Proteome-Wide Survey Reveals Norbornene Is Complementary to Dimedone and Related Nucleophilic Probes for Cysteine Sulfenic Acid

<p>Detection of cysteine sulfenic acid in live cells is critical in advancing our understanding of cysteine redox chemistry and its biological function. Accordingly, there is a need to develop sulfenic acid-specific chemical probes with distinct reaction mechanisms to facilitate proteome-wide detection of this important posttranslational modification. Herein, we report the first whole-cell proteomics analysis using a norbornene probe to detect cysteine sulfenic acid in live HeLa cells. Comparison of the enriched proteins to those identified using dimedone and other <i>C</i>-nucleophilic probes revealed a complementary reactivity profile. Remarkably, 148 new members of the sulfenome were identified. These discoveries highlight how subtle differences in chemical reactivity of both the probes and cysteine residues influence detection. Overall, this study expands our understanding of protein oxidation at cysteine and reveals new proteins to consider for future studies of cysteine oxidation, redox regulation and signaling, and the biochemistry of oxidative stress. </p>

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Norbornene Probes for the Detection of Cysteine Sulfenic Acid in Cells

10.26434/chemrxiv.7396958.v1 ◽

2018 ◽

Author(s):

Lisa Alcock ◽

Bruno Oliveira ◽

Michael Deery ◽

Tara Pukala ◽

Michael Perkins ◽

...

Keyword(s):

Biological Systems ◽

Live Cells ◽

Cysteine Oxidation ◽

Sulfenic Acid ◽

Sulfenic Acids ◽

Norbornene Derivatives ◽

In Cells

Norbornene derivatives were validated as probes for cysteine sulfenic acid on proteins and in live cells. Trapping sulfenic acids with norbornene probes is highly selective and revealed a different reactivity profile than the traditional dimedone reagent. The norbornene probe also revealed a superior chemoselectivity when compared to a commonly used dimedone probe. Together, these results advance the study of cysteine oxidation in biological systems.

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Norbornene Probes for the Detection of Cysteine Sulfenic Acid in Cells

10.26434/chemrxiv.7396958 ◽

2018 ◽

Author(s):

Lisa Alcock ◽

Bruno Oliveira ◽

Michael Deery ◽

Tara Pukala ◽

Michael Perkins ◽

...

Keyword(s):

Biological Systems ◽

Live Cells ◽

Cysteine Oxidation ◽

Sulfenic Acid ◽

Sulfenic Acids ◽

Norbornene Derivatives ◽

In Cells

Norbornene derivatives were validated as probes for cysteine sulfenic acid on proteins and in live cells. Trapping sulfenic acids with norbornene probes is highly selective and revealed a different reactivity profile than the traditional dimedone reagent. The norbornene probe also revealed a superior chemoselectivity when compared to a commonly used dimedone probe. Together, these results advance the study of cysteine oxidation in biological systems.

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α-Methylene-β-Lactone Probe for Measuring Live-Cell Reactions of Small Molecules

10.26434/chemrxiv.12078582.v2 ◽

2020 ◽

Author(s):

Lei Wang ◽

Louis Riel ◽

Bekim Bajrami ◽

Bin Deng ◽

Amy Howell ◽

...

Keyword(s):

Mass Spectra ◽

Live Cell ◽

Live Cells ◽

The Novel ◽

Proteomics Analysis ◽

Chemical Proteomics ◽

Tandem Mass Spectra ◽

Modified Peptides ◽

Covalent Probes ◽

Ht 29

The novel use of the α-methylene-β-lactone (MeLac) moiety as a warhead of multiple electrophilic sites is reported. In this study, we demonstrate that a MeLac-alkyne is a competent covalent probe and reacts with diverse proteins in live cells. Proteomics analysis of affinity-enriched samples identifies probe-reacted proteins, resolves their modified peptides/residues, and thus characterizes probe-protein reactions. Unique methods are developed to evaluate confidence in the identification of the reacted proteins and modified peptides. Tandem mass spectra of the peptides reveal that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael addition or acyl addition. A peptide-centric proteomics platform, using MeLac-alkyne as the measurement probe, successfully analyzes the Orlistat selectivity in live HT-29 cells. MeLac is a versatile warhead demonstrating enormous potential to expedite the development of covalent probes and inhibitors in interrogating protein (re)activity. MeLac-empowered platforms in chemical proteomics are widely adaptable for measuring the live-cell action of reactive molecules.

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Parallel evaluation of nucleophilic and electrophilic chemical probes for sulfenic acid: Reactivity, selectivity and biocompatibility

Redox Biology ◽

10.1016/j.redox.2021.102072 ◽

2021 ◽

pp. 102072

Author(s):

Yunlong Shi ◽

Kate S. Carroll

Keyword(s):

Chemical Probes ◽

Sulfenic Acid ◽

Parallel Evaluation

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Studies on the structure and function of 16S ribosomal RNA using structure-specific chemical probes

Journal of Biosciences ◽

10.1007/bf02702773 ◽

1985 ◽

Vol 8 (3-4) ◽

pp. 747-755 ◽

Cited By ~ 1

Author(s):

Harry F. Noller ◽

Barbara J. Van Stolk ◽

Danesh Moazed ◽

Stephen Douthwaite ◽

Robin R. Gutell

Keyword(s):

Ribosomal Rna ◽

Structure And Function ◽

16S Ribosomal Rna ◽

Chemical Probes ◽

Specific Chemical ◽

And Function

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Cell signalling by reactive lipid species: new concepts and molecular mechanisms

Biochemical Journal ◽

10.1042/bj20111752 ◽

2012 ◽

Vol 442 (3) ◽

pp. 453-464 ◽

Cited By ~ 181

Author(s):

Ashlee Higdon ◽

Anne R. Diers ◽

Joo Yeun Oh ◽

Aimee Landar ◽

Victor M. Darley-Usmar

Keyword(s):

Cell Death ◽

Redox Regulation ◽

Molecular Mechanisms ◽

Chemical Reactivity ◽

Cell Signalling ◽

Significant Proportion ◽

Covalent Modification ◽

Lipid Species ◽

Electrophilic Stress ◽

Cellular Antioxidants

The process of lipid peroxidation is widespread in biology and is mediated through both enzymatic and non-enzymatic pathways. A significant proportion of the oxidized lipid products are electrophilic in nature, the RLS (reactive lipid species), and react with cellular nucleophiles such as the amino acids cysteine, lysine and histidine. Cell signalling by electrophiles appears to be limited to the modification of cysteine residues in proteins, whereas non-specific toxic effects involve modification of other nucleophiles. RLS have been found to participate in several physiological pathways including resolution of inflammation, cell death and induction of cellular antioxidants through the modification of specific signalling proteins. The covalent modification of proteins endows some unique features to this signalling mechanism which we have termed the ‘covalent advantage’. For example, covalent modification of signalling proteins allows for the accumulation of a signal over time. The activation of cell signalling pathways by electrophiles is hierarchical and depends on a complex interaction of factors such as the intrinsic chemical reactivity of the electrophile, the intracellular domain to which it is exposed and steric factors. This introduces the concept of electrophilic signalling domains in which the production of the lipid electrophile is in close proximity to the thiol-containing signalling protein. In addition, we propose that the role of glutathione and associated enzymes is to insulate the signalling domain from uncontrolled electrophilic stress. The persistence of the signal is in turn regulated by the proteasomal pathway which may itself be subject to redox regulation by RLS. Cell death mediated by RLS is associated with bioenergetic dysfunction, and the damaged proteins are probably removed by the lysosome-autophagy pathway.

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Perturbational actions of barbiturate analogues on erythrocyte and synaptosomal membranes

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y85-155 ◽

1985 ◽

Vol 63 (8) ◽

pp. 937-943 ◽

Cited By ~ 2

Author(s):

David V. Godin ◽

Peter McGinn

Keyword(s):

Membrane Proteins ◽

Local Anaesthetics ◽

Erythrocyte Membranes ◽

Synaptosomal Membranes ◽

Structural Components ◽

Chemical Probes ◽

Trinitrobenzenesulfonic Acid ◽

Specific Chemical ◽

Nitrobenzoic Acid ◽

Acid Incorporation

The membrane perturbational actions of pentobarbital, methohexital, and phenobarbital were analyzed in terms of their effects on the incorporation of group-specific chemical probes into protein and phospholipid structural components and on the activity of various functionally distinct ATPases in erythrocyte and brain synaptosomal membranes. When compared at concentrations producing similar degrees of antihemolysis in red cells, the three analogues differed most markedly in the nature and degree of the structural and functional alterations induced in membrane proteins, with the most highly lipophilic agent, methohexital, being frequently, although not invariably, the most potent. Comparison of the effects of pentobarbital on characteristics of trinitrobenzenesulfonic acid and 5,5′-dithiobis-(2-nitrobenzoic acid) incorporation into erythrocyte membranes with results previously obtained using other anaesthetics at concentrations producing 50% antihemolysis showed that pentobarbital behaves similarly to neutral general anaesthetics but differently from cationic local anaesthetics. Our findings suggest that the membrane perturbational characteristics of barbiturates may contribute to their diverse and complex actions on excitable tissues.

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Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates

Science Advances ◽

10.1126/sciadv.aaz0051 ◽

2020 ◽

Vol 6 (15) ◽

pp. eaaz0051 ◽

Cited By ~ 4

Author(s):

Xinyu Ling ◽

Bingteng Xie ◽

Xiaoqin Gao ◽

Liying Chang ◽

Wei Zheng ◽

...

Keyword(s):

Genome Editing ◽

Therapeutic Potential ◽

Chemical Reactivity ◽

Specific Chemical ◽

Site Specific ◽

Additional Tool ◽

Homology Directed Repair ◽

Chemically Modified ◽

Dna Template ◽

Oligonucleotide Conjugates

Site-specific chemical conjugation of proteins can enhance their therapeutic and diagnostic utility but has seldom been applied to CRISPR-Cas9, which is a rapidly growing field with great therapeutic potential. The low efficiency of homology-directed repair remains a major hurdle in CRISPR-Cas9–mediated precise genome editing, which is limited by low concentration of donor DNA template at the cleavage site. In this study, we have developed methodology to site-specifically conjugate oligonucleotides to recombinant Cas9 protein containing a genetically encoded noncanonical amino acid with orthogonal chemical reactivity. The Cas9-oligonucleotide conjugates recruited an unmodified donor DNA template to the target site through base pairing, markedly increasing homology-directed repair efficiency in both human cell culture and mouse zygotes. These chemically modified Cas9 mutants provide an additional tool, one that is complementary to chemically modified nucleic acids, for improving the utility of CRISPR-Cas9–based genome-editing systems.

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