Nicastrin functions to sterically hinder γ-secretase–substrate interactions driven by substrate transmembrane domain

Abstract

γ-Secretase is an intramembrane-cleaving protease that processes many type-I integral membrane proteins within the lipid bilayer, an event preceded by shedding of most of the substrate's ectodomain by α-or β-secretases. The mechanism by which γ-secretase selectively recognizes and recruits ectodomain-shed substrates for catalysis remains unclear. In contrast to previous reports that substrate is actively recruited for catalysis when its remaining short ectodomain interacts with the nicastrin component of γ-secretase, we find that substrate ectodomain is entirely dispensable for cleavage. Instead, γ-secretase-substrate binding is driven by an apparent tight-binding interaction derived from substrate transmembrane domain, a mechanism in stark contrast to rhomboid-another family of intramembrane-cleaving proteases. Disruption of the nicastrin fold allows for more efficient cleavage of substrates retaining longer ectodomains, indicating that nicastrin actively excludes larger substrates through steric hindrance, thus serving as a molecular gatekeeper for substrate binding and catalysis.

The amyloid-beta forming tripeptide cleavage mechanism of γ-secretase

“…In a recent study, we have demonstrated that substrates with large ectodomains have a reduced binding affinity for γ-secretase due to steric clashing with the nicastrin component of the γ-secretase complex (Bolduc et al, 2016). Based on this, and results from our current study, we might expect that γ-secretase can chose its substrates through a complex interplay between ectodomain length, helical TMD stability and the sequence of amino acids (specifically aromatic amino acids) within substrate TMD.…”

Amyloid precursor protein processing and bioenergetics

“…These include Presenilin 1 (PS1), Presenilin 2 (PS2), nicastrin, anterior pharynx defective (APH-1), and PS enhancer (PEN2) (Francis et al, 2002). PS1 and PS2 form the catalytic domain of γ-secretase while APH-1 may act as a stabilizer, PEN2 acts as a regulator/enhancer of activity, and nicastrin serves as a substrate receptor (Bolduc et al, 2016; Dries and Yu, 2008; Holmes et al, 2014). The cleavage event catalyzed by γ-secretase does not occur at a single site as there are three cleavage sites on APP separated by three amino acids; ε, γ, and δ. γ-secretase assembly, trafficking, and substrates have been reviewed elsewhere (De Strooper et al, 2012; Dries and Yu, 2008; Haapasalo and Kovacs, 2011).…”

An Inducible Reconstitution System for the Real-Time Kinetic Analysis of Protease Activity and Inhibition Inside the Membrane

“…To date, all intramembrane proteases that have been analyzed are very slow enzymes, usually taking minutes (and even hours) to cleave each substrate molecule (Dickey et al, 2013; Kamp et al, 2015; Bolduc et al, 2016). As such, even trace ‘invisible’ amounts of contaminating cellular proteases can create severe interference when establishing a protease assay with such slow enzymes.…”

“…This can be achieved with a simple set of four reconstitution reactions: the wildtype intramembrane protease assayed at pH 7.4 and at pH 4.0, and its catalytic mutant assayed at pH 7.4 (in the absence or presence of proteinase K as a positive cleavage control). For acid proteases, elevated pH instead of pH 4 is likely to be required; pH 8.5 works well in reversibly inactivating the aspartyl intramembrane protease γ-secretase (Bolduc et al, 2016). …”