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

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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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