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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.

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Labeling of recombinant caspase-1 with alkyne-based caspase-1 probeanalyzed by fluorescent scanning of SDS-PAGE gel. Recombinant caspase-1was labeled with two different lengths of caspase probe (left) anddoped in U937 lysate (right), showing selective labeling of caspase-1in lysate.
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fig6: Labeling of recombinant caspase-1 with alkyne-based caspase-1 probeanalyzed by fluorescent scanning of SDS-PAGE gel. Recombinant caspase-1was labeled with two different lengths of caspase probe (left) anddoped in U937 lysate (right), showing selective labeling of caspase-1in lysate.

Mentions: To test whether reaction of alkynes with activesite cysteine nucleophilesis limited to the class of Ub-like proteases or can be applied toother families of cysteine proteases as well, we synthesized propargylanalogues of common peptide aldehyde-based cysteine protease inhibitorsby directly converting the aldehyde to alkyne using Bestmann–Ohirareagent27 (Figure S15). The Prg analogues of the inhibitors of cathepsins (leupeptin)and caspases (Ac-YVAD-CHO) showed no significant inhibitory potentialscompared to the parent aldehydes (Figure S16). We postulated this was due to the low affinity of the short peptidefragments for these proteases. To test this, we synthesized two extendedcyanine 5 (Cy5) fluorophore-labeled (16 and 26 amino acid) peptidefragments derived from pro-IL1-β (a natural substrate of caspase-1),carrying a C-terminal propargylated aspartic acid residue (Figure 6). Notably, both peptides selectively labeled caspase-1,and this labeling could readily be abolished by the addition of thegeneral cysteine protease inhibitor iodoacetamide.


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)

Labeling of recombinant caspase-1 with alkyne-based caspase-1 probeanalyzed by fluorescent scanning of SDS-PAGE gel. Recombinant caspase-1was labeled with two different lengths of caspase probe (left) anddoped in U937 lysate (right), showing selective labeling of caspase-1in lysate.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585465&req=5

fig6: Labeling of recombinant caspase-1 with alkyne-based caspase-1 probeanalyzed by fluorescent scanning of SDS-PAGE gel. Recombinant caspase-1was labeled with two different lengths of caspase probe (left) anddoped in U937 lysate (right), showing selective labeling of caspase-1in lysate.
Mentions: To test whether reaction of alkynes with activesite cysteine nucleophilesis limited to the class of Ub-like proteases or can be applied toother families of cysteine proteases as well, we synthesized propargylanalogues of common peptide aldehyde-based cysteine protease inhibitorsby directly converting the aldehyde to alkyne using Bestmann–Ohirareagent27 (Figure S15). The Prg analogues of the inhibitors of cathepsins (leupeptin)and caspases (Ac-YVAD-CHO) showed no significant inhibitory potentialscompared to the parent aldehydes (Figure S16). We postulated this was due to the low affinity of the short peptidefragments for these proteases. To test this, we synthesized two extendedcyanine 5 (Cy5) fluorophore-labeled (16 and 26 amino acid) peptidefragments derived from pro-IL1-β (a natural substrate of caspase-1),carrying a C-terminal propargylated aspartic acid residue (Figure 6). Notably, both peptides selectively labeled caspase-1,and this labeling could readily be abolished by the addition of thegeneral cysteine protease inhibitor iodoacetamide.

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