Substrate Selection Criteria in Regulated Intramembrane Proteolysis

Alzheimer's disease is the most common form of dementia encountered in an aging population. Characteristic amyloid deposits of Aβ peptides in the brain are generated through cleavage of amyloid precursor protein (APP) by γ-secretase, an intramembrane protease. Cryo-EM structures of substrate γ-secretase complexes revealed details of the process, but how substrates are recognized and enter the catalytic site is still largely ignored. γ-Secretase cleaves a diverse range of substrate sequences without a common consensus sequence, but strikingly, single point mutations within the transmembrane domain (TMD) of specific substrates may greatly affect cleavage efficiencies. Previously, conformational flexibility was hypothesized to be the main criterion for substrate selection. Here we review the 3D structure and dynamics of several γ-secretase substrate TMDs and compare them with mutants shown to affect the cleavage efficiency. In addition, we present structural and dynamic data on ITGB1, a known nonsubstrate of γ-secretase. A comparison of biophysical details between these TMDs and changes generated by introducing crucial mutations allowed us to unravel common principles that differ between substrates and nonsubstrates. We identified three motifs in the investigated substrates: a highly flexible transmembrane domain, a destabilization of the cleavage region, and a basic signature at the end of the transmembrane helix. None of these appears to be exclusive. While conformational flexibility on its own may increase cleavage efficiency in well-known substrates like APP or Notch1, our data suggest that the three motifs seem to be rather variably combined to determine whether a transmembrane helix is efficiently recognized as a γ-secretase substrate.