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

Xulun Zhang, Robert J. Hoey, Guoqing Lin, Akiko Koide, Brenda Leung, Kwangwook Ahn, Georgia Dolios, Marcin Paduch, Takeshi Ikeuchi, Rong Wang, Yue Ming Li, Shohei Koide, Sangram S. Sisodia

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

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.

Original languageEnglish
Pages (from-to)8534-8539
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number22
DOIs
StatePublished - 29 May 2012

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