The effect of HOCl-induced modifications on phosphatase and tensin homolog (PTEN) structure and function

Research output: Contribution to journalArticle

Abstract

Oxidation by reactive species can cause changes in protein function and affect cell signaling pathways. Phosphatase and tensin homolog (PTEN) is a negative regulator of the PI3K/AKT pathway and is known to be inhibited by oxidation, but its oxidation by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl) has not previously been investigated. PTEN-GST was treated with HOCl:protein ratios from 15:1 to 300:1. Decreases in PTEN phosphatase activity were observed at treatment ratios of 60:1 and higher, which correlated with the loss of the intact protein band and appearance of high molecular weight aggregates in SDS-PAGE. LC-MSMS was used to map oxidative modifications (oxPTMs) in PTEN-GST tryptic peptides and label-free quantitative proteomics used to determine their relative abundance. Twenty different oxPTMs of PTEN were identified, of which 14 were significantly elevated upon HOCl treatment in a dose-dependent manner. Methionine and cysteine residues were the most heavily oxidized; the percentage modification depended on their location in the sequence, reflecting differences in susceptibility. Other modifications included tyrosine chlorination and dichlorination, and hydroxylations of tyrosine, tryptophan, and proline. Much higher levels of oxidation occurred in the protein aggregates compared to the monomeric protein for certain methionine and tyrosine residues located in the C2 and C-terminal domains, suggesting that their oxidation promoted protein destabilization and aggregation; many of the residues modified were classified as buried according to their solvent accessibility. This study provides novel information on the susceptibility of PTEN to the inflammatory oxidant HOCl and its effects on the structure and activity of the protein.
Original languageEnglish
Pages (from-to)1-471
JournalFree Radical Research
Early online date3 Jan 2018
DOIs
Publication statusPublished - 3 Jan 2018

Fingerprint

Hypochlorous Acid
Phosphoric Monoester Hydrolases
Oxidation
Tyrosine
Proteins
Oxidants
Methionine
Cell signaling
Hydroxylation
Chlorination
Halogenation
Tensins
Phosphatidylinositol 3-Kinases
Proline
Tryptophan
Proteomics
Peroxidase
Cysteine
Labels
Polyacrylamide Gel Electrophoresis

Bibliographical note

© 2018 Informa UK Limited, publishing as Taylor & Francis. This is an Accepted Manuscript of an article published by Taylor & Francis in Free Radical Research on 3rd January 2018, available online: http://www.tandfonline.com/10.1080/10715762.2018.1424333.

Funding: Engineering and Physical Sciences Research Council (EP/I017887/1 Cross-Disciplinary Research Landscape Award)

Keywords

  • Cysteine Oxidation
  • hypochlorous acid
  • mass spectrometry
  • oxidative post-translational modifications
  • protein aggregation
  • tyrosine chlorination

Cite this

@article{6b5baed291d6418fa7f679204bf37db8,
title = "The effect of HOCl-induced modifications on phosphatase and tensin homolog (PTEN) structure and function",
abstract = "Oxidation by reactive species can cause changes in protein function and affect cell signaling pathways. Phosphatase and tensin homolog (PTEN) is a negative regulator of the PI3K/AKT pathway and is known to be inhibited by oxidation, but its oxidation by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl) has not previously been investigated. PTEN-GST was treated with HOCl:protein ratios from 15:1 to 300:1. Decreases in PTEN phosphatase activity were observed at treatment ratios of 60:1 and higher, which correlated with the loss of the intact protein band and appearance of high molecular weight aggregates in SDS-PAGE. LC-MSMS was used to map oxidative modifications (oxPTMs) in PTEN-GST tryptic peptides and label-free quantitative proteomics used to determine their relative abundance. Twenty different oxPTMs of PTEN were identified, of which 14 were significantly elevated upon HOCl treatment in a dose-dependent manner. Methionine and cysteine residues were the most heavily oxidized; the percentage modification depended on their location in the sequence, reflecting differences in susceptibility. Other modifications included tyrosine chlorination and dichlorination, and hydroxylations of tyrosine, tryptophan, and proline. Much higher levels of oxidation occurred in the protein aggregates compared to the monomeric protein for certain methionine and tyrosine residues located in the C2 and C-terminal domains, suggesting that their oxidation promoted protein destabilization and aggregation; many of the residues modified were classified as buried according to their solvent accessibility. This study provides novel information on the susceptibility of PTEN to the inflammatory oxidant HOCl and its effects on the structure and activity of the protein.",
keywords = "Cysteine Oxidation, hypochlorous acid, mass spectrometry, oxidative post-translational modifications, protein aggregation, tyrosine chlorination",
author = "Ivan Verrastro and {Tveen Jensen}, Karina and Spickett, {Corinne M} and Pitt, {Andrew R}",
note = "{\circledC} 2018 Informa UK Limited, publishing as Taylor & Francis. This is an Accepted Manuscript of an article published by Taylor & Francis in Free Radical Research on 3rd January 2018, available online: http://www.tandfonline.com/10.1080/10715762.2018.1424333. Funding: Engineering and Physical Sciences Research Council (EP/I017887/1 Cross-Disciplinary Research Landscape Award)",
year = "2018",
month = "1",
day = "3",
doi = "10.1080/10715762.2018.1424333",
language = "English",
pages = "1--471",
journal = "Free Radical Research",
issn = "1071-5762",
publisher = "Informa Healthcare",

}

TY - JOUR

T1 - The effect of HOCl-induced modifications on phosphatase and tensin homolog (PTEN) structure and function

AU - Verrastro, Ivan

AU - Tveen Jensen, Karina

AU - Spickett, Corinne M

AU - Pitt, Andrew R

N1 - © 2018 Informa UK Limited, publishing as Taylor & Francis. This is an Accepted Manuscript of an article published by Taylor & Francis in Free Radical Research on 3rd January 2018, available online: http://www.tandfonline.com/10.1080/10715762.2018.1424333. Funding: Engineering and Physical Sciences Research Council (EP/I017887/1 Cross-Disciplinary Research Landscape Award)

PY - 2018/1/3

Y1 - 2018/1/3

N2 - Oxidation by reactive species can cause changes in protein function and affect cell signaling pathways. Phosphatase and tensin homolog (PTEN) is a negative regulator of the PI3K/AKT pathway and is known to be inhibited by oxidation, but its oxidation by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl) has not previously been investigated. PTEN-GST was treated with HOCl:protein ratios from 15:1 to 300:1. Decreases in PTEN phosphatase activity were observed at treatment ratios of 60:1 and higher, which correlated with the loss of the intact protein band and appearance of high molecular weight aggregates in SDS-PAGE. LC-MSMS was used to map oxidative modifications (oxPTMs) in PTEN-GST tryptic peptides and label-free quantitative proteomics used to determine their relative abundance. Twenty different oxPTMs of PTEN were identified, of which 14 were significantly elevated upon HOCl treatment in a dose-dependent manner. Methionine and cysteine residues were the most heavily oxidized; the percentage modification depended on their location in the sequence, reflecting differences in susceptibility. Other modifications included tyrosine chlorination and dichlorination, and hydroxylations of tyrosine, tryptophan, and proline. Much higher levels of oxidation occurred in the protein aggregates compared to the monomeric protein for certain methionine and tyrosine residues located in the C2 and C-terminal domains, suggesting that their oxidation promoted protein destabilization and aggregation; many of the residues modified were classified as buried according to their solvent accessibility. This study provides novel information on the susceptibility of PTEN to the inflammatory oxidant HOCl and its effects on the structure and activity of the protein.

AB - Oxidation by reactive species can cause changes in protein function and affect cell signaling pathways. Phosphatase and tensin homolog (PTEN) is a negative regulator of the PI3K/AKT pathway and is known to be inhibited by oxidation, but its oxidation by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl) has not previously been investigated. PTEN-GST was treated with HOCl:protein ratios from 15:1 to 300:1. Decreases in PTEN phosphatase activity were observed at treatment ratios of 60:1 and higher, which correlated with the loss of the intact protein band and appearance of high molecular weight aggregates in SDS-PAGE. LC-MSMS was used to map oxidative modifications (oxPTMs) in PTEN-GST tryptic peptides and label-free quantitative proteomics used to determine their relative abundance. Twenty different oxPTMs of PTEN were identified, of which 14 were significantly elevated upon HOCl treatment in a dose-dependent manner. Methionine and cysteine residues were the most heavily oxidized; the percentage modification depended on their location in the sequence, reflecting differences in susceptibility. Other modifications included tyrosine chlorination and dichlorination, and hydroxylations of tyrosine, tryptophan, and proline. Much higher levels of oxidation occurred in the protein aggregates compared to the monomeric protein for certain methionine and tyrosine residues located in the C2 and C-terminal domains, suggesting that their oxidation promoted protein destabilization and aggregation; many of the residues modified were classified as buried according to their solvent accessibility. This study provides novel information on the susceptibility of PTEN to the inflammatory oxidant HOCl and its effects on the structure and activity of the protein.

KW - Cysteine Oxidation

KW - hypochlorous acid

KW - mass spectrometry

KW - oxidative post-translational modifications

KW - protein aggregation

KW - tyrosine chlorination

UR - http://doi.org/10.17036/researchdata.aston.ac.uk.00000317

U2 - 10.1080/10715762.2018.1424333

DO - 10.1080/10715762.2018.1424333

M3 - Article

SP - 1

EP - 471

JO - Free Radical Research

JF - Free Radical Research

SN - 1071-5762

ER -