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-pharynxdefective 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 conformationspecific 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.A lzheimer's disease (AD), a progressive neurodegenerative disease, is the most prevalent cause of dementia in humans. The principal neuropathological hallmark of AD is the presence of senile plaques composed of dystrophic neurites surrounding extracellular aggregates of Aβ peptides (1). Aβ peptides are liberated from amyloid precursor proteins (APP) by the concerted action of β-site APP cleaving enzyme 1 and γ-secretase (2, 3). γ-Secretase is a macromolecular complex consisting of presenilin 1 or presenilin 2 (PS1 or PS2), anterior-pharynx-defective 1 (APH-1), nicastrin (NCT), and presenilin enhancer 2 (PEN-2) that catalyzes intramembranous proteolysis of several membrane-tethered substrates (4). Evidence has emerged to reveal the functions of each subunit: PS is the catalytic subunit (5); APH-1 serves as a scaffold for the complex assembly (6); NCT seems to be responsible for substrate binding (7); and PEN-2 promotes endoproteolytic cleavage of PS and "activation" of the enzyme complex (5, 8). Although γ-secretase cleaves a multiplicity of substrates at heterogeneous sites within individual transmembrane domains, the molecular mechanism(s) underlying substrate recognition and processing remain elusive.NCT, a 709-aa type I transmembrane glycoprotein with a large, heavily glycosylated ectodomain (ECD), was first identified as a PS-inter...

Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme

“…To do so, we biotinylated IDE in Escherichia coli, immobilized IDE on streptavidin-coated plates, used it to probe a phage-display library, and identified a synthetic antibody in the form of an antigen-binding fragment (Fab). This crystallization chaperone system has effectively facilitated crystallization of membrane proteins and RNA that are otherwise difficult to crystallize (23)(24)(25). The final synthetic antibody, termed Fab (IDE) , bound human IDE with a K D value of 4 nM, as determined by surface plasmon resonance (Fig.…”

“…Fab was expressed in E. coli strain 55244 and purified using a HiTrap protein G HP column, as previously described (25). Surface plasmon resonance measurements were carried out at 20°C on a Biacore 3000 by immobilizing His-tagged IDE onto a Ni-NTA chip and then injecting 1.2-100 nM of Fab at a flow rate of 30 μL/min (25,34).…”

“…We isolated an IDE-binding synthetic antibody as the antigen-binding fragment (Fab) from a phage-display library as previously described, using 100, 100, 50, and 20 nM biotinylated IDE for sorting in rounds 1, 2, 3, and 4, respectively (33). Fab was expressed in E. coli strain 55244 and purified using a HiTrap protein G HP column, as previously described (25). Surface plasmon resonance measurements were carried out at 20°C on a Biacore 3000 by immobilizing His-tagged IDE onto a Ni-NTA chip and then injecting 1.2-100 nM of Fab at a flow rate of 30 μL/min (25,34).…”

Retention in Endoplasmic Reticulum 1 (RER1) Modulates Amyloid-β (Aβ) Production by Altering Trafficking of γ-Secretase and Amyloid Precursor Protein (APP)

“…Other subunits of the complex, NCT, APH-1, and PEN-2 have been known to play roles in its trafficking and maturation. It has been suggested that NCT may be critical for substrate recognition (14,15), although some evidence suggests that NCT may also have a more indirect role in regulation of the localization and activity of the complex (16). Steady-state accumulation of each component of the complex is coordinately regulated and, in large part, is dependent on the expression of the other members of the complex (17)(18)(19).…”

Structural Biology of Presenilins and Signal Peptide Peptidases

“…This extracellular domain has been implicated in the stability of the enzyme as well as substrate binding (63), although the latter has been opposed by results showing that recombinant PS (34) and SPP (64) are able to cleave peptide bonds without any cofactor proteins. However, we and others have shown that antibodies against the Nct extracellular domain are capable of inhibiting ␥-secretase activity by competition with substrates (33,65,66), suggesting the possibility that Nct assists in capturing ␥-substrates on the membrane (Fig. 3).…”