Fig 1: ßA3/A1-crystallin interacts with PITPß and may regulate PIP levels in RPE cells.a RPE flatmounts isolated from wildtype mice were cultured and infected either with Ad-mCherry-Pitpnb or Ad-mCherry-control and Ad-Cryba1-A3 or Ad-Cryba1-A1, respectively. RPE flatmounts were starved in FBS-free medium for 2 h with or without 30 min of EGF stimulation. RPE cell lysates were subjected to co-immunoprecipitation using RFP magnetic beads and the immunoprecipitants were analyzed by western blotting. Data show interaction of PITPß with ßA3/A1-crystallin, especially during EGF stimulation. b Protein complexes of ßA3-crystallin/PITPß and ßA1-crystallin/PITPß as predicted by molecular modeling. ßA1-crystallin (left) and ßA3-crystallin (right) are shown in blue. PITPß is shown in orange. Both ßA3- and ßA1-crystallins are represented as the dimers formed by two “open” conformers. c. ARPE19 cells were infected with Ad-mCherry-Pitpnb or Ad-mCherry-control and Ad-Cryba1-GFP. Ad-Cryba1-GFP vector contains the wildtype Cryba1 gene, and produces both ßA3-crystallin and ßA1-crystallin. Cells were treated with or without 10 ng/mL EGF for 10 min and fixed for imaging and colocalization analysis. Data show the colocalization of PITPß and ßA3/A1-crystallin, especially during EGF stimulation. Scale bar: 10 µm. d PI(4,5)P2 and PI(3,4,5)P3 were analyzed on RPE–choroid complexes of 2-month-old control and cKO mice. Data indicate a markedly reduced amount of PI(4,5)P2 and PI(3,4,5)P3 in cKO RPE–choroid complexes compared to that of control. n = 6. e Phosphorylated PLC?1 levels were determined in RPE lysates of 3-week-old control and cKO mice by western blot. Data suggest increased PLC?1 activity in cKO RPE cells. n = 4. f Phosphorylated PKC (pan) (ßII Ser660) levels were determined in RPE lysates of 3-week-old control and cKO mice. Data suggest the activation of PKC in cKO RPE cells. n = 4. g Myo-inositol was analyzed on RPE–choroid complexes of 2-month-old control and cKO mice. Data indicate reduced myo-inositol level in cKO RPE–choroid complexes compared to that of control. n = 4. Statistical analysis was performed using two-tailed unpaired Student’s t-test (d–g) and one-way ANOVA (c). *P < 0.05, ***P < 0.001, ****P < 0.0001.
Fig 2: ßA1-crystallin regulates STAT3 nuclear localization.a Representative western blot and b, c densitometry graphs showing decreased phosphorylation of STAT3 at tyrosine 705 (p-STAT3Y705) in ßA1 KD astrocytes b untreated or c treated with 30 mM mannitol or high glucose (HG; 30 mM) for 6 h, relative to WT cells (c). ßA3 KO cells did not show such changes (a–c); n = 4. **P < 0.01. d, e Overexpression of ßA1-crystallin (using ßA1-mCherry construct) in ßA1 KD astrocytes or treatment with 10 µM PTP1B inhibitor (MSI-1436) for 1 h, prior to HG (30 mM) exposure for 6 h rescued the levels of p-STAT3Y705, as compared to ßA1 KD cells transfected with blank-mCherry construct. ßA1-crystallin overexpression was confirmed by mCherry western blot. f Western blot and g densitometry graph showing decrease in p-STAT3Y705 expression in WT cells infected with Adenovirus-PTP1B overexpression construct or Cryba1 shRNA for 48 h, followed by HG treatment. h Cryba1 knockdown was confirmed by qPCR, which showed about 70% downregulation compared to control. n = 4. *P < 0.05, **P < 0.01. i, j Representative images from live-cell confocal microscopy of human iPSC-derived astrocytes after overexpression of i ßA1-mCherry or j ßA3-mCherry constructs and k quantitative assessment of nuclear translocation, showing nuclear localization of ßA1-crystallin in these cells (i, k), whereas ßA3-crystallin construct transfected cells showed less nuclear localization (j, k). Scale bar, 10 µm. **P < 0.01 (n = 16).
Fig 3: ßA1-crystallin is a binding partner of PTP1B.Representative western blot and densitometry graph from co-immunoprecipitation studies in mouse WT (a, b) or Cryba1 KO (c, d) astrocytes transfected with blank-mCherry, ßA3-crystallin-mCherry (ßA3-mCherry), ßA1-crystallin-mCherry (ßA1-mCherry), and ßA3/A1-crystallin-mCherry (ßA3/A1-mCherry) show interaction of PTP1B with both ßA3- and ßA1-crystallin; n = 4. e, f Co-immunoprecipitation assay showing ßA3/A1-crystallin levels in the Co-IP eluent by western blot analysis and densitometry, indicating binding to PTP1B, upon pull down with mNeonGreen antibody-bound magnetic beads from lysates of astrocytes overexpressing PTP1B (Ad-CMV-mNeonGreen-mPtpn1) and Cryba1 (Ad-CMV-RFP-mCryba1). Pull down with mouse IgG showed no binding for ßA3/A1-crystallin; n = 4. g Ribbon diagram obtained by molecular modeling showing superimposed ßA1-crystallin (orange), ßA3-crystallin (blue), and PTP1B (gray). Neither isoform is able to bind to the pocket of PTP1B active site (Cys215, Red). The ßA1, but not the ßA3 isoform (due to a steric bump of terminal extension) interacts with an allosteric binding site (green) on the surface of PTP1B.
Fig 4: Compromised EGFR activation in Cryba1 cKO RPE.a RPE lysates from 2-month-old control and cKO mice fasted or fasted and then refed were isolated for a phospho-RTK array assay. Phosphorylated EGFR and ErbB2 were decreased in refed cKO RPE cells compared to refed floxed controls. n = 4. b A diagram showing RPE–choroid–sclera flatmount preparation. c RPE flatmounts of 2-month-old control and cKO mice stimulated with EGF for times indicated were analyzed by western blot. cKO RPE cells presented less phosphorylated EGFR in response to EGF stimulation at each time point. While total EGFR gradually decreased after ligand stimulation in control RPE cells, EGFR was not reduced in cKO RPE cells. Graphs show the EGFR phosphorylation level and total EGFR level in both cell types. Immunoblots are representative of two independent experiments. #: non-specific band. d Phosphorylated AKT and MAPK levels in RPE from fasted or refed mice were measured by western blot. Both phosphorylated AKT and MAPK were significantly upregulated in control RPE after refeeding, but not in cKO RPE. n = 3. Statistical analysis was performed using two-way ANOVA (a, d). *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.
Fig 5: ßA1-crystallin regulates PTP1B activity.a Lineweaver-Burk plot showing increasing doses (0, 0.5, 1, and 2 nM) of ßA3/A1-crystallin decreases both Vmax and KM of PTP1B activity for different concentrations of p-Nitrophenyl Phosphate (pNPP). n = 3. *P < 0.05. **P < 0.05. b The N-terminal sequence of Cryba1; ßA1-crystallin KD mice were generated by knocking in 5 base pairs (CCACC, red) before the first start codon to strengthen the Kozak consensus sequence. For generating ßA3-crystallin KO, the first start codon was removed by a single nucleotide mutation in the mouse Cryba1 gene (A > G, red). Another silent mutation (C > G; red) was also introduced to prevent the binding and re-cutting of the sequence by gRNA after homology-directed repair. c Representative western blot and d graph showing densitometry analysis for the expression level of ßA3/A1-crystallin in astrocyte lysates from WT (black bar), ßA3 KO (blue bar), and ßA1 KD (green bar) mice, respectively, showing complete loss of ßA3-crystallin in the ßA3 KO cells and a notable decrease in ßA1-crystallin expression in the ßA1 KD cells, relative to WT astrocytes. In ßA3 KO astrocytes, there is an increase in expression of ßA1-crystallin; n = 4. *P < 0.05, **P < 0.01. e Increased levels of lactate in mouse WT, ßA3 KO and ßA1 KD astrocytes treated with HG (25 or 30 mM for 6 h) relative to untreated cells. Lactate levels in ßA1 KD astrocytes were higher in all experimental conditions, compared to WT and ßA3 KO cells; n = 3. *P < 0.05, **P < 0.01. f Elevated glycolytic flux is evident from increased glycolytic capacity in WT, ßA3 KO and ßA1 KD astrocytes treated with high glucose (HG; 30 mM for 6 h), relative to untreated cells (cultured in 5 mM d-glucose containing medium). Glycolytic capacity in ßA1 KD astrocytes was drastically higher compared to WT and ßA3 KO cells; n = 4. *P < 0.05, **P < 0.01. g Cultured ßA1 astrocytes either untreated or exposed to mannitol (30 mM for 6 h) have elevated PTP1B activity compared to WT and ßA3 KO cells, which increases further with HG (30 mM for 6 h). The elevation in PTP1B activity was rescued by ßA1-crystallin overexpression in untreated or HG-exposed ßA1 KD cells; n = 5. *P < 0.05, **P < 0.01.
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