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.
The co-localization of β-secretase, γ-secretase, and APP in ~5MDa high-molecular-weight complexes is clear and indeed raises the possibility of a coupled regulated intermembrane proteolysis (RIP) process. Interestingly, confocal microscopy (proximity ligation analysis-data) evidences a short distance between the secretases (< 40 nM), suggesting a direct interaction. Whether this comes with functional consequences is less clear. The final proof will likely require the reconstitution of the β-secretase/γ-secretase/APP complex and functional/structural analyses.
It would be very exciting to look at the tripartite complex at atomic resolution!
This is an elegant follow-up of the authors’ previous paper on the interaction between α- and γ-secretase. This is an important study with a very interesting concept, in that β-secretase forms a complex with γ-secretase, which may allow easy proteolytic processing of a substrate by both proteases. 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 and may then come up with more proteins being part of such a complex. Using mass spec on the purified complexes may be one way to go.
It is a very interesting study, and does provide a logical explanation of how Aβ can be efficiently processed by both β- and γ-secretase in a large complex. One intriguing question is how many such high molecular weight (HMW) complexes will exist, because most BACE1 substrates, such as Neuregulin-1, can also be processed by γ-secretase. How stable will such a HMW complex be in cells or brains?
This carefully conducted study demonstrates that Aβ generation in the brain occurs in an endogenous high-molecular-weight complex (HMW) containing the substrate full-length APP and proteolytically active β- and γ-secretases. In this endogenous complex, which was identified by Liu and colleges using non-denaturing FPLC combined with novel Aβ ELISAs in wild-type mouse and human brain, APP undergoes sequential β- and γ-secretase cleavages. In additional in vitro studies with HEK cells overexpressing Swedish-mutant APP, Liu and colleagues provide further evidence that BACE1 and γ-secretase are the most important enzymes to generate Aβ peptides from this FAD mutation. In general, these HMW fractions could be very useful tools to test and optimize secretase inhibitors and modulators and to further develop safe and effective AD drugs.
Although BACE1 most likely acts as the major β-secretase, several N-terminally truncated Aβ variants including peptides starting at position p2 have been described in the CSF and brains of sporadic AD patients and cannot be attributed to BACE 1 activity (Wiltfang et al., 2001; Bibl et al., 2012).
I am intrigued that Liu and colleagues were able to demonstrate the presence of the metalloprotease meprin β in the same mouse brain HMW fractions containing holo-APP, BACE1, and γ-secretase. We have shown that meprin β is able to generate N-terminally truncated Aβ-peptides starting at position p2 (Bien et al., 2012), and that this only occurs when wild-type and not Swedish-mutant APP is expressed (Schoenherr et al., 2016). Additionally, Schlenzig and colleagues have recently demonstrated an increase in meprin β expression in AD brains (Schlenzig et al., 2018). Nevertheless, so far, proof that endogenously expressed meprin β plays a role in the generation of N-terminal truncated Aβ2-x peptides in vivo or in AD is still missing. Therefore, we suggest that 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.
References:
Bien J, Jefferson T, Causevic M, Jumpertz T, Munter L, Multhaup G, Weggen S, Becker-Pauly C, Pietrzik CU.
The Metalloprotease Meprin β Generates Amino Terminal-truncated Amyloid β Peptide Species.
J Biol Chem. 2012 Sep 28;287(40):33304-13.
PubMed.
Schönherr C, Bien J, Isbert S, Wichert R, Prox J, Altmeppen H, Kumar S, Walter J, Lichtenthaler SF, Weggen S, Glatzel M, Becker-Pauly C, Pietrzik CU.
Generation of aggregation prone N-terminally truncated amyloid β peptides by meprin β depends on the sequence specificity at the cleavage site.
Mol Neurodegener. 2016 Feb 19;11:19.
PubMed.
Schlenzig D, Cynis H, Hartlage-Rübsamen M, Zeitschel U, Menge K, Fothe A, Ramsbeck D, Spahn C, Wermann M, Roßner S, Buchholz M, Schilling S, Demuth HU.
Dipeptidyl-Peptidase Activity of Meprin β Links N-truncation of Aβ with Glutaminyl Cyclase-Catalyzed pGlu-Aβ Formation.
J Alzheimers Dis. 2018;66(1):359-375.
PubMed.
Wiltfang J, Esselmann H, Cupers P, Neumann M, Kretzschmar H, Beyermann M, Schleuder D, Jahn H, Rüther E, Kornhuber J, Annaert W, De Strooper B, Saftig P.
Elevation of beta-amyloid peptide 2-42 in sporadic and familial Alzheimer's disease and its generation in PS1 knockout cells.
J Biol Chem. 2001 Nov 16;276(46):42645-57.
PubMed.
Bibl M, Gallus M, Welge V, Esselmann H, Wolf S, Rüther E, Wiltfang J.
Cerebrospinal fluid amyloid-β 2-42 is decreased in Alzheimer's, but not in frontotemporal dementia.
J Neural Transm. 2012 Jul;119(7):805-13.
PubMed.
This is really interesting work. From a general viewpoint, it appears absolutely conceivable to me that the secretase activities act to process or degrade substrates such as APP in a concerted manner. Indeed, such “substrate tunneling” is a conserved biochemical principle, which is, e.g., 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 rather are 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. Unfortunately, the authors do not comment on meprin β in the complex. Our data and that from other labs support APP cleavage by this protease. It would have been interesting to study the N-terminus of the Aβ peptides, but this was certainly beyond the scope of the study.
Congratulations to Dr. Selkoe's lab for their wonderful work! I am really impressed by their delicate and systematic approaches to convincingly demonstrate the β- and γ-secretase complex and its unique catalytic activity.
In our earlier discovery (Cui et al., 2015), we found that β- and γ-secretases indeed functionally interacted with each other to carry out the well-known sequential cleavage of APP. Though in some of our experiments, such as co-localization and fractioning, the complex of β- and γ-secretases (also with other partners) was observed, our efforts then focused on proving the concept that their interaction could be a potential drug target for disrupting Aβ generation and for AD therapy. Through high-throughput screening and chemical modification, 3-α-akebonoic acid (3AA) and its derivatives were identified as effectively reducing Aβ production and further as alleviating cognitive dysfunction and Aβ-related pathology in an AD mouse model.
In this wonderful work by Liu et al., the complex containing β- and γ-secretases was not only identified in cultured cells but also in mouse and human brain, and the complex’s catalytic ability to generate a full array of Aβ peptides was compellingly shown in vitro. This might serve as a convenient assay for further large-scale screening and more mechanistic study. Most interesting, they found that Roburic acid, an analogue of 3-α-akebonoic acid, dose-dependently reduced Aβ production by interfering with β-/γ-secretase complexes and modulating γ-secretase. Their results really encourage and also facilitate us to continue our exploration for better and more druggable disruptor candidates.
It may be worth pointing out here that in our recent research we found that α-secretase (ADAM10) physically interacted with BACE1 in neurons with some proteolytic regulatory function (Wang et al., 2018). Further study by fluorescence resonance energy transfer showed that α-/β-/γ-secretase likely exists in a ternary complex (even with more partners) (Wang and Pei, 2018), suggesting that α- and β-secretases may compete adjacent each other and dynamically, with physical and functional involvement of γ-secretase, APP, and other stakeholders. We hope that by those delicate methodologies in Dr. Selkoe's work and by the up-to-date Cryo-electron microscopy, the structure of the complexes will be solved soon, and the underlying mechanisms of various modifiers such as genetic, epigenetic, environmental, as well as aging would be better understood.
Aging seems inevitable for living creatures, including ourselves, but AD could be prevented or at least delayed. The disappointing outcome of AD treatment for the last decades probably teaches us to study more and to try alternative and more creative approaches.
Comments
K.U.Leuven and V.I.B.
The co-localization of β-secretase, γ-secretase, and APP in ~5MDa high-molecular-weight complexes is clear and indeed raises the possibility of a coupled regulated intermembrane proteolysis (RIP) process. Interestingly, confocal microscopy (proximity ligation analysis-data) evidences a short distance between the secretases (< 40 nM), suggesting a direct interaction. Whether this comes with functional consequences is less clear. The final proof will likely require the reconstitution of the β-secretase/γ-secretase/APP complex and functional/structural analyses.
It would be very exciting to look at the tripartite complex at atomic resolution!
View all comments by Lucia Chavez-GutierrezGerman Center for Neurodegenerative Diseases (DZNE)
This is an elegant follow-up of the authors’ previous paper on the interaction between α- and γ-secretase. This is an important study with a very interesting concept, in that β-secretase forms a complex with γ-secretase, which may allow easy proteolytic processing of a substrate by both proteases. 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 and may then come up with more proteins being part of such a complex. Using mass spec on the purified complexes may be one way to go.
View all comments by Stefan LichtenthalerUniversity of Connecticut Health
It is a very interesting study, and does provide a logical explanation of how Aβ can be efficiently processed by both β- and γ-secretase in a large complex. One intriguing question is how many such high molecular weight (HMW) complexes will exist, because most BACE1 substrates, such as Neuregulin-1, can also be processed by γ-secretase. How stable will such a HMW complex be in cells or brains?
View all comments by Riqiang YanJohannes Gutenberg University Mainz
This carefully conducted study demonstrates that Aβ generation in the brain occurs in an endogenous high-molecular-weight complex (HMW) containing the substrate full-length APP and proteolytically active β- and γ-secretases. In this endogenous complex, which was identified by Liu and colleges using non-denaturing FPLC combined with novel Aβ ELISAs in wild-type mouse and human brain, APP undergoes sequential β- and γ-secretase cleavages. In additional in vitro studies with HEK cells overexpressing Swedish-mutant APP, Liu and colleagues provide further evidence that BACE1 and γ-secretase are the most important enzymes to generate Aβ peptides from this FAD mutation. In general, these HMW fractions could be very useful tools to test and optimize secretase inhibitors and modulators and to further develop safe and effective AD drugs.
Although BACE1 most likely acts as the major β-secretase, several N-terminally truncated Aβ variants including peptides starting at position p2 have been described in the CSF and brains of sporadic AD patients and cannot be attributed to BACE 1 activity (Wiltfang et al., 2001; Bibl et al., 2012).
I am intrigued that Liu and colleagues were able to demonstrate the presence of the metalloprotease meprin β in the same mouse brain HMW fractions containing holo-APP, BACE1, and γ-secretase. We have shown that meprin β is able to generate N-terminally truncated Aβ-peptides starting at position p2 (Bien et al., 2012), and that this only occurs when wild-type and not Swedish-mutant APP is expressed (Schoenherr et al., 2016). Additionally, Schlenzig and colleagues have recently demonstrated an increase in meprin β expression in AD brains (Schlenzig et al., 2018). Nevertheless, so far, proof that endogenously expressed meprin β plays a role in the generation of N-terminal truncated Aβ2-x peptides in vivo or in AD is still missing. Therefore, we suggest that 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.
References:
Bien J, Jefferson T, Causevic M, Jumpertz T, Munter L, Multhaup G, Weggen S, Becker-Pauly C, Pietrzik CU. The Metalloprotease Meprin β Generates Amino Terminal-truncated Amyloid β Peptide Species. J Biol Chem. 2012 Sep 28;287(40):33304-13. PubMed.
Schönherr C, Bien J, Isbert S, Wichert R, Prox J, Altmeppen H, Kumar S, Walter J, Lichtenthaler SF, Weggen S, Glatzel M, Becker-Pauly C, Pietrzik CU. Generation of aggregation prone N-terminally truncated amyloid β peptides by meprin β depends on the sequence specificity at the cleavage site. Mol Neurodegener. 2016 Feb 19;11:19. PubMed.
Schlenzig D, Cynis H, Hartlage-Rübsamen M, Zeitschel U, Menge K, Fothe A, Ramsbeck D, Spahn C, Wermann M, Roßner S, Buchholz M, Schilling S, Demuth HU. Dipeptidyl-Peptidase Activity of Meprin β Links N-truncation of Aβ with Glutaminyl Cyclase-Catalyzed pGlu-Aβ Formation. J Alzheimers Dis. 2018;66(1):359-375. PubMed.
Wiltfang J, Esselmann H, Cupers P, Neumann M, Kretzschmar H, Beyermann M, Schleuder D, Jahn H, Rüther E, Kornhuber J, Annaert W, De Strooper B, Saftig P. Elevation of beta-amyloid peptide 2-42 in sporadic and familial Alzheimer's disease and its generation in PS1 knockout cells. J Biol Chem. 2001 Nov 16;276(46):42645-57. PubMed.
Bibl M, Gallus M, Welge V, Esselmann H, Wolf S, Rüther E, Wiltfang J. Cerebrospinal fluid amyloid-β 2-42 is decreased in Alzheimer's, but not in frontotemporal dementia. J Neural Transm. 2012 Jul;119(7):805-13. PubMed.
View all comments by Claus PietrzikFraunhofer Institute for Cell Therapy and Immunology
This is really interesting work. From a general viewpoint, it appears absolutely conceivable to me that the secretase activities act to process or degrade substrates such as APP in a concerted manner. Indeed, such “substrate tunneling” is a conserved biochemical principle, which is, e.g., 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 rather are 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. Unfortunately, the authors do not comment on meprin β in the complex. Our data and that from other labs support APP cleavage by this protease. It would have been interesting to study the N-terminus of the Aβ peptides, but this was certainly beyond the scope of the study.
View all comments by Stephan SchillingTongji University/Chinese Academy of Sciences
Congratulations to Dr. Selkoe's lab for their wonderful work! I am really impressed by their delicate and systematic approaches to convincingly demonstrate the β- and γ-secretase complex and its unique catalytic activity.
In our earlier discovery (Cui et al., 2015), we found that β- and γ-secretases indeed functionally interacted with each other to carry out the well-known sequential cleavage of APP. Though in some of our experiments, such as co-localization and fractioning, the complex of β- and γ-secretases (also with other partners) was observed, our efforts then focused on proving the concept that their interaction could be a potential drug target for disrupting Aβ generation and for AD therapy. Through high-throughput screening and chemical modification, 3-α-akebonoic acid (3AA) and its derivatives were identified as effectively reducing Aβ production and further as alleviating cognitive dysfunction and Aβ-related pathology in an AD mouse model.
In this wonderful work by Liu et al., the complex containing β- and γ-secretases was not only identified in cultured cells but also in mouse and human brain, and the complex’s catalytic ability to generate a full array of Aβ peptides was compellingly shown in vitro. This might serve as a convenient assay for further large-scale screening and more mechanistic study. Most interesting, they found that Roburic acid, an analogue of 3-α-akebonoic acid, dose-dependently reduced Aβ production by interfering with β-/γ-secretase complexes and modulating γ-secretase. Their results really encourage and also facilitate us to continue our exploration for better and more druggable disruptor candidates.
It may be worth pointing out here that in our recent research we found that α-secretase (ADAM10) physically interacted with BACE1 in neurons with some proteolytic regulatory function (Wang et al., 2018). Further study by fluorescence resonance energy transfer showed that α-/β-/γ-secretase likely exists in a ternary complex (even with more partners) (Wang and Pei, 2018), suggesting that α- and β-secretases may compete adjacent each other and dynamically, with physical and functional involvement of γ-secretase, APP, and other stakeholders. We hope that by those delicate methodologies in Dr. Selkoe's work and by the up-to-date Cryo-electron microscopy, the structure of the complexes will be solved soon, and the underlying mechanisms of various modifiers such as genetic, epigenetic, environmental, as well as aging would be better understood.
Aging seems inevitable for living creatures, including ourselves, but AD could be prevented or at least delayed. The disappointing outcome of AD treatment for the last decades probably teaches us to study more and to try alternative and more creative approaches.
View all comments by Gang PeiMake a Comment
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