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Detergent resistant membrane-associated IDE in brain tissue and cultured cells: Relevance to Abeta and insulin degradation.

Bulloj A, Leal MC, Surace EI, Zhang X, Xu H, Ledesma MD, Castaño EM, Morelli L - Mol Neurodegener (2008)

Bottom Line: DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo.We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele.Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fundación Instituto Leloir, IIBBA-CONICET, Ave, Patricias Argentinas 435, Ciudad de Buenos Aires C1405BWE, Argentina. xuh@burnham.org.

ABSTRACT

Background: Insulin degrading enzyme (IDE) is implicated in the regulation of amyloid beta (Abeta) steady-state levels in the brain, and its deficient expression and/or activity may be a risk factor in sporadic Alzheimer's disease (AD). Although IDE sub-cellular localization has been well studied, the compartments relevant to Abeta degradation remain to be determined.

Results: Our results of live immunofluorescence, immuno gold electron-microscopy and gradient fractionation concurred to the demonstration that endogenous IDE from brain tissues and cell cultures is, in addition to its other localizations, a detergent-resistant membrane (DRM)-associated metallopeptidase. Our pulse chase experiments were in accordance with the existence of two pools of IDE: the cytosolic one with a longer half-life and the membrane-IDE with a faster turn-over. DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo. When IDE was mis-located from DRMs by treating cells with methyl-beta-cyclodextrin (MbetaCD), endogenous Abeta accumulated in the extracellular space and exogenous Abeta proteolysis was impaired. We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele. We confirmed that a moderate shift of IDE from DRMs induced a substantial decrement on IDE-mediated insulin and Abeta degradation in vitro.

Conclusion: Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance. Structural integrity of DRMs may also be required to tightly retain insulin receptor and IDE for insulin proteolysis. The concept that mis-location of Abeta degrading proteases away from DRMs may impair the physiological turn-over of Abeta in vivo deserves further investigation in light of therapeutic strategies based on enhancing Abeta proteolysis in which DRM protease-targeting may need to be taken into account.

No MeSH data available.


Related in: MedlinePlus

DRM- and DSM-associated IDE pools are proteolytically active in vitro. A- Western blotting with BC2 anti-IDE polyclonal antibody of immunoprecipated IDE from isolated DRM and DSM fractions of rat brain using 1C1/3A2 anti-IDE monoclonal antibodies. B- Representative phosphorimage scan showed degradation of [125I]-insulin and [125I]-Aβ after incubation with anti-IDE immunoprecipitates from DRM and DSM in the presence of a protease inhibitor cocktail (as defined in Antibodies and Chemicals) with or without metalloprotease inhibitors (EDTA/1,10 phenantroline). IgG, unrelated immunoglobulin used as a negative control for the immunoprecipitation. The intensity of the band in the presence of unrelated IgG and after incubation in degradation buffer was referred as intact substrate (0% degradation). C- Bars represent the semi-quantitative analysis of the percentage of [125I]-insulin and [125I]-Aβ degradation by IDE from DRMs and DSMs in the presence and absence of 1,10-Phe/EDTA (n = 3; *p < 0.001).
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Figure 4: DRM- and DSM-associated IDE pools are proteolytically active in vitro. A- Western blotting with BC2 anti-IDE polyclonal antibody of immunoprecipated IDE from isolated DRM and DSM fractions of rat brain using 1C1/3A2 anti-IDE monoclonal antibodies. B- Representative phosphorimage scan showed degradation of [125I]-insulin and [125I]-Aβ after incubation with anti-IDE immunoprecipitates from DRM and DSM in the presence of a protease inhibitor cocktail (as defined in Antibodies and Chemicals) with or without metalloprotease inhibitors (EDTA/1,10 phenantroline). IgG, unrelated immunoglobulin used as a negative control for the immunoprecipitation. The intensity of the band in the presence of unrelated IgG and after incubation in degradation buffer was referred as intact substrate (0% degradation). C- Bars represent the semi-quantitative analysis of the percentage of [125I]-insulin and [125I]-Aβ degradation by IDE from DRMs and DSMs in the presence and absence of 1,10-Phe/EDTA (n = 3; *p < 0.001).

Mentions: To determine if DRM-associated IDE was proteolytically active, we examined degradation of insulin (the most specific substrate for IDE) and Aβ (a relevant substrate for AD pathology) by an immunoprecipitation-based assay. DRM and DSM fractions were isolated from rat brain cortex, and endogenous IDE was immunoprecipitated using 1C1 and 3A2 monoclonal antibodies. The presence of IDE in immunoprecipitates was confirmed by western blotting using BC2 anti-IDE antibody (Fig. 4A). Immunoprecipitates were then incubated with [125I]-insulin or [125I]-Aβ40, and degradation analyzed by SDS-PAGE followed by phosphorimaging. The results showed substantial degradation of substrates when reactions were performed in the absence of 1,10 phenanthroline/EDTA (Fig. 4B). Conversely, addition of inhibitor strongly blocked degradation of insulin and Aβ, indicating substrate specificity of IDE in the reactions. The partial resistance of Aβ to degradation was likely due to the biophysical state of radiolabeled peptide and the reported resistance of aggregated Aβ to IDE proteolysis. Semi-quantitative analysis of the degradation assays indicated that insulin degradation (Fig. 4C, upper panel) in the absence of 1,10 phenantroline/EDTA was 83.8 ± 0.6% and 88.2 ± 0.3% in DRM and DSM, respectively, while a significant degradation impairment was detected in both fractions after IDE inhibition (11.3 ± 0.3% and 1.2 ± 0.6% in DRM and DSM, respectively; n = 3; p < 0.001). In addition, f [125I]-Aβ degradation (Fig. 4C, lower panel) in the absence of 1,10 phenantroline/EDTA was 41.2 ± 0.9% and 37.3 ± 0.9% in DRM and DSM, respectively and values were significantly decreased after the addition of metalloproteases inhibitors (2.0 ± 1.0% and 1.5 ± 0.9% in DRM and DSM, respectively, n = 3, p < 0.001). Our results clearly indicate that DRM and DSM -associated IDE are proteolitically active in vitro.


Detergent resistant membrane-associated IDE in brain tissue and cultured cells: Relevance to Abeta and insulin degradation.

Bulloj A, Leal MC, Surace EI, Zhang X, Xu H, Ledesma MD, Castaño EM, Morelli L - Mol Neurodegener (2008)

DRM- and DSM-associated IDE pools are proteolytically active in vitro. A- Western blotting with BC2 anti-IDE polyclonal antibody of immunoprecipated IDE from isolated DRM and DSM fractions of rat brain using 1C1/3A2 anti-IDE monoclonal antibodies. B- Representative phosphorimage scan showed degradation of [125I]-insulin and [125I]-Aβ after incubation with anti-IDE immunoprecipitates from DRM and DSM in the presence of a protease inhibitor cocktail (as defined in Antibodies and Chemicals) with or without metalloprotease inhibitors (EDTA/1,10 phenantroline). IgG, unrelated immunoglobulin used as a negative control for the immunoprecipitation. The intensity of the band in the presence of unrelated IgG and after incubation in degradation buffer was referred as intact substrate (0% degradation). C- Bars represent the semi-quantitative analysis of the percentage of [125I]-insulin and [125I]-Aβ degradation by IDE from DRMs and DSMs in the presence and absence of 1,10-Phe/EDTA (n = 3; *p < 0.001).
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Related In: Results  -  Collection

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Figure 4: DRM- and DSM-associated IDE pools are proteolytically active in vitro. A- Western blotting with BC2 anti-IDE polyclonal antibody of immunoprecipated IDE from isolated DRM and DSM fractions of rat brain using 1C1/3A2 anti-IDE monoclonal antibodies. B- Representative phosphorimage scan showed degradation of [125I]-insulin and [125I]-Aβ after incubation with anti-IDE immunoprecipitates from DRM and DSM in the presence of a protease inhibitor cocktail (as defined in Antibodies and Chemicals) with or without metalloprotease inhibitors (EDTA/1,10 phenantroline). IgG, unrelated immunoglobulin used as a negative control for the immunoprecipitation. The intensity of the band in the presence of unrelated IgG and after incubation in degradation buffer was referred as intact substrate (0% degradation). C- Bars represent the semi-quantitative analysis of the percentage of [125I]-insulin and [125I]-Aβ degradation by IDE from DRMs and DSMs in the presence and absence of 1,10-Phe/EDTA (n = 3; *p < 0.001).
Mentions: To determine if DRM-associated IDE was proteolytically active, we examined degradation of insulin (the most specific substrate for IDE) and Aβ (a relevant substrate for AD pathology) by an immunoprecipitation-based assay. DRM and DSM fractions were isolated from rat brain cortex, and endogenous IDE was immunoprecipitated using 1C1 and 3A2 monoclonal antibodies. The presence of IDE in immunoprecipitates was confirmed by western blotting using BC2 anti-IDE antibody (Fig. 4A). Immunoprecipitates were then incubated with [125I]-insulin or [125I]-Aβ40, and degradation analyzed by SDS-PAGE followed by phosphorimaging. The results showed substantial degradation of substrates when reactions were performed in the absence of 1,10 phenanthroline/EDTA (Fig. 4B). Conversely, addition of inhibitor strongly blocked degradation of insulin and Aβ, indicating substrate specificity of IDE in the reactions. The partial resistance of Aβ to degradation was likely due to the biophysical state of radiolabeled peptide and the reported resistance of aggregated Aβ to IDE proteolysis. Semi-quantitative analysis of the degradation assays indicated that insulin degradation (Fig. 4C, upper panel) in the absence of 1,10 phenantroline/EDTA was 83.8 ± 0.6% and 88.2 ± 0.3% in DRM and DSM, respectively, while a significant degradation impairment was detected in both fractions after IDE inhibition (11.3 ± 0.3% and 1.2 ± 0.6% in DRM and DSM, respectively; n = 3; p < 0.001). In addition, f [125I]-Aβ degradation (Fig. 4C, lower panel) in the absence of 1,10 phenantroline/EDTA was 41.2 ± 0.9% and 37.3 ± 0.9% in DRM and DSM, respectively and values were significantly decreased after the addition of metalloproteases inhibitors (2.0 ± 1.0% and 1.5 ± 0.9% in DRM and DSM, respectively, n = 3, p < 0.001). Our results clearly indicate that DRM and DSM -associated IDE are proteolitically active in vitro.

Bottom Line: DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo.We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele.Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fundación Instituto Leloir, IIBBA-CONICET, Ave, Patricias Argentinas 435, Ciudad de Buenos Aires C1405BWE, Argentina. xuh@burnham.org.

ABSTRACT

Background: Insulin degrading enzyme (IDE) is implicated in the regulation of amyloid beta (Abeta) steady-state levels in the brain, and its deficient expression and/or activity may be a risk factor in sporadic Alzheimer's disease (AD). Although IDE sub-cellular localization has been well studied, the compartments relevant to Abeta degradation remain to be determined.

Results: Our results of live immunofluorescence, immuno gold electron-microscopy and gradient fractionation concurred to the demonstration that endogenous IDE from brain tissues and cell cultures is, in addition to its other localizations, a detergent-resistant membrane (DRM)-associated metallopeptidase. Our pulse chase experiments were in accordance with the existence of two pools of IDE: the cytosolic one with a longer half-life and the membrane-IDE with a faster turn-over. DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo. When IDE was mis-located from DRMs by treating cells with methyl-beta-cyclodextrin (MbetaCD), endogenous Abeta accumulated in the extracellular space and exogenous Abeta proteolysis was impaired. We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele. We confirmed that a moderate shift of IDE from DRMs induced a substantial decrement on IDE-mediated insulin and Abeta degradation in vitro.

Conclusion: Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance. Structural integrity of DRMs may also be required to tightly retain insulin receptor and IDE for insulin proteolysis. The concept that mis-location of Abeta degrading proteases away from DRMs may impair the physiological turn-over of Abeta in vivo deserves further investigation in light of therapeutic strategies based on enhancing Abeta proteolysis in which DRM protease-targeting may need to be taken into account.

No MeSH data available.


Related in: MedlinePlus