Cobley JN et al. (2019), Catalyst-free Click PEGylation reveals substant...

XB-ART-56066

Redox Biol 2019 Sep 01;26:101258. doi: 10.1016/j.redox.2019.101258.

Show Gene links Show Anatomy links

Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation.

Cobley JN , Noble A , Jimenez-Fernandez E , Valdivia Moya MT , Guille M , Husi H .

???displayArticle.abstract???
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.

???displayArticle.pubmedLink??? 31234016
???displayArticle.pmcLink??? PMC6597785
???displayArticle.link??? Redox Biol
???displayArticle.grants??? [+]

???attribute.lit??? ???displayArticles.show???


	Fig. 1. Catalyst-free trans-cyclooctene-methyltetrazine (TCO-Tz) Click PEGylation schematic. The left side of the circle depicts the TCO-Tz Click PEGylation reduction (Click-PEGRED) protocol wherein reduced thiols are labelled with TCO-3 polyethylene glycol (PEG)-maleimide (TCO-PEG3-NEM, TPN); (2) before 6-methyltetrazine PEG 5 kDa (Tz-PEG5) is added to initiate the inverse electron demand Diels alder reaction and thereby mass shift reduced thiols. An optional Tris (2-carboxyethyl)phosphine hydrochloride (TCEP) reduction step to reduce reversibly oxidised thiols before labelling them with N-ethylmaleimide (NEM) is depicted. The right side of the circle depicts the TCO-Tz Click PEGylation oxidation (Click-PEGOX) protocol wherein (1) reduced thiols are labelled with NEM; (2) reversibly oxidised thiols are reduced with TCEP; (3) before being labelled with TCO-PEG3-NEM (TPN); and (4) 6 Tz-PEG5 is added to initiate the inverse electron demand Diels Alder reaction and thereby mass shift reversibly oxidised thiols. The insets on the far right depicts NEM mediated Michael addition (top), TPN mediated Michael addition to a reduced thiol (middle) and the inverse electron demand TCO-Tz Diels Alder reaction (bottom).
	Fig. 2. Cysteine residues in X. laevis ATP-α-F1are evolutionary conserved. Top. Phylogenetic tree of the common model organisms selected. Middle. Multiple sequence alignments of ATP-α-F1 derived from Clustal Omega (an open access online tool: https://www.ebi.ac.uk/Tools/msa/clustalo/). Bottom. Amino acid mark-up showing conserved cysteine residues in yellow. C294 occurs earlier in X. laevis due to a small deletion (see main text). Neighbouring amino acids surrounding C244 and C294 are highly conserved across phyla. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 3. ATP-α-F1is subject to substantial reversible oxidation before and after fertilisation in X. laevis. Left. Western blot image showing reversibly oxidised (i.e., mass shifted 5 and 10 kDa bands) relative to reduced ATP-α-F1 (bottom band) before and after (15 and 90 min) fertilisation in X. laevis. Right. Percent reversible ATP-α-F1 oxidation before (n = 12) and after fertilisation (n = 12) quantified. Each n = a pool of 5 eggs/embryos. The positive control refers to a diamide (50 mM for 30 min) treated sample. A one way ANOVA revealed no significant differences between time-points (P = 0.8306).
	Fig. 4. Trans-cyclooctene-methyltetrazine (TCO-Tz) Click PEGylation reduction (Click-PEGRED) confirms substantial reversible ATP-α-F1before and after fertilisation in X. laevis. Left. Western blot image showing reduced (i.e., mass shifted 5 and 10 kDa bands) relative to reversibly oxidised ATP-α-F1 (bottom band) before and after (15 and 90 min) fertilisation in X. laevis. Right. Percent ATP-α-F1 reduction before (n = 9) and after fertilisation (n = 9) in X. laevis quantified. Each n = a pool of 5 eggs/embryos. The positive control refers to a TCEP (50 mM for 30 min) treated sample. A one way ANOVA revealed no significant differences between time-points (P = 0.6366).
	Fig. 5. ATP-α-F1is reversibly S-glutathionylated before and after fertilisation in X. laevis. Top. A reaction scheme depicting the selected chemical reduction strategy for S-glutathionylated proteins. In this scheme, GRX2 reduces S-glutathionylated proteins using glutathione (GSH) derived electrons. Glutathione reductase (GR) is used to reduce oxidised glutathione (GSSG) using NADPH. Left. Western blot image showing the S-glutathionylated (i.e., mass shifted 5 and 10 kDa bands) relative to total ATP-α-F1 (bottom band) before and after (90 min) fertilisation in X. laevis. Right. Percent reversible ATP-α-F1 S-glutathionylation (i.e., PSSG) before (n = 10) and after fertilisation (n = 10) in X. laevis quantified. Each n = a pool of 5 eggs/embryos. An independent unpaired Student's t-test revealed no statistically significant difference in reversible S-glutathionylation between time-points (P = 0.8901).
	Fig. 6. Three dimensional molecular model of mitochondrial F1-FoATP synthase in state 3a derived fromRef. [65]in Bos taurus. State 3a corresponds to a catalytic rotary state of the F1 domain. Cysteine residues are highlighted in yellow, with C244 on the left and C294 on the right in each model display (left: surface; centre: space fill; right: ribbon). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

References [+] :

Abrahams, Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. 1994, Pubmed