The production of Aβ peptides from the amyloid precursor protein is a two-step affair: A first cut by the β-secretase (BACE) releases the extracellular portion of APP, leaving the C-terminal fragment (CTF) to find its way to γ-secretase for a final clipping to release Aβ. New data suggest the protease-to-protease handoff may be more intimate than previously thought. Work from the lab of Dennis Selkoe reveals that BACE, γ-secretase, and full-length APP can be isolated from mouse and human brain tissue in a high-molecular-weight complex that is capable of de novo production of Aβ peptides. The work, published January 9 in the Journal of Cell Biology, suggests the complex generates Aβ in vivo. A small molecule that destabilizes the union reduces Aβ production by cells, without directly inhibiting either BACE or γ-secretase catalytic activity.
- BACE and γ-secretase co-purify from mouse and human brain.
- Large complex processes full-length APP to Aβ peptides in vitro.
- Breaking it up may reduce Aβ while allowing processing of other substrates.
Stefan Lichtenthaler, German Center for Neurodegenerative Diseases, Munich, called the work an elegant follow-up of the lab’s previous studies on the interaction between α- and γ-secretases. “This is an important study with a very interesting concept, in that β-secretase forms a complex with γ-secretase that may allow easy proteolytic processing of substrate by both proteases,” he wrote to Alzforum. “Given the huge size of the complex, I am wondering whether β- and γ-secretase directly interact or whether they are co-purifying because they are in the same membrane microdomain, but without a direct interaction. I am sure the authors will investigate this further,” he wrote (see comment below).
“The presence of active multi-protease complexes is an exciting discovery,” said Gopal Thinakaran, University of Chicago. However, the location of the complexes, and their exact contribution to Aβ production in intact cells, remains to be clarified, he said.
To access γ-secretase, APP needs to shed its large extracelluar domain. That can be achieved by several membrane-bound proteases, most prominently BACE or α-secretase, also known as ADAM10. Previously, Selkoe’s lab discovered that α-secretase paired up with γ-secretase in large, multi-protease complexes that seemed to facilitate sequential proteolysis (Jan 2016 news). In the new study, first author Lei Liu asked if the same was true for BACE. Liu started with microsomes prepared from mouse brain, then solubilized the membrane proteins with mild detergent. In those preparations, antibodies to BACE1 consistently co-precipitated components of γ-secretase, including nicastrin and the N- and C-terminal fragments of presenilin1 (PS1). When Liu separated the solubilized membrane proteins using non-denaturing size-exclusion chromatography, he identified a complex of more than 5 million Daltons that harbored the γ-secretase proteins and full-length APP. Immunoprecipitation of this complex with anti-nicastrin antibodies pulled down BACE activity, suggesting the investigators had isolated a BACE1/γ-secretase complex analogous to their previously described α-secretase/γ-secretase pair. The complex contained only a fraction of the BACE in the cell: Most of the BACE1 protein, and protease activity, eluted in lower-molecular-weight fractions, without PS1.
Still, would the complex make Aβ? To measure this in vitro, scientists usually spike enzyme preps with recombinant APP. In this case, Liu took advantage of the endogenous APP already in the complex. He size-fractionated microsomes from HEK293 cells stably overexpressing human APP with the Swedish mutation, incubated the high-molecular-weight fractions at 37 degrees for 12 hours, solubilized them with detergent, and measured Aβ by ELISA. He found the complex had made Aβx-42 and Aβx-40 in a ratio of 0.1-0.2, which is within physiological norms. In contrast, the lower-molecular-weight fractions, with abundant BACE1 but no PS1, produced little or no Aβ. “This told us the BACE1/γ-secretase complex was functionally relevant, because the tiny amount of BACE1 in the high molecular weight fractions was, in fact, contributing to Aβ generation,” Liu told Alzforum. Complexes from cells expressing the familial AD PS1 mutants Y115H or L286V produced Aβ with a 42/40 ratio of 0.5 or higher, supporting the idea that these complexes are a physiological source of the peptides. Based on the cell fractionation experiments, Selkoe thinks most of the Aβ in cells could be derived from the high-molecular-weight BACE1/γ-secretase assemblies.
Surprisingly, the γ-secretase active site inhibitor L685,458 did not diminish Aβ production by the high-molecular-weight complex, though L685,458 suppresses Aβ production in human neuronal cells (Tagami et al., 2017). Liu and Selkoe speculated it had no effect on the complexes because APP was already bound and prevented the inhibitor from accessing the PS1 active site. A PS1 allosteric modulator that binds outside the catalytic site did have the expected effect: JNJ-40418677 attenuated production of Aβx-42 by the complex, and boosted Aβx-37.
To prove the complex sequentially cleaved full-length APP, and not simply APP CTFs bound in the complex, the investigators depleted CTFs in HEK293-swAPP cells by treatment with the BACE inhibitor AZD3293. This increased the amount of full-length APP in the cell and when Liu isolated the high-molecular-weight BACE/γ-secretase, now sans AZD3293, it made 1.5- to fourfold more Aβ than complexes from untreated cells.
Can the same complex be isolated from human brain? Apparently, yes. Liu size-fractionated preparations of fresh human brain tissue, and showed the HMW fractions produced the bulk of Aβ from endogenous APP. Aβ production by the complex was only marginally inhibited by three different γ-secretase inhibitors, but, as they saw with HEK293 cells, the modulator JNJ-40418677 shifted cleavage toward the smaller Aβx-37.
Close companions. Proximity ligation (red puncta) links PS1 to BACE1 (middle panel), but not to a transferrin receptor control (left panel). Super-resolution STED microscopy shows PS1 (red) and BACE1 (green) in individual puncta (right). [Courtesy of Liu et al., JCB 2019.]
The scientists next asked if they could target the complex to reduce Aβ production as a potential therapeutic strategy. Previously, Chinese researchers had discovered that the natural product 3-α-akebonoic acid (3AA) disrupted BACE/γ-secretase interactions in cells and crippled Aβ secretion (Cui et al., 2015). Liu showed that a related compound, roburic acid, inhibited Aβ production from HEK293-swAPP cells. This was tied to reduced γ-secretase and BACE activity in the large complex. Roburic acid did not directly inhibit BACE1 or γ-secretase enzymatic activity, although it did show some γ-secretase modulatory activity, decreasing the Aβx-42/x-40 ratio and increasing the Aβx-38/x-42 ratio. “Roburic acid or other compounds that separate the BACE and γ-secretases, without affecting their active sites, could offer an alternative approach to BACE inhibitors,” Selkoe said. With BACE-inhibitor clinical trials turning up discouraging cognitive and other side effects, possibly due to decreased processing of other important substrates, new approaches are clearly needed, he added (Nov 2018 conference news).
Given that BACE and γ-secretase travel in different circles in the cell, with distinct targeting and trafficking pathways, Thinakaran wondered where they would meet up to form a large complex. “The next challenges are to determine where within the neurons the multi-protease α/γ and β/γ complexes are assembled, and characterize the trafficking motifs in the individual components of the complexes that cooperate or act as predominant signals to direct the complexes to their subcellular sites within neurons,” he said. By several methods, including proximity ligation (see image above), Selkoe’s group detected close association of BACE1 and γ-secretase near nuclei of HEK cells. Selkoe said their techniques establish that the proteins are together, but they do not yet know precisely where they sit in the cell. "We believe there must be physical loci where this complex resides, but we’re not sure where that is yet,” he said.
“It is absolutely conceivable that the secretase activities act to process or degrade substrates such as APP in a concerted manner,” said Stephan Schilling, Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany. “Indeed, such ‘substrate tunneling’ is a conserved biochemical principle, which is, for example, known for enzymes acting in amino-acid synthesis in the cytosol. The principle is that products of one processing step are not released into bulk but are rather guided to another enzyme within a complex, which makes these processes much more efficient. That this principle might also apply to membrane processing by proteases is an intriguing finding, and the authors collected compelling evidence for formation of such a multi-enzyme complex,” he wrote to Alzforum.
Liu spied another potential APP sheddase in the high-molecular-weight fraction. The metalloprotease meprin β has been proposed as an alternative BACE that cleaves APP at the same site and thus might also feed presenilin (Becker-Pauly and Pietrzik, 2016). “The localization of meprin β to HMW fractions containing APP and γ-secretase provides another important piece of evidence that this enzyme contributes to Aβ generation in the brain,” wrote Claus Pietrzik, Johannes Gutenberg University Mainz, Germany, to Alzforum. However, Liu said the presence of the enzyme does not mean it’s necessarily a partner of γ-secretase. They have not yet done the co-immunoprecipitation experiments to determine that.—Pat McCaffrey
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No Available Further Reading
- Liu L, Ding L, Rovere M, Wolfe MS, Selkoe DJ. A cellular complex of BACE1 and γ-secretase sequentially generates Aβ from its full-length precursor. J Cell Biol. 2019 Feb 4;218(2):644-663. Epub 2019 Jan 9 PubMed.