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Cysteine S-nitrosylation protects protein-tyrosine phosphatase 1B against oxidation-induced permanent inactivation.

ABSTRACT: Protein S-nitrosylation mediated by cellular nitric oxide (NO) plays a primary role in executing biological functions in cGMP-independent NO signaling. Although S-nitrosylation appears similar to Cys oxidation induced by reactive oxygen species, the molecular mechanism and biological consequence remain unclear. We investigated the structural process of S-nitrosylation of protein-tyrosine phosphatase 1B (PTP1B). We treated PTP1B with various NO donors, including S-nitrosothiol reagents and compound-releasing NO radicals, to produce site-specific Cys S-nitrosylation identified using advanced mass spectrometry (MS) techniques. Quantitative MS showed that the active site Cys-215 was the primary residue susceptible to S-nitrosylation. The crystal structure of NO donor-reacted PTP1B at 2.6 A resolution revealed that the S-NO state at Cys-215 had no discernible irreversibly oxidized forms, whereas other Cys residues remained in their free thiol states. We further demonstrated that S-nitrosylation of the Cys-215 residue protected PTP1B from subsequent H(2)O(2)-induced irreversible oxidation. Increasing the level of cellular NO by pretreating cells with an NO donor or by activating ectopically expressed NO synthase inhibited reactive oxygen species-induced irreversible oxidation of endogenous PTP1B. These findings suggest that S-nitrosylation might prevent PTPs from permanent inactivation caused by oxidative stress.

SUBMITTER: Chen YY

PROVIDER: S-EPMC3259880 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Cysteine S-nitrosylation protects protein-tyrosine phosphatase 1B against oxidation-induced permanent inactivation.

Chen Yi-Yun YY Chu Hsing-Mao HM Pan Kuan-Ting KT Teng Chun-Hung CH Wang Danny-Ling DL Wang Andrew H-J AH Khoo Kay-Hooi KH Meng Tzu-Ching TC

The Journal of biological chemistry 20081007 50

Protein S-nitrosylation mediated by cellular nitric oxide (NO) plays a primary role in executing biological functions in cGMP-independent NO signaling. Although S-nitrosylation appears similar to Cys oxidation induced by reactive oxygen species, the molecular mechanism and biological consequence remain unclear. We investigated the structural process of S-nitrosylation of protein-tyrosine phosphatase 1B (PTP1B). We treated PTP1B with various NO donors, including S-nitrosothiol reagents and compou ...[more]

PMID: 18840608

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Project description:One hallmark of acute lung injury is the disruption of the pulmonary endothelial barrier. Such disruption correlates with increased endothelial permeability, partly through the disruption of cell-cell contacts. Protein tyrosine phosphatases (PTPs) are known to affect the stability of both cell-extracellular matrix adhesions and intercellular adherens junctions (AJs). However, evidence for the role of select PTPs in regulating endothelial permeability is limited. Our investigations noted that the inhibition of PTP1B in cultured pulmonary endothelial cells (ECs), as well as in the vasculature of intact murine lungs via the transient overexpression of a catalytically inactive PTP1B, decreased the baseline resistance of cultured EC monolayers and increased the formation of edema in murine lungs, respectively. In addition, we observed that the overexpression of wild-type PTP1B enhanced basal barrier function in vitro. Immunohistochemical analyses of pulmonary ECs and the coimmunoprecipitation of murine lung homogenates demonstrated the association of PTP1B with the AJ proteins ?-catenin, p120-catenin, and VE-cadherin both in vitro and ex vivo. Using LPS in a model of sepsis-induced acute lung injury, we showed that reactive oxygen species were generated in response to LPS, which correlated with enhanced PTP1B oxidation, inhibited phosphatase activity, and attenuation of the interactions between PTP1B and ?-catenin, as well as enhanced ?-catenin tyrosine phosphorylation. Finally, the overexpression of a cytosolic PTP1B fragment, shown to be resistant to nicotinamide adenine dinucleotide phosphate-reduced oxidase-4 (Nox4)-mediated oxidation, significantly attenuated LPS-induced endothelial barrier dysfunction and the formation of lung edema, and preserved the associations of PTP1B with AJ protein components, independent of PTP1B phosphatase activity. We conclude that PTP1B plays an important role in maintaining the pulmonary endothelial barrier, and PTP1B oxidation appears to contribute to sepsis-induced pulmonary vascular dysfunction, possibly through the disruption of AJs.

Project description:A new paradigm in metallobiochemistry describes the activation of inactive metalloenzymes by metal ion removal. Protein tyrosine phosphatases (PTPs) do not seem to require a metal ion for enzymatic activity. However, both metal cations and metal anions modulate their enzymatic activity. One binding site is the phosphate binding site at the catalytic cysteine residue. Oxyanions with structural similarity to phosphate, such as vanadate, inhibit the enzyme with nanomolar to micromolar affinities. In addition, zinc ions (Zn2+) inhibit with picomolar to nanomolar affinities. We mapped the cation binding site close to the anion binding site and established a specific mechanism of inhibition occurring only in the closed conformation of the enzyme when the catalytic cysteine is phosphorylated and the catalytic aspartate moves into the active site. We discuss this dual inhibition by anions and cations here for PTP1B, the most thoroughly investigated protein tyrosine phosphatase. The significance of the inhibition in phosphorylation signaling is becoming apparent only from the functions of PTP1B in the biological context of metal cations as cellular signaling ions. Zinc ion signals complement redox signals but provide a different type of control and longer lasting inhibition on a biological time scale owing to the specificity and affinity of zinc ions for coordination environments. Inhibitor design for PTP1B and other PTPs is a major area of research activity and interest owing to their prominent roles in metabolic regulation in health and disease, in particular cancer and diabetes. Our results explain the apparent dichotomy of both cations (Zn2+) and oxyanions such as vanadate inhibiting PTP1B and having insulin-enhancing ("anti-diabetic") effects and suggest different approaches, namely targeting PTPs in the cell by affecting their physiological modulators and considering a metallodrug approach that builds on the knowledge of the insulin-enhancing effects of both zinc and vanadium compounds.

Dataset Information

Cysteine S-nitrosylation protects protein-tyrosine phosphatase 1B against oxidation-induced permanent inactivation.

Publications

Cysteine S-nitrosylation protects protein-tyrosine phosphatase 1B against oxidation-induced permanent inactivation.

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