Increased H-Bond Stability Relates to Altered ε-Cleavage Efficiency and Aβ Levels in the I45T Familial Alzheimer’s Disease Mutant of APP

Götz, Alexander; Högel, Philipp; Silber, Mara; Chaitoglou, Iro; Luy, Burkhard; Muhle‐Goll, Claudia; Scharnagl, Christina; Langosch, Dieter

doi:10.1101/372698

Cited by 4 publications

(9 citation statements)

References 78 publications

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“…The C99 26-55 WT, C99 26-55 G38L, and C99 26-55 G38P model peptide (for sequences, see Table 1) were investigated in a fully hydrated POPC bilayer and in a mixture of 80% TFE with 20% TIP3 water (v/v). Because no experimental structures were available for the G38 mutants, we used a stochastic sampling protocol to generate a set of 78 initial start conformations (for details, see (75)).…”

Section: Simulationsmentioning

confidence: 99%

“…All-atom simulations in 80% TFE and 20% TIP3 (v/v) were set up as described previously (75). Each start conformation was simulated for 200 ns using settings as described in (46).…”

Section: Simulationsmentioning

confidence: 99%

“…To obtain insight into local amide H-bond strength, we next measured residue-specific amide DHX rate constants (k exp,DHX ) using ETD of our TMD peptides in the gas phase after various periods of exchange (MS-ETD-DHX) (47,72). To enhance fragmentation efficiency, we substituted the N-terminal SNK sequence of the C99 26-55 TMD by KKK (75). The rate constants shown in Fig.…”

Section: G38 Mutant Tmd Helices Display Altered Hydrogen-bond Stability Around the G 37 G 38 Hingementioning

confidence: 99%

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Modulating Hinge Flexibility in the APP Transmembrane Domain Alters γ-Secretase Cleavage

Götz

Mylonas

Högel

et al. 2019

Biophysical Journal

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Intramembrane cleavage of the b-amyloid precursor protein C99 substrate by g-secretase is implicated in Alzheimer's disease pathogenesis. Biophysical data have suggested that the N-terminal part of the C99 transmembrane domain (TMD) is separated from the C-terminal cleavage domain by a di-glycine hinge. Because the flexibility of this hinge might be critical for g-secretase cleavage, we mutated one of the glycine residues, G38, to a helix-stabilizing leucine and to a helix-distorting proline. Both mutants impaired g-secretase cleavage and also altered its cleavage specificity. Circular dichroism, NMR, and backbone amide hydrogen/deuterium exchange measurements as well as molecular dynamics simulations showed that the mutations distinctly altered the intrinsic structural and dynamical properties of the substrate TMD. Although helix destabilization and/or unfolding was not observed at the initial ε-cleavage sites of C99, subtle changes in hinge flexibility were identified that substantially affected helix bending and twisting motions in the entire TMD. These resulted in altered orientation of the distal cleavage domain relative to the N-terminal TMD part. Our data suggest that both enhancing and reducing local helix flexibility of the di-glycine hinge may decrease the occurrence of enzyme-substrate complex conformations required for normal catalysis and that hinge mobility can thus be conducive for productive substrate-enzyme interactions.

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Section: Simulationsmentioning

confidence: 99%

“…All-atom simulations in 80% TFE and 20% TIP3 (v/v) were set up as described previously (75). Each start conformation was simulated for 200 ns using settings as described in (46).…”

Section: Simulationsmentioning

confidence: 99%

Section: G38 Mutant Tmd Helices Display Altered Hydrogen-bond Stability Around the G 37 G 38 Hingementioning

confidence: 99%

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Modulating Hinge Flexibility in the APP Transmembrane Domain Alters γ-Secretase Cleavage

Götz

Mylonas

Högel

et al. 2019

Biophysical Journal

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“…However, artificial structural constraints were included that could affect the enzyme-substrate interactions. Molecular dynamics (MD) simulations have proven useful in understanding the structural dynamics of γ-secretase, notably the enzyme-substrate interactions (13)(14)(15)(16)(17). Recently, we computationally restored the wildtype (WT) enzyme-substrate co-structure and applied all-atom simulations using the Gaussian accelerated molecular dynamics (GaMD) method to build the first dynamic model of γ-secretase activation (18).…”

Section: Introductionmentioning

confidence: 99%

Mechanism of Tripeptide Trimming by γ-Secretase

Bhattarai

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.

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“…Indeed, mutations that affect transmembrane domain flexibility in the cleavage site region of APP can markedly alter γ‐secretase cleavage (Fernandez et al , ). Likewise, flexibility via a naturally occurring hinge in the TMH as in the case of APP can also be critical for γ‐secretase cleavage (Götz et al , ). Recent structural studies of γ‐secretase in complex with APP or Notch1 substrates suggest that unfolding of the cleavage region is caused by formation of a hybrid β‐strand between substrate and enzyme (Yang et al , ; Zhou et al , ).…”

Section: Introductionmentioning

confidence: 99%

γ‐Secretase cleavage of the Alzheimer risk factor TREM 2 is determined by its intrinsic structural dynamics

et al. 2020

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Sequence variants of the microglial expressed TREM2 (triggering receptor expressed on myeloid cells 2) are a major risk factor for late onset Alzheimer's disease. TREM2 requires a stable interaction with DAP12 in the membrane to initiate signaling, which is terminated by TREM2 ectodomain shedding and subsequent intramembrane cleavage by c-secretase. To understand the structural basis for the specificity of the intramembrane cleavage event, we determined the solution structure of the TREM2 transmembrane helix (TMH). Caused by the presence of a charged amino acid in the membrane region, the TREM2-TMH adopts a kinked structure with increased flexibility. Charge removal leads to TMH stabilization and reduced dynamics, similar to its structure in complex with DAP12. Strikingly, these dynamical features match with the site of the initial c-secretase cleavage event. These data suggest an unprecedented cleavage mechanism by c-secretase where flexible TMH regions act as key determinants of substrate cleavage specificity.

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Increased H-Bond Stability Relates to Altered ε-Cleavage Efficiency and Aβ Levels in the I45T Familial Alzheimer’s Disease Mutant of APP

Cited by 4 publications

References 78 publications

Modulating Hinge Flexibility in the APP Transmembrane Domain Alters γ-Secretase Cleavage

Modulating Hinge Flexibility in the APP Transmembrane Domain Alters γ-Secretase Cleavage

Mechanism of Tripeptide Trimming by γ-Secretase

γ‐Secretase cleavage of the Alzheimer risk factor TREM 2 is determined by its intrinsic structural dynamics

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