P3-322: The Nicastrin peptidase-like domain is crucially involved in the assembly of the γ-secretase complex

Abstract

The identification of Nicastrin (NCT) as the substrate receptor of the γ-secretase complex raised the possibility of considering it as a drug target in AD. It has been reported that hNCT-E333 is crucial in the recognition mechanism by interacting with the substrate's alpha amino terminal group. In the absence of structural information for the complex, we used the NCT similarity to peptidases with known crystal structure, as a guide to further explore and characterize NCT's role. We performed alanine-scanning mutagenesis to further investigate the role of the NCT “peptidase-like” domain in the activity-specificity of the γ-secretase complex. We evaluated residues located in the putative substrate's binding pocket of the NCT model. Nicastrin knock-out mouse fibroblast were rescued with mouse wild type NCT or mutants. The expression levels were similar among mutants and at a similar extent to the endogenous NCT. However, not all of them rescued NCT maturation. Unexpectedly, the E332A-NCT mutant severely affected NCT maturation and complex assembly, stabilizing only about 10% of the wt-NCT complex. We next investigated whether any of these substitutions influences the γ-secretase processing of APP or Notch substrates. In general, mutations the NCT-peptidase like domain slightly regulated the processing of these substrates. Interestingly, we observed that the specific activity of the E332A- γ-secretase complex was comparable with the wt enzyme suggesting that the substrate recognition mechanism is mainly unaffected. In conclusion, our data show that the NCT-peptidase like domain is mainly implicated in the assembly of the complex, whereas its contribution to the activity-specificity of the complex is less obvious. Most importantly, our data strongly shows that the substrate recognition mechanism is not critically dependent on the E332 residue, as previously reported. However, we cannot discard a dual mechanism for NCT-ectodomain: initially involved in the complex assembly and that could lead to a contribution in substrate recognition.