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A Versatile Strategy for the Semisynthetic Production of Ser65 Phosphorylated Ubiquitin and Its Biochemical and Structural Characterisation.

Han C, Pao KC, Kazlauskaite A, Muqit MM, Virdee S - Chembiochem (2015)

Bottom Line: Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form.The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function.Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH (UK).

No MeSH data available.


Related in: MedlinePlus

Biochemical analysis of activation of Parkin E3 ligase by phospho-Ser65-ubiquitin. A) Top: Ub (anti-Flag) immunoblot of Parkin-mediated polyubiquitin chain formation in the presence of the indicated Ub species. Ub: wild-type ubiquitin; Ub-pSer65: wild-type ubiquitin enzymatically phosphorylated by recombinant PINK1;10 UbC46 and UbC46-pSer65: synthetic non-phosphorylated and phosphorylated ubiquitin generated by ligation of biosynthetic and synthetic precursors. Middle: Miro1 (anti-SUMO) immunoblot demonstrates Parkin-mediated ubiquitylation of His-SUMO-Miro1 in the presence of the various Ub species. Bottom: anti-Parkin immunoblot demonstrates the extent of autoubiquitylation in the presence of the indicated Ub species. (B) Top: quantification of Parkin-dependent E2 discharge mediated by the indicated Ub species. The activity was assessed by change in UbcH7/UbcH7-Ub ratio. Bottom: the E2-charging reaction was performed in the presence of Ube1, UbcH7 and FLAG-ubiquitin with magnesium acetate and ATP followed by addition of indicated Ub species. Reaction mixtures were subjected to SDS-PAGE and Coomassie staining.
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fig02: Biochemical analysis of activation of Parkin E3 ligase by phospho-Ser65-ubiquitin. A) Top: Ub (anti-Flag) immunoblot of Parkin-mediated polyubiquitin chain formation in the presence of the indicated Ub species. Ub: wild-type ubiquitin; Ub-pSer65: wild-type ubiquitin enzymatically phosphorylated by recombinant PINK1;10 UbC46 and UbC46-pSer65: synthetic non-phosphorylated and phosphorylated ubiquitin generated by ligation of biosynthetic and synthetic precursors. Middle: Miro1 (anti-SUMO) immunoblot demonstrates Parkin-mediated ubiquitylation of His-SUMO-Miro1 in the presence of the various Ub species. Bottom: anti-Parkin immunoblot demonstrates the extent of autoubiquitylation in the presence of the indicated Ub species. (B) Top: quantification of Parkin-dependent E2 discharge mediated by the indicated Ub species. The activity was assessed by change in UbcH7/UbcH7-Ub ratio. Bottom: the E2-charging reaction was performed in the presence of Ube1, UbcH7 and FLAG-ubiquitin with magnesium acetate and ATP followed by addition of indicated Ub species. Reaction mixtures were subjected to SDS-PAGE and Coomassie staining.

Mentions: We next tested the cysteine mutant UbC46C and UbC46-pSer65 in biochemical assays for their ability to activate the E3 ligase Parkin.8 We employed a Parkin E3 ligase activity assay that monitors ubiquitylation of the substrate Miro1 as well as the formation of free polyubiquitin chains; this assay has previously been used to demonstrate activation of full- length Parkin E3 ligase activity upon addition of enzyme-derived Ub-pSer65.8a As expected, UbC46 did not lead to appreciable activation of Parkin; however, UbC46-pSer65 induced marked Parkin activation, as determined by polyubiquitin chain formation, ubiquitylation of Miro1 and Parkin autoubiquitylation (Figure 2 A). We also assessed UbC46 and UbC46-pSer65 for their ability to stimulate Parkin-mediated discharge of ubiquitin from a ubiquitin-charged E2 (UbcH7∼Ub) as previously observed for enzymatically derived Ub-pSer65.8a This assay decouples the loading of E3 with ubiquitin from transfer of ubiquitin from E3 to substrate. As expected, UbC46-pSer65 promoted Parkin-mediated ubiquitin discharge from UbcH7∼Ub, whereas UbC46 did not (Figure 2 B). In both the ubiquitylation assay and the UbcH7∼Ub discharge assay, Parkin activation was not as high as observed with wild-type Ub that had been phosphorylated by PINK1.8a The minimal activation of Parkin by UbC46 is most likely attributable to the Ala46Cys mutation (which can be converted to the native Ala).12e


A Versatile Strategy for the Semisynthetic Production of Ser65 Phosphorylated Ubiquitin and Its Biochemical and Structural Characterisation.

Han C, Pao KC, Kazlauskaite A, Muqit MM, Virdee S - Chembiochem (2015)

Biochemical analysis of activation of Parkin E3 ligase by phospho-Ser65-ubiquitin. A) Top: Ub (anti-Flag) immunoblot of Parkin-mediated polyubiquitin chain formation in the presence of the indicated Ub species. Ub: wild-type ubiquitin; Ub-pSer65: wild-type ubiquitin enzymatically phosphorylated by recombinant PINK1;10 UbC46 and UbC46-pSer65: synthetic non-phosphorylated and phosphorylated ubiquitin generated by ligation of biosynthetic and synthetic precursors. Middle: Miro1 (anti-SUMO) immunoblot demonstrates Parkin-mediated ubiquitylation of His-SUMO-Miro1 in the presence of the various Ub species. Bottom: anti-Parkin immunoblot demonstrates the extent of autoubiquitylation in the presence of the indicated Ub species. (B) Top: quantification of Parkin-dependent E2 discharge mediated by the indicated Ub species. The activity was assessed by change in UbcH7/UbcH7-Ub ratio. Bottom: the E2-charging reaction was performed in the presence of Ube1, UbcH7 and FLAG-ubiquitin with magnesium acetate and ATP followed by addition of indicated Ub species. Reaction mixtures were subjected to SDS-PAGE and Coomassie staining.
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Related In: Results  -  Collection

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fig02: Biochemical analysis of activation of Parkin E3 ligase by phospho-Ser65-ubiquitin. A) Top: Ub (anti-Flag) immunoblot of Parkin-mediated polyubiquitin chain formation in the presence of the indicated Ub species. Ub: wild-type ubiquitin; Ub-pSer65: wild-type ubiquitin enzymatically phosphorylated by recombinant PINK1;10 UbC46 and UbC46-pSer65: synthetic non-phosphorylated and phosphorylated ubiquitin generated by ligation of biosynthetic and synthetic precursors. Middle: Miro1 (anti-SUMO) immunoblot demonstrates Parkin-mediated ubiquitylation of His-SUMO-Miro1 in the presence of the various Ub species. Bottom: anti-Parkin immunoblot demonstrates the extent of autoubiquitylation in the presence of the indicated Ub species. (B) Top: quantification of Parkin-dependent E2 discharge mediated by the indicated Ub species. The activity was assessed by change in UbcH7/UbcH7-Ub ratio. Bottom: the E2-charging reaction was performed in the presence of Ube1, UbcH7 and FLAG-ubiquitin with magnesium acetate and ATP followed by addition of indicated Ub species. Reaction mixtures were subjected to SDS-PAGE and Coomassie staining.
Mentions: We next tested the cysteine mutant UbC46C and UbC46-pSer65 in biochemical assays for their ability to activate the E3 ligase Parkin.8 We employed a Parkin E3 ligase activity assay that monitors ubiquitylation of the substrate Miro1 as well as the formation of free polyubiquitin chains; this assay has previously been used to demonstrate activation of full- length Parkin E3 ligase activity upon addition of enzyme-derived Ub-pSer65.8a As expected, UbC46 did not lead to appreciable activation of Parkin; however, UbC46-pSer65 induced marked Parkin activation, as determined by polyubiquitin chain formation, ubiquitylation of Miro1 and Parkin autoubiquitylation (Figure 2 A). We also assessed UbC46 and UbC46-pSer65 for their ability to stimulate Parkin-mediated discharge of ubiquitin from a ubiquitin-charged E2 (UbcH7∼Ub) as previously observed for enzymatically derived Ub-pSer65.8a This assay decouples the loading of E3 with ubiquitin from transfer of ubiquitin from E3 to substrate. As expected, UbC46-pSer65 promoted Parkin-mediated ubiquitin discharge from UbcH7∼Ub, whereas UbC46 did not (Figure 2 B). In both the ubiquitylation assay and the UbcH7∼Ub discharge assay, Parkin activation was not as high as observed with wild-type Ub that had been phosphorylated by PINK1.8a The minimal activation of Parkin by UbC46 is most likely attributable to the Ala46Cys mutation (which can be converted to the native Ala).12e

Bottom Line: Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form.The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function.Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH (UK).

No MeSH data available.


Related in: MedlinePlus