Dimeric Transmembrane Orientations of APP/C99 Regulate γ-Secretase Processing Line Impacting Signaling and Oligomerization

Abstract

Summary Amyloid precursor protein (APP) cleavage by the β-secretase produces the C99 transmembrane (TM) protein, which contains three dimerization-inducing Gly-x-x-x-Gly motifs. We demonstrate that dimeric C99 TM orientations regulate the precise cleavage lines by γ-secretase. Of all possible dimeric orientations imposed by a coiled-coil to the C99 TM domain, the dimer containing the 33 Gly-x-x-x-Gly 37 motif in the interface promoted the Aβ 42 processing line and APP intracellular domain-dependent gene transcription, including the induction of BACE1 mRNA, enhancing amyloidogenic processing and signaling. Another orientation exhibiting the 25 Gly-x-x-x-Gly 29 motif in the interface favored processing to Aβ 43/40 . It induced significantly less gene transcription, while promoting formation of SDS-resistant “Aβ-like” oligomers, reminiscent of Aβ peptide oligomers. These required both Val24 of a pro-β motif and the 25 Gly-x-x-x-Gly 29 interface. Thus, crossing angles imposed by precise dimeric orientations control γ-secretase initial cleavage at Aβ 48 or Aβ 49, linking the former to enhanced signaling and Aβ 42 production.

Mechanism of Cellular Formation and In Vivo Seeding Effects of Hexameric β-Amyloid Assemblies

“…Importantly, extracellular monomeric Aβ was found exclusively in the soluble proteins fraction while hexameric Aβ was confined exclusively in vesicles (Fig. 4c), in agreement with the recent observations on "Aβ-like" oligomeric species [44]. To note, EVs size distribution was similar between all conditions but the number of EVs was higher in PS1-KD and PS2-KD as compared to respective controls (Fig.…”

Structural Determinant of β-Amyloid Formation: From Transmembrane Protein Dimerization to β-Amyloid Aggregates

“…Using a system in which dimerization is imposed from the intracellular domain, Eggert and colleagues observed that controlled dimerization resulted in decreased Aβ generation [105]. But more recently, the acquisition of the cryo-EM structure of 𝛾-secretase in complex with C83 hinted that processing of C-terminal fragments (CTF) could indeed not occur in their dimeric form [106], in line with our own observations using a cell-free 𝛾-secretase processing assay [96]. A first answer to these apparently contradicting observations was provided by our finding that GxxxG motifs were critical in regulating the orientations of TM dimerization and that a specific interface was required for amyloidogenic processing [90].…”

“…A first answer to these apparently contradicting observations was provided by our finding that GxxxG motifs were critical in regulating the orientations of TM dimerization and that a specific interface was required for amyloidogenic processing [90]. Using a system of fusion proteins that force dimerization of TM helices in all possible orientations, we demonstrated that precise dimeric orientations of C99 controlled 𝛾-secretase processing by regulating the initial 𝜀cleavage and therefore influencing AICD-dependent signaling and generation of Aβ42 [96]. Noteworthy, the dimeric orientation resulting in enhanced Aβ42 production and AICD-dependent signaling contained GxxxG motifs in the interface, but unfolding is mandatory for γ-processing since covalently bound APP CTFs are not further processed [96].…”

“…Using a model of forced dimerization of full-length APP, Eggert and collaborators analyzed in more detail the role of APP dimerization on subcellular localization and observed that APP dimerization resulted in increased localization in the endoplasmic reticulum (ER) and in endosomes, where the generation of pathogenic Aβ is believed to occur, by modulating its interaction with LRP1 and SorLA [107] (reviewed in [108]). The role of dimerization in intracellular trafficking was extended by Perrin and colleagues who observed that different dimeric orientations of C99 resulted in distinct subcellular localization in primary neurons and that the Aβ42 processing line was favored by a dimeric conformation that resulted in decreased cell surface and increased intracellular localization [96].…”

Specific Mutations in the Cholesterol-Binding Site of APP Alter Its Processing and Favor the Production of Shorter, Less Toxic Aβ Peptides

“…To clarify how the CBS regulates APP processing, we produced seven single mutants in the APP 751 protein either in the juxtamembrane region at aa positions 22, 26, and 28 of the Aβ sequence (positions 674, 678, and 680 of APP 751 ) that exhibited the greatest chemical shift perturbation in response to cholesterol in NMR studies [ 23 ] or in the TMD at aa positions 29, 33, and 39 (positions 681, 685 and 691 of APP 751 ) involved in APP dimerization and orientation [ 26 ]. In addition, two double mutants, 26/28 and 29/33, were constructed.…”