An immunochemical approach to detect oxidized protein tyrosine phosphatases using a selective C-nucleophile tag

García, Francisco J.; Carroll, Kate S.

doi:10.1039/c5mb00847f

Cited by 7 publications

(10 citation statements)

References 46 publications

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“…In addition, the dimedone scaffold (Figure ), when incorporated into an appropriate binding module, has also been demonstrated to exhibit selectivity for the sulfenic acid generated in the PTP active site . More recently, a novel immunochemical approach consisting of an antibody designed to recognize the conserved sequence of the PTP active site (VHCDMDSAG) harboring the catalytic cysteine modified with dimedone has been developed to directly profile oxidized PTPs . It is anticipated that future work in this area will lead to novel therapeutic molecules that can covalently capture the oxidized forms of PTPs generated inside the cell under disease conditions.…”

Section: Therapeutic Targeting Of Protein Tyrosine Phosphatase Regula...mentioning

confidence: 99%

Regulatory Mechanisms and Novel Therapeutic Targeting Strategies for Protein Tyrosine Phosphatases

Zhang

2017

Chem. Rev.

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Appropriate level of protein phosphorylation on tyrosine is essential for cells to react to extracellular stimuli and keep cellular homeostasis. Faulty operation of signal pathways mediated by protein tyrosine phosphorylation causes numerous human diseases, which presents enormous opportunities for therapeutic interventions. While the importance of protein tyrosine kinases in orchestrating the tyrosine phosphorylation networks and in target-based drug discovery has long been recognized, the significance of protein tyrosine phosphatases (PTPs) in cellular signaling and disease biology has historically been underappreciated, due to a large extent an erroneous assumption that they are largely constitutive and housekeeping enzymes. Here, we provide a comprehensive examination on a number of regulatory mechanisms including redox modulation, allosteric regulation, and protein oligomerization, in controlling PTP activity. These regulatory mechanisms are integral to the myriad PTP-mediated biochemical events and reinforce the concept that PTPs are indispensable and specific modulators of cellular signaling. We also discuss how disruption of these PTP regulatory mechanisms can cause human diseases and how these diverse regulatory mechanisms can be exploited for novel therapeutic development.

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Section: Therapeutic Targeting Of Protein Tyrosine Phosphatase Regula...mentioning

confidence: 99%

Regulatory Mechanisms and Novel Therapeutic Targeting Strategies for Protein Tyrosine Phosphatases

Zhang

2017

Chem. Rev.

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“…However, many other types of protein can be oxidized, and in several cases, such oxidation has important physiologic and pathologic consequences (9)(10)(11)(12). More general reduction-oxidation reaction (redox) techniques have been developed to globally detect protein oxidation, such as the isotope-coded affinity tag method (13), the modified cysteinyl-labeling assay (14,15), and dimedone-dimedone antibody-based approaches (16)(17)(18)(19). Each of these approaches has certain technical limitations.…”

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confidence: 99%

Activated Thiol Sepharose‐based proteomic approach to quantify reversible protein oxidation

Xu¹,

Andrade²,

Ueberheide³

et al. 2019

The FASEB Journal

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Reactive oxygen species (ROS) can act as second messengers in various signaling pathways, and abnormal oxidation contributes to multiple diseases, including cancer. Detecting and quantifying protein oxidation is crucial for a detailed understanding of reduction‐oxidation reaction (redox) signaling. We developed an Activated Thiol Sepharose‐based proteomic (ATSP) approach to quantify reversible protein oxidation. ATSP can enrich H2O2‐sensitive thiol peptides, which are more likely to contain reactive cysteines involved in redox signaling. We applied our approach to analyze hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a type of kidney cancer that harbors fumarate hydratase (FH)‐inactivating mutations and has elevated ROS levels. Multiple proteins were oxidized in FH‐deficient cells, including many metabolic proteins such as the pyruvate kinase M2 isoform (PKM2). Treatment of HLRCC cells with dimethyl fumarate or PKM2 activators altered PKM2 oxidation levels. Finally, we found that ATSP could detect Src homology region 2 domain‐containing phosphatase‐2 and PKM2 oxidation in cells stimulated with platelet‐derived growth factor. This newly developed redox proteomics workflow can detect reversible oxidation of reactive cysteines and can be employed to analyze multiple physiologic and pathologic conditions.—Xu, Y., Andrade, J., Ueberheide, B., Neel, B. G. Activated Thiol Sepharose‐based proteomic approach to quantify reversible protein oxidation. FASEB J. 33, 12336–12347 (2019). http://www.fasebj.org

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“…PSOHs also react with H 2 S to yield persulfides (PSSHs), providing a molecular mechanism for redox-coupled H 2 S signaling. , Further PSOH reaction with excess H 2 O 2 yields protein sulfinic (PSO 2 H) and sulfonic (PSO 3 H) acids, generally indicative of oxidative damage . These multiple and rapid reactions make the tagging of protein sulfenic acids and the subsequent identification of the protein and site of modification under biological conditions challenging. , A number of probes containing acidic carbon nucleophiles (including the 2,4-(dioxocyclohexyl)propoxy (DCP) unit) or strained cyclic alkynes (including the bicyclo[6.1.0]nonyne (BCN) group) trap PSOHs at rates sufficient to reveal information regarding the site of PSOH formation in various proteins and their role in redox-mediated processes. − …”

Section: Introductionmentioning

confidence: 99%

Triphenylphosphonium-Derived Protein Sulfenic Acid Trapping Agents: Synthesis, Reactivity, and Effect on Mitochondrial Function

Forshaw

Holmila

et al. 2019

Chem. Res. Toxicol.

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Redox-mediated protein modifications control numerous processes in both normal and disease metabolism. Protein sulfenic acids, formed from the oxidation of protein cysteine residues, play a critical role in thiol-based redox signaling. The reactivity of protein sulfenic acids requires their identification through chemical trapping, and this paper describes the use of the triphenylphosphonium (TPP) ion to direct known sulfenic acid traps to the mitochondria, a verified source of cellular reactive oxygen species. Coupling of the TPP group with the 2,4-(dioxocyclohexyl)propoxy (DCP) unit and the bicyclo[6.1.0]nonyne (BCN) group produces two new probes, DCP-TPP and BCN-TPP. DCP-TPP and BCN-TPP react with C165A AhpC-SOH, a model protein sulfenic acid, to form the expected adducts with second-order rate constants of k = 1.1 M−1 s−1 and k = 5.99 M−1 s−1, respectively, as determined by electrospray ionization time-of-flight mass spectrometry. The TPP group does not alter the rate of DCP-TPP reaction with protein sulfenic acid compared to dimedone but slows the rate of BCN-TPP reaction compared to a non-TPP-containing BCN-OH control by 4.6-fold. The hydrophobic TPP group may interact with the protein, preventing an optimal reaction orientation for BCN-TPP. Unlike BCN-OH, BCN-TPP does not react with the protein persulfide, C165A AhpC-SSH. Extracellular flux measurements using A549 cells show that DCP-TPP and BCN-TPP influence mitochondrial energetics, with BCN-TPP producing a drastic decrease in basal respiration, perhaps due to its faster reaction kinetics with sulfenylated proteins. Further control experiments with BCN-OH, TPP-COOH, and dimedone provide strong evidence for mitochondrial localization and accumulation of DCP-TPP and BCN-TPP. These results reveal the compatibility of the TPP group with reactive sulfenic acid probes as a mitochondrial director and support the use of the TPP group in the design of sulfenic acid traps.

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An immunochemical approach to detect oxidized protein tyrosine phosphatases using a selective C-nucleophile tag

Cited by 7 publications

References 46 publications

Regulatory Mechanisms and Novel Therapeutic Targeting Strategies for Protein Tyrosine Phosphatases

Regulatory Mechanisms and Novel Therapeutic Targeting Strategies for Protein Tyrosine Phosphatases

Activated Thiol Sepharose‐based proteomic approach to quantify reversible protein oxidation

Triphenylphosphonium-Derived Protein Sulfenic Acid Trapping Agents: Synthesis, Reactivity, and Effect on Mitochondrial Function

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