36 h in the absence of cystamine. The cystamine-mediated rescue of CFTR at the epithelial surface persisted for 36 h after cystamine withdrawal and was abrogated by 3-MA ( Fig.…”

Early tissue transglutaminase–mediated response underlies K562(S)-cell gliadin-dependent agglutination

“…Following electrophoresis, the proteins were transferred to a polyvinylidene fluoride membrane (Biorad). Membranes were blocked for 1 h with 5% nonfat milk in Tris-buffered saline (100 mmol/l NaCl, 5 mmol/l KCl, 100 mmol/l TrisHCl, pH 7.4, and 0.05% Tween 20, Biorad) and incubated overnight TG2 activity and actin rearrangement were detected as previously described (34). Briefly, for the detection of TG2 intracellular enzymatic activity, a drop of cellular suspension of K562(S) cells treated for 30 min was put on coated slides, preincubated with TG assay buffer (965 L of 100 mmol/l Tris-HCl, pH 7.4, 25 ml of 200 mmol/l CaCl 2 , both from Sigma) for 15 min, and then preincubated with the same triglyceride assay buffer added with 10 µl of 10 mmol/l biotinylated monodansylcadaverine (Molecular Probes, Leiden, The Netherlands) for 1 h at room temperature.…”