Transmembrane Substrate Determinants For Gamma-Secretase Processing Of App Ctf Beta

The amyloid beta-peptide (A beta) of Alzheimer's disease (AD) is generated by proteolysis within the trans membrane domain (TMD) of a C-terminal fragment of the amyloid beta protein-precursor (APP CTF beta) by the gamma-secretase complex. This processing produces A beta ranging from 38 to 49 residues in length. Evidence suggests that this spectrum of A beta peptides is the result of successive gamma-secretase cleavages, with endoproteolysis first occurring at the epsilon sites to generate A beta 48 or A beta 49, followed by C-terminal trimming mostly every three residues along two product lines to generate shorter, secreted forms of A beta: the primary A beta 49-46-43-40 line and a minor A beta 48-45-42-38 line. The major secreted A beta species are A beta 40 and A beta 42, and an increased proportion of the longer, aggregation-prone A beta 42 compared to A beta 40 is widely thought to be important in AD pathogenesis. We examined TMD substrate determinants of the specificity and efficiency of epsilon site endoproteolysis and carboxypeptidase trimming of CTF beta by gamma-secretase. We determined that the C-terminal negative charge of the intermediate A beta 49 does not play a role in its trimming by gamma-secretase. Peptidomimetic probes suggest that gamma-secretase has S1', S2', and S3' pockets, through which trimming by tripeptides may be determined. However, deletion of residues around the epsilon sites demonstrates that a depth of three residues within the TMD is not a determinant of the location of endoproteolytic epsilon cleavage of CTF beta. We also show that instability of the CTF beta TMD helix near the epsilon site significantly increases endoproteolysis, and that helical instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by gamma-secretase. In addition, we found that CTF beta dimers are not endoproteolyzed by gamma-secretase. These results support a model in which initial interaction of the array of residues along the undimerized single helical TMD of substrates dictates the site of initial epsilon cleavage and that helix unwinding is essential for both endoproteolysis and carboxypeptidase trimming.