Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFβ

Area of Science:

• Molecular Biology
• Neuroscience
• Biochemistry

Background:

• Alzheimer's disease (AD) pathogenesis is linked to the production of amyloid-beta (Aβ) peptides.
• Aβ peptides are generated by sequential cleavage of the amyloid precursor protein (APP) within its transmembrane domain (TMD) by the γ-secretase complex.
• The spectrum of Aβ lengths, particularly the ratio of Aβ42 to Aβ40, is critical in AD.

Purpose of the Study:

• To investigate the determinants of γ-secretase specificity and efficiency in cleaving APP C-terminal fragments (CTFβ).
• To elucidate the mechanisms underlying the generation of different Aβ peptide lengths.
• To understand the role of substrate structure, specifically the TMD helix, in γ-secretase activity.

Main Methods:

• Analysis of CTFβ substrate determinants, including C-terminal charge and TMD helical properties.
• Use of peptidomimetic probes to explore γ-secretase active site pockets (S1', S2', S3').
• Site-directed mutagenesis (deletions) around ε sites and assessment of helical instability.

Main Results:

• The C-terminal negative charge of intermediate Aβ49 does not influence its trimming.
• Helical instability within the CTFβ TMD significantly enhances both endoproteolysis (ε-site cleavage) and carboxypeptidase trimming.
• CTFβ dimers are not substrates for γ-secretase endoproteolysis, and helix unwinding is essential for both cleavage steps.

Conclusions:

• Substrate TMD helical instability is a key factor promoting γ-secretase activity and Aβ peptide generation.
• Initial ε-site cleavage is dictated by the interaction of residues along the undimerized, single helical TMD.
• Helix unwinding is a prerequisite for both the initial endoproteolytic cleavage and subsequent C-terminal trimming by γ-secretase.