Alexandra Endrizzi, Pauline Grunst, Silvia Rudloff, Jan De Laffolie, Klaus-Peter Zimmer, Sebastian Stricker
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Furthermore, we investigated the role of ERp57 and TRX in the context of CD by using siRNA-mediated knockdown in Caco-2 cells.</p><p><strong>Methods: </strong>The effect of TG2 inhibitors on recombinant and extracellular TG2 activity was investigated by using photometric and fluorometric quantitation of the cross-linking of biotinylated gliadin peptide P56-88 or 5-(biotinamido)-pentylamine. After siRNA knockdown, the protein levels of ERp57, TRX, and TG2 as well as TG2 activity were investigated by using Western blotting and fluorometry in Caco-2 cells.</p><p><strong>Results: </strong>The active-site-directed inhibitors ERW1041, KCC009, and cysteamine as well as the allosteric inhibitor LDN27219 revealed the most prominent reduction in recombinant and cellular (35%-50%) TG2 activity. In contrast, PX12, <i>S</i>-Nitroso-<i>N</i>-acetyl-DL-penicillamine, zinc chloride, and ascorbic acid either did not affect TG2 activity or had only moderate effects at high doses close to cytotoxic concentrations. SiRNA knockdown of TG2 resulted in a prominent reduction (63%) in TG2 activity, whereas knockdown of ERp57 did not; knockdown of TRX only slightly (27%) reduced TG2 activity.</p><p><strong>Conclusion: </strong>Active-site-directed inhibitors, LDN27219 and knockdown of TG2 expression significantly reduced extracellular TG2 activity and represent potential alternative treatment targets in the context of CD.</p>","PeriodicalId":54275,"journal":{"name":"Gastroenterology Report","volume":"13 ","pages":"goaf086"},"PeriodicalIF":4.2000,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12496130/pdf/","citationCount":"0","resultStr":"{\"title\":\"Targeting transglutaminase 2: pathways to celiac disease therapies.\",\"authors\":\"Alexandra Endrizzi, Pauline Grunst, Silvia Rudloff, Jan De Laffolie, Klaus-Peter Zimmer, Sebastian Stricker\",\"doi\":\"10.1093/gastro/goaf086\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Transglutaminase 2 (TG2)-mediated enzymatic modification of gliadin peptides plays a major role in the pathogenesis of celiac disease (CD). Different inhibitory mechanisms have been reported to reduce TG2 activity but comparative data on the cellular level are lacking. Furthermore, recent evidence suggested that endogenous redox proteins such as endoplasmic reticulum resident protein 57 (ERp57, inhibits TG2) and thioredoxin-1 (TRX, activates TG2) may regulate TG2 activity. In this study, we aimed to compare the effects and applicability of different inhibitors on the activity of recombinant and cellular TG2. Furthermore, we investigated the role of ERp57 and TRX in the context of CD by using siRNA-mediated knockdown in Caco-2 cells.</p><p><strong>Methods: </strong>The effect of TG2 inhibitors on recombinant and extracellular TG2 activity was investigated by using photometric and fluorometric quantitation of the cross-linking of biotinylated gliadin peptide P56-88 or 5-(biotinamido)-pentylamine. After siRNA knockdown, the protein levels of ERp57, TRX, and TG2 as well as TG2 activity were investigated by using Western blotting and fluorometry in Caco-2 cells.</p><p><strong>Results: </strong>The active-site-directed inhibitors ERW1041, KCC009, and cysteamine as well as the allosteric inhibitor LDN27219 revealed the most prominent reduction in recombinant and cellular (35%-50%) TG2 activity. In contrast, PX12, <i>S</i>-Nitroso-<i>N</i>-acetyl-DL-penicillamine, zinc chloride, and ascorbic acid either did not affect TG2 activity or had only moderate effects at high doses close to cytotoxic concentrations. SiRNA knockdown of TG2 resulted in a prominent reduction (63%) in TG2 activity, whereas knockdown of ERp57 did not; knockdown of TRX only slightly (27%) reduced TG2 activity.</p><p><strong>Conclusion: </strong>Active-site-directed inhibitors, LDN27219 and knockdown of TG2 expression significantly reduced extracellular TG2 activity and represent potential alternative treatment targets in the context of CD.</p>\",\"PeriodicalId\":54275,\"journal\":{\"name\":\"Gastroenterology Report\",\"volume\":\"13 \",\"pages\":\"goaf086\"},\"PeriodicalIF\":4.2000,\"publicationDate\":\"2025-10-03\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12496130/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Gastroenterology Report\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1093/gastro/goaf086\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2025/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q2\",\"JCRName\":\"GASTROENTEROLOGY & HEPATOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Gastroenterology Report","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1093/gastro/goaf086","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/1/1 0:00:00","PubModel":"eCollection","JCR":"Q2","JCRName":"GASTROENTEROLOGY & HEPATOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
背景:转谷氨酰胺酶2 (TG2)介导的麦胶蛋白肽酶修饰在乳糜泻(CD)的发病机制中起重要作用。据报道,不同的抑制机制可以降低TG2活性,但缺乏细胞水平的比较数据。此外,最近的证据表明内源性氧化还原蛋白如内质网驻留蛋白57 (ERp57,抑制TG2)和硫氧还蛋白1 (TRX,激活TG2)可能调节TG2活性。在本研究中,我们旨在比较不同抑制剂对重组和细胞TG2活性的影响和适用性。此外,我们通过在Caco-2细胞中使用sirna介导的敲低来研究ERp57和TRX在CD背景下的作用。方法:采用光度法和荧光法定量测定生物素化麦胶蛋白肽P56-88或5-(生物素胺)-戊胺的交联,研究TG2抑制剂对重组蛋白和细胞外TG2活性的影响。siRNA敲除后,采用Western blotting和荧光法检测Caco-2细胞中ERp57、TRX和TG2蛋白水平及TG2活性。结果:活性位点抑制剂ERW1041、KCC009和半胱胺以及变构抑制剂LDN27219显示重组和细胞TG2活性(35%-50%)降低最为显著。相比之下,PX12、s -亚硝基- n -乙酰基- dl -青霉胺、氯化锌和抗坏血酸在接近细胞毒性浓度的高剂量下要么不影响TG2活性,要么只有适度的影响。SiRNA敲低TG2导致TG2活性显著降低(63%),而ERp57敲低则没有显著降低;TRX的敲除仅轻微(27%)降低TG2活性。结论:活性位点定向抑制剂、LDN27219和TG2表达下调可显著降低细胞外TG2活性,是CD背景下潜在的替代治疗靶点。
Targeting transglutaminase 2: pathways to celiac disease therapies.
Background: Transglutaminase 2 (TG2)-mediated enzymatic modification of gliadin peptides plays a major role in the pathogenesis of celiac disease (CD). Different inhibitory mechanisms have been reported to reduce TG2 activity but comparative data on the cellular level are lacking. Furthermore, recent evidence suggested that endogenous redox proteins such as endoplasmic reticulum resident protein 57 (ERp57, inhibits TG2) and thioredoxin-1 (TRX, activates TG2) may regulate TG2 activity. In this study, we aimed to compare the effects and applicability of different inhibitors on the activity of recombinant and cellular TG2. Furthermore, we investigated the role of ERp57 and TRX in the context of CD by using siRNA-mediated knockdown in Caco-2 cells.
Methods: The effect of TG2 inhibitors on recombinant and extracellular TG2 activity was investigated by using photometric and fluorometric quantitation of the cross-linking of biotinylated gliadin peptide P56-88 or 5-(biotinamido)-pentylamine. After siRNA knockdown, the protein levels of ERp57, TRX, and TG2 as well as TG2 activity were investigated by using Western blotting and fluorometry in Caco-2 cells.
Results: The active-site-directed inhibitors ERW1041, KCC009, and cysteamine as well as the allosteric inhibitor LDN27219 revealed the most prominent reduction in recombinant and cellular (35%-50%) TG2 activity. In contrast, PX12, S-Nitroso-N-acetyl-DL-penicillamine, zinc chloride, and ascorbic acid either did not affect TG2 activity or had only moderate effects at high doses close to cytotoxic concentrations. SiRNA knockdown of TG2 resulted in a prominent reduction (63%) in TG2 activity, whereas knockdown of ERp57 did not; knockdown of TRX only slightly (27%) reduced TG2 activity.
Conclusion: Active-site-directed inhibitors, LDN27219 and knockdown of TG2 expression significantly reduced extracellular TG2 activity and represent potential alternative treatment targets in the context of CD.
期刊介绍:
Gastroenterology Report is an international fully open access (OA) online only journal, covering all areas related to gastrointestinal sciences, including studies of the alimentary tract, liver, biliary, pancreas, enteral nutrition and related fields. The journal aims to publish high quality research articles on both basic and clinical gastroenterology, authoritative reviews that bring together new advances in the field, as well as commentaries and highlight pieces that provide expert analysis of topical issues.