TY - JOUR
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 A.
AU - Klimek, John
AU - Monnier, Vincent M.
AU - David, Larry
AU - Fan, Xingjun
N1 - Funding Information:
Research funding provided by NIH grants EY07099 to VMM, EY024553 to XF, T32 EY007157 to the Visual Sciences Research Center at Case Western Reserve University , T32 EY024236 to the Cole Eye Institute at the Cleveland Clinic , P30 EY10572 to the Casey Eye Institute at the Oregon Health & Science University , and S10 OD012246 to LLD . We are very grateful to Dr. Graeme Wistow for his generosity in providing us with the lengsin (LGSN) antibody. Appendix A
Publisher Copyright:
© 2017 Elsevier Inc.
PY - 2017/12
Y1 - 2017/12
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.
KW - Cataract
KW - Epithelial-mesenchymal transition
KW - Glutathione
KW - Isobaric tagging
KW - LEGSKO mouse
KW - NRF2
KW - Oxidative stress
KW - Proteomics
KW - TMT
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U2 - 10.1016/j.freeradbiomed.2017.09.019
DO - 10.1016/j.freeradbiomed.2017.09.019
M3 - Article
C2 - 28951044
AN - SCOPUS:85030481564
SN - 0891-5849
VL - 113
SP - 84
EP - 96
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
ER -