Substrate ectodomain is critical for substrate preference and inhibition of γ-secretase

저자: Funamoto S, Sasaki T, Ishihara S, Nobuhara M, Nakano M, Watanabe-Takahashi M, Saito T, Kakuda N, Miyasaka T, Nishikawa K, Saido TC, Ihara Y.

저널: Nat Commun. 2013;4:2529

사용한 제품: Dynabeads M-280 Streptavidin (Cat# 11205D, 11206D, 60210)

Abstract

Understanding the substrate recognition mechanism of γ-secretase is a key step for establishing substrate-specific inhibition of amyloid β-protein (Aβ) production. However, it is widely believed that γ-secretase is a promiscuous protease and that its substrate-specific inhibition is elusive. Here we show that γ-secretase distinguishes the ectodomain length of substrates and preferentially captures and cleaves substrates containing a short ectodomain. We also show that a subset of peptides containing the CDCYCxxxxCxCxSC motif binds to the amino terminus of C99 and inhibits Aβ production in a substrate-specific manner. Interestingly, these peptides suppress β-secretase-dependent cleavage of APP, but not that of sialyltransferase 1. Most importantly, intraperitoneal administration of peptides into mice results in a significant reduction in cerebral Aβ levels. This report provides direct evidence of the substrate preference of γ-secretase and its mechanism. Our results demonstrate that the ectodomain of C99 is a potent target for substrate-specific anti-Aβ therapeutics to combat Alzheimer's disease.

제품 사용법

Selection of binding peptides

A DNA library was constructed from synthetic oligonucleotides, which included ATG(NNT)20 or ATG(NNK)20 (N is A, C, G or T, and K is G or T) as a randomized peptide-encoding region. The NNT-type was added in the same amount as the NNK-type to increase the proportion of Cys and Tyr and to decrease the appearance of stop codons. The DNA library (100 pmol) was used for transcription and the resultant mRNA library was employed as a template for a covalently linked peptide or protein-displaying system38. Peptidedisplaying molecules were incubated with 2.6 mM of Ab1–28 immobilized at its biotinylated C terminus on Dynabeads M-280 Streptavidin (Invitrogen) for 1 h at ambient temperature. After washing the beads, the binding molecules were recovered by heating at 95 ℃ and subjected to PCR for preparation of templates for the next round. The concentration of binding molecules was repeated six times in this manner and was followed by three rounds with the fourfold-diluted bait. Recovery of templates increased with increasing number of selection rounds, and five clones after the 6th round and seven clones after the 9th round were sequenced. Two sequences sharing a CDCYCxxxxCxCxCxSC motif were semi-randomized by the secondary DNA library including ATG(NNT)4TGTGATTGTTATTGT (NNT)4TGTTHTTGTBATTHTTGT(NNT)3, where H is A/C/T and B is C/G/T. Selection from the secondary library was performed in the same way as done in the 7th round in the initial selection except that the incubation temperature was 37℃. After five rounds of selection and cloning, four derivative peptides were identified (Fig. 6a).