Chemical and biological regulation of extracellular human transglutaminase 2 activity [electronic resource]

Transglutaminase 2 (TG2) is a ubiquitously expressed, Ca2+-activated enzyme in mammals that catalyzes the formation of cross-links between glutamine and lysine residues on protein or peptide substrates. While chiefly a cytosolic enzyme, TG2 can localize to the extracellular environment through a poorly understood non-classical secretory mechanism. Aberrant extracellular TG2 activity has been implicated in several human diseases. Most notable is celiac disease (CD; celiac sprue), a widespread lifelong autoimmune disorder that affects the small intestine and is driven by the consumption of dietary gluten. In the context of CD, TG2 is responsible for the deamidation of immunogenic gluten-derived peptides, resulting in their increased antigenicity in genetically susceptible individuals (HLA-DQ2 or HLA-DQ8). However, extracellular TG2 is predominantly catalytically inactive in most organs under normal physiological conditions and the precise mechanism of activation in the celiac gut is unknown. A clearer understanding of how TG2 activity is regulated at the post-translational level could shed light not only on our understanding of celiac disease pathogenesis but lead to the development of novel therapeutic strategies to combat diseases affected by abnormal extracellular TG2 activity. In the first part of this thesis, we identify a novel class of compounds that exhibits unusual dual antagonist and agonist action on TG2. Acylideneoxoindoles were first identified in our lab as reversible inhibitors but closer examination has revealed that these molecules behave as activators under sub-saturating but physiologically relevant Ca2+ concentrations. Detailed analysis of a lead compound, CK-IV-55, revealed that these class of molecules target low-affinity Ca2+ binding sites on the catalytic core of TG2. This discovery sheds light on potential dietary triggers of TG2 activation as indoles are abundant in cruciferous vegetables and can also be produced by commensal gut bacteria. Until recently, the mechanisms that lead to inactive extracellular TG2 remained a fundamental mystery; the extracellular environment fosters conditions that favor constitutive TG2 activation due to the abundance of Ca2+. Identification of a vicinal disulfide bond (Cys370-Cys371) that inhibits enzymatic function and acts as a protein redox switch has provided the missing link. While disulfide bonds play a large role in maintaining tertiary and quaternary protein structure, allosteric disulfide bonds that control the function of mature proteins have recently been identified. Previous efforts in our lab have established that the redox protein cofactor thioredoxin-1 (TRX) could switch 'on' TG2 in vitro and in vivo through cleavage of the vicinal disulfide bond but it is unclear how TG2 is switched 'off.' Here, we systematically evaluated biologically relevant oxidants for their ability to oxidatively inactivate TG2 and identified the thiol-disulfide oxidoreductase, ERp57, as a suitable candidate. This discovery presents the first example of an allosteric disulfide bond redox switch that is dynamically regulated by two distinct proteins. Lastly, we validate the redox regulation of human TG2 through mutagenetic analysis and develop a robust tissue culture model that displays constitutive extracellular TG2 activity. This powerful tool overcomes limitations of previous models and could elucidate the biological consequences of extracellular TG2 activity. In summary, this dissertation provides insights into the complex post-translational regulation of TG2 using a variety of chemical biology techniques. We identify acylideneoxoindoles as allosteric chemical activators of TG2 that can be derived from the diet or other exogenous sources. We also provide a mechanism for the oxidative inactivation of TG2 through a unique redox switch controlled by two distinct proteins. Lastly, tools for developing robust cell culture models to assess the biological consequences of extracellular TG2 activity are discussed. These findings serve as the much-needed foundation to understanding the pathophysiological implications of this enigmatic protein.

Keywords: Chemical Engineering.
Advisor: Khosla Chaitan
Dunn Alexander Robert advisor. Thesis advisor
Sattely Elizabeth advisor. Thesis advisor