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Mutations in the transmembrane domain of APP altering gamma-secretase specificity
S F Lichtenthaler1, N Ida, G Multhaup
1Center for Molecular Biology Heidelberg (ZMBH), University of Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany. ir2@ix.urz.uni-heidelberg.de
Abstract:
Alzheimer's disease (AD) beta-amyloid peptide (Abeta and betaA4) is derived from the amyloid precursor protein (APP) by the subsequent action of the so-far unidentified beta- and gamma-secretases. gamma-secretase, which generates the C-terminus of Abeta, cleaves within the transmembrane domain of APP, preferentially after Abeta-residue 40 (Abeta 40) but also after residue 42 (Abeta 42). This Abeta 42 represents the major subunit of the plaques in AD. Since the position of gamma-secretase cleavage is crucial for understanding the pathogenic pathway, we investigated the effect of different point mutations at Thr43 on gamma-secretase specificity in SPA4CT (SPC99)-expressing COS7 cells. These constructs only require gamma-cleavage for Abeta release. We observed that all Thr43 mutations altered the specificity of gamma-secretase. Small hydrophobic residues favored the generation of Abeta 42, leading to an increase in the 42/40 ratio of Abeta (1.6-2.8-fold). The increase was even stronger (5.6-5.8-fold) when combined with the familial mutation Val46Phe. Thus, these constructs might be highly valuable for the generation of animal models for AD. Processing of full-length APP or SPA4CT yielded the same 42/40 ratio of Abeta (4. 7%). Both constructs, bearing the familial AD mutation Val46Phe, led to a similar increase in the 42/40 ratio (3.3- versus 3.6-fold). The p3 fragment, produced by alpha- and gamma-secretase, showed 42/40 ratios similar to Abeta when derived from wild-type and mutant proteins. These results suggest that the different Abeta- and p3-species are generated by gamma-cleavage activities with a similar enzymatic mechanism.
Insights
Investigating mutations in amyloid precursor protein (APP) revealed how gamma-secretase activity influences Alzheimer's disease (AD) pathology. Specific mutations increase the production of amyloid-beta 42 (Abeta 42), a key component of AD plaques.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Alzheimer's disease (AD) is characterized by amyloid plaques, primarily composed of amyloid-beta 42 (Abeta 42).
- Abeta peptides are generated from amyloid precursor protein (APP) by beta- and gamma-secretase enzymes.
- Gamma-secretase cleavage site determines the ratio of Abeta 40 to Abeta 42, influencing AD pathogenesis.
Purpose of the Study:
- To investigate the impact of mutations at Thr43 within APP on gamma-secretase specificity.
- To understand how these mutations affect the production ratio of Abeta 42 versus Abeta 40.
- To explore the potential of these findings for developing AD animal models.
Main Methods:
- Utilized SPA4CT constructs in COS7 cells, requiring only gamma-cleavage for Abeta release.
- Introduced point mutations at Thr43 and combined them with the familial Val46Phe mutation.
- Analyzed the resulting Abeta 42/40 ratios and compared them to full-length APP processing.
Main Results:
- All Thr43 mutations altered gamma-secretase specificity, favoring Abeta 42 generation.
- Small hydrophobic residues at Thr43 increased the Abeta 42/40 ratio (1.6-2.8-fold).
- Combined Thr43 mutations with Val46Phe further elevated the Abeta 42/40 ratio (5.6-5.8-fold).
- Similar effects on Abeta 42/40 ratios were observed for p3 fragments generated by alpha- and gamma-secretase.
Conclusions:
- Gamma-secretase cleavage specificity is significantly influenced by mutations within the APP transmembrane domain.
- These findings provide insights into the enzymatic mechanism of Abeta and p3 fragment generation.
- The developed constructs and observed mutation effects offer valuable tools for creating more accurate AD animal models.