Abstract
Transglutaminase type 2 (TG2) is a ubiquitously expressed member of the transglutaminase family, capable of mediating a transamidation reaction between a variety of protein substrates. TG2 also has a unique role as a G-protein with GTPase activity. In response to GDP/GTP binding and increases in intracellular calcium levels, TG2 can undergo a large conformational change that reciprocally modulates the enzymatic activities of TG2. We have generated a TG2 biosensor that allows for quantitative assessment of TG2 conformational changes in live cells using Förster resonance energy transfer (FRET), as measured by fluorescence lifetime imaging microscopy (FLIM). Quantifying FRET efficiency with this biosensor provides a robust assay to quickly measure the effects of cell stress, changes in calcium levels, point mutations and chemical inhibitors on the conformation and localization of TG2 in living cells. The TG2 FRET biosensor was validated using established TG2 conformational point mutants, as well as cell stress events known to elevate intracellular calcium levels. We demonstrate in live cells that inhibitors of TG2 transamidation activity can differentially influence the conformation of the enzyme. The irreversible inhibitor of TG2, NC9, forces the enzyme into an open conformation, whereas the reversible inhibitor CP4d traps TG2 in the closed conformation. Thus, this biosensor provides new mechanistic insights into the action of two TG2 inhibitors and defines two new classes based on ability to alter TG2 conformation in addition to inhibiting transamidation activity. Future applications of this biosensor could be to discover small molecules that specifically alter TG2 conformation to affect GDP/GTP or calcium binding.
Highlights
Transglutaminase type 2 (TG2; EC 2.3.2.13) is a multifunctional enzyme capable of catalyzing several calcium-dependent reactions, including a transamidation reaction between the c-carboxamide group of a peptide bound glutamine and a variety of amine substrates [1], in both an intraand extracellular context [2]
The various activities of TG2 have been directly implicated in the progression of celiac disease, arthrosclerosis, diabetes, cancer, glaucoma and the formation of cataracts [27].TG2 has a role in multiple neurodegenerative diseases including Huntington’s (HD) [28], Parkinson’s (PD) as well as Alzheimer’s disease (AD) [29,30].Studies of patient brains have shown that expression levels and transamidating activity of TG2 are highly elevated in these diseases, where it has been proposed that cell death may be caused by the aberrant cross-linking of substrates [30]
The role of TG2 in the cell death process is dependent on cell type, the stressor, its subcellular localization, its enzymatic role, and its conformation [24]
Summary
Transglutaminase type 2 (TG2; EC 2.3.2.13) is a multifunctional enzyme capable of catalyzing several calcium-dependent reactions, including a transamidation reaction (covalent cross-link) between the c-carboxamide group of a peptide bound glutamine and a variety of amine substrates [1], in both an intraand extracellular context [2]. TG2 can hydrolyze GTP, where it acts as a G-protein mediating the phospholipase C signalling cascade [3,4] These cellular roles of TG2 are reciprocally regulated by a large conformational change [5,6]. An additional crystal structure shows that a substrate-mimicking peptide inhibitor bound to TG2 extends the enzyme to an ‘open’ conformation [6]. This suggests that the open conformation represents the enzymatically active version of the enzyme, yet to date no crystal has been solved with both calcium ions and a substrate bound to TG2. Under specific cell stress conditions, calcium levels rise dramatically causing a shift in the TG2 population towards its open and enzymatically active cross-linking conformation
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