Regulated intramembrane proteolysis (RIP) is currently the hot topic in the field of proteases, and what this new paper is telling us is that there is more to it than just the four subunits of active γ-secretase, which has become famous for its role in amyloid-β production in Alzheimer disease (AD).

He et al., from Paul Greengard’s laboratory, describe a novel, so-called γ-secretase activating protein (GSAP) in their current publication. Importantly, the authors show that when it is processed into a 16-kDa fragment, GSAP—the direct target of an anticancer drug that inhibits amyloid formation—directly interacts with the γ-secretase substrate APP. When cellular expression of GSAP was reduced by 72 percent with RNAi, a dramatic reduction of APP-derived amyloid peptides with 38, 40, and 42 residues was observed. Thus, this finding strengthens the idea of substrate targeting to lower amyloid production, which surfaced when NSAIDs were characterized as γ-secretase modulators (GSMs) and found to directly bind to the GxxxG interaction motif of the transmembrane sequence of APP (  Read more

Regulated intramembrane proteolysis (RIP) is currently the hot topic in the field of proteases, and what this new paper is telling us is that there is more to it than just the four subunits of active γ-secretase, which has become famous for its role in amyloid-β production in Alzheimer disease (AD).

He et al., from Paul Greengard’s laboratory, describe a novel, so-called γ-secretase activating protein (GSAP) in their current publication. Importantly, the authors show that when it is processed into a 16-kDa fragment, GSAP—the direct target of an anticancer drug that inhibits amyloid formation—directly interacts with the γ-secretase substrate APP. When cellular expression of GSAP was reduced by 72 percent with RNAi, a dramatic reduction of APP-derived amyloid peptides with 38, 40, and 42 residues was observed. Thus, this finding strengthens the idea of substrate targeting to lower amyloid production, which surfaced when NSAIDs were characterized as γ-secretase modulators (GSMs) and found to directly bind to the GxxxG interaction motif of the transmembrane sequence of APP (Kukar et al., 2008; Richter et al., 2010).

GSAP could have been regarded erroneously as a novel γ-secretase inhibitor, since AICD is reduced. This occurs most likely because it binds to AICD and thereby inhibits its degradation. However, since it affects both product lines and enhances Aβ42, which was suggested to be the precursor of Aβ38, we can be confident that it is a real activator. This explanation is based on the current model of the γ-secretase mechanism. In a consecutive cleavage process, two product lines containing either Aβ40 or Aβ42 are generated in the degradation of remnants of membrane APP. Although we do not know much about GSAP and its 16-kDa active fragment, which makes the story even more complicated, the work clearly shows that an understanding of the fine-tuning of the γ-mediated cleavage by cofactors is very important. This means that results from in vitro assays of the γ-secretase should be considered with great care when they are obtained in the absence of important cofactors. The loss of cofactors (when purified away during the enrichment of the γ-secretase module) may explain why findings from in vitro assays do not always match to those obtained with living cells. Unfortunately, researchers sometimes assume that in vivo results will parallel in vitro findings.

Given that regulatory factors such as TMP21 (Chen et al., 2006), GSAP, and others exist (for a review see De Strooper and Annaert, 2010), it is evident how dangerous it can be to extrapolate from the minimal composition of a protein module with four subunits to a fully active unit containing all cofactors (many of which might have been otherwise lost during the preparation of the module for the in vitro assay). In addition, other factors, such as the dimerization of the APP-derived substrate β-CTF, have been recognized as an important mechanism to regulate Aβ42 production. Production of Aβ42 is reduced in favor of an increase of Aβ38 when dimerization of the substrate is attenuated (Munter et al., 2007; Munter et al., 2010). This nicely fitted into findings from Yasuo Ihara’s group (see Qi-Takahara et al., 2005 and Takami et al., 2009), from which we have learned that γ-secretase is able to process Aβ49 or Aβ48 at every third residue, leading to the stepwise generation of shorter Aβ peptides representing intermediate products of the γ-secretase-driven degradation process.

Another point which I would like to stress is that major recent findings regarding the γ-secretase module have been made by classical biochemical approaches. These were based on thorough analyses of interactions with γ-secretase subunits (e.g., by Chen et al., who identified TMP21), or with compounds such as NSAIDs and Gleevec, elegantly used as photoactivatable derivatives (Kukar et al., 2008; He et al., 2010) to characterize binding partners of secretase substrates or cofactors. Together with complementing studies that were oriented to understand enzyme-substrate interactions (Munter et al., 2007; Munter et al., 2010; Richter et al., 2010), the knowledge of the substrate conformation (i.e., dimer or monomer) and an understanding of regulatory factors such as GSAP will help to find answers to questions regarding the physiology and the structure/ function relation within the γ-secretase module. All this tells us that there is an additional level of activity modulation that is more complex than previously thought and which has a high potential for the development of novel therapeutic strategies, which are urgently needed for AD.

View all comments by Gerd Multhaup

I am very enthusiastic about this paper. The authors identify a 16-kDa protein that apparently binds to APP-CTF at the juxtamembrane area—close to the ε cleavage site—and which is able to modulate the different cleavages of the APP-CTF. Blocking the interaction with APP-CTF (either using siRNA or imatinib) lowers Aβ generation and increases AICD formation. The protein does not seem to affect Notch signaling (in vitro or in vivo), although further work is needed to see whether it affects other substrates or whether the effect is entirely APP selective. In addition, if GSAP inhibition stimulates AICD formation and inhibits γ-cleavage, then long Aβ49 should be observed. The question of what happens with Aβ49 has surprisingly not been addressed in the paper.

The fact that its activity can be modulated by imatinib provides proof of concept that the protein is a drug target (although it will not be easy to generate drugs that are specific enough and brain permeable to interfere with the proposed protein-protein interaction). Interestingly, several γ-secretase modulators have been...  Read more

I am very enthusiastic about this paper. The authors identify a 16-kDa protein that apparently binds to APP-CTF at the juxtamembrane area—close to the ε cleavage site—and which is able to modulate the different cleavages of the APP-CTF. Blocking the interaction with APP-CTF (either using siRNA or imatinib) lowers Aβ generation and increases AICD formation. The protein does not seem to affect Notch signaling (in vitro or in vivo), although further work is needed to see whether it affects other substrates or whether the effect is entirely APP selective. In addition, if GSAP inhibition stimulates AICD formation and inhibits γ-cleavage, then long Aβ49 should be observed. The question of what happens with Aβ49 has surprisingly not been addressed in the paper.

The fact that its activity can be modulated by imatinib provides proof of concept that the protein is a drug target (although it will not be easy to generate drugs that are specific enough and brain permeable to interfere with the proposed protein-protein interaction). Interestingly, several γ-secretase modulators have been proposed to bind also to APP-CTF and thus modulate γ-cleavages of this substrate. It is unclear whether they act via a similar mechanism to γ-secretase activating protein.

Many other exciting questions remain to be answered in follow-up studies: How precisely does this protein interact with γ-secretase? How do mutations in presenilin affect this interaction? What is the role of the large precursor protein of GSAP, and are the proteases that process this precursor candidate drug targets themselves?

View all comments by Bart De Strooper

The identification of GSAP as a specific stimulator of APP processing is important in understanding how γ-secretase is regulated and which mechanism enables selection among the various substrates of the γ-secretase activity. Although more than 30 single transmembrane proteins have been reported as γ-secretase substrates, including APP, Notch, and E- and N-cadherins (see Marambaud et al., 2002, and 2003 for cadherins), no consensus sequence is recognized among these substrates. Accordingly, substrate recognition and broad specificity for γ-secretase remain an enigma. It is also noted that the γ-secretase complex detected by glycerol velocity gradient, or blue native-PAGE (Georgakopoulos et al., 1999; Gu et al., 2004; Evin et al., 2005; Kiss et al., 2008), shows an apparent molecular weight larger than 400 kDa, which exceeds the simple sum of the molecular weights of the γ-secretase core components, presenilin (PS)/N- and C-terminal fragments (28 and 18 kDa, respectively), Nicastrin (120 kDa), APH-1 (24 kDa) and PEN-2 (12 kDa). One interesting concept is that adapter proteins act...  Read more

The identification of GSAP as a specific stimulator of APP processing is important in understanding how γ-secretase is regulated and which mechanism enables selection among the various substrates of the γ-secretase activity. Although more than 30 single transmembrane proteins have been reported as γ-secretase substrates, including APP, Notch, and E- and N-cadherins (see Marambaud et al., 2002, and 2003 for cadherins), no consensus sequence is recognized among these substrates. Accordingly, substrate recognition and broad specificity for γ-secretase remain an enigma. It is also noted that the γ-secretase complex detected by glycerol velocity gradient, or blue native-PAGE (Georgakopoulos et al., 1999; Gu et al., 2004; Evin et al., 2005; Kiss et al., 2008), shows an apparent molecular weight larger than 400 kDa, which exceeds the simple sum of the molecular weights of the γ-secretase core components, presenilin (PS)/N- and C-terminal fragments (28 and 18 kDa, respectively), Nicastrin (120 kDa), APH-1 (24 kDa) and PEN-2 (12 kDa). One interesting concept is that adapter proteins act as recruiting factors specific for one, or a subgroup of, substrate(s) and regulate γ-secretase cleavages. We proposed this model in our paper published two years ago (Kouchi et al., 2009).

We have reported p120 catenin, an isoform of δ-catenin, as such a recruiting protein specific for N- and E-cadherins since it has binding affinity to PS1, as well as to these cadherins, and also mediates PS1-dependent cleavage of these substrates (Kouchi et al., 2009; also see Kiss et al., 2008, for investigations on γ-secretase catenin supercomplex). In this model, various recruiting factors could be indispensible for substrate recognition and explain the high-molecular-weight γ-secretase supercomplex(es) with broad substrate specificity.

GSAP reported by He et al. seems to be another example of this kind of protein, but specific for the APP substrate. Interestingly, the interaction between p120 catenin and the cadherins involves the juxtamembrane region of the substrate, i.e., cadherin, just as in the case of GSAP and APP-CTFs. And, just like GSAP, p120 catenin plays the role as a bridge between the γ-secretase and the substrate. We have further identified a p120 binding site in PS1, amino acids 330-360 (Kouchi et al., 2009), although the GSAP binding site has yet to be identified. It is possibly in the PS1 CTF.

Whereas p120 binds to cadherins, but not to APP, expression of p120 catenin not only promoted E-cadherin cleavage but also partially suppressed Aβ and AICD production (Kouchi et al., 2009). We explained this by a possible competition between substrates, i.e., E-cadherin and APP, for limited availability of γ-secretase, but now we can give an alternative interpretation—that p120 catenin and GSAP may competitively bind to the cytoplasmic loop region of PS1CTF. Since cadherins and p120 are implicated in dendritic spine formation and synaptic transmission, it seems to be a crucial issue whether GSAP affects cadherin/p120-catenin/PS interaction in order to validate GSAP as an attractive target for treatment of AD.

References:
Evin G, Canterford LD, Hoke DE, Sharples RA, Culvenor JG, Masters CL. Transition-state analogue gamma-secretase inhibitors stabilize a 900 kDa presenilin/nicastrin complex. Biochemistry. 2005 Mar 22;44(11):4332-41. Abstract

Georgakopoulos A, Marambaud P, Efthimiopoulos S, Shioi J, Cui W, Li HC, Schütte M, Gordon R, Holstein GR, Martinelli G, Mehta P, Friedrich VL Jr, Robakis NK. Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts. Mol Cell. 1999 Dec;4(6):893-902. Abstract

Gu Y, Sanjo N, Chen F, Hasegawa H, Petit A, Ruan X, Li W, Shier C, Kawarai T, Schmitt-Ulms G, Westaway D, St George-Hyslop P, Fraser PE. The presenilin proteins are components of multiple membrane-bound complexes that have different biological activities. J Biol Chem. 2004 Jul 23;279(30):31329-36. Abstract

Kiss A, Troyanovsky RB, Troyanovsky SM. p120-catenin is a key component of the cadherin-gamma-secretase supercomplex. Mol Biol Cell. 2008 Oct;19(10):4042-50. Abstract

Kouchi Z, Barthet G, Serban G, Georgakopoulos A, Shioi J, Robakis NK. p120 catenin recruits cadherins to gamma-secretase and inhibits production of Abeta peptide. J Biol Chem. 2009 Jan 23;284(4):1954-61. Abstract

Marambaud P, Wen PH, Dutt A, Shioi J, Takashima A, Siman R, Robakis NK. A CBP binding transcriptional repressor produced by the PS1/epsilon-cleavage of N-cadherin is inhibited by PS1 FAD mutations. Cell. 2003 Sep 5;114(5):635-45. Abstract

Marambaud P, Shioi J, Serban G, Georgakopoulos A, Sarner S, Nagy V, Baki L, Wen P, Efthimiopoulos S, Shao Z, Wisniewski T, Robakis NK. A presenilin-1/gamma-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions. EMBO J. 2002 Apr 15;21(8):1948-56. Abstract

View all comments by Gael Barthet
View all comments by Junichi Shioi

In this paper, He and colleagues work revealed that the γ-secretase processing mechanisms of Aβ generation from APP C-terminal fragments (CTFs) by γ-site cleavage were distinct from the mechanisms of ε-site cleavage. This result is consistent with our previous reports (Kume et al., 2004; Kume and Kametani, 2006). These show that APP CTFs are cleaved in two γ-secretase processing pathways and clarify the relationship between the processing sites and their products in these pathways.

One is the so-called γ-secretase regulated pathway. γ-secretase is responsible for processing not only APP CTFs, but also various type 1 membrane proteins, including Notch and cadherins (Wolfe and Kopan, 2004). In general, this cleavage occurs near the cytoplasmic membrane boundary region and releases the intracellular cytoplasmic domain for intracellular signaling. In APP CTFs, γ-secretase cleaves at the ε-site near the cytoplasmic membrane boundary region and produces AICDε (C50) (Gu et al., 2001; Sastre et al., 2001; Weidemann et al., 2002). As previously reported, ε-site cleavage preferentially...  Read more

In this paper, He and colleagues work revealed that the γ-secretase processing mechanisms of Aβ generation from APP C-terminal fragments (CTFs) by γ-site cleavage were distinct from the mechanisms of ε-site cleavage. This result is consistent with our previous reports (Kume et al., 2004; Kume and Kametani, 2006). These show that APP CTFs are cleaved in two γ-secretase processing pathways and clarify the relationship between the processing sites and their products in these pathways.

One is the so-called γ-secretase regulated pathway. γ-secretase is responsible for processing not only APP CTFs, but also various type 1 membrane proteins, including Notch and cadherins (Wolfe and Kopan, 2004). In general, this cleavage occurs near the cytoplasmic membrane boundary region and releases the intracellular cytoplasmic domain for intracellular signaling. In APP CTFs, γ-secretase cleaves at the ε-site near the cytoplasmic membrane boundary region and produces AICDε (C50) (Gu et al., 2001; Sastre et al., 2001; Weidemann et al., 2002). As previously reported, ε-site cleavage preferentially occurs on the α-secretase processing product C83, which is the major APP CTF (Kume et al., 2004; Kume and Kametani, 2006). Furthermore, it was recently reported that the β-secretase processing product C99, which is the minor APP CTF, is an inefficient substrate for proteolysis by γ-secretase (Funamoto et al., 2010). Therefore, in the γ-secretase processing pathway, trace amounts of Aβ49 may be produced from C99, while AICDε (C50) is mostly produced from C83. Long p3 is also produced in this pathway (Kametani, 2004).

The other regulatory pathway is via GSAP regulation processing. As this paper describes, this processing involves GSAP binding to the cytoplasmic domain of APP CTFs. GSAP/γ-secretase/APP CTF ternary complex alters the structural relationship between γ-secretase and APP CTFs. In the presence of GSAP, γ-secretase may directly cleave at γ-site in the middle of the membrane domain of APP CTFs. Thus, γ-site processing may produce Aβ from C99, p3 from C83, and AICDγ (C57/59) from C99 and C83.

Under normal conditions, γ-secretase processing of APP CTFs occurs in these two pathways. An increased level of expression of GSAP induces an increase in Aβ production and a decrease in AICDε production, according to the authors. Therefore, GSAP binding to the cytoplasmic domain of APP CTF and GSAP regulated processing may act upstream of γ-secretase processing. Aβ may be mostly produced by the GSAP regulated processing, and GSAP regulated processing is a key event of AD.

References:
Kume, H., Maruyama, K., Kametani, F. Intracellular domain generation of amyloid precursor protein by ε-cleavage depends on C-terminal fragment by alpha-secretase cleavage. Int J Mol Med 13, 121-125 (2004). Abstract

Kume, H., Kametani, F. Abeta 11-40/42 production without gamma-secretase epsilon-site cleavage. Biochem Biophys Res Commun 349, 1356-1360 (2006). Abstract

Wolfe, M.S., Kopan, R. Intramembrane Proteolysis: Theme and Variations. Science 305, 1119-1123 (2004). Abstract

Gu, Y. et al. Distinct intramembrane cleavage of the beta-amyloid precursor protein family resembling gamma-secretase-like cleavage of Notch. J Biol Chem 276, 35235-35238 (2001). Abstract

Sastre, M. et al. Presenilin-dependent gamma-secretase processing of beta-amyloid precursor protein at a site corresponding to the S3 cleavage of Notch. EMBO Rep 2, 835-841 (2001). Abstract

Weidemann, A. et al. A novel epsilon-cleavage within the transmembrane domain of the Alzheimer amyloid precursor protein demonstrates homology with Notch processing. Biochemistry 41, 2825-2835 (2002). Abstract

Funamoto, S., Nobuhara, M., Ishihara, S., Ihara, Y. betaCTF is an inefficient substrate for proteolysis by gamma-secretase. Alzheimer's & Dementia: J Alzheimer's Association 6, S395 (2010).

Kametani, F. Secretion of long Abeta-related peptides processed at epsilon-cleavage site is dependent on the alpha-secretase pre-cutting. FEBS Lett 570, 73-76 (2004). Abstract

View all comments by Fuyuki Kametani