The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function

Elizabeth Escobar-Álvarez; Fabian Leinisch; Gissela Araya; Octavio Monasterio; Lasse G. Lorentzen; Eduardo Silva; Michael J. Davies; Camilo López-Alarcón

doi:10.1016/j.freeradbiomed.2017.07.014

The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function

Elizabeth Escobar-Álvarez, Fabian Leinisch, Gissela Araya, Octavio Monasterio, Lasse G. Lorentzen, Eduardo Silva, Michael J. Davies, Camilo López-Alarcón^*

^*Corresponding author for this work

Inflammation, Metabolism and Oxidation

6 Citations (Scopus)

Abstract

FtsZ (filamenting temperature-sensitive mutant Z) is a key protein in bacteria cell division. The wild-type Escherichia coli FtsZ sequence (FtsZwt) contains three tyrosine (Tyr, Y) and sixteen methionine (Met, M) residues. The Tyr at position 222 is a key residue for FtsZ polymerization. Mutation of this residue to tryptophan (Trp, W; mutant Y222W) inhibits GTPase activity resulting in an extended time in the polymerized state compared to FtsZwt. Protein oxidation has been highlighted as a determinant process for bacteria resistance and consequently oxidation of FtsZwt and the Y222W mutant, by peroxyl radicals (ROO•) generated from AAPH (2,2′-azobis(2-methylpropionamidine) dihydrochloride) was studied. The non-oxidized proteins showed differences in their polymerization behavior, with this favored by the presence of Trp at position 222. AAPH-treatment of the proteins inhibited polymerization. Protein integrity studies using SDS-PAGE revealed the presence of both monomers and oligomers (dimers, trimers and high mass material) on oxidation. Western blotting indicated the presence of significant levels of protein carbonyls. Amino acid analysis showed that Tyr, Trp (in the Y222W mutant), and Met were consumed by ROO•. Quantification of the number of moles of amino acid consumed per mole of ROO• shows that most of the initial oxidant can be accounted for at low radical fluxes, with Met being a major target. Western blotting provided evidence for di-tyrosine cross-links in the dimeric and trimeric proteins, confirming that oxidation of Tyr residues, at positions 339 and/or 371, are critical to ROO•-mediated crosslinking of both the FtsZwt and Y222W mutant protein. These findings are in agreement with di-tyrosine, N-formyl kynurenine, and kynurenine quantification assessed by UPLC, and with LC-MS data obtained for AAPH-treated protein samples.

Original language	English
Journal	Free Radical Biology and Medicine
Volume	112
Pages (from-to)	60-68
Number of pages	9
ISSN	0891-5849
DOIs	https://doi.org/10.1016/j.freeradbiomed.2017.07.014
Publication status	Published - Nov 2017

Keywords

AAPH
Di-tyrosine
FtsZ
Peroxyl radicals
Protein oxidation
Tryptophan
Tyrosine

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10.1016/j.freeradbiomed.2017.07.014

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Escobar-Álvarez, E., Leinisch, F., Araya, G., Monasterio, O., Lorentzen, L. G., Silva, E., Davies, M. J., & López-Alarcón, C. (2017). The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function. Free Radical Biology and Medicine, 112, 60-68. https://doi.org/10.1016/j.freeradbiomed.2017.07.014

The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function. / Escobar-Álvarez, Elizabeth; Leinisch, Fabian; Araya, Gissela et al.

In: Free Radical Biology and Medicine, Vol. 112, 11.2017, p. 60-68.

Research output: Contribution to journal › Journal article › Research › peer-review

Escobar-Álvarez, E, Leinisch, F, Araya, G, Monasterio, O, Lorentzen, LG, Silva, E, Davies, MJ & López-Alarcón, C 2017, 'The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function', Free Radical Biology and Medicine, vol. 112, pp. 60-68. https://doi.org/10.1016/j.freeradbiomed.2017.07.014

Escobar-Álvarez E, Leinisch F, Araya G, Monasterio O, Lorentzen LG, Silva E et al. The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function. Free Radical Biology and Medicine. 2017 Nov;112:60-68. doi: 10.1016/j.freeradbiomed.2017.07.014

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title = "The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function",

abstract = "FtsZ (filamenting temperature-sensitive mutant Z) is a key protein in bacteria cell division. The wild-type Escherichia coli FtsZ sequence (FtsZwt) contains three tyrosine (Tyr, Y) and sixteen methionine (Met, M) residues. The Tyr at position 222 is a key residue for FtsZ polymerization. Mutation of this residue to tryptophan (Trp, W; mutant Y222W) inhibits GTPase activity resulting in an extended time in the polymerized state compared to FtsZwt. Protein oxidation has been highlighted as a determinant process for bacteria resistance and consequently oxidation of FtsZwt and the Y222W mutant, by peroxyl radicals (ROO•) generated from AAPH (2,2′-azobis(2-methylpropionamidine) dihydrochloride) was studied. The non-oxidized proteins showed differences in their polymerization behavior, with this favored by the presence of Trp at position 222. AAPH-treatment of the proteins inhibited polymerization. Protein integrity studies using SDS-PAGE revealed the presence of both monomers and oligomers (dimers, trimers and high mass material) on oxidation. Western blotting indicated the presence of significant levels of protein carbonyls. Amino acid analysis showed that Tyr, Trp (in the Y222W mutant), and Met were consumed by ROO•. Quantification of the number of moles of amino acid consumed per mole of ROO• shows that most of the initial oxidant can be accounted for at low radical fluxes, with Met being a major target. Western blotting provided evidence for di-tyrosine cross-links in the dimeric and trimeric proteins, confirming that oxidation of Tyr residues, at positions 339 and/or 371, are critical to ROO•-mediated crosslinking of both the FtsZwt and Y222W mutant protein. These findings are in agreement with di-tyrosine, N-formyl kynurenine, and kynurenine quantification assessed by UPLC, and with LC-MS data obtained for AAPH-treated protein samples.",

keywords = "AAPH, Di-tyrosine, FtsZ, Peroxyl radicals, Protein oxidation, Tryptophan, Tyrosine",

author = "Elizabeth Escobar-{\'A}lvarez and Fabian Leinisch and Gissela Araya and Octavio Monasterio and Lorentzen, {Lasse G.} and Eduardo Silva and Davies, {Michael J.} and Camilo L{\'o}pez-Alarc{\'o}n",

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T1 - The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function

AU - Escobar-Álvarez, Elizabeth

AU - Leinisch, Fabian

AU - Araya, Gissela

AU - Monasterio, Octavio

AU - Lorentzen, Lasse G.

AU - Silva, Eduardo

AU - Davies, Michael J.

AU - López-Alarcón, Camilo

PY - 2017/11

Y1 - 2017/11

N2 - FtsZ (filamenting temperature-sensitive mutant Z) is a key protein in bacteria cell division. The wild-type Escherichia coli FtsZ sequence (FtsZwt) contains three tyrosine (Tyr, Y) and sixteen methionine (Met, M) residues. The Tyr at position 222 is a key residue for FtsZ polymerization. Mutation of this residue to tryptophan (Trp, W; mutant Y222W) inhibits GTPase activity resulting in an extended time in the polymerized state compared to FtsZwt. Protein oxidation has been highlighted as a determinant process for bacteria resistance and consequently oxidation of FtsZwt and the Y222W mutant, by peroxyl radicals (ROO•) generated from AAPH (2,2′-azobis(2-methylpropionamidine) dihydrochloride) was studied. The non-oxidized proteins showed differences in their polymerization behavior, with this favored by the presence of Trp at position 222. AAPH-treatment of the proteins inhibited polymerization. Protein integrity studies using SDS-PAGE revealed the presence of both monomers and oligomers (dimers, trimers and high mass material) on oxidation. Western blotting indicated the presence of significant levels of protein carbonyls. Amino acid analysis showed that Tyr, Trp (in the Y222W mutant), and Met were consumed by ROO•. Quantification of the number of moles of amino acid consumed per mole of ROO• shows that most of the initial oxidant can be accounted for at low radical fluxes, with Met being a major target. Western blotting provided evidence for di-tyrosine cross-links in the dimeric and trimeric proteins, confirming that oxidation of Tyr residues, at positions 339 and/or 371, are critical to ROO•-mediated crosslinking of both the FtsZwt and Y222W mutant protein. These findings are in agreement with di-tyrosine, N-formyl kynurenine, and kynurenine quantification assessed by UPLC, and with LC-MS data obtained for AAPH-treated protein samples.

AB - FtsZ (filamenting temperature-sensitive mutant Z) is a key protein in bacteria cell division. The wild-type Escherichia coli FtsZ sequence (FtsZwt) contains three tyrosine (Tyr, Y) and sixteen methionine (Met, M) residues. The Tyr at position 222 is a key residue for FtsZ polymerization. Mutation of this residue to tryptophan (Trp, W; mutant Y222W) inhibits GTPase activity resulting in an extended time in the polymerized state compared to FtsZwt. Protein oxidation has been highlighted as a determinant process for bacteria resistance and consequently oxidation of FtsZwt and the Y222W mutant, by peroxyl radicals (ROO•) generated from AAPH (2,2′-azobis(2-methylpropionamidine) dihydrochloride) was studied. The non-oxidized proteins showed differences in their polymerization behavior, with this favored by the presence of Trp at position 222. AAPH-treatment of the proteins inhibited polymerization. Protein integrity studies using SDS-PAGE revealed the presence of both monomers and oligomers (dimers, trimers and high mass material) on oxidation. Western blotting indicated the presence of significant levels of protein carbonyls. Amino acid analysis showed that Tyr, Trp (in the Y222W mutant), and Met were consumed by ROO•. Quantification of the number of moles of amino acid consumed per mole of ROO• shows that most of the initial oxidant can be accounted for at low radical fluxes, with Met being a major target. Western blotting provided evidence for di-tyrosine cross-links in the dimeric and trimeric proteins, confirming that oxidation of Tyr residues, at positions 339 and/or 371, are critical to ROO•-mediated crosslinking of both the FtsZwt and Y222W mutant protein. These findings are in agreement with di-tyrosine, N-formyl kynurenine, and kynurenine quantification assessed by UPLC, and with LC-MS data obtained for AAPH-treated protein samples.

KW - AAPH

KW - Di-tyrosine

KW - FtsZ

KW - Peroxyl radicals

KW - Protein oxidation

KW - Tryptophan

KW - Tyrosine

U2 - 10.1016/j.freeradbiomed.2017.07.014

DO - 10.1016/j.freeradbiomed.2017.07.014

M3 - Journal article

C2 - 28733212

AN - SCOPUS:85025085472

SN - 0891-5849

VL - 112

SP - 60

EP - 68

JO - Free Radical Biology & Medicine

JF - Free Radical Biology & Medicine

ER -

The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function

Abstract

Keywords

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