Fluorescent Assay to Demonstrate the Interaction of Thioredoxin Reductase and Protein Disulfide Isomerase Tomazzolli R.1, Guella G.2, Bellisola G.3, Colombatti M.3 and Menestrina G.1 1: ITC-CNR IBF Unit at Trento, Via Sommarive 18, 38050 Povo (TN), Italy 2: Lab Bioorg Chem, Dep Physics, Uni Trento, Via Sommarive 14, 38050 Povo, Italy 3: Dep Pathology, Sect of Immunol, Uni Verona, L.go Scuro 10, 37134 Verona, Italy In eukaryotic cells, the ubiquitous thioredoxin reductase-thioredoxin system (TrxR-Trx) and the glutathione reductase-glutathione system (GSHR-GSSG) catalyse substrate disulfide reduction using NADPH as a source of reducing equivalents. TrxR reduces the redox protein Trx as well as other endogenous and exogenous compounds, while the glutathione system plays a key role in protecting cellular macromolecules from damage caused by reactive oxygen species. Due to a second redox-active site, mammalian TrxR has a broader substrate specificity than glutathione reductase and Escherichia coli TrxR [1]. Thioredoxin forms a superfamily with several other proteins sharing little sequence similarity but possessing a common active site [2]. Among these, protein disulfide isomerase (PDI) is colocalized, in cells, with TrxR. In vitro there are now some evidences that PDI can be a substrate of TrxR [3, 4]. To further demonstrate this interaction we have produced a fluorogenic substrate suitable to test TrxR-dependent PDI activity. The substrate is constituted by two identical cys-containing tri-peptides linked at the N-terminus to a molecule of fluorescein-5-isothiocyanate (FITC) and held together by a disulfide bridge between the cysteines. The preparation follows a few steps: the tri-peptide is labelled with FITC; it is separated from FITC excess by two reverse phase liquid chromatography elutions and analysed by electrospray ionization mass spectrometry (ESI-MS); oxidation occurs spontaneously or is induced by DMSO. The final substrate, named pep-FITC, is characterized by NMR and ESI-MS. The reduction of pep-FITC was measured at 25°C in a Fluoromax spectrofluorimeter and was indicated by an increase of the FITC fluorescence at 520nm with excitation at 494nm. All the fluorescence measurements were taken in NaP 0.1M, EDTA 2mM pH 7.5. We demonstrated that the TrxR (both mammalian and bacterial)-PDI system successfully reduced pep-FITC while the single components didn’t. GSHR didn’t reduce pep-FITC, in spite of its aminoacidic sequence similarity to GSSG but was active on a fluorogenic GSSG, labelled at N-termini with FITC, thus demonstrating the narrow substrate specificity of GSHR. Further proofs of the interaction between PDI and TrxR we obtained by fluorescence resonant energy transfer (FRET). In this case we labelled the enzymes with Alexa Fluor 546 and Alexa Fluor 488 respectively, two dyes forming a donor/acceptor pair with a Forster distance of 58 Å [5]. A FRET was observed under suitable conditions confirming tight interaction. (1) Mustacich D, Powis G. 2000. Biochem. J., 346: 1-8. (2). Hirota K, Nakamura H, Masutani H, Yodoi J. 2002. Ann. NY Acad. Sci., 957:189-99. (3). Lundstrom J, Holmgren A. 1990. J. Biol. Chem., 265: 9114-9120. (4). Bellisola G, Fracasso G, Ippoliti R, Menestrina G, Rosen A, Solda S, Udali S, Tomazzolli R, Tridente G, Colombatti M. 2004. Biochem. Pharmacol., 67: 1721-1731. (5). Epe B, Steinhauser KG, Woolley P. 1983. Proc. Natl. Acad. Sci. USA 80 (May), 2579-2583.

Fluorescent Assay to Demonstrate the Interaction of Thioredoxin Reductase and Protein Disulfide Isomerase.

COLOMBATTI, Marco;
2004

Abstract

Fluorescent Assay to Demonstrate the Interaction of Thioredoxin Reductase and Protein Disulfide Isomerase Tomazzolli R.1, Guella G.2, Bellisola G.3, Colombatti M.3 and Menestrina G.1 1: ITC-CNR IBF Unit at Trento, Via Sommarive 18, 38050 Povo (TN), Italy 2: Lab Bioorg Chem, Dep Physics, Uni Trento, Via Sommarive 14, 38050 Povo, Italy 3: Dep Pathology, Sect of Immunol, Uni Verona, L.go Scuro 10, 37134 Verona, Italy In eukaryotic cells, the ubiquitous thioredoxin reductase-thioredoxin system (TrxR-Trx) and the glutathione reductase-glutathione system (GSHR-GSSG) catalyse substrate disulfide reduction using NADPH as a source of reducing equivalents. TrxR reduces the redox protein Trx as well as other endogenous and exogenous compounds, while the glutathione system plays a key role in protecting cellular macromolecules from damage caused by reactive oxygen species. Due to a second redox-active site, mammalian TrxR has a broader substrate specificity than glutathione reductase and Escherichia coli TrxR [1]. Thioredoxin forms a superfamily with several other proteins sharing little sequence similarity but possessing a common active site [2]. Among these, protein disulfide isomerase (PDI) is colocalized, in cells, with TrxR. In vitro there are now some evidences that PDI can be a substrate of TrxR [3, 4]. To further demonstrate this interaction we have produced a fluorogenic substrate suitable to test TrxR-dependent PDI activity. The substrate is constituted by two identical cys-containing tri-peptides linked at the N-terminus to a molecule of fluorescein-5-isothiocyanate (FITC) and held together by a disulfide bridge between the cysteines. The preparation follows a few steps: the tri-peptide is labelled with FITC; it is separated from FITC excess by two reverse phase liquid chromatography elutions and analysed by electrospray ionization mass spectrometry (ESI-MS); oxidation occurs spontaneously or is induced by DMSO. The final substrate, named pep-FITC, is characterized by NMR and ESI-MS. The reduction of pep-FITC was measured at 25°C in a Fluoromax spectrofluorimeter and was indicated by an increase of the FITC fluorescence at 520nm with excitation at 494nm. All the fluorescence measurements were taken in NaP 0.1M, EDTA 2mM pH 7.5. We demonstrated that the TrxR (both mammalian and bacterial)-PDI system successfully reduced pep-FITC while the single components didn’t. GSHR didn’t reduce pep-FITC, in spite of its aminoacidic sequence similarity to GSSG but was active on a fluorogenic GSSG, labelled at N-termini with FITC, thus demonstrating the narrow substrate specificity of GSHR. Further proofs of the interaction between PDI and TrxR we obtained by fluorescence resonant energy transfer (FRET). In this case we labelled the enzymes with Alexa Fluor 546 and Alexa Fluor 488 respectively, two dyes forming a donor/acceptor pair with a Forster distance of 58 Å [5]. A FRET was observed under suitable conditions confirming tight interaction. (1) Mustacich D, Powis G. 2000. Biochem. J., 346: 1-8. (2). Hirota K, Nakamura H, Masutani H, Yodoi J. 2002. Ann. NY Acad. Sci., 957:189-99. (3). Lundstrom J, Holmgren A. 1990. J. Biol. Chem., 265: 9114-9120. (4). Bellisola G, Fracasso G, Ippoliti R, Menestrina G, Rosen A, Solda S, Udali S, Tomazzolli R, Tridente G, Colombatti M. 2004. Biochem. Pharmacol., 67: 1721-1731. (5). Epe B, Steinhauser KG, Woolley P. 1983. Proc. Natl. Acad. Sci. USA 80 (May), 2579-2583.
PDI; Fluorescence; Assay
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/20303
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact