Identification of a tetratricopeptide repeat-like domain in the nicastrin subunit of γ-secretase using synthetic antibodies.

Abstract

The γ-secretase complex, composed of presenilin, anterior-pharynx-defective 1, nicastrin, and presenilin enhancer 2, catalyzes the intramembranous processing of a wide variety of type I membrane proteins, including amyloid precursor protein (APP) and Notch. Earlier studies have revealed that nicastrin, a type I membrane-anchored glycoprotein, plays a role in γ-secretase assembly and trafficking and has been proposed to bind substrates. To gain more insights regarding nicastrin structure and function, we generated a conformation-specific synthetic antibody and used it as a molecular probe to map functional domains within nicastrin ectodomain. The antibody bound to a conformational epitope within a nicastrin segment encompassing residues 245-630 and inhibited the processing of APP and Notch substrates in in vitro γ-secretase activity assays, suggesting that a functional domain pertinent to γ-secretase activity resides within this region. Epitope mapping and database searches revealed the presence of a structured segment, located downstream of the previously identified DAP domain (DYIGS and peptidase; residues 261-502), that is homologous to a tetratricopeptide repeat (TPR) domain commonly involved in peptide recognition. Mutagenesis analyses within the predicted TPR-like domain showed that disruption of the signature helical structure resulted in the loss of γ-secretase activity but not the assembly of the γ-secretase and that Leu571 within the TPR-like domain plays an important role in mediating substrate binding. Taken together, these studies offer provocative insights pertaining to the structural basis for nicastrin function as a "substrate receptor" within the γ-secretase complex.

DOI: 10.1073/pnas.1202691109
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@article{Zhang2012IdentificationOA, title={Identification of a tetratricopeptide repeat-like domain in the nicastrin subunit of γ-secretase using synthetic antibodies.}, author={Xulun Zhang and Robert J. Hoey and Guoqing Lin and Akiko Koide and Brenda M. Y. Leung and Kwangwook Ahn and Georgia Dolios and Marcin Paduch and T Ikeuchi and Rong Wang and Yue-Ming Li and Shohei Koide and Sangram S. Sisodia}, journal={Proceedings of the National Academy of Sciences of the United States of America}, year={2012}, volume={109 22}, pages={8534-9} }