Odorčić I, Hamed MB, Lismont S, Chávez-Gutiérrez L, Efremov RG. Apo and Aβ46-bound γ-secretase structures provide insights into amyloid-β processing by the APH-1B isoform. Nat Commun. 2024 May 27;15(1):4479. PubMed.
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University of Kansas
This new study is another tour de force in structure elucidation of the γ-secretase complex from the labs of Rui Zhou in Beijing and Yigong Shi in Hangzhou. The snapshots of the protease bound to APP-derived C99, Aβ49, Aβ46, and Aβ43 reveal critical details that, taken together, provide important insight into the mechanism of processive proteolysis. The adjustment of the helical region of bound Aβ intermediates—into the hydrophobic presenilin cavity and toward the active site to set up tripeptide trimming—offers incontrovertible proof for the “piston” model of processivity (Yang et al., 2019). The alternative “unwinding” model (Yang et al., 2019; Szaruga et al., 2017), with a static helical region, is incompatible with the new set of cryoEM structures, which show movement of this region by roughly one turn of a helix with each processing step.
This mechanism is likely operative for the Aβ48→Aβ45→Aβ42→Aβ38 pathway of C99 processing, as well as for the many other substrates of γ-secretase (Güner and Lichtenthaler, 2020), such as Notch receptors. I would suggest, however, calling this a “ratchet” model, rather than a “piston” model, as the helical region moves in only one direction during processive proteolysis, into the hydrophobic cavity of presenilin, and not back and forth as a piston.
The study, of course, has its limitations in providing snapshots of bound Aβ intermediates. Most notably, it does not inform about how ratcheting takes place. Presenilin and the rest of the γ-secretase complex appear static from one Aβ-bound state to the next, but there must be conformational adjustments in the enzyme, to allow movement of the helical domain of Aβ intermediate and setting up of the next cleavage step. Conformational adjustments of the enzyme might involve fleeting dissociation from the substrate of presenilin loop 1 and widening of the presenilin hydrophobic channel and active site. Such dynamic processes are not captured by the new cryoEM structures.
This same limitation makes it difficult to extrapolate the effects of FAD mutations in APP and presenilin on γ-secretase processing based on the new structures. In this regard, the suggestion in the discussion that FAD mutations would be expected to destabilize enzyme-substrate complexes does not consider dynamics. Indeed, molecular dynamics simulations suggest reduced conformational flexibility—with increased stabilization—of FAD-mutant enzyme-substrate complexes, and this idea is supported experimentally by fluorescence lifetime imaging microscopy in intact cells (Devkota et al., 2024).
References:
Yang G, Zhou R, Zhou Q, Guo X, Yan C, Ke M, Lei J, Shi Y. Structural basis of Notch recognition by human γ-secretase. Nature. 2019 Jan;565(7738):192-197. Epub 2018 Dec 31 PubMed.
Szaruga M, Munteanu B, Lismont S, Veugelen S, Horré K, Mercken M, Saido TC, Ryan NS, De Vos T, Savvides SN, Gallardo R, Schymkowitz J, Rousseau F, Fox NC, Hopf C, De Strooper B, Chávez-Gutiérrez L. Alzheimer's-Causing Mutations Shift Aβ Length by Destabilizing γ-Secretase-Aβn Interactions. Cell. 2017 Jul 27;170(3):443-456.e14. PubMed. Correction.
Güner G, Lichtenthaler SF. The substrate repertoire of γ-secretase/presenilin. Semin Cell Dev Biol. 2020 Sep;105:27-42. Epub 2020 Jun 29 PubMed.
Devkota S, Zhou R, Nagarajan V, Maesako M, Do H, Noorani A, Overmeyer C, Bhattarai S, Douglas JT, Saraf A, Miao Y, Ackley BD, Shi Y, Wolfe MS. Familial Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes. Cell Rep. 2024 Feb 27;43(2):113761. Epub 2024 Feb 13 PubMed.
View all comments by Michael WolfeK.U.Leuven and V.I.B.
VIB-VUB Center for Structural Biology and VIB-KU Leuven Center for Brain & Disease Research
We read with great interest the report by Xuefei Guo et al. We just published structures of γ-secretase in an apo state as well as in complex with the intermediate Aβ46 substrate. Comparing these two independent studies has been particularly exciting.
The various structures reported by Guo et al. on the complexes between γ-secretase and both the initial and intermediate APP-derived substrates reveal a β-sheet structure between presenilin 1 (PSEN1) and both the initial and intermediate substrates (located C-terminal to the cleavage site) that is a common feature during the sequential proteolysis. This hybrid structure likely stabilizes the distinct enzyme-substrate (E-S) complexes prior to proteolysis.
Regarding the recognition of Aβ peptides, it has been very interesting to look at the structures with different Aβs, and to contrast the two structures with the intermediate Aβ46 peptide.
Both our and Guo et al.’s structures consistently show H-bonding intermolecular interactions involving the backbone of the substrate, Aβ, and PSEN1-Tyr115 (loop 1), Ser169, and Trp165. In addition, our data pointed to the PSEN1-Gly384 as another H-bonding interactor with the substrate. Our analysis further adds to the functional significance of the intermolecular H-bonding interactions by showing that removal of the H-bonding capability at the PSEN1 Tyr115 (loop 1), Ser169, and Trp165 positions results in impaired γ-secretase processivity, i.e., the generation of longer Aβ peptides from APPC99. Similarly, the G384A substitution promotes generation of longer Aβ peptides from APP by destabilizing enzyme-substrate (E-S) interactions (Szaruga et al., 2017).
Our study and Guo et al. highlight the importance of specific H-bonding interactions for the recognition and processing of Aβ. As discussed in both reports, the formation of these intermolecular enzyme-substrate H-bonds enables the recognition and stabilization of substrates presenting low sequence homology. Their disruption (through mutations), leading to enhanced production of longer Aβ peptides, likely explains the pathogenicity of early onset Alzheimer’s disease variants at these positions, as discussed by Guo et al. and ourselves.
The comparison also highlights differences in the E-S complexes. In our structure with Aβ46, the N-terminal part of the substrate is unstructured and rests on the PSEN1 loop 1, indicating that a close interaction with this loop reduces the flexibility of this substrate region and makes it detectable in cryo-EM. This interaction is absent in all structures reported by Guo et al., and its functional significance is unclear, but the presence of Alzheimer’s-disease-linked mutations in this loop support its involvement in efficient enzyme processivity.
Regarding the mechanisms of cleavage, the available structural and functional data seems to reach a consensus on the following: i) initial substrate cleaving and subsequent cuts are preceded by unwinding of the C-terminal part of the substrate helix and formation of a hybrid β-sheet that facilitates filling of key S’1-3 pockets (Bolduc et al., 2016); ii) in subsequent cuts, this is accompanied by a piston-like movement of the helical substrate towards the active site. The successive cuts thus result in the embedding of the polar ectodomain of the substrates (Lys28, Asn27, Asp23, etc. in APP) into the hydrophobic substrate channel, which limits the number of cuts (Koch et al., 2023).
That two independent scientific analyses highlight similar conclusions while also identifying differences is exciting and significant, as they offer valuable insights into the complexities and nuances of an enzyme that plays a major role in AD pathogenicity and represents a potential target for therapeutic intervention.
Figure 5 in our paper illustrates this:
Structural comparison of GSEC1B-Aβ46 with GSEC1A-APPC83 and experimental validation of potential hydrogen bonds between PSEN1 and APP. A) Structural alignment of GSEC1B-Aβ46 and GSEC1A-APPC83 (PDB: 6IYC; shown in gray) complexes. PSEN1 TMs are indicated with circled numbers. B) Close-up of extracellular side of the substrate and loop 1. The GSEC1A-APPC83 complex was stabilized by disulphide cross-link between V7C APPC83 (unresolved) and Q112C PSEN1. C) Closeup view on intracellular side of substrate binding site. D) Details of PSEN1-Aβ interactions in the trans-membrane region. Potential hydrogen bond interactions between the substrates and W165, S169 and G384 are indicated. E) Western blot analysis of solubilized membranes from Psen1-/-/Psen2-/- (dKO) mouse embryonic fibroblast cell lines rescued with WT or mutant PSEN1. NCTm and NCTi indicate mature glycosylated and immature NCT, respectively. Molecular weights of protein standards are indicated on the left. F) GSEC processivity of APPC99 in Psen1-/-Psen2-/- MEFs rescued with WT or mutated PSEN1. Data are presented as mean ± SD, n=6 for the WT and n=3 for the mutants. Multiple comparison ANOVA was used to determine statistical significance (P < 0.05); P(WT vs Y115A)<0.0001, P(WT vs Y115F) )<0.0001, P(WT vs W165F) )<0.0001, P(WT vs S169A) )=0.0001, P(Y115A vs Y115F)=0.0115. Source data are provided as a Source Data file.
References:
Bolduc DM, Montagna DR, Seghers MC, Wolfe MS, Selkoe DJ. The amyloid-beta forming tripeptide cleavage mechanism of γ-secretase. Elife. 2016 Aug 31;5 PubMed.
Koch M, Enzlein T, Chen SY, Petit D, Lismont S, Zacharias M, Hopf C, Chávez-Gutiérrez L. APP substrate ectodomain defines amyloid-β peptide length by restraining γ-secretase processivity and facilitating product release. EMBO J. 2023 Dec 1;42(23):e114372. Epub 2023 Oct 18 PubMed.
Szaruga M, Munteanu B, Lismont S, Veugelen S, Horré K, Mercken M, Saido TC, Ryan NS, De Vos T, Savvides SN, Gallardo R, Schymkowitz J, Rousseau F, Fox NC, Hopf C, De Strooper B, Chávez-Gutiérrez L. Alzheimer's-Causing Mutations Shift Aβ Length by Destabilizing γ-Secretase-Aβn Interactions. Cell. 2017 Jul 27;170(3):443-456.e14. PubMed. Correction.
View all comments by Ivica OdorčićUCL Queen Square Institute of Neurology
Institute of Neurology, University College London
Guo and colleagues advance our understanding of the biochemical processes behind γ-secretase-mediated cleavage of APP. Using cryo-EM of γ-secretase bound to APP-C99, Aβ49, Aβ46, and Aβ43, we are presented with the greatest insight yet of the sequential generation of Aβ species.
The work confirms the established tripeptide cleavage pathway pattern of Aβ49>46>43>40 at a molecular resolution (Matsumura et al., 2014; Takami et al., 2009). The stereology of the active site also greatly supports previous in vitro work using amino acid substitutions to unravel substrate loading into the active site (Fernandez et al., 2016; Lichtenthaler et al., 1999). Evidence suggests an unwinding of the α-helix within the active site by one turn, helping to explain the tripeptide cleavage phenomenon. The data strongly support the piston model of γ-secretase cleavage, rather than an unwinding model (Yang et al., 2019).
This detailed picture helps us understand the effect of familial AD mutations. In PSEN1, Guo et al. show that Y115, S169, and W165 are crucial residues that form hydrogen bonds with the substrate. Mutations around these three residues are some with the earliest ages at onset (e.g., Y115D AAO = 29, S169L AAO = 31, W165G AAO = 36, and L166P AAO = 15). Additionally, the PALP motif appears important for flexibility and progression to the next cleavage event. We have recently discussed the importance of this motif in reference to the novel P436S mutation (Arber et al., 2024). In APP, residues such as V717 are also crucial for progression to the subsequent cleavage site, supporting our previous finding of a roadblock-like effect in the Aβ49>46>43>40 pathway caused by APP V717I (Arber et al., 2020). It will be interesting to determine the relevance of pathway switching in fAD, as shown (Kakuda et al., 2021).
The complexity of Aβ generation is fascinating, and we are now able to visualize this biochemical process thanks to this elegant work.
AAO as presented in Liu et al., 2022.
References:
Arber C, Belder CR, Tomczuk F, Gabriele R, Buhidma Y, Farrell C, O'Connor A, Rice H, Lashley T, Fox NC, Ryan NS, Wray S. The presenilin 1 mutation P436S causes familial Alzheimer's disease with elevated Aβ43 and atypical clinical manifestations. Alzheimers Dement. 2024 Jul;20(7):4717-4726. Epub 2024 Jun 2 PubMed.
Arber C, Toombs J, Lovejoy C, Ryan NS, Paterson RW, Willumsen N, Gkanatsiou E, Portelius E, Blennow K, Heslegrave A, Schott JM, Hardy J, Lashley T, Fox NC, Zetterberg H, Wray S. Familial Alzheimer's disease patient-derived neurons reveal distinct mutation-specific effects on amyloid beta. Mol Psychiatry. 2020 Nov;25(11):2919-2931. Epub 2019 Apr 12 PubMed.
Fernandez MA, Biette KM, Dolios G, Seth D, Wang R, Wolfe MS. Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFβ. Biochemistry. 2016 Oct 11;55(40):5675-5688. Epub 2016 Sep 30 PubMed.
Kakuda N, Takami M, Okochi M, Kasuga K, Ihara Y, Ikeuchi T. Switched Aβ43 generation in familial Alzheimer's disease with presenilin 1 mutation. Transl Psychiatry. 2021 Nov 3;11(1):558. PubMed.
Lichtenthaler SF, Wang R, Grimm H, Uljon SN, Masters CL, Beyreuther K. Mechanism of the cleavage specificity of Alzheimer's disease gamma-secretase identified by phenylalanine-scanning mutagenesis of the transmembrane domain of the amyloid precursor protein. Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):3053-8. PubMed.
Liu L, Lauro BM, He A, Lee H, Bhattarai S, Wolfe MS, Bennett DA, Karch CM, Young-Pearse T, Dominantly Inherited Alzheimer Network (DIAN), Selkoe DJ. Identification of the Aβ37/42 peptide ratio in CSF as an improved Aβ biomarker for Alzheimer's disease. Alzheimers Dement. 2022 Mar 12; PubMed.
Matsumura N, Takami M, Okochi M, Wada-Kakuda S, Fujiwara H, Tagami S, Funamoto S, Ihara Y, Morishima-Kawashima M. γ-Secretase associated with lipid rafts: multiple interactive pathways in the stepwise processing of β-carboxyl-terminal fragment. J Biol Chem. 2014 Feb 21;289(8):5109-21. Epub 2013 Dec 28 PubMed.
Takami M, Nagashima Y, Sano Y, Ishihara S, Morishima-Kawashima M, Funamoto S, Ihara Y. gamma-Secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment. J Neurosci. 2009 Oct 14;29(41):13042-52. PubMed.
Yang G, Zhou R, Zhou Q, Guo X, Yan C, Ke M, Lei J, Shi Y. Structural basis of Notch recognition by human γ-secretase. Nature. 2019 Jan;565(7738):192-197. Epub 2018 Dec 31 PubMed.
View all comments by Selina WrayLudwig-Maximilians-University Munich and German Center for Neurodegenerative Diseases, Munich
This is beautiful work. The question of whether the mechanism—the formation of a stabilizing hybrid β-sheet that allows unwinding of the preceding small part of the transmembrane helix to expose the cleavage sites—would also be seen for the subsequent stepwise trimming cleavages, had been around since the first publication by Zhou et al., 2019, showing γ-secretase in complex with APP C83. Now, these structural snapshots by Guo et al. nicely show that this is the case, and they allow us to marvel at how systematically γ-secretase does its work.
Truly fascinating. It will be interesting to see if other substrates follow the same processing path as APP, with the same repetitive structural features. Based on cleavage site analyses, this is very likely, even if corresponding small peptide byproducts have not yet been identified for substrates other than APP.
References:
Zhou R, Yang G, Guo X, Zhou Q, Lei J, Shi Y. Recognition of the amyloid precursor protein by human γ-secretase. Science. 2019 Feb 15;363(6428) Epub 2019 Jan 10 PubMed.
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