An Isotopically Tagged Azobenzene‐Based Cleavable Linker for Quantitative Proteomics

Yu, Qian; Martell, Julianne; Pace, Nicholas J.; Ballard, T. Eric; Johnson, Douglas S.; Weerapana, Eranthie

doi:10.1002/cbic.201300396

Cited by 69 publications

(65 citation statements)

References 35 publications

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“…Therefore we used competitive cysteine reactivity profiling with an iodoacetamide-alkyne (IA-alkyne) probe (100 μM) to more thoroughly investigate the effects of ITCs on the global cysteine reactome of HELA cells (18). Over 1000 IA-alkyne-labeled cysteine residues were identified in HELA cells using mass-spectrometry analysis.…”

Section: Resultsmentioning

confidence: 99%

Naturally Occurring Isothiocyanates Exert Anticancer Effects by Inhibiting Deubiquitinating Enzymes

et al. 2015

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The anticancer properties of cruciferous vegetables are well known and attributed to an abundance of isothiocyanates (ITCs) such as benzyl ITC (BITC) and phenethyl ITC (PEITC). While many potential targets of ITCs have been proposed, a full understanding of the mechanisms underlying their anticancer activity has remained elusive. Here we report that BITC and PEITC effectively inhibit deubiquitinating enzymes (DUBs), including the enzymes USP9x and UCH37, which are associated with tumorigenesis, at physiologically relevant concentrations and time scales. USP9x protects the anti-apoptotic protein Mcl-1 from degradation, and cells dependent on Mcl-1 were especially sensitive to BITC and PEITC. These ITCs increased Mcl-1 ubiquitination and either ITC treatment or RNAi-mediated silencing of USP9x decreased Mcl-1 levels, consistent with the notion that USP9x is a primary target of ITC activity. These ITCs also increased ubiquitination of the oncogenic fusion protein Bcr-Abl, resulting in degradation under low ITC concentrations and aggregation under high ITC concentrations. USP9x inhibition paralleled the decrease in Bcr-Abl levels induced by ITC treatment, and USP9x silencing was sufficient to decrease Bcr-Abl levels, further suggesting that Bcr-Abl is a USP9x substrate. Overall, our findings suggest that USP9x targeting is critical to the mechanism underpinning the well established anticancer activity of ITC. We propose that the ITC-induced inhibition of DUB may also explain how ITCs affect inflammatory and DNA repair processes, thus offering a unifying theme in understanding the function and useful application of ITCs to treat cancer as well as a variety of other pathological conditions.

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Section: Resultsmentioning

confidence: 99%

Naturally Occurring Isothiocyanates Exert Anticancer Effects by Inhibiting Deubiquitinating Enzymes

et al. 2015

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“…A third limitation of the current method is its bias towards highly abundant proteins, which may obfuscate the detection of comparatively rare signaling proteins that are subject to regulation by non-enzymatic acylation. In the future this may be addressed by integrating thioester probes with affinity enrichment strategies that use isotopically-tagged cleavable linkers (Qian et al, 2013; Weerapana et al, 2007), which would enable detection and quantification of non-enzymatic acylation at the peptide level, or integration with recombinant protein array technologies, which can detect the ex situ thioester reactivity of many proteins in parallel independent of protein abundance (Olia et al, 2015). The development of such methods have the potential to further broaden and enrich our understanding of non-enzymatic acylation in biology.…”

Section: Discussionmentioning

confidence: 99%

Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling

Kulkarni

Worth

Zengeya

et al. 2017

Cell Chemical Biology

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SUMMARY Non-enzymatic protein modification driven by thioester reactivity is thought to play a major role in the establishment of cellular lysine acylation. However, the specific protein targets of this process are largely unknown. Here we report an experimental strategy to investigate non-enzymatic acylation in cells. Specifically, we develop a chemoproteomic method that separates thioester reactivity from enzymatic utilization, allowing selective enrichment of non-enzymatic acylation targets. Applying this method to cancer cell lines identifies numerous candidate targets of non-enzymatic acylation, including several enzymes in lower glycolysis. Functional studies highlight malonyl-CoA as a reactive thioester metabolite that can modify and inhibit glycolytic enzyme activity. Finally, we show that synthetic thioesters can be used as novel reagents to probe non-enzymatic acylation in living cells. Our studies provide new insights into the targets and drivers of non-enzymatic acylation, and demonstrate the utility of reactivity-based methods to experimentally investigate this phenomenon in biology and disease.

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“…The platform was extended by applying a promiscuous cysteine-reactive probe to globally identify putative Zn 2+ -binding cysteines across ~900 cysteines in the human proteome (Figure 5b). This strategy employs isotopic, chemically cleavable azobenzene biotin tags (Azo-H & Azo-L) [74] that are conjugated to the Zn 2+ -treated and control proteomes. The populations are enriched on streptavidin, mixed, and digested with trypsin.…”

Section: Methods Of Identification Of Zn2+-cysteine Complexesmentioning

confidence: 99%

Zinc-Binding Cysteines: Diverse Functions and Structural Motifs

2014

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Cysteine residues are known to perform essential functions within proteins, including binding to various metal ions. In particular, cysteine residues can display high affinity toward zinc ions (Zn2+), and these resulting Zn2+-cysteine complexes are critical mediators of protein structure, catalysis and regulation. Recent advances in both experimental and theoretical platforms have accelerated the identification and functional characterization of Zn2+-bound cysteines. Zn2+-cysteine complexes have been observed across diverse protein classes and are known to facilitate a variety of cellular processes. Here, we highlight the structural characteristics and diverse functional roles of Zn2+-cysteine complexes in proteins and describe structural, computational and chemical proteomic technologies that have enabled the global discovery of novel Zn2+-binding cysteines.

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An Isotopically Tagged Azobenzene‐Based Cleavable Linker for Quantitative Proteomics

Cited by 69 publications

References 35 publications

Naturally Occurring Isothiocyanates Exert Anticancer Effects by Inhibiting Deubiquitinating Enzymes

Naturally Occurring Isothiocyanates Exert Anticancer Effects by Inhibiting Deubiquitinating Enzymes

Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling

Zinc-Binding Cysteines: Diverse Functions and Structural Motifs

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