Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation
Using non-reducing Western blotting to assess protein thiol redox state is challenging because most reduced and oxidised forms migrate at the same molecular weight and are, therefore, indistinguishable. While copper catalysed Click chemistry can be used to ligate a polyethylene glycol (PEG) moiety t...
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doaj-d18f44a516b8445893e1d7c561771be62020-11-25T01:20:47ZengElsevierRedox Biology2213-23172019-09-0126Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisationJames N. Cobley0Anna Noble1Eduardo Jimenez-Fernandez2Manuel-Thomas Valdivia Moya3Matthew Guille4Holger Husi5Free Radical Research Group, University of the Highlands and Islands, Centre for Health Sciences, Inverness, IV2 3JH, UK; Corresponding author.European Xenopus Resource Centre, University of Portsmouth, School of Biological Sciences, King Henry Building, Portsmouth, PO1 2DY, UKFree Radical Research Group, University of the Highlands and Islands, Centre for Health Sciences, Inverness, IV2 3JH, UKFree Radical Research Group, University of the Highlands and Islands, Centre for Health Sciences, Inverness, IV2 3JH, UKEuropean Xenopus Resource Centre, University of Portsmouth, School of Biological Sciences, King Henry Building, Portsmouth, PO1 2DY, UKFree Radical Research Group, University of the Highlands and Islands, Centre for Health Sciences, Inverness, IV2 3JH, UKUsing non-reducing Western blotting to assess protein thiol redox state is challenging because most reduced and oxidised forms migrate at the same molecular weight and are, therefore, indistinguishable. While copper catalysed Click chemistry can be used to ligate a polyethylene glycol (PEG) moiety termed Click PEGylation to mass shift the reduced or oxidised form as desired, the potential for copper catalysed auto-oxidation is problematic. Here we define a catalyst-free trans-cyclooctene-methyltetrazine (TCO-Tz) inverse electron demand Diels Alder chemistry approach that affords rapid (k ~2000 M−1 s−1), selective and bio-orthogonal Click PEGylation. We used TCO-Tz Click PEGylation to investigate how fertilisation impacts reversible mitochondrial ATP synthase F1-Fo sub-unit alpha (ATP-α-F1) oxidation—an established molecular correlate of impaired enzyme activity—in Xenopus laevis. TCO-Tz Click PEGylation studies reveal substantial (~65%) reversible ATP-α-F1 oxidation at evolutionary conserved cysteine residues (i.e., C244 and C294) before and after fertilisation. A single thiol is, however, preferentially oxidised likely due to greater solvent exposure during the catalytic cycle. Selective reduction experiments show that: S-glutathionylation accounts for ~50–60% of the reversible oxidation observed, making it the dominant oxidative modification type. Intermolecular disulphide bonds may also contribute due to their relative stability. Substantial reversible ATP-α-F1 oxidation before and after fertilisation is biologically meaningful because it implies low mitochondrial F1-Fo ATP synthase activity. Catalyst-free TCO-Tz Click PEGylation is a valuable new tool to interrogate protein thiol redox state in health and disease. Keywords: Mitochondria, ATP synthase, Fertilisation, Development, Redox signallinghttp://www.sciencedirect.com/science/article/pii/S2213231719305634 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
James N. Cobley Anna Noble Eduardo Jimenez-Fernandez Manuel-Thomas Valdivia Moya Matthew Guille Holger Husi |
spellingShingle |
James N. Cobley Anna Noble Eduardo Jimenez-Fernandez Manuel-Thomas Valdivia Moya Matthew Guille Holger Husi Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation Redox Biology |
author_facet |
James N. Cobley Anna Noble Eduardo Jimenez-Fernandez Manuel-Thomas Valdivia Moya Matthew Guille Holger Husi |
author_sort |
James N. Cobley |
title |
Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation |
title_short |
Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation |
title_full |
Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation |
title_fullStr |
Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation |
title_full_unstemmed |
Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation |
title_sort |
catalyst-free click pegylation reveals substantial mitochondrial atp synthase sub-unit alpha oxidation before and after fertilisation |
publisher |
Elsevier |
series |
Redox Biology |
issn |
2213-2317 |
publishDate |
2019-09-01 |
description |
Using non-reducing Western blotting to assess protein thiol redox state is challenging because most reduced and oxidised forms migrate at the same molecular weight and are, therefore, indistinguishable. While copper catalysed Click chemistry can be used to ligate a polyethylene glycol (PEG) moiety termed Click PEGylation to mass shift the reduced or oxidised form as desired, the potential for copper catalysed auto-oxidation is problematic. Here we define a catalyst-free trans-cyclooctene-methyltetrazine (TCO-Tz) inverse electron demand Diels Alder chemistry approach that affords rapid (k ~2000 M−1 s−1), selective and bio-orthogonal Click PEGylation. We used TCO-Tz Click PEGylation to investigate how fertilisation impacts reversible mitochondrial ATP synthase F1-Fo sub-unit alpha (ATP-α-F1) oxidation—an established molecular correlate of impaired enzyme activity—in Xenopus laevis. TCO-Tz Click PEGylation studies reveal substantial (~65%) reversible ATP-α-F1 oxidation at evolutionary conserved cysteine residues (i.e., C244 and C294) before and after fertilisation. A single thiol is, however, preferentially oxidised likely due to greater solvent exposure during the catalytic cycle. Selective reduction experiments show that: S-glutathionylation accounts for ~50–60% of the reversible oxidation observed, making it the dominant oxidative modification type. Intermolecular disulphide bonds may also contribute due to their relative stability. Substantial reversible ATP-α-F1 oxidation before and after fertilisation is biologically meaningful because it implies low mitochondrial F1-Fo ATP synthase activity. Catalyst-free TCO-Tz Click PEGylation is a valuable new tool to interrogate protein thiol redox state in health and disease. Keywords: Mitochondria, ATP synthase, Fertilisation, Development, Redox signalling |
url |
http://www.sciencedirect.com/science/article/pii/S2213231719305634 |
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