Our broad goal is to understand the role of transglutaminase 2 (TG2) in celiac disease pathogenesis. TG2 is known to deamidate dietary gluten peptides, a chemical modification that greatly enhances their recognition by inflammatory, disease-associated T cells. TG2 is also the primary target of autoantibodies in celiac disease. The most significant findings of the past funding cycle were that: (A) extracellula TG2, while ubiquitous in the intestine and most other organs, is held inactive by a C371-372 disulfide bond;and (B) TG2 activity is switched on through contact with thioredoxin (Trx), whose release into the extracellular matrix is promoted by interferon- g. In the course of those studies, we also identified the first small molecule TG2 inhibitor with activity in vivo. Building on these results, our extensively revised application focuses on understanding the biochemical mechanism and physiological relevance of this unusual allosteric regulatory phenomenon. We also propose to exploit this chemical switch to turn TG2 on in a controlled manner, thereby enabling quantitative identification of its endogenous substrates in systems of relevance to celiac disease.
Aim 1 proposes to study the mechanistic principles by which Trx recognizes and reduces the C371-C372 disulfide bond in TG2. Three sub-Aims are proposed: (A) Mapping TG2-Trx interactions by alanine-scanning mutagenesis;(B) Engineering a Trx mutant to covalently trap and structurally characterize the transient TG2- Trx complex;and (C) Engineering small molecule inhibitors to inactivate extracellular Trx without affecting its intracellular counterpart. These insights and tools will not only inform our own studies under Aim 2, but will also have broader biological relevance.
Aim 2 will test in vivo our hypothesis that Trx activates extracellular TG2 via two complementary approaches. In sub-Aim A, inhibitors from Aim 1C will be tested in one constitutive and two induced animal models where TG2 activity has been observed. In sub-Aim B, covalent TG2-Trx complexes will be trapped and visualized using the Trx mutant from Aim 1B. If successful, our results will substantially strengthen the case for Trx being the missing link between inflammatory T cell activity and TG2 activity in celiac disease.
Aim 3 will identify endogenous proteins that undergo post-translational modification when TG2 activity is switched on in the extracellular matrix. In sub-Aim A, we will use cultured enterocytes and fibroblasts to identify such substrates. Sub-Aim 2 seeks to validate their identity in the mouse intestine. Sub-Aim C presents an example of a simple biological assay to understand the role of these post-translational modifications in cell adhesion. These molecular insights are expected to broaden our view of the pathophysiological consequences of up-regulated TG2 activity in celiac disease beyond generation of gluten-derived T cell epitopes.

Public Health Relevance

Celiac disease is a widespread, lifelong disease of the small intestine. There is growing awareness of the central role of transglutaminase 2 (TG2) in celiac disease pathogenesis. Having contributed to this emerging picture in the past funding cycle, the goal of the present proposal is to strengthen this knowledge base and to develop innovative molecular tools to investigate the role of TG2 in complex biological systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK063158-12
Application #
8691788
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Grey, Michael J
Project Start
2002-10-01
Project End
2018-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
12
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Stanford
State
CA
Country
United States
Zip Code
94304
Plugis, Nicholas M; Palanski, Brad A; Weng, Chih-Hisang et al. (2017) Thioredoxin-1 Selectively Activates Transglutaminase 2 in the Extracellular Matrix of the Small Intestine: IMPLICATIONS FOR CELIAC DISEASE. J Biol Chem 292:2000-2008
Bouziat, Romain; Hinterleitner, Reinhard; Brown, Judy J et al. (2017) Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease. Science 356:44-50
Hilmer, Andrew J; Jeffrey, R Brooke; Park, Walter G et al. (2017) Cholestyramine as a promising, strong anion exchange resin for direct capture of genetic biomarkers from raw pancreatic fluids. Biotechnol Bioeng 114:934-938
Garber, Mitchell E; Saldanha, Alok; Parker, Joel S et al. (2017) A B-Cell Gene Signature Correlates With the Extent of Gluten-Induced Intestinal Injury in Celiac Disease. Cell Mol Gastroenterol Hepatol 4:1-17
Syage, Jack A; Murray, Joseph A; Green, Peter H R et al. (2017) Latiglutenase Improves Symptoms in Seropositive Celiac Disease Patients While on a Gluten-Free Diet. Dig Dis Sci 62:2428-2432
Wibowo, Arif; Park, Jae Mo; Liu, Shie-Chau et al. (2017) Real-Time in Vivo Detection of H2O2 Using Hyperpolarized 13C-Thiourea. ACS Chem Biol 12:1737-1742
Guo, Shunling; Palanski, Brad A; Kloeck, Cornelius et al. (2017) Intracellular TG2 Activity Increases Microtubule Stability but is not Sufficient to Prompt Neurite Growth. Neurosci Bull 33:103-106
Lebwohl, Benjamin; Murray, Joseph A; VerdĂș, Elena F et al. (2016) Gluten Introduction, Breastfeeding, and Celiac Disease: Back to the Drawing Board. Am J Gastroenterol 111:12-4
Yi, Michael C; Khosla, Chaitan (2016) Thiol-Disulfide Exchange Reactions in the Mammalian Extracellular Environment. Annu Rev Chem Biomol Eng 7:197-222
Plugis, Nicholas M; Khosla, Chaitan (2015) Therapeutic approaches for celiac disease. Best Pract Res Clin Gastroenterol 29:503-21

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