Institute of Pediatrics, University of Foggia, viale Pinto 1, Foggia 71100, Italy.
Gut. 2010 Mar;59(3):311-9. doi: 10.1136/gut.2009.183608. Epub 2009 Dec 1.
An unresolved question in coeliac disease is to understand how some toxic gliadin peptides, in particular p31-43, can initiate an innate response and lead to tissue transglutaminase (TG2) upregulation in coeliac intestine and gliadin sensitive epithelial cell lines. Aim We addressed whether the epithelial uptake of p31-43 induces an intracellular pro-oxidative envoronment favouring TG2 activation and leading to the innate immune response.
The time course of intracellular delivery to lysosomes of p31-43, palpha-2 or palpha-9 gliadin peptides was analysed in T84 and Caco-2 epithelial cells. The effects of peptide challenge on oxidative stress, TG2 and peroxisome proliferator-activated receptor (PPAR)gamma ubiquitination and p42/44-mitogen activated protein (MAP) kinase or tyrosine phosphorylation were investigated in cell lines and cultured coeliac disease biopsies with/without anti-oxidant treatment or TG2 gene silencing by immunoprecipitation, western blot, confocal microscopy and Fluorenscence Transfer Resonance Energy (FRET) analysis.
After 24 h of challenge p31-43, but not palpha-2 or palpha-9, is still retained within LAMP1-positive perinuclear vesicles and leads to increased levels of reactive oxygen species (ROS) that inhibit TG2 ubiquitination and lead to increases of TG2 protein levels and activation. TG2 induces cross-linking, ubiquitination and proteasome degradation of PPARgamma. Treatment with the antioxidant EUK-134 as well as TG2 gene silencing restored PPARgamma levels and reversed all monitored signs of innate activation, as indicated by the dramatic reduction of tyrosine and p42/p44 phosphorylation.
p31-43 accumulation in lysosomes leads to epithelial activation via the ROS-TG2 axis. TG2 works as a rheostat of ubiquitination and proteasome degradation and drives inflammation via PPARgamma downregulation.
在乳糜泻中，一个悬而未决的问题是如何理解某些有毒的麦醇溶蛋白肽，特别是 p31-43，如何引发先天反应，并导致乳糜泻肠和麦醇溶蛋白敏感上皮细胞系中组织转谷氨酰胺酶 (TG2) 的上调。目的 我们旨在确定 p31-43 的上皮摄取是否会诱导有利于 TG2 激活的细胞内促氧化环境，从而导致先天免疫反应。
在 T84 和 Caco-2 上皮细胞中分析 p31-43、palpha-2 或 palpha-9 麦醇溶蛋白肽向溶酶体的细胞内递呈的时间过程。通过免疫沉淀、Western blot、共聚焦显微镜和 Fluorenscence Transfer Resonance Energy (FRET) 分析，研究了肽挑战对氧化应激、TG2 和过氧化物酶体增殖物激活受体 (PPAR)γ泛素化以及 p42/44-有丝分裂原激活蛋白 (MAP) 激酶或酪氨酸磷酸化的影响在细胞系和培养的乳糜泻活检中，进行了抗氧化治疗或 TG2 基因沉默。
在挑战后的 24 小时内，p31-43（而不是 palpha-2 或 palpha-9）仍保留在 LAMP1 阳性核周囊泡内，并导致活性氧 (ROS) 水平升高，抑制 TG2 泛素化，并导致 TG2 蛋白水平和活性增加。TG2 诱导 PPARγ的交联、泛素化和蛋白酶体降解。用抗氧化剂 EUK-134 治疗以及 TG2 基因沉默恢复了 PPARγ水平，并逆转了所有监测到的先天激活迹象，酪氨酸和 p42/44 磷酸化明显减少。
p31-43 在溶酶体中的积累通过 ROS-TG2 轴导致上皮细胞激活。TG2 作为泛素化和蛋白酶体降解的变阻器，通过下调 PPARγ 驱动炎症。
