Crystal structure of the γ-secretase component nicastrin

Xie, Tian; Yan, Chuangye; Zhou, Rui; Zhao, Yanyu; Sun, Linfeng; Yang, Guanghui; Lu, Pinyan; Ma, Dan; Shi, Yigong

doi:10.1073/pnas.1414837111

Cited by 65 publications

(93 citation statements)

References 42 publications

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“…Because mutations of nicastrin can lead to reduced γ-secretase complex formation and stability (36,37,52) and have led to contradictory results in the literature (35-37), we hoped to find a way to disrupt nicastrin after mature wild-type γ-secretase had been formed and purified in its native state. Nicastrin is the only component of γ-secretase known to contain any disulfide bonds (24,34). We therefore decided to take advantage of this and attempt to disrupt nicastrin function by reducing these highly conserved disulfide bridges.…”

Section: Resultsmentioning

confidence: 99%

“…The seven-TMD protein Aph-1 is believed to play a scaffolding role in complex formation (30,31). Nicastrin is a type-I integral membrane protein with a large, heavily glycosylated ectodomain (32)(33)(34) that contains multiple stabilizing disulfide bridges (24,34).…”

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confidence: 99%

“…The ectodomain of nicastrin is structurally homologous to a bacterial amino peptidase (34). Although nicastrin lacks the specific amino acids required for peptidase activity, it has been proposed to bind the N terminus of ectodomain-shed substrate, thereby directing substrate TMD to the γ-secretase active site for cleavage (35,36).…”

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confidence: 99%

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

Bolduc

Montagna

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

129

152

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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.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Bolduc

Montagna

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

129

152

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“…Unfortunately, such effort has been hampered by the difficulty in expression, purification, and manipulation of the complex protease. This problem persists despite recent breakthroughs in structural elucidation of γ-secretase and nicastrin (7,8).The intramembrane aspartate protease PSH from the archaeon Methanoculleus marisnigri JR1 shares 19% sequence identity and 53% sequence similarity with human PS1 (hPS1). The signature motifs for catalysis, ΦYDΦΦ (Φ for a hydrophobic residue) on transmembrane segment 6 (TM6) and ΦGΦGD on TM7, are identical between PSH and hPS1.…”

mentioning

confidence: 99%

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confidence: 99%

Cleavage of amyloid precursor protein by an archaeal presenilin homologue PSH

Dang

Wang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Aberrant cleavage of amyloid precursor protein (APP) by γ-secretase contributes to the development of Alzheimer's disease. More than 200 disease-derived mutations have been identified in presenilin (the catalytic subunit of γ-secretase), making modulation of γ-secretase activity a potentially attractive therapeutic opportunity. Unfortunately, the technical challenges in dealing with intact γ-secretase have hindered discovery of modulators and demand a convenient substitute approach. Here we report that, similar to γ-secretase, the archaeal presenilin homolog PSH faithfully processes the substrate APP C99 into Aβ42, Aβ40, and Aβ38. The molar ratio of the cleavage products Aβ42 over Aβ40 by PSH is nearly identical to that by γ-secretase. The proteolytic activity of PSH is specifically suppressed by presenilin-specific inhibitors. Known modulators of γ-secretase also modulate PSH similarly in terms of the Aβ42/Aβ40 ratio. Structural analysis reveals association of a known γ-secretase inhibitor with PSH between its two catalytic aspartate residues. These findings identify PSH as a surrogate protease for the screening of agents that may regulate the protease activity and the cleavage preference of γ-secretase.A myloid precursor protein (APP) is initially cleaved by β-secretase in the extracellular space, producing a membrane-tethered, 99-residue carboxyl-terminal fragment known as APP C99 (1). APP C99 then undergoes sequential cleavages by γ-secretase, first at the e sites, yielding Aβ49/Aβ48, and eventually at the γ-sites, generating Aβ42/Aβ40/Aβ38 (2-4) (Fig. 1A). The 230-kDa γ-secretase contains four components: presenilin (PS), Pen-2, Aph-1, and nicastrin (NCT), of which PS1 is the target of most mutations derived from early-onset familial Alzheimer's disease (FAD) patients. Rather than abolishing the protease activity of γ-secretase, these mutations are thought to increase the molar ratio of Aβ42 over Aβ40 (2).Among the cleavage products of γ-secretase, Aβ42 is particularly prone to aggregation, leading to formation of β-amyloid plaque in the brain and presumably causing Alzheimer's disease (3). Other FAD-derived mutations map to APP and PS2, lending support to the causal relationship between formation of β-amyloid plaque and Alzheimer's disease (5). Therapeutic intervention of Alzheimer's disease may directly benefit from in vitro investigation of γ-secretase and discovery of its potential modulators (6). Unfortunately, such effort has been hampered by the difficulty in expression, purification, and manipulation of the complex protease. This problem persists despite recent breakthroughs in structural elucidation of γ-secretase and nicastrin (7,8).The intramembrane aspartate protease PSH from the archaeon Methanoculleus marisnigri JR1 shares 19% sequence identity and 53% sequence similarity with human PS1 (hPS1). The signature motifs for catalysis, ΦYDΦΦ (Φ for a hydrophobic residue) on transmembrane segment 6 (TM6) and ΦGΦGD on TM7, are identical between PSH and hPS1. PSH can be readily overexpressed in Escheri...

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Production of active glycosylation‐deficient γ‐secretase complex for crystallization studies

López

Dimitrov

Gerber

et al. 2015

Biotech & Bioengineering

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Alzheimer's disease (AD)-associated γ-secretase is a ubiquitously expressed multi-subunit protease complex embedded in the lipid bilayer of cellular compartments including endosomes and the plasma membrane. Although γ-secretase is of crucial interest for AD drug discovery, its atomic structure remains unresolved. γ-Secretase assembly and maturation is a multistep process, which includes extensive glycosylation on nicastrin (NCT), the only γ-secretase subunit having a large extracellular domain. These posttranslational modifications lead to protein heterogeneity that likely prevents the three-dimensional (3D) crystallization of the protease complex. To overcome this issue, we have engineered a Chinese hamster ovary (CHO) cell line deficient in complex sugar modifications (CHO lec1) to overexpress the four subunits of γ-secretase as a functional complex. We purified glycosylation-deficient γ-secretase from this recombinant cell line (CL1-9) and fully glycosylated γ-secretase from a recombinant CHO DG44-derived cell line (SS20). We characterized both complexes biochemically and pharmacologically in vitro. Interestingly, we found that the complex oligosaccharides, which largely decorate the extracellular domain of fully glycosylated NCT, are not involved in the proper assembly and maturation of the complex, and are dispensable for the specific generation, in physiological ratios, of the amyloid precursor protein (APP) cleavage products. In conclusion, we propose a novel bioengineering approach for the production of functional glycosylation-deficient γ-secretase, which may be suitable for crystallization studies. We expect that these findings will contribute both to solving the high-resolution 3D structure of γ-secretase and to structure-based drug discovery for AD.

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Crystal structure of the γ-secretase component nicastrin

Cited by 65 publications

References 42 publications

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

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

Cleavage of amyloid precursor protein by an archaeal presenilin homologue PSH

Production of active glycosylation‐deficient γ‐secretase complex for crystallization studies

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