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On terminal alkynes that can react with active-site cysteine nucleophiles in proteases.

Ekkebus R, van Kasteren SI, Kulathu Y, Scholten A, Berlin I, Geurink PP, de Jong A, Goerdayal S, Neefjes J, Heck AJ, Komander D, Ovaa H - J. Am. Chem. Soc. (2013)

Bottom Line: By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed.The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography.The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

ABSTRACT
Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward de-ubiquitinating enzymes. The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.

Show MeSH
Quantificationof DUBs precipitated from EL-4 lysate after on-beadreaction with Ub-Prg. Ratios of ion intensities of proteins retrievedfrom Ub-Prg-resin pulldown vs pulldown with a control resin are shown.
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fig5: Quantificationof DUBs precipitated from EL-4 lysate after on-beadreaction with Ub-Prg. Ratios of ion intensities of proteins retrievedfrom Ub-Prg-resin pulldown vs pulldown with a control resin are shown.

Mentions: Identification of the isolated DUBs by LC-MS/MS(Figures 5 and S13) showed thatmembers of all four known classes of cysteine DUBs were retrievedin a single simple experiment. Using a dimethyl-labeling-based quantitativeproteomics approach,24 all recovered DUBsdisplayed a selective enrichment over background (Figure 5). For all recovered DUBs, with the exception ofUCHL3, no signal was observed in the control sample. Nonspecific interactorswere not found to be significantly enriched. Only the E3-ligase HUWE1was found, which has also been reported using other active-site directedprobes.25 The general applicability ofthis reaction was tested on another member of the family of Ub-likeproteins, Nedd8 (Figure S14). A syntheticpropargylated Nedd8 reacted efficiently with UCHL3, which isknown to cross-react with Nedd8.26


On terminal alkynes that can react with active-site cysteine nucleophiles in proteases.

Ekkebus R, van Kasteren SI, Kulathu Y, Scholten A, Berlin I, Geurink PP, de Jong A, Goerdayal S, Neefjes J, Heck AJ, Komander D, Ovaa H - J. Am. Chem. Soc. (2013)

Quantificationof DUBs precipitated from EL-4 lysate after on-beadreaction with Ub-Prg. Ratios of ion intensities of proteins retrievedfrom Ub-Prg-resin pulldown vs pulldown with a control resin are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585465&req=5

fig5: Quantificationof DUBs precipitated from EL-4 lysate after on-beadreaction with Ub-Prg. Ratios of ion intensities of proteins retrievedfrom Ub-Prg-resin pulldown vs pulldown with a control resin are shown.
Mentions: Identification of the isolated DUBs by LC-MS/MS(Figures 5 and S13) showed thatmembers of all four known classes of cysteine DUBs were retrievedin a single simple experiment. Using a dimethyl-labeling-based quantitativeproteomics approach,24 all recovered DUBsdisplayed a selective enrichment over background (Figure 5). For all recovered DUBs, with the exception ofUCHL3, no signal was observed in the control sample. Nonspecific interactorswere not found to be significantly enriched. Only the E3-ligase HUWE1was found, which has also been reported using other active-site directedprobes.25 The general applicability ofthis reaction was tested on another member of the family of Ub-likeproteins, Nedd8 (Figure S14). A syntheticpropargylated Nedd8 reacted efficiently with UCHL3, which isknown to cross-react with Nedd8.26

Bottom Line: By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed.The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography.The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

ABSTRACT
Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward de-ubiquitinating enzymes. The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.

Show MeSH