Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFβ

Abstract

The amyloid β-peptide (Aβ) of Alzheimer’s disease (AD) is generated by proteolysis within the transmembrane domain (TMD) of a C-terminal fragment of the amyloid β protein-precursor (APP CTFβ) by the γ-secretase complex. This processing produces Aβ ranging from 38 to 49 residues in length. Evidence suggests that this spectrum of Aβ peptides is the result of successive γ-secretase cleavages, with endoproteolysis first occurring at the ε sites to generate Aβ48 or Aβ49, followed by C-terminal trimming mostly every three residues along two product lines to generate shorter, secreted forms of Aβ: the primary Aβ49-46-43-40 line and a minor Aβ48-45-42-38 line. The major secreted Aβ species are Aβ40 and Aβ42, and an increased proportion of the longer, aggregation-prone Aβ42 compared to Aβ40 is widely thought to be important in AD pathogenesis. We examined TMD substrate determinants of the specificity and efficiency of ε site endoproteolysis and carboxypeptidase trimming of CTFβ by γ-secretase. We determined that the C-terminal negative charge of the intermediate Aβ49 does not play a role in its trimming by γ-secretase. Peptidomimetic probes suggest that γ-secretase has S1’, S2’, and S3’ pockets, through which trimming by tripeptides may be determined. However, deletion of residues around the ε sites demonstrates that a depth of three residues within the TMD is not a determinant of the location of endoproteolytic ε cleavage of CTFβ. We also show that instability of the CTFβ TMD helix near the ε site significantly increases endoproteolysis, and that helical instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by γ-secretase. In addition, we found that CTFβ dimers are not endoproteolyzed by γ-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 ε cleavage and that helix unwinding is essential for both endoproteolysis and carboxypeptidase trimming.

Intramembrane cleavage of TREM2 is determined by its intrinsic structural dynamics

“…1b). Estimation of the secondary structure content using secondary 13 Ca and 13 Cb chemical shifts yielded an a-helical secondary structure between residues 174 and 198, corresponding to an a-helical content of 50% ( Fig. 1c), in excellent agreement with the results obtained by CD spectroscopy (Fig.…”

“…Such TMH sampling may explain the observation that helix-destabilizing diglycine mutations introduced between the gand e-sites increase the cleavage efficiency and processivity of g-secretase 13 .…”

“…S1a and b) that was suitable for further structural investigations. Next, using a U-2 H, 13 C, 15 N, -labeled TREM2-TMH (aa 161-206) sample in 300 mM DPC micelles, backbone resonance assignments were obtained for 88% or all non-proline residues using a set of 3D-triple resonance experiments at 37 °C ( Fig. 1b).…”

“…However, it is still unclear how g-secretase selects its substrates, since not every protein with a small enough ectodomain can be cleaved by g-secretase 11 . Thus, in addition to membrane orientation and size of the ectodomain other molecular signatures must exist allowing interaction with the g-secretase complex and recruitment to the active site 12,13 . Unlike soluble proteases g-secretase and other intramembrane cleaving enzymes usually do not recognize their substrates via short linear sequence motifs 14 .…”

“…Indeed, mutations that affect transmembrane domain flexibility in the cleavage site region of APP can markedly alter g-secretase cleavage 13 . Likewise, flexibility via a naturally occurring hinge in the TMH as in the case of APP can also be critical for g-secretase cleavage 15 .…”