Major carboxyl terminal fragments generated by γ-secretase processing of the Alzheimer amyloid precursor are 50 and 51 amino acids long.

OBJECTIVES: To understand the cleavage of the amyloid ß protein (Aß) precursor (APP) by γ-secretase and to determine its changes in a representative familial Alzheimer disease (FAD) mutation. METHODS: Transfected cells expressing wild-type and FAD mutant APP were analyzed for changes in the levels of the major secreted Aß species and of the corresponding intracellular C-terminal APP fragments (APP intracellular domain, AICD) generated by γ-secretase, whereas radio-sequencing was used to precisely identify the resulting cleavage site(s). RESULTS: The AICD fragment(s) generated by γ-secretase cleavage comigrated in gels with a 50-residue synthetic peptide used as control, which is smaller than the 59 and 57 residues predicted from Aß ending at positions 40 (Aß40) and 42 (Aß42), respectively. In agreement with previous findings, an FAD mutant form of presenilin 1 (PS1-M139V) significantly increased the longer Aß42 while showing trends toward reducing Aß40. AICD levels were reduced by the mutation, suggesting that γ-secretase activity may be actually impaired by the mutation. Radiosequence analysis in cells expressing wild-type PS1 detected γ-secretase cleavage sites at the Aß peptide bond L(49)-V(50) to generate a 50-amino acid (aa) AICD fragment (AICD50) and the Aß peptide bond T(48)-L(49), generating an AICD of 51 aa (AICD51). No other cleavage sites were reliably detected. CONCLUSIONS: Based on findings that the FAD mutation that increases Aß42 also reduces AICD, we propose that γ-secretase activity is impaired by FAD mutations and predict that physiologic and environmental agents that inhibit γ-secretase will actually induce AD pathogenesis rather that prevent it. Furthermore, we propose that the cleavage site to generate AICD is naturally ragged and occurs predominantly at two sites 48 and 49 aa from the start of the Aß sequence. Thus, end specific antibodies to these two sites will need to be generated to study the quantitative relationships between these two cleavages in sporadic AD and FAD.

Lipid rafts play an important role in A beta biogenesis by regulating the beta-secretase pathway.

The Alzheimer's amyloid beta protein (A beta) precursor (APP) is proteolytically cleaved by beta-secretase to N- and C-terminal fragments sAPPbeta and CTFbeta, respectively. Subsequently, CTFbeta is cleaved by gamma-secretase to generate A beta. We previously showed that the levels of secreted A beta and sAPPbeta were significantly reduced upon removal of glycosylphosphatidylinositol (GPI)-anchored proteins from either primary brain cells or Chinese hamster ovary cultures. The results indicated that GPI-anchored proteins facilitated beta-secretase activity. In this report, we strengthen the previous findings by demonstrating that CTFbeta, like sAPPbeta, is also reduced upon removal of GPI-anchored proteins and that sAPPbeta does not accumulate in an intracellular compartment. This facilitation pathway does not appear to be important for the processing of a disease-linked mutant form of APP (670NL), known to be a superior beta-secretase substrate. A novel aspartyl protease, BACE, responsible for beta-secretase activity in the brain is not GPI-anchored. However, BACE in brain membranes accumulate in lipid rafts, a compartment marked by the accumulation of GPI-anchored proteins. This finding is consistent with the hypothesis that BACE interacts with GPI-anchored proteins that facilitate its activity possibly by chaperoning it into lipid rafts.