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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

APP-TM peptides oligomerize within phospolipid bilayers. Different forms of fluorescent donor (Trp) and acceptor (Edans) labeled APP-TM peptides were incorporated in DOPG phospholipid vesicles. Purified peptide-incorporated vesicle fractions were subjected to fluorescence measurements. Typical donor quenching and acceptor sensitization was observed and is indicative of FRET. The fluorescence spectra of donor-labeled APP-TM wild-type peptide (□), acceptor-labeled APP-TM wild-type peptide (○), and a mixed sample (●) are shown. Note: arrows show donor quenching and acceptor sensitization and indicate that the peptides oligomerize in phospholipid bilayers. Inset: FRET decreases with increasing concentration of unlabeled peptide in phospholipids bilayers. Equimolar mixtures of donor- and acceptor-labeled APP-TM peptides were mixed with increasing concentrations of unlabeled peptide. Total peptide concentration was held constant at 100 μM. The unlabeled peptides act as competitors and reduce the likelihood of forming dimers containing both donor- and acceptor-labeled peptides, which results in a decrease in FRET and A/D ratio.
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Figure 2: APP-TM peptides oligomerize within phospolipid bilayers. Different forms of fluorescent donor (Trp) and acceptor (Edans) labeled APP-TM peptides were incorporated in DOPG phospholipid vesicles. Purified peptide-incorporated vesicle fractions were subjected to fluorescence measurements. Typical donor quenching and acceptor sensitization was observed and is indicative of FRET. The fluorescence spectra of donor-labeled APP-TM wild-type peptide (□), acceptor-labeled APP-TM wild-type peptide (○), and a mixed sample (●) are shown. Note: arrows show donor quenching and acceptor sensitization and indicate that the peptides oligomerize in phospholipid bilayers. Inset: FRET decreases with increasing concentration of unlabeled peptide in phospholipids bilayers. Equimolar mixtures of donor- and acceptor-labeled APP-TM peptides were mixed with increasing concentrations of unlabeled peptide. Total peptide concentration was held constant at 100 μM. The unlabeled peptides act as competitors and reduce the likelihood of forming dimers containing both donor- and acceptor-labeled peptides, which results in a decrease in FRET and A/D ratio.

Mentions: To determine whether the APP-TM peptides show similar behavior in phospholipid bilayers as they do in SDS micelles, we examined APP-TM peptides incorporated into dioleoyl phosphatidylglycerol (DOPG) vesicles using fluorescence resonance energy transfer (FRET) analysis. The compositions of these samples were as follows: (a) vesicles containing donor-labeled and unlabeled peptide mixtures, (b) vesicles containing acceptor-labeled and unlabeled peptide mixtures, and (c) vesicles containing equimolar mixtures of donor- and acceptor-labeled peptides. The fluorescence emission spectra of these peptide preparations are shown in Figure 2. In the preparation containing both donor and acceptor, the emission of the Trp fluorophore is significantly quenched compared to the emission of vesicles with only Trp-labeled peptide. The EDANS emission, on the other hand, is significantly enhanced in the mixed fluorophore preparation relative to the emission with acceptor only. Certain unavoidable factors, such as differential incorporation of peptides in vesicles, trapping of peptides within vesicle lumens, and asymmetric distribution of parallel and antiparallel orientations of peptides in the bilayer, can affect the fluorescence emission of the donor and acceptor. However, the observed donor quenching and acceptor sensitization are hallmark signatures of FRET and indicate that the peptides incorporated in DOPG vesicles are oligomeric in structure. A quantitative measure of the extent of FRET was obtained by measuring the ratio of acceptor fluorescence to donor fluorescence (i.e. A/D ratio; see Materials and Methods). To confirm that the observed spectral changes are caused by FRET, the A/D ratio was measured in the presence of increasing amounts of unlabeled peptide. The addition of excess unlabeled peptide caused a continuous decrease in the A/D ratio (Fig. 2 inset). This reduction in FRET suggests that the unlabeled peptides were forming mixed oligomers with the labeled peptides and preventing association between donor and acceptor fluorophores. The similarity of the FRET results obtained with DOPG vesicles and those obtained with SDS micelles (see below) suggests that the APP-TM peptides associate into similar structures in both systems.


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)

APP-TM peptides oligomerize within phospolipid bilayers. Different forms of fluorescent donor (Trp) and acceptor (Edans) labeled APP-TM peptides were incorporated in DOPG phospholipid vesicles. Purified peptide-incorporated vesicle fractions were subjected to fluorescence measurements. Typical donor quenching and acceptor sensitization was observed and is indicative of FRET. The fluorescence spectra of donor-labeled APP-TM wild-type peptide (□), acceptor-labeled APP-TM wild-type peptide (○), and a mixed sample (●) are shown. Note: arrows show donor quenching and acceptor sensitization and indicate that the peptides oligomerize in phospholipid bilayers. Inset: FRET decreases with increasing concentration of unlabeled peptide in phospholipids bilayers. Equimolar mixtures of donor- and acceptor-labeled APP-TM peptides were mixed with increasing concentrations of unlabeled peptide. Total peptide concentration was held constant at 100 μM. The unlabeled peptides act as competitors and reduce the likelihood of forming dimers containing both donor- and acceptor-labeled peptides, which results in a decrease in FRET and A/D ratio.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: APP-TM peptides oligomerize within phospolipid bilayers. Different forms of fluorescent donor (Trp) and acceptor (Edans) labeled APP-TM peptides were incorporated in DOPG phospholipid vesicles. Purified peptide-incorporated vesicle fractions were subjected to fluorescence measurements. Typical donor quenching and acceptor sensitization was observed and is indicative of FRET. The fluorescence spectra of donor-labeled APP-TM wild-type peptide (□), acceptor-labeled APP-TM wild-type peptide (○), and a mixed sample (●) are shown. Note: arrows show donor quenching and acceptor sensitization and indicate that the peptides oligomerize in phospholipid bilayers. Inset: FRET decreases with increasing concentration of unlabeled peptide in phospholipids bilayers. Equimolar mixtures of donor- and acceptor-labeled APP-TM peptides were mixed with increasing concentrations of unlabeled peptide. Total peptide concentration was held constant at 100 μM. The unlabeled peptides act as competitors and reduce the likelihood of forming dimers containing both donor- and acceptor-labeled peptides, which results in a decrease in FRET and A/D ratio.
Mentions: To determine whether the APP-TM peptides show similar behavior in phospholipid bilayers as they do in SDS micelles, we examined APP-TM peptides incorporated into dioleoyl phosphatidylglycerol (DOPG) vesicles using fluorescence resonance energy transfer (FRET) analysis. The compositions of these samples were as follows: (a) vesicles containing donor-labeled and unlabeled peptide mixtures, (b) vesicles containing acceptor-labeled and unlabeled peptide mixtures, and (c) vesicles containing equimolar mixtures of donor- and acceptor-labeled peptides. The fluorescence emission spectra of these peptide preparations are shown in Figure 2. In the preparation containing both donor and acceptor, the emission of the Trp fluorophore is significantly quenched compared to the emission of vesicles with only Trp-labeled peptide. The EDANS emission, on the other hand, is significantly enhanced in the mixed fluorophore preparation relative to the emission with acceptor only. Certain unavoidable factors, such as differential incorporation of peptides in vesicles, trapping of peptides within vesicle lumens, and asymmetric distribution of parallel and antiparallel orientations of peptides in the bilayer, can affect the fluorescence emission of the donor and acceptor. However, the observed donor quenching and acceptor sensitization are hallmark signatures of FRET and indicate that the peptides incorporated in DOPG vesicles are oligomeric in structure. A quantitative measure of the extent of FRET was obtained by measuring the ratio of acceptor fluorescence to donor fluorescence (i.e. A/D ratio; see Materials and Methods). To confirm that the observed spectral changes are caused by FRET, the A/D ratio was measured in the presence of increasing amounts of unlabeled peptide. The addition of excess unlabeled peptide caused a continuous decrease in the A/D ratio (Fig. 2 inset). This reduction in FRET suggests that the unlabeled peptides were forming mixed oligomers with the labeled peptides and preventing association between donor and acceptor fluorophores. The similarity of the FRET results obtained with DOPG vesicles and those obtained with SDS micelles (see below) suggests that the APP-TM peptides associate into similar structures in both systems.

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