Using FLIM-FRET to measure conformational changes of transglutaminase type 2 in live cells - PubMed (original) (raw)

Using FLIM-FRET to measure conformational changes of transglutaminase type 2 in live cells

Nicholas S Caron et al. PLoS One. 2012.

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.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. The Transglutaminase type 2 (TG2) Conformational FRET Sensor.

(A and B) Speculative models of mCerulean-TG2-eYFP FRET sensor in a GDP/GTP bound closed conformation (A) and of mCerulean-TG2-eYFP sensor in a Ca2+ and substrate bound open conformation (B). (C) Sample FLIM image of mCerulean-TG2 with eYFP alone negative control. Intensity, intensity-weighted lifetime images and lifetime histograms are shown for every construct. Lifetimes shown in the intensity-weighted lifetime images are pseudo-coloured using a rainbow LUT that corresponds to the lifetime scale represented in the histogram. Dashed white lines within the histograms represents the approximate lifetime with the highest number of pixels. (D) Sample FLIM image of mCerulean-TG2 co-expressed with TG2-eYFP, a negative control for intermolecular FRET. (E) Sample FLIM image of mCerulean-TG2-eYFP conformational FRET sensor. (F) Quantitative FLIM-FRET data shown as percent efficiency of FRET for TG2 negative controls, as well as the mCerulean-TG2-eYFP FRET sensor. (G) Quantification of the effect on percent FRET efficiency of adding 4 glycines as linkers between the fluorophores and TG2. The boxes encompass the 25% to 75% confidence intervals, the line within indicates the data median and the whiskers represent the 5% to 95% confidence intervals. *p<0.001. N = 15 for 6 replicates. Scale bar represents 10 µm.

Figure 2

Figure 2. Point Mutations and Cell Stresses Affect TG2 Conformation in Live Cells.

(A) Quantitative FLIM-FRET data shown as percent efficiency of FRET for the mCerulean-TG2-eYFP FRET sensor, GDP/GTP insensitive R580A mutant and catalytically inactive W241A mutant. Black line represents median values, boxes encompass 25% and 75% confidence intervals and whiskers indicate the 5% to 95% confidence intervals. *p<0.001. N = 15 for 4 replicates. (B–D) Representative FLIM images of data in (A). (E) Quantitative FLIM-FRET data shown as percent efficiency of FRET for the mCerulean-TG2-eYFP FRET sensor, or the sensor following either a 30 minute heat shock (42.5°C) treatment or a treatment with Ca2+ ionophore A23187 for 10 minutes. (F, G) Representative FLIM images of data in (E). *p<0.001. N>10 for 4 replicates. Scale bar represents 10 µm.

Figure 3

Figure 3. TG2 Inhibitors Differentially Affect TG2 Conformation.

(A) Quantitative FLIM-FRET data shown as percent efficiency of FRET for the mCerulean-TG2-eYFP FRET sensor after a 16 hour treatment with either the irreversible TG2 inhibitor, NC9, or the reversible TG2 inhibitor, CP4d. (B) Percent efficiency of FRET graph generated following treatment of TG2 sensor with 5 increasing concentrations of CP4d. (C) Percent efficiency of FRET graph for TG2 sensor following treatment with 10 µM NC9 for 5 different time points from 0 to 24 hours. Black line represents median values, boxes encompass 25% and 75% confidence intervals and whiskers indicate the 5% to 95% confidence intervals. *p<0.001, **p<0.05 and N.S = not significant. N>10 for 4 replicates.

References

    1. Folk JE, Finlayson JS (1977) The epsilon-(gamma-glutamyl)lysine crosslink and the catalytic role of transglutaminases. AdvProtein Chem 31: 1–133. - PubMed
    1. Griffin M, Casadio R, Bergamini CM (2002) Transglutaminases: nature’s biological glues. BiochemJ 368: 377–396. - PMC - PubMed
    1. Chen JS, Mehta K (1999) Tissue transglutaminase: an enzyme with a split personality. IntJBiochemCell Biol 31: 817–836. - PubMed
    1. Nakaoka H, Perez DM, Baek KJ, Das T, Husain A, et al. (1994) Gh: a GTP-binding protein with transglutaminase activity and receptor signaling function. Science 264: 1593–1596. - PubMed
    1. Begg GE, Holman SR, Stokes PH, Matthews JM, Graham RM, et al. (2006) Mutation of a critical arginine in the GTP-binding site of transglutaminase 2 disinhibits intracellular cross-linking activity. JBiol Chem 281: 12603–12609. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources