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


ABSTRACT: The ?-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 ?-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 ?-secretase activity assays, suggesting that a functional domain pertinent to ?-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 ?-secretase activity but not the assembly of the ?-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 ?-secretase complex.

SUBMITTER: Zhang X 

PROVIDER: S-EPMC3365189 | biostudies-other | 2012 May

REPOSITORIES: biostudies-other

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

Zhang Xulun X   Hoey Robert J RJ   Lin Guoqing G   Koide Akiko A   Leung Brenda B   Ahn Kwangwook K   Dolios Georgia G   Paduch Marcin M   Ikeuchi Takeshi T   Wang Rong R   Li Yue-Ming YM   Koide Shohei S   Sisodia Sangram S SS  

Proceedings of the National Academy of Sciences of the United States of America 20120514 22


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 func  ...[more]

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