Submitted to the Department of Chemical Engineering.
Thesis (Ph.D.)--Stanford University, 2013.
ABSTRACT Celiac disease (CD; celiac sprue) is a widespread inflammatory disease of the small intestine. The primary genetic (HLA-DQ2 or, less frequently, -DQ8) and environmental (dietary gluten) factors responsible for the onset of this lifelong illness have been well established. It has also been demonstrated that toxic gluten peptides elicit a strong immune response in the celiac intestine following posttranslational modification by an endogenous extracellular enzyme, transglutaminase 2 (TG2). However, under normal physiological conditions, extracellular TG2 in the small intestinal mucosa is predominantly inactive, and must therefore be activated before gluten peptides can be deamidated. A better understanding of the disease relevant mechanisms as well as the physiological consequences of TG2 activation could facilitate the discovery of drugs that protect celiac patients from gluten-induced immunotoxicity. Despite the growing prevalence of CD patients worldwide along with the incentive to translate the fundamental aspects of CD pathogenesis into clinical practices that facilitate improved quality of life for these patients, serious inadequacies in the available tools for long term therapy of CD still persist. Currently, the only clinically accepted treatment of CD is a gluten free diet (GFD) that, with the ubiquity of gluten in food, is extremely difficult to maintain and likely contributes to the incomplete recovery of many CD patients. Therefore, non-dietary methods of CD management are of considerable interest. However, the discovery and development of potential CD therapies critically depends on the use of improved CD relevant models. The research described in this dissertation was motivated by the need for improved chemical and biological tools to aid the discovery of novel CD targets and elucidate critical pathogenic mechanisms behind the activation of TG2. At present, a causative link between the transamidating activity of transglutaminase 2 (TG2) and celiac disease (CD) pathogenesis has not yet been definitively established. Nonetheless, a large body of evidence supports this hypothesis. First, TG2 has remarkably high substrate specificity for immunopathogenic gluten peptides. Second, recognition of these gluten peptides by the disease associated HLA-DQ2 or -DQ8 is strongly enhanced by TG2-catalyzed deamidation. Third, interferon-[gamma] (IFN-[gamma]) is the predominant cytokine secreted when DQ2-restricted, gluten responsive T cells derived from the celiac intestine are activated. IFN-[gamma] also triggers phosphatidylinositol-3-kinase (PI3K) and TRX mediated activation of extracellular TG2, thereby plausibly establishing an auto-amplificatory loop for gluten-mediated inflammation. Last but not least, chronic exposure of celiac patients to dietary gluten is invariably accompanied by the production of autoantibodies against TG2. Whereas the mechanistic underpinnings of some of these phenomena are relatively well understood, others are not. A deeper understanding of these TG2-related events will not only enable definitive verification that transglutaminase activity is necessary for gluten induced pathogenesis in CD, but could also cast fundamentally new light on the biological function(s) of TG2. A number of lines of evidence suggest that TG2 may be one of the earliest disease-relevant proteins to encounter immunotoxic gluten in the celiac gut. These and other investigations also suggest that the reaction catalyzed by TG2 on dietary gluten peptides is essential for the pathogenesis of CD. If so, several questions are of critical significance. How is TG2 activated in the celiac gut? What are the disease-specific and general consequences of activating TG2? Can local inhibition of TG2 in the celiac intestine suppress gluten induced pathogenesis? And what are the long-term consequences of suppressing TG2 activity? The main goal of this project and the subject of this dissertation were the design, development, and application of disease relevant models and biochemical tools for the study of TG2 activity and its potential physiological roles in CD pathogenesis. Innovative technologies advanced toward this goal have the potential of yielding powerful next-generation drug candidates for treating this widespread chronic disease.