Dataset Information

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

ABSTRACT: Intramembrane cleavage of the ?-amyloid precursor protein C99 substrate by ?-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 ?-secretase cleavage, we mutated one of the glycine residues, G38, to a helix-stabilizing leucine and to a helix-distorting proline. Both mutants impaired ?-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 ?-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.

SUBMITTER: Gotz A

PROVIDER: S-EPMC6554489 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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

Götz Alexander A Mylonas Nadine N Högel Philipp P Silber Mara M Heinel Hannes H Menig Simon S Vogel Alexander A Feyrer Hannes H Huster Daniel D Luy Burkhard B Langosch Dieter D Scharnagl Christina C Muhle-Goll Claudia C Kamp Frits F Steiner Harald H

Biophysical journal 20190503 11

Intramembrane cleavage of the β-amyloid precursor protein C99 substrate by γ-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 γ-secretase cleavage, we mutated one of the glycine residues, G38, to a helix-stabilizing leucine and to a helix-distorting proline. Bo ...[more]

PMID: 31130234

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Project description:Alzheimer's disease (AD) is characterized by an accumulation of Amyloid-β (Aβ), released by sequential proteolytic processing of the amyloid precursor protein (APP) by β - and γ-secretase. Aβ peptides can aggregate, leading to toxic Aβ oligomers and amyloid plaque formation. Aβ accumulation is not only dependent on de novo synthesis but also on Aβ degradation. Neprilysin (NEP) is one of the major enzymes involved in Aβ degradation. Here we investigate the molecular mechanism of NEP regulation, which is up to now controversially discussed to be affected by APP processing itself. We found that NEP expression is highly dependent on the APP intracellular domain (AICD), released by APP processing. Mouse embryonic fibroblasts devoid of APP processing, either by the lack of the catalytically active subunit of the γ-secretase complex [presenilin (PS) 1/2] or by the lack of APP and the APP-like protein 2 (APLP2), showed a decreased NEP expression, activity and protein level. Similar results were obtained by utilizing cells lacking a functional AICD domain (APPΔCT15) or expressing mutations in the genes encoding for PS1. AICD supplementation or retransfection with an AICD encoding plasmid could rescue the down-regulation of NEP further strengthening the link between AICD and transcriptional NEP regulation, in which Fe65 acts as an important adaptor protein. Especially AICD generated by the amyloidogenic pathway seems to be more involved in the regulation of NEP expression. In line, analysis of NEP gene expression in vivo in six transgenic AD mouse models (APP and APLP2 single knock-outs, APP/APLP2 double knock-out, APP-swedish, APP-swedish/PS1Δexon9, and APPΔCT15) confirmed the results obtained in cell culture. In summary, in the present study we clearly demonstrate an AICD-dependent regulation of the Aβ-degrading enzyme NEP in vitro and in vivo and elucidate the underlying mechanisms that might be beneficial to develop new therapeutic strategies for the treatment of AD.

Project description:The amyloid ?-peptide (A?) of Alzheimer's disease (AD) is generated by proteolysis within the transmembrane domain (TMD) of a C-terminal fragment of the amyloid ? protein-precursor (APP CTF?) by the ?-secretase complex. This processing produces A? ranging from 38 to 49 residues in length. Evidence suggests that this spectrum of A? peptides is the result of successive ?-secretase cleavages, with endoproteolysis first occurring at the ? sites to generate A?48 or A?49, followed by C-terminal trimming mostly every three residues along two product lines to generate shorter, secreted forms of A?: the primary A?49-46-43-40 line and a minor A?48-45-42-38 line. The major secreted A? species are A?40 and A?42, and an increased proportion of the longer, aggregation-prone A?42 compared to A?40 is widely thought to be important in AD pathogenesis. We examined TMD substrate determinants of the specificity and efficiency of ? site endoproteolysis and carboxypeptidase trimming of CTF? by ?-secretase. We determined that the C-terminal negative charge of the intermediate A?49 does not play a role in its trimming by ?-secretase. Peptidomimetic probes suggest that ?-secretase has S1', S2', and S3' pockets, through which trimming by tripeptides may be determined. However, deletion of residues around the ? sites demonstrates that a depth of three residues within the TMD is not a determinant of the location of endoproteolytic ? cleavage of CTF?. We also show that instability of the CTF? TMD helix near the ? site significantly increases endoproteolysis, and that helical instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by ?-secretase. In addition, we found that CTF? dimers are not endoproteolyzed by ?-secretase. These results support a model in which initial interaction of the array of residues along the undimerized single helical TMD of substrates dictates the site of initial ? cleavage and that helix unwinding is essential for both endoproteolysis and carboxypeptidase trimming.

Project description:BACKGROUND:γ-Secretase is a multiprotein protease that cleaves amyloid protein precursor (APP) and other type I transmembrane proteins. It has two catalytic subunits, presenilins 1 and 2 (PS1 and 2). In our previous report, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates and slightly different potencies of PS1 versus PS2 inhibition for select γ-secretase inhibitors (GSIs) on various substrates. In this study, we investigated whether γ-secretase modulators (GSMs) and inverse γ-secretase modulators (iGSMs) modulate γ-secretase processivity using multiple different substrates. We next used HEK 293T cell lines in which PSEN1 or PSEN2 was selectively knocked out to investigate processivity and response to GSMs and iGSMs. METHODS:For cell-free γ-secretase cleavage assay, recombinant substrates were incubated with CHAPSO-solubilized CHO or HEK 293T cell membrane with GSMs or iGSMs in suitable buffer. For cell-based assay, cDNA encoding substrates were transfected into HEK 293T cells. Cells were then treated with GSMs or iGSMs, and conditioned media were collected. Aβ and Aβ-like peptide production from cell-free and cell-based assay were measured by ELISA and mass spectrometry. RESULT:These studies demonstrated that GSMs are highly selective for effects on APP, whereas iGSMs have a more promiscuous effect on many substrates. Surprisingly, iGSMs actually appear to act as like GSIs on select substrates. The data with PSEN1 or PSEN2 knocked out HEK 293T reveal that PS1 has higher processivity and response to GSMs than PS2, but PS2 has higher response to iGSM. CONCLUSION:Collectively, these data indicate that GSMs are likely to have limited target-based toxicity. In addition, they show that iGSMs may act as substrate-selective GSIs providing a potential new route to identify leads for substrate-selective inhibitors of certain γ-secretase-mediated signaling events. With growing concerns that long-term β-secretase inhibitor is limited by target-based toxicities, such data supports continued development of GSMs as AD prophylactics.

Project description:APP, whose mutations cause familial Alzheimer's disease (FAD), modulates neurotransmission via interaction of its cytoplasmic tail with the synaptic release machinery. Here we identified an intravesicular domain of APP, called intraluminal SV-APP interacting domain (ISVAID), which interacts with glutamatergic, but not GABAergic, synaptic vesicle proteins. ISVAID contains the ?- and ?-secretase cleavage sites of APP: proteomic analysis of the interactome of ISVAID suggests that ?- and ?-secretase cleavage of APP cuts inside the interaction domain of ISVAID and destabilizes protein-protein interactions. We have tested the functional significance of the ISVAID and of ?-/?-secretase-processing of APP using various ISVAID-derived peptides in competition experiments on both female and male mouse and rats hippocampal slices. A peptide encompassing the entire ISVAID facilitated glutamate, but not GABA, release acting as dominant negative inhibitor of the functions of this APP domain in acute hippocampal slices. In contrast, peptides representing the product of ?-/?-secretase-processing of ISVAID did not alter excitatory neurotransmitter release. These findings suggest that cleavage of APP by either ?- or ?-secretase may inactivate the ISVAID, thereby enhancing glutamate release. Our present data support the notion that APP tunes glutamate release, likely through intravesicular and extravesicular interactions with synaptic vesicle proteins and the neurotransmitter release machinery, and that ?-/? cleavage of APP facilitates the release of excitatory neurotransmitter.SIGNIFICANCE STATEMENT Alzheimer's disease has been linked to mutations in APP. However, the biological function of APP is poorly understood. Here we show that an intravesicular APP domain interacts with the proteins that control the release of glutamate, but not GABA. Interfering with the function of this domain promotes glutamate release. This APP domain contains the sites cleaved by ?- and ?-secretases: our data suggest that ?-/? cleavage of APP inactivates this functional APP domain promoting excitatory neurotransmitter release.

Dataset Information

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

Publications

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

Similar Datasets

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