Ditryptophan Cross-Links as Novel Products of Protein Oxidation

Protein oxidation is an unavoidable consequence of aerobic metabolism. The oxidation of most proteins residues is non-repairable and may affect protein structure and function. In particular, protein cross-links arising from oxidative modifications are presumably toxic to cells because they may accumulate and induce protein aggregation. However, most of these irreversible protein cross-links remain partially characterized. Up to very recently, ditryptophan cross-links (Trp-Trp), in particular, have been largely disregarded in the literature. Here, we briefly review studies showing that Trp-Trp cross-links can be formed in proteins exposed to a variety of oxidants. The challenges to fully characterize Trp-Trp cross-links are discussed as well as their potential roles in protein dysfunction and aggregation.

Keywords:
ditryptophan; cross-links; protein oxidation; free radicals

Authorship

Verônica Paviani

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Avenida Lineu Prestes, 748, 05508-000 São Paulo-SP, BrazilUniversidade de São PauloBrazilSão Paulo, SP, BrazilDepartamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Avenida Lineu Prestes, 748, 05508-000 São Paulo-SP, Brazil

Veronica Paviani graduated in Pharmacy (2010) and obtained the Master (2013) and the PhD degree (2016) in Biochemistry. Currently, she is a postdoctoral fellow at the Department of Biochemistry, Chemistry Institute, USP, where she employs mass spectrometry, among other techniques, to characterize oxidative modifications of proteins in vitro and in biological samples.

Gabriel T. Galdino

Gabriel Tiago Galdino is an undergraduate research student of the Chemistry Institute (IQ-USP) and he is working in the elucidation of posttranslational modifications of tryptophan since 2016. He is now completing the Molecular Sciences course and has experience in organic synthesis since high school when he developed a new larvicide from the cashew shell nut oil. He is a two-times champion of the Brazilian Science and Engineering Fair (FEBRACE) and obtained the third place in the Intel-International Science and Engineering Fair (Intel ISEF 2013), the biggest science competition for high school students in the world.

Janaina N. dos Prazeres

Janaina N. dos Prazeres is graduated in Biological Sciences at Universidade Estadual de Campinas (UNICAMP) in 1999, and obtained the Master and PhD degrees in 2002 and in 2006, respectively, in the Food Science Institute at the same University. Her experience includes protein biochemistry and enzyme expression in bacteria and yeast. Since 2009, she works as a biochemical specialist at Instituto de Química, Universidade de São Paulo (USP). Her current focus is analytical biochemistry, working with a wide variety of techniques such as liquid chromatography, 2D electrophoresis and mass spectrometry. She also has experience in working with laboratory animals.

Raphael F. Queiroz

Departamento de Ciências Naturais, Universidade Estadual do Sudoeste da Bahia, 45083-900 Vitória da Conquista-BA, BrazilUniversidade Estadual do Sudoeste da BahiaBrazilVitória da Conquista, BA, BrazilDepartamento de Ciências Naturais, Universidade Estadual do Sudoeste da Bahia, 45083-900 Vitória da Conquista-BA, Brazil

Raphael F. Queiroz received the bachelor's degree in Pharmacy 10 years ago from Universidade Federal de Alfenas, Minas Gerais, Brazil. He completed the PhD in Biochemistry in 2012 at the Instituto de Química, Universidade de São Paulo, São Paulo, Brazil. Since then, he is an Associate Professor of Pharmacology and Biochemistry at the Medical School of the Universidade do Sudoeste da Bahia. Currently, his research interests are focused on redox processes related to infection and inflammation, and pharmacology of natural products.

Ohara Augusto ^**e-mail: oaugusto@iq.usp.br

Ohara Augusto received her undergraduate (BSc in Chemistry in 1972) and graduate (PhD in Biochemistry in 1975) titles at the Instituto de Química, Universidade de São Paulo (IQ-USP) and did postdoctoral work at the University of California (Berkeley, 1980) and University of California (San Francisco, 1981-1982). She is Full Professor of Biochemistry at the IQ-USP since 1993. Her research interests have always been focused in the understanding of the multiple roles of free radicals and oxidants in physiology and pathophysiology. She has received several prizes and honors for her contributions to redox biochemistry.

*e-mail: oaugusto@iq.usp.br

Veronica Paviani graduated in Pharmacy (2010) and obtained the Master (2013) and the PhD degree (2016) in Biochemistry. Currently, she is a postdoctoral fellow at the Department of Biochemistry, Chemistry Institute, USP, where she employs mass spectrometry, among other techniques, to characterize oxidative modifications of proteins in vitro and in biological samples.

Gabriel Tiago Galdino is an undergraduate research student of the Chemistry Institute (IQ-USP) and he is working in the elucidation of posttranslational modifications of tryptophan since 2016. He is now completing the Molecular Sciences course and has experience in organic synthesis since high school when he developed a new larvicide from the cashew shell nut oil. He is a two-times champion of the Brazilian Science and Engineering Fair (FEBRACE) and obtained the third place in the Intel-International Science and Engineering Fair (Intel ISEF 2013), the biggest science competition for high school students in the world.

Janaina N. dos Prazeres is graduated in Biological Sciences at Universidade Estadual de Campinas (UNICAMP) in 1999, and obtained the Master and PhD degrees in 2002 and in 2006, respectively, in the Food Science Institute at the same University. Her experience includes protein biochemistry and enzyme expression in bacteria and yeast. Since 2009, she works as a biochemical specialist at Instituto de Química, Universidade de São Paulo (USP). Her current focus is analytical biochemistry, working with a wide variety of techniques such as liquid chromatography, 2D electrophoresis and mass spectrometry. She also has experience in working with laboratory animals.

Raphael F. Queiroz received the bachelor's degree in Pharmacy 10 years ago from Universidade Federal de Alfenas, Minas Gerais, Brazil. He completed the PhD in Biochemistry in 2012 at the Instituto de Química, Universidade de São Paulo, São Paulo, Brazil. Since then, he is an Associate Professor of Pharmacology and Biochemistry at the Medical School of the Universidade do Sudoeste da Bahia. Currently, his research interests are focused on redox processes related to infection and inflammation, and pharmacology of natural products.

Ohara Augusto received her undergraduate (BSc in Chemistry in 1972) and graduate (PhD in Biochemistry in 1975) titles at the Instituto de Química, Universidade de São Paulo (IQ-USP) and did postdoctoral work at the University of California (Berkeley, 1980) and University of California (San Francisco, 1981-1982). She is Full Professor of Biochemistry at the IQ-USP since 1993. Her research interests have always been focused in the understanding of the multiple roles of free radicals and oxidants in physiology and pathophysiology. She has received several prizes and honors for her contributions to redox biochemistry.

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (4)

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Figure 1
Some of the characterized products of the oxidation of protein-Trp residues.¹⁴14 Haywood, J.; Mozziconacci, O.; Allegre, K. M.; Kerwin, B. A.; Schöneich, C.; Mol. Pharmaceutics 2013, 10, 1146.^,¹⁹19 Medinas, D. B.; Gozzo, F. C.; Santos, L. F. A.; Iglesias, A. H.; Augusto, O.; Free Radical Biol. Med. 2010, 49, 1046.^,²⁶26 Plowman, J. E.; Deb-Choudhury, S.; Grosvenor, A. J.; Dyer, J. M.; Photochem. Photobiol. Sci. 2013, 12, 1960.

27 Simat, T. J.; Steinhart, H.; J. Agric. Food Chem. 1998, 46, 490.

28 Sala, A.; Nicolis, S.; Roncone, R.; Casella, L.; Monzani, E.; Eur. J. Biochem. 2004, 271, 2841.

29 Yamakura, F.; Ikeda, K.; Nitric Oxide 2006, 14, 152.

30 Ehrenshaft, M.; Deterding, L. J.; Mason, R. P.; Free Radical Biol. Med. 2015, 89, 220.

31 Pattison, D. I.; Rahmanto, A. S.; Davies, M. J.; Photochem. Photobiol. Sci. 2011, 11, 38.^-³²32 Ronsein, G. E.; de Oliveira, M. C. B.; de Medeiros, M. H. G.; Di Mascio, P.; J. Am. Soc. Mass Spectrom. 2009, 20, 188.

Thumbnail

Figure 2
Schematic representation of the mechanism proposed for hSOD1 dimerization during its bicarbonate-dependent peroxidase activity. This scheme also illustrates the fragmentation and rearrangement of the hSOD1Trp-TrphSOD1 dimer during MS/MS analysis. The inset shows a stereochemical view of the ditryptophan cross-link in the hSOD1 structure. The figure was adapted from reference 19 with permission.

Thumbnail

Figure 3
Time-dependent dimerization of lysozyme upon UV irradiation (a) and MS/MS characterization of the produced dimer (b) and (c). (a) Representative SDS-PAGE analysis of the time-dependent lysozyme dimerization and aggregation in samples of lysozyme (0.14 mM) irradiated with UV light (254 nm; radiance of 6.3 mW cm^-2) for the specified times in phosphate buffer (20 mM) containing DTPA (0.1 mM), pH 7.0. Aliquots corresponding to 30 µg protein were removed and analyzed by SDS-PAGE. The gel was stained with Coomassie Blue; (b) nano-ESI-Q-TOF-MS and (c) MS/MS analysis of the dimeric tryptic peptide (²²GYSLGNWVCAAK³³)₂ obtained from lysozyme treated with UV light for 10 min. The MS/MS sequencing of the peak at m/z 883.41, which corresponds to the peptide (²²GYSLGNWVCAAK³³)₂ (monoisotopic mass 2647.24 Da) with 3 charges is shown in (c) and its MS is shown in (b). The lysozyme dimer was isolated from the enzyme irradiated for 10 min as shown in (a); the samples were applied in 8 gel lanes, and the spots (at approximately 28 kDa) were excised from the gel, digested with trypsin, and subjected to ESI-Q-TOF-MS/MS analysis. Reprinted from reference 21 with permission.

Thumbnail

Figure 4
Characterization of ditryptophan crosslinks produced by treatmente of Trp with the CO₃^•-. Trp (1 mM), DTPA (0.1 mM) and the carbonatotetramminecobalt(III) complex (4 mM) in phosphate buffer (20 mM), pH 7.0 were irradiated for 1 min with UV light at a wavelength of 254 nm (radiance of 6.3 mW cm^-2) (abbreviated as Co/UVC).²¹21 Paviani, V.; Queiroz, R. F.; Marques, E. F.; Di Mascio, P.; Augusto, O.; Free Radical Biol. Med. 2015, 89, 72. The control corresponds to the same solution without irradiation. (a) Extraction ion chromatogram corresponding to ditryptophan (m/z 407.17 with one charge); (b) HPLC-ESI-Q-TOF-MS/MS analysis of the ion with m/z 407.17 corresponding to peaks 1, 2 and 3 shown in (a) as specified.

Figure 1 Some of the characterized products of the oxidation of protein-Trp residues.¹⁴14 Haywood, J.; Mozziconacci, O.; Allegre, K. M.; Kerwin, B. A.; Schöneich, C.; Mol. Pharmaceutics 2013, 10, 1146.^,¹⁹19 Medinas, D. B.; Gozzo, F. C.; Santos, L. F. A.; Iglesias, A. H.; Augusto, O.; Free Radical Biol. Med. 2010, 49, 1046.^,²⁶26 Plowman, J. E.; Deb-Choudhury, S.; Grosvenor, A. J.; Dyer, J. M.; Photochem. Photobiol. Sci. 2013, 12, 1960.

27 Simat, T. J.; Steinhart, H.; J. Agric. Food Chem. 1998, 46, 490.

28 Sala, A.; Nicolis, S.; Roncone, R.; Casella, L.; Monzani, E.; Eur. J. Biochem. 2004, 271, 2841.

29 Yamakura, F.; Ikeda, K.; Nitric Oxide 2006, 14, 152.

30 Ehrenshaft, M.; Deterding, L. J.; Mason, R. P.; Free Radical Biol. Med. 2015, 89, 220.

31 Pattison, D. I.; Rahmanto, A. S.; Davies, M. J.; Photochem. Photobiol. Sci. 2011, 11, 38.^-³²32 Ronsein, G. E.; de Oliveira, M. C. B.; de Medeiros, M. H. G.; Di Mascio, P.; J. Am. Soc. Mass Spectrom. 2009, 20, 188.

Figure 2 Schematic representation of the mechanism proposed for hSOD1 dimerization during its bicarbonate-dependent peroxidase activity. This scheme also illustrates the fragmentation and rearrangement of the hSOD1Trp-TrphSOD1 dimer during MS/MS analysis. The inset shows a stereochemical view of the ditryptophan cross-link in the hSOD1 structure. The figure was adapted from reference 19 with permission.

Figure 3 Time-dependent dimerization of lysozyme upon UV irradiation (a) and MS/MS characterization of the produced dimer (b) and (c). (a) Representative SDS-PAGE analysis of the time-dependent lysozyme dimerization and aggregation in samples of lysozyme (0.14 mM) irradiated with UV light (254 nm; radiance of 6.3 mW cm^-2) for the specified times in phosphate buffer (20 mM) containing DTPA (0.1 mM), pH 7.0. Aliquots corresponding to 30 µg protein were removed and analyzed by SDS-PAGE. The gel was stained with Coomassie Blue; (b) nano-ESI-Q-TOF-MS and (c) MS/MS analysis of the dimeric tryptic peptide (²²GYSLGNWVCAAK³³)₂ obtained from lysozyme treated with UV light for 10 min. The MS/MS sequencing of the peak at m/z 883.41, which corresponds to the peptide (²²GYSLGNWVCAAK³³)₂ (monoisotopic mass 2647.24 Da) with 3 charges is shown in (c) and its MS is shown in (b). The lysozyme dimer was isolated from the enzyme irradiated for 10 min as shown in (a); the samples were applied in 8 gel lanes, and the spots (at approximately 28 kDa) were excised from the gel, digested with trypsin, and subjected to ESI-Q-TOF-MS/MS analysis. Reprinted from reference 21 with permission.

Figure 4 Characterization of ditryptophan crosslinks produced by treatmente of Trp with the CO₃^•-. Trp (1 mM), DTPA (0.1 mM) and the carbonatotetramminecobalt(III) complex (4 mM) in phosphate buffer (20 mM), pH 7.0 were irradiated for 1 min with UV light at a wavelength of 254 nm (radiance of 6.3 mW cm^-2) (abbreviated as Co/UVC).²¹21 Paviani, V.; Queiroz, R. F.; Marques, E. F.; Di Mascio, P.; Augusto, O.; Free Radical Biol. Med. 2015, 89, 72. The control corresponds to the same solution without irradiation. (a) Extraction ion chromatogram corresponding to ditryptophan (m/z 407.17 with one charge); (b) HPLC-ESI-Q-TOF-MS/MS analysis of the ion with m/z 407.17 corresponding to peaks 1, 2 and 3 shown in (a) as specified.

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[1] *e-mail: oaugusto@iq.usp.br