Proteomic analysis of the glutathione-deficient LEGSKO mouse lens reveals activation of EMT signaling, loss of lens specific markers, and changes in stress response proteins

Jeremy A. Whitson, Phillip Wilmarth, John Klimek, Vincent M. Monnier, Larry David, Xingjun Fan

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Purpose To determine global protein expression changes in the lens of the GSH-deficient LEGSKO mouse model of age-related cataract for comparison with recently published gene expression data obtained by RNA-Seq transcriptome analysis. Methods Lenses were separated into epithelial and cortical fiber sections, digested with trypsin, and labeled with isobaric tags (10-plex TMTTM). Peptides were analyzed by LC-MS/MS (Orbitrap Fusion) and mapped to the mouse proteome for relative protein quantification. Results 1871 proteins in lens epithelia and 870 proteins in lens fiber cells were quantified. 40 proteins in LEGSKO epithelia, 14 proteins in LEGSKO fiber cells, 22 proteins in buthionine sulfoximine (BSO)-treated LEGSKO epithelia, and 55 proteins in BSO-treated LEGSKO fiber cells had significantly (p<0.05, FDR<0.1) altered protein expression compared to WT controls. HSF4 and MAF transcription factors were the most common upstream regulators of the response to GSH-deficiency. Many detoxification proteins, including aldehyde dehydrogenases, peroxiredoxins, and quinone oxidoreductase, were upregulated but several glutathione S-transferases were downregulated. Several cellular stress response proteins showed regulation changes, including an upregulation of HERPUD1, downregulation of heme oxygenase, and mixed changes in heat shock proteins. NRF2-regulated proteins showed broad upregulation in BSO-treated LEGSKO fiber cells, but not in other groups. Strong trends were seen in downregulation of lens specific proteins, including β- and γ-crystallins, lengsin, and phakinin, and in epithelial-mesenchymal transition (EMT)-related changes. Western blot analysis of LEGSKO lens epithelia confirmed expression changes in several proteins. Conclusions This dataset confirms at the proteomic level many findings from the recently determined GSH-deficient lens transcriptome and provides new insight into the roles of GSH in the lens, how the lens adapts to oxidative stress, and how GSH affects EMT in the lens.

Original languageEnglish (US)
Pages (from-to)84-96
Number of pages13
JournalFree Radical Biology and Medicine
Volume113
DOIs
StatePublished - Dec 1 2017

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Epithelial-Mesenchymal Transition
Heat-Shock Proteins
Proteomics
Lenses
Glutathione
Chemical activation
Crystallins
Buthionine Sulfoximine
Proteins
Epithelium
Down-Regulation
Fibers
Up-Regulation
Peroxiredoxins
Heme Oxygenase (Decyclizing)
Aldehyde Dehydrogenase
Gene Expression Profiling
Proteome
Glutathione Transferase
Transcriptome

Keywords

  • Cataract
  • Epithelial-mesenchymal transition
  • Glutathione
  • Isobaric tagging
  • LEGSKO mouse
  • NRF2
  • Oxidative stress
  • Proteomics
  • TMT

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

Cite this

Proteomic analysis of the glutathione-deficient LEGSKO mouse lens reveals activation of EMT signaling, loss of lens specific markers, and changes in stress response proteins. / Whitson, Jeremy A.; Wilmarth, Phillip; Klimek, John; Monnier, Vincent M.; David, Larry; Fan, Xingjun.

In: Free Radical Biology and Medicine, Vol. 113, 01.12.2017, p. 84-96.

Research output: Contribution to journalArticle

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abstract = "Purpose To determine global protein expression changes in the lens of the GSH-deficient LEGSKO mouse model of age-related cataract for comparison with recently published gene expression data obtained by RNA-Seq transcriptome analysis. Methods Lenses were separated into epithelial and cortical fiber sections, digested with trypsin, and labeled with isobaric tags (10-plex TMTTM). Peptides were analyzed by LC-MS/MS (Orbitrap Fusion) and mapped to the mouse proteome for relative protein quantification. Results 1871 proteins in lens epithelia and 870 proteins in lens fiber cells were quantified. 40 proteins in LEGSKO epithelia, 14 proteins in LEGSKO fiber cells, 22 proteins in buthionine sulfoximine (BSO)-treated LEGSKO epithelia, and 55 proteins in BSO-treated LEGSKO fiber cells had significantly (p<0.05, FDR<0.1) altered protein expression compared to WT controls. HSF4 and MAF transcription factors were the most common upstream regulators of the response to GSH-deficiency. Many detoxification proteins, including aldehyde dehydrogenases, peroxiredoxins, and quinone oxidoreductase, were upregulated but several glutathione S-transferases were downregulated. Several cellular stress response proteins showed regulation changes, including an upregulation of HERPUD1, downregulation of heme oxygenase, and mixed changes in heat shock proteins. NRF2-regulated proteins showed broad upregulation in BSO-treated LEGSKO fiber cells, but not in other groups. Strong trends were seen in downregulation of lens specific proteins, including β- and γ-crystallins, lengsin, and phakinin, and in epithelial-mesenchymal transition (EMT)-related changes. Western blot analysis of LEGSKO lens epithelia confirmed expression changes in several proteins. Conclusions This dataset confirms at the proteomic level many findings from the recently determined GSH-deficient lens transcriptome and provides new insight into the roles of GSH in the lens, how the lens adapts to oxidative stress, and how GSH affects EMT in the lens.",
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T1 - Proteomic analysis of the glutathione-deficient LEGSKO mouse lens reveals activation of EMT signaling, loss of lens specific markers, and changes in stress response proteins

AU - Whitson, Jeremy A.

AU - Wilmarth, Phillip

AU - Klimek, John

AU - Monnier, Vincent M.

AU - David, Larry

AU - Fan, Xingjun

PY - 2017/12/1

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N2 - Purpose To determine global protein expression changes in the lens of the GSH-deficient LEGSKO mouse model of age-related cataract for comparison with recently published gene expression data obtained by RNA-Seq transcriptome analysis. Methods Lenses were separated into epithelial and cortical fiber sections, digested with trypsin, and labeled with isobaric tags (10-plex TMTTM). Peptides were analyzed by LC-MS/MS (Orbitrap Fusion) and mapped to the mouse proteome for relative protein quantification. Results 1871 proteins in lens epithelia and 870 proteins in lens fiber cells were quantified. 40 proteins in LEGSKO epithelia, 14 proteins in LEGSKO fiber cells, 22 proteins in buthionine sulfoximine (BSO)-treated LEGSKO epithelia, and 55 proteins in BSO-treated LEGSKO fiber cells had significantly (p<0.05, FDR<0.1) altered protein expression compared to WT controls. HSF4 and MAF transcription factors were the most common upstream regulators of the response to GSH-deficiency. Many detoxification proteins, including aldehyde dehydrogenases, peroxiredoxins, and quinone oxidoreductase, were upregulated but several glutathione S-transferases were downregulated. Several cellular stress response proteins showed regulation changes, including an upregulation of HERPUD1, downregulation of heme oxygenase, and mixed changes in heat shock proteins. NRF2-regulated proteins showed broad upregulation in BSO-treated LEGSKO fiber cells, but not in other groups. Strong trends were seen in downregulation of lens specific proteins, including β- and γ-crystallins, lengsin, and phakinin, and in epithelial-mesenchymal transition (EMT)-related changes. Western blot analysis of LEGSKO lens epithelia confirmed expression changes in several proteins. Conclusions This dataset confirms at the proteomic level many findings from the recently determined GSH-deficient lens transcriptome and provides new insight into the roles of GSH in the lens, how the lens adapts to oxidative stress, and how GSH affects EMT in the lens.

AB - Purpose To determine global protein expression changes in the lens of the GSH-deficient LEGSKO mouse model of age-related cataract for comparison with recently published gene expression data obtained by RNA-Seq transcriptome analysis. Methods Lenses were separated into epithelial and cortical fiber sections, digested with trypsin, and labeled with isobaric tags (10-plex TMTTM). Peptides were analyzed by LC-MS/MS (Orbitrap Fusion) and mapped to the mouse proteome for relative protein quantification. Results 1871 proteins in lens epithelia and 870 proteins in lens fiber cells were quantified. 40 proteins in LEGSKO epithelia, 14 proteins in LEGSKO fiber cells, 22 proteins in buthionine sulfoximine (BSO)-treated LEGSKO epithelia, and 55 proteins in BSO-treated LEGSKO fiber cells had significantly (p<0.05, FDR<0.1) altered protein expression compared to WT controls. HSF4 and MAF transcription factors were the most common upstream regulators of the response to GSH-deficiency. Many detoxification proteins, including aldehyde dehydrogenases, peroxiredoxins, and quinone oxidoreductase, were upregulated but several glutathione S-transferases were downregulated. Several cellular stress response proteins showed regulation changes, including an upregulation of HERPUD1, downregulation of heme oxygenase, and mixed changes in heat shock proteins. NRF2-regulated proteins showed broad upregulation in BSO-treated LEGSKO fiber cells, but not in other groups. Strong trends were seen in downregulation of lens specific proteins, including β- and γ-crystallins, lengsin, and phakinin, and in epithelial-mesenchymal transition (EMT)-related changes. Western blot analysis of LEGSKO lens epithelia confirmed expression changes in several proteins. Conclusions This dataset confirms at the proteomic level many findings from the recently determined GSH-deficient lens transcriptome and provides new insight into the roles of GSH in the lens, how the lens adapts to oxidative stress, and how GSH affects EMT in the lens.

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KW - Isobaric tagging

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KW - TMT

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