Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature

The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Nature (Impact Factor: 41.46). 11/2010; 468(7325):790-5. DOI: 10.1038/nature09472
Source: PubMed


Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulphur protein biogenesis. We also demonstrate that quantitative reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.

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    • "Cysteine (Cys), an amino acid in living organisms plays a crucial role in numerous biological systems. Cys is a component of proteins, a source of sulfide in iron–sulfur clusters, and a part of the antioxidant glutathione (GSH) [1] [2]. Abnormal levels of Cys have been linked to several serious diseases, such as edema, hair depigmentation, liver damage, slowed growth and skin lesions [3]. "
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    ABSTRACT: BODIPY functionalized Au nanoparticles (PyBDP-AuNPs) have been developed for detecting cysteine. Gold nanoparticles were prepared by reducing HAuCl4 with sodium citrate, using PyBDP as the capping agent. Upon addition of cysteine, PyBDP molecules are liberated from the AuNP surface through ligand-exchange displacement based on the strong affinity of Au with the thiol group and result in green fluorescence from PyBDP molecules. This cysteine-induced displacement of PyBDP-AuNPs was monitored by fluorescent spectroscopy with a detection limit of 1.2 μM. Optimal detection of cysteine was achieved over a pH range from 5 to 12. In addition, PyBDP-AuNPs can be used as a fluorescent probe for detecting cysteine in living cells.
    Sensors and Actuators B Chemical 12/2015; 221:1366–1371. DOI:10.1016/j.snb.2015.08.015 · 4.10 Impact Factor
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    • "The DUB inhibitory activity is likely to be due to the electrophilic nature of these compounds coupled with the fact that the vast majority of DUBs are cysteine proteases that are sensitive to electrophilic attack. Generally, cysteine residues in proteins are nucleophilic and tend to show high levels of variation with regard to their reactivity to electrophiles (Codreanu et al., 2014; Weerapana et al., 2010). ␣,␤-unsaturated ketones are generally considered as relatively soft electrophiles and are speculated to display selective reactivity towards a specific subset of cysteine thiolates in proteins (Aldini et al., 2006). "
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    ABSTRACT: Although more traditionally associated with degradation and maintenance of protein homeostasis, the ubiquitin-proteasome system (UPS) has emerged as a critical component in the regulation of cancer cell growth and survival. The development of inhibitors that block the proteolytic activities of the proteasome have highlighted its suitability as a bona fide anti-cancer drug target. However, key determinants including the development of drug resistance and dose-limiting toxicity call for the identification of alternative components of the UPS for novel drug targeting. Recently the deubiquitinases (DUBs), a diverse family of enzymes that catalyze ubiquitin removal, have attracted significant interest as targets for the development of next generation UPS inhibitors. In particular, pharmacological inhibition of the proteasomal cysteine DUBs (i.e., USP14 and UCHL5) has been shown to be particularly cytotoxic to cancer cells and inhibit tumour growth in several in vivo models. In the current review we focus on the modes of action of proteasome DUB inhibitors and discus the potential of DUB inhibitors to circumvent acquired drug resistance and provide a therapeutic option for the treatment of cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.
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    • "Redox-Modified Cysteine Residues Occur Outside Common Functional Domains To further explore possible underlying biochemical and biophysical factors that promote specificity of post-translational cysteine modifications, an in-depth structural and biochemical analysis of the proteomes was performed. We did not identify a specific primary sequence motif associated with the cysteine modifications , consistent with previous analyses of large datasets including those of reactive cysteine residues (defined by the reactivity toward iodoacetamide) (Marino and Gladyshev, 2010b; Weerapana et al., 2010). However, the frequency of certain amino acids within a 13-amino-acid window surrounding the modified cysteine residues revealed that cysteine, glutamine, serine, and tryptophan are generally under-represented while alanine, histidine, isoleucine, lysine, methionine, and valine are generally over-represented amino acids (Figure S1). "
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    ABSTRACT: S-Acylation, S-glutathionylation, S-nitrosylation, and S-sulfenylation are prominent, chemically distinct modifications that regulate protein function, redox sensing, and trafficking. Although the biological significance of these modifications is increasingly appreciated, their integration in the proteome remains unknown. Novel mass spectrometry-based technologies identified 2,596 predominately unique sites in 1,319 mouse liver proteins under physiological conditions. Structural analysis localized the modifications in unique, evolutionary conserved protein segments, outside commonly annotated functional regions. Contrary to expectations, propensity for modification did not correlate with biophysical properties that regulate cysteine reactivity. However, the in vivo chemical reactivity is fine-tuned for specificity, demonstrated by the nominal complementation between the four modifications and quantitative proteomics which showed that a reduction in S-nitrosylation is not correlated with increased S-glutathionylation. A comprehensive survey uncovered clustering of modifications within biologically related protein networks. The data provide the first evidence for the occurrence of distinct, endogenous protein networks that undergo redox signaling through specific cysteine modifications. Copyright © 2015 Elsevier Ltd. All rights reserved.
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