The Impact of the ‘Austrian’ Mutation of the Amyloid Precursor Protein Transmembrane Helix is Communicated to the Hinge Region

Stelzer, Walter; Scharnagl, Christina; Leurs, Ulrike; Rand, Kasper D.; Langosch, Dieter

doi:10.1002/slct.201601090

Cited by 12 publications

(36 citation statements)

References 28 publications

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“…Furthermore, a detailed recent analysis showed that helix-instability of the C99 TMD caused by the introduction of helix-destabilizing di-glycine motifs in the domain near the ε-sites enhance the initial cleavage and subsequent carboxy-terminal trimming (38). Consistent with this view, biophysical studies indicate that substrate backbone dynamics could play a role in substrate selection (39). In particular the di-glycine hinge region in the C99 TMD (residues G37 and G38; Aβ numbering, see Figure 1A) has been suggested to provide the necessary flexibility for the interaction with the enzyme (25,(40)(41)(42)(43).…”

Section: Instroductionmentioning

confidence: 85%

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Modulating hinge flexibility in the APP transmembrane domain alters γ-secretase cleavage

Götz¹,

Mylonas²,

Högel³

et al. 2018

Preprint

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Intramembrane cleavage of the β-amyloid precursor protein C99 substrate by γsecretase is implicated in Alzheimer´s disease pathogenesis. Since conformational flexibility of a di-glycine hinge in the C99 transmembrane domain (TMD) might be critical for γ-secretase cleavage, we mutated one of the glycine residues, G38, to a helixstabilizing leucine and to a helix-distorting proline. CD, NMR and hydrogen/deuterium exchange measurements as well as MD simulations showed that the mutations distinctly altered the intrinsic structural and dynamical properties of the TMD. However, although helix destabilization/unfolding was not observed at the initial ε-cleavage sites of C99, both mutants impaired γ-secretase cleavage and altered its cleavage specificity. Moreover, helix flexibility enabled by the di-glycine hinge translated to motions of other helix parts. Our data suggest that both local helix stabilization and destabilization in the di-glycine hinge may decrease the occurrence of enzyme-substrate complex conformations required for normal catalysis and that hinge mobility can be conducive for productive substrate-enzyme interactions.

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Section: Instroductionmentioning

confidence: 85%

“…We note that base-catalyzed exchange is responsible for at least 95 % of total deuteron exchange at pH 4.0 and above. The resulting ETD c and z fragment spectra were evaluated using a semi-automated procedure (MassMap_2017-11-16_LDK Software, MassMap GmbH & Co. KG, Wolfratshausen)(39).…”

mentioning

confidence: 99%

Modulating hinge flexibility in the APP transmembrane domain alters γ-secretase cleavage

Götz¹,

Mylonas²,

Högel³

et al. 2018

Preprint

Self Cite

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“…For example, TM domain mutations in the amyloid precursor protein, a substrate of g-secretase, can lead to Alzheimer's disease and have an influence on processing. Such mutations have been proposed to change the global flexibility of the TM helix and thus to affect its positioning within the enzyme (Langosch et al, 2015;Stelzer et al, 2016). In addition, introducing glycines near the site where g-secretase performs the first endoproteolytic cut, referred to as e-site, facilitates cleavage (Fernandez et al, 2016) and solid-state NMR analysis revealed a helix-to-coil transition near the e-site (Sato et al, 2009).…”

Section: Spp-catalyzed Cleavage Is Governed By Conformational Controlmentioning

confidence: 99%

“…An incubation time of 0.1 min at pH 5 was simulated by incubation for 1 min at pH 4 and an incubation time of 30 days at pH 5 was simulated by incubating for 22 hour at pH 6.5. ETD was conducted as described (Stelzer et al, 2016) at a flow rate of 3 µl/min using a cooled syringe. ETD was started by decreasing the wave height from 1.5 V to 0.2 V. Hydrogen scrambling was < 5-10%, as determined by the ammonia loss method (Rand et al, 2012).…”

Section: Mass Spectrometry and Dhx Experimentsmentioning

confidence: 99%

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The metastable XBP1u transmembrane domain defines determinants for intramembrane proteolysis by signal peptide peptidase

Yücel

Stelzer

Lorenzoni

et al. 2018

Preprint

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The unspliced XBP1 mRNA encodes the transcriptionally inert variant of the unfolded protein response (UPR) transcription factor XBP1u. Besides acting as a negative regulator for UPR, XBP1u targets its mRNA-ribosome-nascent-chain-complex to the endoplasmic reticulum (ER) facilitating IRE1-mediated splicing. Yet, its membrane association is controversial. Here, we use cell-free translocation assays and living cells to define a moderately hydrophobic stretch in XBP1u that is sufficient for insertion into the ER membrane. Mutagenesis of this transmembrane (TM) region reveals residues that determine XBP1u for an ER-associated degradation route centered around the signal peptide peptidase (SPP). Furthermore, the impact of these mutations on TM helix dynamics was assessed by recording residue-specific amide exchange kinetics, where data was evaluated by a novel semi-automated method. As compared to the previously employed procedure, this method reduces the time needed for data processing by approximately an order of magnitude. Based on our results, we suggest that SPP-catalyzed intramembrane proteolysis of TM helices is not only determined by its conformational flexibility, but also by side chain interactions near the cleavage site with the enzyme's active site.All rights reserved. No reuse allowed without permission.

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Using mass spectrometry‐based methods to understand amyloid formation and inhibition of alpha‐synuclein and amyloid beta

Wagner

Gross

2022

Mass Spectrometry Reviews

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Amyloid fibrils, insoluble β‐sheets structures that arise from protein misfolding, are associated with several neurodegenerative disorders. Many small molecules have been investigated to prevent amyloid fibrils from forming; however, there are currently no therapeutics to combat these diseases. Mass spectrometry (MS) is proving to be effective for studying the high order structure (HOS) of aggregating proteins and for determining structural changes accompanying protein–inhibitor interactions. When combined with native MS (nMS), gas‐phase ion mobility, protein footprinting, and chemical cross‐linking, MS can afford regional and sometimes amino acid spatial resolution of the aggregating protein. The spatial resolution is greater than typical low‐resolution spectroscopic, calorimetric, and the traditional ThT fluorescence methods used in amyloid research today. High‐resolution approaches can struggle when investigating protein aggregation, as the proteins exist as complex oligomeric mixtures of many sizes and several conformations or polymorphs. Thus, MS is positioned to complement both high‐ and low‐resolution approaches to studying amyloid fibril formation and protein–inhibitor interactions. This review covers basics in MS paired with ion mobility, continuous hydrogen‐deuterium exchange (continuous HDX), pulsed hydrogen‐deuterium exchange (pulsed HDX), fast photochemical oxidation of proteins (FPOP) and other irreversible labeling methods, and chemical cross‐linking. We then review the applications of these approaches to studying amyloid‐prone proteins with a focus on amyloid beta and alpha‐synuclein. Another focus is the determination of protein–inhibitor interactions. The expectation is that MS will bring new insights to amyloid formation and thereby play an important role to prevent their formation.

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The Impact of the ‘Austrian’ Mutation of the Amyloid Precursor Protein Transmembrane Helix is Communicated to the Hinge Region

Cited by 12 publications

References 28 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

The metastable XBP1u transmembrane domain defines determinants for intramembrane proteolysis by signal peptide peptidase

Using mass spectrometry‐based methods to understand amyloid formation and inhibition of alpha‐synuclein and amyloid beta

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