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

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 TMT^TM). 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.

Keywords: Cataract; Epithelial-mesenchymal transition; Glutathione; Isobaric tagging; LEGSKO mouse; NRF2; Oxidative stress; Proteomics; TMT.

Figure 1. Overview of the GSH-Deficient Lens Proteome

(A) Total number of mapped proteins in lens epithelia and fiber cells. (B). Number of significantly (FDR < 0.1) up- and down-regulated proteins for each comparison. (C) FDR ranking of significantly altered proteins. (D) Fold change ranking of significantly altered proteins. All comparisons are relative to WT control.

Figure 2. Cellular Localization of Significantly Modulated Proteins (FDR< 0.1) in the GSH-Deficient Lens Proteome

(A) LEGSKO epithelia. (B) BSO-treated LEGSKO epithelia. (C) LEGSKO fiber cells. (D) BSO-treated LEGSKO fiber cells. All comparisons are relative to WT control.

Figure 3. Schematic Representation of Major Trends in the GSH-Deficient Lens Proteome

Color and direction of arrows indicate direction of regulation change. Green arrows = downregulation, red arrows = upregulation. Line graphs represent lenticular GSH content which decreases from top to bottom of the figure.

Figure 4. Relative Abundance Changes in Detoxification and Cellular Stress Response Proteins

(A–K) Protein expression changes relating to detoxification. (L–R) Protein expression changes relating to the cellular stress response. (A) aldehyde dehydrogenase 1 family member A1 (ALDH1A1), (B) aldehyde dehydrogenase family 3 member A1 (ALDH3A1), (C) quinone oxidoreductase (CRYZ), (D) glutathione S-transferase alpha 4 (GSTA4), (E) glutathione S-transferase mu 1 (GSTM1), (F) glutathione S-transferase mu 2 (GSTM2), (G) glutathione S-transferase omega 1 (GSTO1), (H) glutathione S-transferase pi 1 (GSTP1), (I) omega-amidase (NIT2), (J) peroxiredoxin 5 (PRDX5), (K) peroxiredoxin 6 (PRDX6), (L) carbonic anhydrase 2 (CAR2), (M) homocysteine inducible ER protein with ubiquitin like domain 1 (HERPUD1), (N) heme oxygenase 1 (HMOX1), (O) heat shock protein family A (HSP70) member 1B) HSPA1B, (P) heat shock protein family B member 1 (HSPB1), (Q) heat shock protein family B member 6 (HSPB6), (R) Parkinson disease protein (PARK7). Values are means +/− SD. 3.8 mM GSH = WT, 1.3 mM GSH = LEGSKO, 0.35 mM GSH = BSO-treated LEGSKO. * = significant (FDR < 0.1) change. Fold change and significance is relative to WT.

Figure 5. NRF2-Regulated Protein Expression Changes

(A) LEGSKO lens epithelia. (B) BSO-treated LEGSKO lens epithelia. (C) LEGSKO lens fiber cells. (D) BSO-treated LEGSKO lens fiber cells. Arrows indicate activation. Color indicates fold change of gene relative to WT, as indicated by the color bar. Diagram created using Ingenuity Pathway Analysis software (Qiagen, Hilgen, Germany).

Figure 6. Relative Abundance Changes in Lens Specific and EMT-Related Proteins

(A–K) Protein expression changes in lens specific proteins. (L–S) Protein expression changes relating to EMT. (A) beaded filament structural protein/phakinin (BFSP2), (B) β-crystallin B1 (CRYBB1), (C) β-crystallin B2 (CRYBB2), (D) β-crystallin B3 (CRYBB3), (E) γ-crystallin A (CRYGA), (F) γ-crystallin B (CRYGB), (G) γ-crystallin C (CRYGC), (H) γ-crystallin D (CRYGD), (I) γ-crystallin E (CRYGE), (J) γ-crystallin N (CRYGN), (K) lengsin (LGSN), (L) cellular retinoic acid binding protein 1 (CRABP1), (M) fascin actin-bundling protein (FSCN1), (N) MARVEL domain containing 3 (MARVELD3), (O) neuron navigator 3 (NAV3), (P) programmed cell death protein 4 (PCDC4), (W) astrocytic phosphoprotein PEA-15 (PEA15A), (R) vimentin (VIM). Values are means +/− SD. 3.8 mM GSH = WT, 1.3 mM GSH = LEGSKO, 0.35 mM GSH = BSO-treated LEGSKO. * = significant (FDR < 0.1) change. Fold change and significance is relative to WT.

Figure 7. Confirmation of proteomic changes by Western blot analysis

Left = Representative images of Western blot analysis. Right = Quantification of relative protein expression. All values are normalized to GAPDH loading control and WT values are set to 1.0. Values are means ± SD. n = 3. * = p < 0.05, *** = p < 0.001.