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Dimerization of the transmembrane domain of amyloid precursor proteins and familial Alzheimer's disease mutants.

Gorman PM, Kim S, Guo M, Melnyk RA, McLaurin J, Fraser PE, Bowie JU, Chakrabartty A - BMC Neurosci (2008)

Bottom Line: We find that FAD-APP mutations destabilize the APP-TM dimer and increase the population of APP peptide monomers.The dissociation constants are correlated to both the Abeta42/Abeta40 ratio and the mean age of disease onset in AD patients.We also show that these TM-peptides reduce Abeta production and Abeta42/Abeta40 ratios when added to HEK293 cells overexpressing the Swedish FAD mutation and gamma-secretase components, potentially revealing a new class of gamma-secretase inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M5G 2M9, Canada. pgorman@westwindpartners.ca

ABSTRACT

Background: Amyloid precursor protein (APP) is enzymatically cleaved by gamma-secretase to form two peptide products, either Abeta40 or the more neurotoxic Abeta42. The Abeta42/40 ratio is increased in many cases of familial Alzheimer's disease (FAD). The transmembrane domain (TM) of APP contains the known dimerization motif GXXXA. We have investigated the dimerization of both wild type and FAD mutant APP transmembrane domains.

Results: Using synthetic peptides derived from the APP-TM domain, we show that this segment is capable of forming stable transmembrane dimers. A model of a dimeric APP-TM domain reveals a putative dimerization interface, and interestingly, majority of FAD mutations in APP are localized to this interface region. We find that FAD-APP mutations destabilize the APP-TM dimer and increase the population of APP peptide monomers.

Conclusion: The dissociation constants are correlated to both the Abeta42/Abeta40 ratio and the mean age of disease onset in AD patients. We also show that these TM-peptides reduce Abeta production and Abeta42/Abeta40 ratios when added to HEK293 cells overexpressing the Swedish FAD mutation and gamma-secretase components, potentially revealing a new class of gamma-secretase inhibitors.

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Related in: MedlinePlus

Hetero-oligomerization of wild-type and mutant APP-TM peptides monitored by FRET. Trp-labeled wild-type APP-TM peptides were incubated with equimolar amounts of Edans-labeled V717G APP-TM peptides in the presence of 20 mM SDS. The A/D ratio is plotted as a function of total peptide concentration. Inset: The solid bars represent the A/D ratio of APP-TM homodimers at 50 μM total peptide concentration. The open bars represent the A/D ratio of wild-type and mutant heterodimers at 50 μM total peptide concentration. The gray line indicates the A/D ratio for a mixture of monomeric peptides.
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Figure 5: Hetero-oligomerization of wild-type and mutant APP-TM peptides monitored by FRET. Trp-labeled wild-type APP-TM peptides were incubated with equimolar amounts of Edans-labeled V717G APP-TM peptides in the presence of 20 mM SDS. The A/D ratio is plotted as a function of total peptide concentration. Inset: The solid bars represent the A/D ratio of APP-TM homodimers at 50 μM total peptide concentration. The open bars represent the A/D ratio of wild-type and mutant heterodimers at 50 μM total peptide concentration. The gray line indicates the A/D ratio for a mixture of monomeric peptides.

Mentions: With the autosomal dominant inheritance pattern of APP-FAD [9] both wild-type and mutant APP are expressed in heterozygotes. Combined with the dimerization propensity of APP, there is a possibility that heterodimerization of mutant and wild-type APP could contribute to FAD. To determine whether the APP-TM domain may mediate the formation of hetero-oligomers, we employed FRET experiments using mixed preparations of wild-type and mutant APP-TM peptides. The FRET signal (i.e. the A/D ratio) between the Trp fluorophore on wild-type APP-TM and the EDANS fluorophore on V717G APP-TM peptides was measured in a series of samples. These samples contained increasing amounts of wild-type and mutant peptide but a constant 1:1 ratio of wild-type to mutant peptide was maintained (Fig. 5). The results show that the A/D ratio increases with total peptide concentration and shows signs of saturation above 50 μM total peptide concentration. While it is not possible to extract the apparent equilibrium constant for hetero-oligomerization from this data, the similarities in the shapes of the FRET curves for self-association (Fig. 4) and mixed-association (Fig. 5) suggest that both processes are energetically similar. To determine whether the wild type peptide can form mixed oligomers with the other FAD mutant peptides, the A/D ratio of APP-TM homodimers were compared with the A/D ratio of equimolar mixtures of wild-type and mutant peptides at 50 μM total peptide concentration (Fig. 5 inset). The A/D ratios of the mixed peptide samples are significantly higher than 0.24, which is the baseline A/D ratio for non-interacting peptides, indicating that wild-type APP-TM can hetero-oligomerize with all mutant APP-TM peptides tested. The A/D ratios of the homodimeric samples differ from those of the mixed peptide samples. These differences may be the result of altered orientation of the fluorophores in the mixed samples leading to changes in the dipole moment orientation factor and FRET efficiency.


Dimerization of the transmembrane domain of amyloid precursor proteins and familial Alzheimer's disease mutants.

Gorman PM, Kim S, Guo M, Melnyk RA, McLaurin J, Fraser PE, Bowie JU, Chakrabartty A - BMC Neurosci (2008)

Hetero-oligomerization of wild-type and mutant APP-TM peptides monitored by FRET. Trp-labeled wild-type APP-TM peptides were incubated with equimolar amounts of Edans-labeled V717G APP-TM peptides in the presence of 20 mM SDS. The A/D ratio is plotted as a function of total peptide concentration. Inset: The solid bars represent the A/D ratio of APP-TM homodimers at 50 μM total peptide concentration. The open bars represent the A/D ratio of wild-type and mutant heterodimers at 50 μM total peptide concentration. The gray line indicates the A/D ratio for a mixture of monomeric peptides.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266763&req=5

Figure 5: Hetero-oligomerization of wild-type and mutant APP-TM peptides monitored by FRET. Trp-labeled wild-type APP-TM peptides were incubated with equimolar amounts of Edans-labeled V717G APP-TM peptides in the presence of 20 mM SDS. The A/D ratio is plotted as a function of total peptide concentration. Inset: The solid bars represent the A/D ratio of APP-TM homodimers at 50 μM total peptide concentration. The open bars represent the A/D ratio of wild-type and mutant heterodimers at 50 μM total peptide concentration. The gray line indicates the A/D ratio for a mixture of monomeric peptides.
Mentions: With the autosomal dominant inheritance pattern of APP-FAD [9] both wild-type and mutant APP are expressed in heterozygotes. Combined with the dimerization propensity of APP, there is a possibility that heterodimerization of mutant and wild-type APP could contribute to FAD. To determine whether the APP-TM domain may mediate the formation of hetero-oligomers, we employed FRET experiments using mixed preparations of wild-type and mutant APP-TM peptides. The FRET signal (i.e. the A/D ratio) between the Trp fluorophore on wild-type APP-TM and the EDANS fluorophore on V717G APP-TM peptides was measured in a series of samples. These samples contained increasing amounts of wild-type and mutant peptide but a constant 1:1 ratio of wild-type to mutant peptide was maintained (Fig. 5). The results show that the A/D ratio increases with total peptide concentration and shows signs of saturation above 50 μM total peptide concentration. While it is not possible to extract the apparent equilibrium constant for hetero-oligomerization from this data, the similarities in the shapes of the FRET curves for self-association (Fig. 4) and mixed-association (Fig. 5) suggest that both processes are energetically similar. To determine whether the wild type peptide can form mixed oligomers with the other FAD mutant peptides, the A/D ratio of APP-TM homodimers were compared with the A/D ratio of equimolar mixtures of wild-type and mutant peptides at 50 μM total peptide concentration (Fig. 5 inset). The A/D ratios of the mixed peptide samples are significantly higher than 0.24, which is the baseline A/D ratio for non-interacting peptides, indicating that wild-type APP-TM can hetero-oligomerize with all mutant APP-TM peptides tested. The A/D ratios of the homodimeric samples differ from those of the mixed peptide samples. These differences may be the result of altered orientation of the fluorophores in the mixed samples leading to changes in the dipole moment orientation factor and FRET efficiency.

Bottom Line: We find that FAD-APP mutations destabilize the APP-TM dimer and increase the population of APP peptide monomers.The dissociation constants are correlated to both the Abeta42/Abeta40 ratio and the mean age of disease onset in AD patients.We also show that these TM-peptides reduce Abeta production and Abeta42/Abeta40 ratios when added to HEK293 cells overexpressing the Swedish FAD mutation and gamma-secretase components, potentially revealing a new class of gamma-secretase inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M5G 2M9, Canada. pgorman@westwindpartners.ca

ABSTRACT

Background: Amyloid precursor protein (APP) is enzymatically cleaved by gamma-secretase to form two peptide products, either Abeta40 or the more neurotoxic Abeta42. The Abeta42/40 ratio is increased in many cases of familial Alzheimer's disease (FAD). The transmembrane domain (TM) of APP contains the known dimerization motif GXXXA. We have investigated the dimerization of both wild type and FAD mutant APP transmembrane domains.

Results: Using synthetic peptides derived from the APP-TM domain, we show that this segment is capable of forming stable transmembrane dimers. A model of a dimeric APP-TM domain reveals a putative dimerization interface, and interestingly, majority of FAD mutations in APP are localized to this interface region. We find that FAD-APP mutations destabilize the APP-TM dimer and increase the population of APP peptide monomers.

Conclusion: The dissociation constants are correlated to both the Abeta42/Abeta40 ratio and the mean age of disease onset in AD patients. We also show that these TM-peptides reduce Abeta production and Abeta42/Abeta40 ratios when added to HEK293 cells overexpressing the Swedish FAD mutation and gamma-secretase components, potentially revealing a new class of gamma-secretase inhibitors.

Show MeSH
Related in: MedlinePlus