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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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Detalles Bibliográficos
Autores principales: Cobley, James N., Noble, Anna, Jimenez-Fernandez, Eduardo, Valdivia Moya, Manuel-Thomas, Guille, Matthew, Husi, Holger
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597785/
https://www.ncbi.nlm.nih.gov/pubmed/31234016
http://dx.doi.org/10.1016/j.redox.2019.101258
Descripción
Sumario: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 F(1)-F(o) sub-unit alpha (ATP-α-F(1)) oxidation—an established molecular correlate of impaired enzyme activity—in Xenopus laevis. TCO-Tz Click PEGylation studies reveal substantial (~65%) reversible ATP-α-F(1) oxidation at evolutionary conserved cysteine residues (i.e., C(244) and C(294)) 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-F(1) oxidation before and after fertilisation is biologically meaningful because it implies low mitochondrial F(1)-F(o) ATP synthase activity. Catalyst-free TCO-Tz Click PEGylation is a valuable new tool to interrogate protein thiol redox state in health and disease.