Familial Alzheimer’s disease mutations in amyloid protein precursor alter proteolysis by γ-secretase to increase amyloid β-peptides of ≥45 residues

Devkota

Nguyen

et al. 2021

Preprint

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The membrane-embedded γ-secretase complex processively cleaves within the transmembrane domain of amyloid precursor protein (APP) to produce 37-to-43-residue amyloid β-peptides (Aβ) of Alzheimer’s disease (AD). Despite its importance in pathogenesis, the mechanism of processive proteolysis by γ-secretase remains poorly understood. Here, mass spectrometry and western blotting were used to quantify the efficiency of the first tripeptide trimming step (Aβ49→Aβ46) of wildtype (WT) and familial AD (FAD) mutant APP substrate. In comparison to WT APP, the efficiency of this first trimming step was slightly higher for the I45F, A42T and V46F APP FAD mutants, but substantially diminished for the I45T and T48P mutants. In parallel with biochemical experiments, all-atom simulations using a novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method were applied to investigate tripeptide trimming of Aβ49 by γ-secretase. The starting structure was active γ-secretase bound to Aβ49 and APP intracellular domain (AICD), as generated from our previous study that captured activation of γ-secretase for the initial endoproteolytic cleavage of APP (Bhattarai et al., ACS Cent Sci, 2020, 6:969-983). Pep-GaMD simulations captured remarkable structural rearrangements of both the enzyme and substrate, in which hydrogen-bonded catalytic aspartates and water became poised for tripeptide trimming of Aβ49 to Aβ46. These structural changes required a positively charged N-terminus of endoproteolytic coproduct AICD, which could dissociate during conformational rearrangements of the protease and Aβ49. The simulation findings were highly consistent with biochemical experimental data. Taken together, our complementary biochemical experiments and Pep-GaMD simulations have enabled elucidation of the mechanism of tripeptide trimming by γ-secretase.Significance statementProduction of amyloid β-peptide (Aβ) of Alzheimer’s disease (AD) from its precursor protein requires a series of proteolytic events carried out by the membrane-embedded γ-secretase complex. Mutations in the substrate and enzyme that produce Aβ cause hereditary AD and these mutations affect tripeptide trimming of initially formed long Aβ peptides by γ-secretase. Little is known about the structural mechanism of this trimming process. Here we have developed a molecular dynamics model for the first step of trimming (Aβ49 to Aβ46) by γ-secretase. Conformational changes in the enzyme-substrate complex to set up this trimming step require the presence of N-terminally charged endoproteolytic cleavage co-product. Computational effects of AD-causing mutations in Aβ49 on the trimming process were validated through biochemical experiments.

Section: Introductionsupporting

confidence: 53%

Section: Discussionmentioning

confidence: 99%

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Mechanism of Tripeptide Trimming by γ-Secretase

Devkota

Nguyen

et al. 2021

Preprint

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“…In particular, the new structures should reveal mechanisms of processive proteolysis of long Aß peptides to secreted shorter forms by γ-secretase; this trimming process is deficient with familial Alzheimer's disease (FAD) mutations found in APP and presenilins. [33][34][35] Understanding of specific structural and functional changes caused by FAD mutations may suggest strategies to identify small drug-like molecules that bind allosterically and restore normal function, as both chemical tools and therapeutic prototypes.…”

Section: Figure 2 Design Of Substrate Transmembrane Mimetic Inhibitors Of γ-Secretase Substrate A)mentioning

confidence: 99%

Design of Transmembrane Mimetic Structural Probes to Trap Different Stages of γ-Secretase-Substrate Interaction

Devkota

Wolfe

2021

Preprint

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ABSTRACTγ-Secretase, a membrane-embedded aspartyl protease complex with presenilin as the catalytic component, cleaves within the transmembrane domain (TMD) of its many substrates, which include the amyloid precursor protein (APP) of Alzheimer’s disease (AD). APP TMD is processively cut by γ-secretase through endoproteolysis followed by tricarboxypeptidase “trimming”, the latter function being deficient with mutations causing hereditary AD. Toward better understanding of substrate recognition and hydrolytic reactions catalyzed by this enzyme within its hydrophobic milieu, we recently developed a prototype substrate TMD mimetic as a chemical tool for structural analysis (Bhattarai S et al. J. Am. Chem. Soc., 2020, 142(7): 3351-3355). This TMD mimetic—composed of a helical peptide inhibitor (HPI) connected through a linker to a transition-state analog inhibitor (TSA)— simultaneously engages the substrate docking exosite and the active site and is pre-organized to trap the protease transition state of carboxypeptidase trimming. In this study, we developed variants of this prototype designed to allow visualization of transition states for endoproteolysis, TMD helix unwinding, and lateral gating of substrate. New HPI-TSA conjugates were synthesized using an earlier established divergent strategy, which involved the construction of a tripeptidomimetic building block followed by solid-phase peptide synthesis (SPPS). For each class of structural probe, SAR analysis led to discovery of highly potent prototypes with stoichiometric or low-nanomolar inhibition. These TMD mimetics exhibited non-competitive inhibition of γ-secretase activity and occupy both docking and active site as demonstrated by enzyme cross competition experiments and photoaffinity probe binding assays. The new probes should be important structural tools for trapping different stages of substrate recognition and processing via ongoing cryo-electron microscopy with γ-secretase, ultimately aiding rational drug design.Abstract Graphic

“…8,9 In addition, deficiencies in carboxypeptidase trimming can contribution to increased Aβ42/Aβ40 while also increasing levels of longer Aβ peptide intermediates. [10][11][12] Remarkably, the effects of GSMs on Aβ production appear to be limited to the final trimming step. Most reported GSMs lower Aβ42 levels and increase Aβ38 levels by stimulating the Aβ42→Aβ38 cleavage event.…”

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confidence: 99%

Discovery of aryl aminothiazole γ-secretase modulators with novel effects on amyloid β-peptide production

Liu

Wolfe

2021

Preprint

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A series of analogs based on a prototype aryl aminothiazole γ-secretase modulator (GSM) were synthesized and tested for their effects on the profile of 37-to-42-residue amyloid β-peptides (Aβ) generated through processive proteolysis of precursor protein substrate by γ-secretase. Certain substitutions on the terminal aryl D ring resulted in an altered profile of Aβ production compared to that seen with the parent molecule. Small structural changes led to concentration-dependent increases in Aβ37 and Aβ38 production without parallel decreases in their precursors Aβ40 and Aβ42, respectively. The new compounds therefore apparently also stimulate carboxypeptidase trimming of Aβ peptides > 43 residues, providing novel chemical tools for mechanistic studies of processive proteolysis by γ-secretase.