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Structural basis of glycogen branching enzyme deficiency and pharmacologic rescue by rational peptide design.

Froese DS, Michaeli A, McCorvie TJ, Krojer T, Sasi M, Melaev E, Goldblum A, Zatsepin M, Lossos A, Álvarez R, Escribá PV, Minassian BA, von Delft F, Kakhlon O, Yue WW - Hum. Mol. Genet. (2015)

Bottom Line: Expression of recombinant GBE1-p.Y329S resulted in drastically reduced protein yield and solubility compared with wild type, suggesting this disease allele causes protein misfolding and may be amenable to small molecule stabilization.We demonstrate intracellular transport of this peptide, its binding and stabilization of GBE1-p.Y329S, and 2-fold increased mutant enzymatic activity compared with untreated patient cells.Together, our data provide the rationale and starting point for the screening of small molecule chaperones, which could become novel therapies for this disease.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, OX3 7DQ, UK.

No MeSH data available.


Related in: MedlinePlus

Peptide rescue of hGBE1 p.Y329S. (A) PBMCs isolated from APBD patients were incubated with FITC-labelled LTKE peptides at 37°C or 4°C. At the indicated times, intracellular peptide uptake was determined by flow cytometry. (B) Isolated PBMCs from an APBD patient (Y329S) or a control subject (WT) were incubated overnight with or without the peptides indicated (20µm). Lysed cells were subjected to SDS–PAGE and immunoblotting with anti-GBE1 and anti-α-tubulin (loading control) antibodies. (C) Isolated PBMCs treated as in (A) were assayed for GBE activity based on (27). (D) Standard curve showing displacement of solid phase FITC by soluble LTKE-FITC. Curve was fit by non-linear regression using the four-parameter logistic equation: % Absorbance (650) = Bottom + (Top-Bottom)/(1+10^((logEC50-log[LTKE-FITC])*Hillslope), where Bottom = 7.996, Top = 100, EC50 = 8.460, Hillslope = −1.015. R2 = 0.9934. (E) FITC-labelled peptide competition experiment. Curve fitting, using the homologous one-site competition model, was found for APBD patient cells competed with LTKE-FITC. APBD patient cells competed with control peptides, or wild-type cells competed with LTKE-FITC did not demonstrate competitive binding of LTKE-FITC. The competition model equation is: % Absorbance (650) = (Bmax*[LTKE])/([LTKE]+peptide-FITC (nM) + Kd (nM)), where Bmax = 5229 nm, [LTKE] = 316 nm, Kd = 18 000 nM, Bottom = 13.24 nm. R2 = 0.9458. In all experiments, cells from n = 3 different APBD patients (or control unaffected subjects) were used. Error bars indicate SEM.
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DDV280F6: Peptide rescue of hGBE1 p.Y329S. (A) PBMCs isolated from APBD patients were incubated with FITC-labelled LTKE peptides at 37°C or 4°C. At the indicated times, intracellular peptide uptake was determined by flow cytometry. (B) Isolated PBMCs from an APBD patient (Y329S) or a control subject (WT) were incubated overnight with or without the peptides indicated (20µm). Lysed cells were subjected to SDS–PAGE and immunoblotting with anti-GBE1 and anti-α-tubulin (loading control) antibodies. (C) Isolated PBMCs treated as in (A) were assayed for GBE activity based on (27). (D) Standard curve showing displacement of solid phase FITC by soluble LTKE-FITC. Curve was fit by non-linear regression using the four-parameter logistic equation: % Absorbance (650) = Bottom + (Top-Bottom)/(1+10^((logEC50-log[LTKE-FITC])*Hillslope), where Bottom = 7.996, Top = 100, EC50 = 8.460, Hillslope = −1.015. R2 = 0.9934. (E) FITC-labelled peptide competition experiment. Curve fitting, using the homologous one-site competition model, was found for APBD patient cells competed with LTKE-FITC. APBD patient cells competed with control peptides, or wild-type cells competed with LTKE-FITC did not demonstrate competitive binding of LTKE-FITC. The competition model equation is: % Absorbance (650) = (Bmax*[LTKE])/([LTKE]+peptide-FITC (nM) + Kd (nM)), where Bmax = 5229 nm, [LTKE] = 316 nm, Kd = 18 000 nM, Bottom = 13.24 nm. R2 = 0.9458. In all experiments, cells from n = 3 different APBD patients (or control unaffected subjects) were used. Error bars indicate SEM.

Mentions: We evaluated the potential of the LTKE peptide to rescue the destabilized mutant protein in vivo, by testing it in APBD patient cells harbouring the p.Y329S mutation. To confirm that the peptide is internalized into cells, we determined its sensitivity to uptake temperature in peripheral blood mononuclear cells (PBMCs) and observed a time-dependent increase in the uptake of the C-terminal fluorescein isothiocyanate (FITC)-labelled peptide (LTKE-FITC) at 37°C but not 4°C, suggesting it is actively transported into cells (Fig. 6A). These peptide levels were sufficient to partially rescue mutant p.Y329S protein level in vivo as determined by Western blot analysis (Fig. 6B). Pre-incubation of PBMCs with the LTKE peptide resulted in detectable mutant GBE1 protein, which was absent when the ‘reverse peptide’ (EKTL) was used, or in patient-derived cells with no peptide treatment. More importantly, the LTKE and LTKE-FITC peptides enhanced GBE1 activity by 2-fold, compared with untreated or EKTL-treated mutant cells (>15% of unaffected control) (Fig. 6C). As these ameliorating effects of LTKE were sequence specific, we conjecture that they arose from the predicted hGBE1-Y329S binding model described in Figure 5, although attempts to prove this directly in vitro were hampered by difficulty in obtaining purified recombinant mutant protein (Fig. 4D). We resolved this by applying the hapten immunoassay (30) (Fig. 6D and E), to show that the LTKE-FITC peptide, but not the FITC-labelled control peptides ATKE, Ac-LTKE and EKTL with predicted inferior binding to hGBE1-Y329S model (Supplementary Material, Fig. S7), were able to out-compete LTKE binding in patient skin fibroblasts. This competitive binding of LTKE, specific to mutant cells and to the peptide amino acid sequence, clearly indicates the binding specificity of the LTKE peptide towards hGBE1 p.Y329S. The apparent Kd of peptide binding determined by the hapten immunoassay was 18 µm (Fig. 6E), within the range of error from the calculated Kd (1.6 µm;Supplementary Material, Table S3). Collectively, our data suggest that the LTKE peptide can potentially function as a stabilizing chaperone for the mutant p.Y329S protein.Figure 6.


Structural basis of glycogen branching enzyme deficiency and pharmacologic rescue by rational peptide design.

Froese DS, Michaeli A, McCorvie TJ, Krojer T, Sasi M, Melaev E, Goldblum A, Zatsepin M, Lossos A, Álvarez R, Escribá PV, Minassian BA, von Delft F, Kakhlon O, Yue WW - Hum. Mol. Genet. (2015)

Peptide rescue of hGBE1 p.Y329S. (A) PBMCs isolated from APBD patients were incubated with FITC-labelled LTKE peptides at 37°C or 4°C. At the indicated times, intracellular peptide uptake was determined by flow cytometry. (B) Isolated PBMCs from an APBD patient (Y329S) or a control subject (WT) were incubated overnight with or without the peptides indicated (20µm). Lysed cells were subjected to SDS–PAGE and immunoblotting with anti-GBE1 and anti-α-tubulin (loading control) antibodies. (C) Isolated PBMCs treated as in (A) were assayed for GBE activity based on (27). (D) Standard curve showing displacement of solid phase FITC by soluble LTKE-FITC. Curve was fit by non-linear regression using the four-parameter logistic equation: % Absorbance (650) = Bottom + (Top-Bottom)/(1+10^((logEC50-log[LTKE-FITC])*Hillslope), where Bottom = 7.996, Top = 100, EC50 = 8.460, Hillslope = −1.015. R2 = 0.9934. (E) FITC-labelled peptide competition experiment. Curve fitting, using the homologous one-site competition model, was found for APBD patient cells competed with LTKE-FITC. APBD patient cells competed with control peptides, or wild-type cells competed with LTKE-FITC did not demonstrate competitive binding of LTKE-FITC. The competition model equation is: % Absorbance (650) = (Bmax*[LTKE])/([LTKE]+peptide-FITC (nM) + Kd (nM)), where Bmax = 5229 nm, [LTKE] = 316 nm, Kd = 18 000 nM, Bottom = 13.24 nm. R2 = 0.9458. In all experiments, cells from n = 3 different APBD patients (or control unaffected subjects) were used. Error bars indicate SEM.
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Related In: Results  -  Collection

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DDV280F6: Peptide rescue of hGBE1 p.Y329S. (A) PBMCs isolated from APBD patients were incubated with FITC-labelled LTKE peptides at 37°C or 4°C. At the indicated times, intracellular peptide uptake was determined by flow cytometry. (B) Isolated PBMCs from an APBD patient (Y329S) or a control subject (WT) were incubated overnight with or without the peptides indicated (20µm). Lysed cells were subjected to SDS–PAGE and immunoblotting with anti-GBE1 and anti-α-tubulin (loading control) antibodies. (C) Isolated PBMCs treated as in (A) were assayed for GBE activity based on (27). (D) Standard curve showing displacement of solid phase FITC by soluble LTKE-FITC. Curve was fit by non-linear regression using the four-parameter logistic equation: % Absorbance (650) = Bottom + (Top-Bottom)/(1+10^((logEC50-log[LTKE-FITC])*Hillslope), where Bottom = 7.996, Top = 100, EC50 = 8.460, Hillslope = −1.015. R2 = 0.9934. (E) FITC-labelled peptide competition experiment. Curve fitting, using the homologous one-site competition model, was found for APBD patient cells competed with LTKE-FITC. APBD patient cells competed with control peptides, or wild-type cells competed with LTKE-FITC did not demonstrate competitive binding of LTKE-FITC. The competition model equation is: % Absorbance (650) = (Bmax*[LTKE])/([LTKE]+peptide-FITC (nM) + Kd (nM)), where Bmax = 5229 nm, [LTKE] = 316 nm, Kd = 18 000 nM, Bottom = 13.24 nm. R2 = 0.9458. In all experiments, cells from n = 3 different APBD patients (or control unaffected subjects) were used. Error bars indicate SEM.
Mentions: We evaluated the potential of the LTKE peptide to rescue the destabilized mutant protein in vivo, by testing it in APBD patient cells harbouring the p.Y329S mutation. To confirm that the peptide is internalized into cells, we determined its sensitivity to uptake temperature in peripheral blood mononuclear cells (PBMCs) and observed a time-dependent increase in the uptake of the C-terminal fluorescein isothiocyanate (FITC)-labelled peptide (LTKE-FITC) at 37°C but not 4°C, suggesting it is actively transported into cells (Fig. 6A). These peptide levels were sufficient to partially rescue mutant p.Y329S protein level in vivo as determined by Western blot analysis (Fig. 6B). Pre-incubation of PBMCs with the LTKE peptide resulted in detectable mutant GBE1 protein, which was absent when the ‘reverse peptide’ (EKTL) was used, or in patient-derived cells with no peptide treatment. More importantly, the LTKE and LTKE-FITC peptides enhanced GBE1 activity by 2-fold, compared with untreated or EKTL-treated mutant cells (>15% of unaffected control) (Fig. 6C). As these ameliorating effects of LTKE were sequence specific, we conjecture that they arose from the predicted hGBE1-Y329S binding model described in Figure 5, although attempts to prove this directly in vitro were hampered by difficulty in obtaining purified recombinant mutant protein (Fig. 4D). We resolved this by applying the hapten immunoassay (30) (Fig. 6D and E), to show that the LTKE-FITC peptide, but not the FITC-labelled control peptides ATKE, Ac-LTKE and EKTL with predicted inferior binding to hGBE1-Y329S model (Supplementary Material, Fig. S7), were able to out-compete LTKE binding in patient skin fibroblasts. This competitive binding of LTKE, specific to mutant cells and to the peptide amino acid sequence, clearly indicates the binding specificity of the LTKE peptide towards hGBE1 p.Y329S. The apparent Kd of peptide binding determined by the hapten immunoassay was 18 µm (Fig. 6E), within the range of error from the calculated Kd (1.6 µm;Supplementary Material, Table S3). Collectively, our data suggest that the LTKE peptide can potentially function as a stabilizing chaperone for the mutant p.Y329S protein.Figure 6.

Bottom Line: Expression of recombinant GBE1-p.Y329S resulted in drastically reduced protein yield and solubility compared with wild type, suggesting this disease allele causes protein misfolding and may be amenable to small molecule stabilization.We demonstrate intracellular transport of this peptide, its binding and stabilization of GBE1-p.Y329S, and 2-fold increased mutant enzymatic activity compared with untreated patient cells.Together, our data provide the rationale and starting point for the screening of small molecule chaperones, which could become novel therapies for this disease.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, OX3 7DQ, UK.

No MeSH data available.


Related in: MedlinePlus