This is an intriguing paper describing the surprising discovery that a stretch of 4-7 amino acids in the APP juxtamembrane region, within the Aβ sequence (residues 17-23, Aβ numbering), is an inhibitory domain for γ-secretase cleavage that appears to work by a novel mechanism. This discovery was made while attempting to unravel the biochemical mechanism by which the Flemish familial AD mutation (A21G) increases Aβ production. Evidence is provided demonstrating that this mutation attenuates the inhibitory activity of the juxtamembrane region: Placing this mutation into a direct APP γ-secretase substrate, recombinant β-CTF, revealed a ~threefold increase in Aβ production by γ-secretase using an in vitro assay, and deletion or replacement of the entire 17-23 region leads to dramatic (12- to 25-fold) increases. Oddly though, no change is seen in the production of APP intracellular domain (AICD), the other product formed during Aβ generation. This is problematic, because an AICD molecule is formed for every Aβ generated. Not only is this straightforward stoichiometry, but it has been...  Read more

This is an intriguing paper describing the surprising discovery that a stretch of 4-7 amino acids in the APP juxtamembrane region, within the Aβ sequence (residues 17-23, Aβ numbering), is an inhibitory domain for γ-secretase cleavage that appears to work by a novel mechanism. This discovery was made while attempting to unravel the biochemical mechanism by which the Flemish familial AD mutation (A21G) increases Aβ production. Evidence is provided demonstrating that this mutation attenuates the inhibitory activity of the juxtamembrane region: Placing this mutation into a direct APP γ-secretase substrate, recombinant β-CTF, revealed a ~threefold increase in Aβ production by γ-secretase using an in vitro assay, and deletion or replacement of the entire 17-23 region leads to dramatic (12- to 25-fold) increases. Oddly though, no change is seen in the production of APP intracellular domain (AICD), the other product formed during Aβ generation. This is problematic, because an AICD molecule is formed for every Aβ generated. Not only is this straightforward stoichiometry, but it has been formally demonstrated by the lab of Yasuo Ihara (Kakuda et al., 2006). Thus, other Aβ variants must be decreased to compensate for the increase of Aβ38, Aβ40, and Aβ42 peptides that were examined.

The authors also show that short peptides based on the 17-23 region are inhibitory for Aβ production (at least of the Aβ variants measured). Conversion of one of these peptides into a photoaffinity labeling reagent led to the tagging of γ-secretase subunits PS1 NTF, PS1 CTF, and nicastrin, but not Aph1 or Pen-2. Competition with the parent peptide demonstrated the specificity of the labeling. In this respect, as well as kinetically, the 17-23 peptide appears to work by a different mechanism than transition-state analogue inhibitors (i.e., that interact with the protease active site). Indeed, two affinity labeling reagents based on a transition-state analogue inhibitor are differentially affected by the 17-23 peptide in their ability to label PS1, consistent with the idea that the 17-23 peptide somehow alters the properties of one of the substrate-recognition pockets in the enzyme active site.

Because the 17-23 peptides do not inhibit Aβ production in cells, retro-inverso peptides were designed and synthesized. Retro-inverso peptides have a similar shape to their L-peptide counterparts, but as they are composed of all D-amino acids, they are more metabolically stable. Two such retro-inverso peptides based on 17-23 inhibitory peptides could inhibit Aβ production from APP in cells, albeit at relatively high concentrations (e.g., 30 micromolar). The compounds had no effect on the processing of a Notch substrate (Notch ΔE) in cells, but Notch ΔE is a direct γ-secretase substrate. The comparison of Aβ generation from full-length APP and Notch intracellular domain (NICD) from Notch ΔE may be misleading, suggesting selectivity when there is really none. Moreover, there is concern that the retro-inverso peptides may not be working by the same mechanism as the parent 17-23 peptides. Competition studies in affinity labeling assays may help address this question.

Although this study suggests a new strategy for the development of Notch-sparing γ-secretase inhibitors, it is unclear that this is a worthwhile approach. Because the Aβ-reducing 17-23 peptides do not affect AICD production, they undoubtedly elevate variants of Aβ that were not examined. If the elevated variants are the shorter, less aggregation-prone form, then this is a promising approach. However, if longer, more aggregation-prone forms are elevated, this would be detrimental. Thus, the jury is still out until we have information on how this juxtamembrane region affects the production of the full spectrum of Aβ peptides.

These concerns notwithstanding, the study by Tian and colleagues has revealed a novel regulatory region in the APP substrate that affects Aβ production, presumably altering the types of Aβ peptides that are produced. A better understanding of how this region modulates γ-secretase activity is definitely warranted and may yet reveal an important new strategy for AD drug discovery.

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