Targeting the APP-Mint2 Protein–Protein Interaction with a Peptide-Based Inhibitor Reduces Amyloid-β Formation

Abstract

There is an urgent need for novel therapeutic approaches to treat Alzheimer’s disease (AD) with the ability to both alleviate the clinical symptoms and halt the progression of the disease. AD is characterized by the accumulation of amyloid-β (Aβ) peptides which are generated through the sequential proteolytic cleavage of the amyloid precursor protein (APP). Previous studies reported that Mint2, a neuronal adaptor protein binding both APP and the γ-secretase complex, affects APP processing and formation of pathogenic Aβ. However, there have been contradicting results concerning whether Mint2 has a facilitative or suppressive effect on Aβ generation. Herein, we deciphered the APP-Mint2 protein–protein interaction (PPI) via extensive probing of both backbone H-bond and side-chain interactions. We also developed a proteolytically stable, high-affinity peptide targeting the APP-Mint2 interaction. We found that both an APP binding-deficient Mint2 variant and a cell-permeable PPI inhibitor significantly reduced Aβ42 levels in a neuronal in vitro model of AD. Together, these findings demonstrate a facilitative role of Mint2 in Aβ formation, and the combination of genetic and pharmacological approaches suggests that targeting Mint2 is a promising therapeutic strategy to reduce pathogenic Aβ levels.

Molecular Details of a Coupled Binding and Folding Reaction between the Amyloid Precursor Protein and a Folded Domain

“…Therefore, we generated a range of amide to ester (A-to-E) modified APP (G756γ, Y757ψ, E758ε, N759ν, and F765φ) peptides and PARM (Y459ψ, I460ι, A461α, and D462δ) domains ( Figure 1 C), and the latter was achieved by protein semisynthesis as described previously ( Tables S1 and S2 ). 22 …”

“… 30 , 31 A two-segment semisynthetic strategy was chosen in which the tyrosine phosphorylation (pY) was introduced synthetically in the ARM segment and the PTB domain was expressed as a thioester using an intein approach as previously described ( Figure 2 A). 22 We generated Δ N PTB-sodium 2-mercaptoethanesulfonate (MesNa) from thiolysis of expressed PTB 364–532 C-terminally fused to GyrA mini and synthesized ΔARM WT 533–560 and ΔARM Y543pY 533–560 using SPPS ( Tables S1 and S2 ). 32 With all fragments in hand, we ligated and refolded the WT semisynthetic (ss) PARM WT as a control and phosphorylated ssPARM Y543pY to generate the desired proteins with good purity ( Figure 2 B,C and Figure S8 ).…”

“…The semisynthetic proteins were characterized by RP-UPLC at 214 nm, 16% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and LC-MS. PARM A-to-E variants were generated as described previously and characterized by LC-MS and RP-UPLC at 214 nm ( Figure 2 and Table S2 ). 22 …”

“… 18 , 19 The APP–Mint interaction modulates the amyloidogenic processing pathway of APP, where β- and γ-secretase proteolytically cleave APP to form amyloid-β peptides leading to extracellular plaques characteristic of Alzheimer’s disease. 20 − 22 It has been shown that the APP–Mint interaction is regulated by an open–closed conformation of a self-inhibitory α-helical linker (ARM) in the C-terminus of the Mint PTB domain ( Figure 1 A). In the closed state, the ARM linker occupies the APP binding site by forming extensive intramolecular hydrophobic interactions with the PTB domain.…”

Rational Design of Peptide-Based Inhibitors Disrupting Protein-Protein Interactions

“…Peptidomimetics design is currently regarded as an eminently practical and promising approach for discovering peptide-based PPI inhibitors. So far, they have achieved fruitful success in taming series of critical but intractable PPI targets such as the MLL1-WDR5 (Karatas et al, 2013 ), APC-Asef (Jiang et al, 2017 ; He et al, 2021 ), DCN1-UBC12 (Zhou et al, 2018 ), and APP-Mint2 (Bartling et al, 2021 ). Some of them have even marched into clinical applications.…”