Presenilin is the catalytic component of γ-secretase, a complex aspartyl protease and a founding member of intramembrane-cleaving proteases. γ-Secretase is involved in the pathogenesis of Alzheimer’s disease and a top target for therapeutic intervention. However, the protease complex processes a variety of transmembrane substrates, including the Notch receptor, raising concerns about toxicity. Nevertheless, γ-secretase inhibitors and modulators have been identified that allow Notch processing and signalling to continue, and promising compounds are entering clinical trials. Molecular and biochemical studies offer a model for how this protease hydrolyzes transmembrane domains in the confines of the lipid bilayer. Progress has also been made toward structure elucidation of presenilin and the γ-secretase complex by electron microscopy as well as by studying cysteine-mutant presenilins. The signal peptide peptidase (SPP) family of proteases are distantly related to presenilins. However, the SPPs work as single polypeptides without the need for cofactors and otherwise appear to be simple model systems for presenilin in the γ-secretase complex. SPP biology, structure, and inhibition will also be discussed.
amyloid; Notch receptor; peptidomimetics; signal peptide peptidase; substrate analogues; substrate recognition
C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by γ-secretase to release the amyloid-β polypeptides, which are associated with Alzheimer’s disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded “N-helix” followed by a short “N-loop” connecting to the transmembrane domain (TMD). The TMD is a flexibly curved α helix, making it well suited for processive cleavage by γ-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer’s therapeutics.
Amyloid-β peptide ending at 42nd residue (Aβ42) is believed as a pathogenic peptide for Alzheimer disease. Although γ-secretase is a responsible protease to generate Aβ through a processive cleavage, the proteolytic mechanism of γ-secretase at molecular level is poorly understood.
We found that the transmembrane domain (TMD) 1 of presenilin (PS) 1, a catalytic subunit for the γ-secretase, as a key modulatory domain for Aβ42 production. Aβ42-lowering and -raising γ-secretase modulators (GSMs) directly targeted TMD1 of PS1 and affected its structure. A point mutation in TMD1 caused an aberrant secretion of longer Aβ species including Aβ45 that are the precursor of Aβ42. We further found that the helical surface of TMD1 is involved in the binding of Aβ45/48 and that the binding was altered by GSMs as well as TMD1 mutation.
Binding between PS1 TMD1 and longer Aβ is critical for Aβ42 production.
Presenilin; Secretases; Alzheimer disease; Intramembrane proteolysis; γ-Secretase modulator
Presenilins (PSs) are catalytic components of the γ-secretase proteolytic complexes that produce Aβ and cell signaling peptides. γ-Secretase substrates are mostly membrane-bound peptides derived following proteolytic cleavage of the extracellular domain of typeI transmembrane proteins. Recent work reveals that γ-secretase substrate processing is regulated by proteins termed γ-Secretase Substrate Recruiting Factors (γSSRFs) that bridge substrates to γ-secretase complexes. These factors constitute novel targets for pharmacological control of specific γ-secretase products such as Aβ and signaling peptides. PS familial Alzheimer’s disease (FAD) mutants cause a loss of γ-secretase cleavage function at epsilon sites of substrates thus inhibiting production of cell signaling peptides while promoting accumulation of uncleaved toxic substrates. Importantly, γ-secretase inhibitors may cause toxicity in vivo by similar mechanisms. Here we review novel mechanisms that control γ-secretase substrate selection and cleavage and examine their relevance to AD.
Alzheimer’s disease; Presenilin; Familial AD mutations; γ-Secretase Substrate Recruiting Factors (γSSRFs); metalloproteinases; ADAMs; toxic substrates; γ-Secretase-produced signaling peptides
γ-Secretase, a multi-subunit transmembrane protease comprised of presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1, participates in the regulated intramembrane proteolysis of Type I membrane proteins including the amyloid precursor protein (APP). Although Aph-1 is thought to play a structural role in the assembly of γ-secretase complex and several transmembrane domains (TMDs) of Aph-1 have been shown to be critical for its function, the importance of the other domains of Aph-1 remains elusive. We screened a series of Aph-1 mutants and focused on 9 mutations distributed in 6 different TMDs of human APH-1aS, assessing their ability to complement mouse embryonic fibroblasts lacking Aph-1. We showed that mutations in TMD4 (G126) and TMD5 (H171) of Aph-1a prevented the formation of the Nct/Aph-1 subcomplex. Importantly, although mutations in TMD3 (Q83/E84/R85) and TMD6 (H197) of APH-1aS did not affect Nct/Aph-1 subcomplex formation, both mutations prevented further association/endoproteolysis of PS1. We propose a model that identifies critical TMDs of Aph-1 for associations with Nct and PS for the stepwise assembly of γ-secretase components.
γ-Secretase; Aph-1; Nct; PS; mutagenesis; transmembrane domain
Presenilin-mediated endoproteolysis of transmembrane proteins plays a key role in physiological signaling and in the pathogenesis of Alzheimer disease and some cancers. Numerous inhibitors have been found via library screens, but their structural mechanisms remain unknown. We used several biophysical techniques to investigate the structure of human presenilin complexes and the effects of peptidomimetic γ-secretase inhibitors. The complexes are bilobed. The head contains nicastrin ectodomain. The membrane-embedded base has a central channel and a lateral cleft, which may represent the initial substrate docking site. Inhibitor binding induces widespread structural changes, including rotation of the head and closure of the lateral cleft. These changes block substrate access to the catalytic pocket and inhibit the enzyme. Intriguingly, peptide substrate docking has reciprocal effects on the inhibitor binding site. Similar reciprocal shifts may underlie the mechanisms of other inhibitors and of the “lateral gate” through which substrates access to the catalytic site.
•The head contains nicastrin ectodomain and overhangs a solute-accessible cavity in base•The base has a central channel and a lateral cleft (putative substrate docking site)•Inhibitors close the cleft and channel and rotate the head, blocking substrate access
Presenilin complexes mediate proteolysis of transmembrane proteins during physiological signaling and disease. Li et al. describe the architecture of human presenilin complex PS1 and inhibitor-induced structural changes. They propose that similar shifts likely underlie substrate access to the catalytic site.
γ-Secretase generates the peptides of Alzheimer's disease, Aβ40 and Aβ42, by cleaving the amyloid precursor protein within its transmembrane domain. γ-Secretase also cleaves numerous other substrates, raising concerns about γ-secretase inhibitor off-target effects. Another important class of drugs, γ-secretase modulators, alter the cleavage site of γ-secretase on amyloid precursor protein, changing the Aβ42/Aβ40 ratio, and are thus a promising therapeutic approach for Alzheimer's disease. However, the target for γ-secretase modulators is uncertain, with some data suggesting that they function on γ-secretase, whereas others support their binding to the amyloid precursor. In this paper we address this controversy by using a fluorescence resonance energy transfer-based assay to examine whether γ-secretase modulators alter Presenilin-1/γ-secretase conformation in intact cells in the absence of its natural substrates such as amyloid precursor protein and Notch. We report that the γ-secretase allosteric site is located within the γ-secretase complex, but substrate docking is needed for γ-secretase modulators to access this site.
γ-Secretase modulators have promise in the treatment of Alzheimer's disease, but their molecular target is uncertain. Here, fluorescence resonance energy transfer is used to determine that the γ-secretase allosteric site is within the γ-secretase complex and that substrate docking is required for modulators to access the site.
Presenilins were first discovered as sites of missense mutations responsible for early-onset Alzheimer disease (AD). The encoded multipass membrane proteins were subsequently found to be the catalytic components of γ-secretases, membrane-embedded aspartyl protease complexes responsible for generating the carboxyl terminus of the amyloid β-protein (Aβ) from the amyloid protein precursor (APP). The protease complex also cleaves a variety of other type I integral membrane proteins, most notably the Notch receptor, signaling from which is involved in many cell differentiation events. Although γ-secretase is a top target for developing disease-modifying AD therapeutics, interference with Notch signaling should be avoided. Compounds that alter Aβ production by γ-secretase without affecting Notch proteolysis and signaling have been identified and are currently at various stages in the drug development pipeline.
γ-Secretases produce amyloid β-protein in Alzheimer disease, but they also regulate Notch signaling. Drugs that alter amyloid β-protein production without affecting Notch signaling are currently under development.
The amyloid β-peptide (Aβ), strongly implicated in the pathogenesis of Alzheimer’s disease (AD), is produced from the amyloid β-protein precursor (APP) through consecutive proteolysis by β- and γ-secretases. The latter protease contains presenilin as the catalytic component of a membrane-embedded aspartyl protease complex. Missense mutations in presenilin are associated with early-onset familial AD, and these mutations generally both decrease Aβ production and increase the proportion of the aggregation-prone 42-residue form (Aβ42) over the 40-residue form (Aβ40). The connection between these two effects is not understood. Besides Aβ40 and Aβ42, γ-secretase produces a range of Aβ peptides, the result of initial cutting at the ε site to form Aβ48 or Aβ49 and subsequent trimming every 3–4 residues. Thus, γ-secretase displays both overall proteolytic activity (ε cutting) and processivity (trimming) toward its substrate APP. Here we tested whether a decrease in total activity correlates with decreased processivity using wild type and AD-mutant presenilin-containing protease complexes. Changes in pH, temperature and salt concentration that reduced overall activity of the wild type enzyme did not consistently result in increased proportions of longer Aβ peptides. Low salt concentrations and acidic pH were notable exceptions that subtly alter the proportion of individual Aβ peptides, suggesting that the charged state of certain residues may influence processivity. Five different AD-mutant complexes, representing a broad range of effects on overall activity, Aβ42-to-Aβ40 ratios, and ages of disease onset were also tested, revealing again that changes in total activity and processivity can be dissociated. Factors that control initial proteolysis of APP at the ε site apparently differ significantly from factors affecting subsequent trimming and the distribution of Aβ peptides.
Presenilin-dependent γ-secretase cleavage of several transmembrane proteins, including amyloid-β precursor protein and Notch, mediates the intramembrane proteolysis to liberate their intracellular domains that are involved in cellular signaling. Considering γ-secretase inhibitors as therapeutics for Alzheimer's disease, understanding the physiologically and biologically important substrate for γ-secretase activity in brains is emerging issue. To elucidate the molecular mechanism and physiological role of γ-secretase, we screened candidate molecules for γ-secretase substrates.
We show that ephrin-B1, that participates in cell-cell repulsive and attractive signaling together with its Eph receptor, constitutively undergoes ectodomain shedding and that the residual membrane-tethered fragment is sequentially cleaved by γ-secretase to release the intracellular domain. Furthermore, overexpression of membrane-tethered ephrin-B1 caused protrusion of numerous cellular processes consisted of F-actin, that required the preservation of the most C-terminal region of ephrin-B1. In contrast, soluble intracellular domain translocated into the nucleus and had no effect on cell morphology.
Our findings suggest that ephrin-B is a genuine substrate for γ-secretase and regulates the cytoskeletal dynamics through intramembrane proteolysis.
γ-Secretase is a membrane embedded aspartyl protease complex with presenilin as the catalytic component. Along with β-secretase, this enzyme produces the amyloid β-protein (Aβ) of Alzheimer’s disease (AD) from the amyloid β-protein precursor (APP). Because of its key role in the pathogenesis of AD, γ-secretase has been a prime target for drug discovery, and many inhibitors of this protease have been developed. The therapeutic potential of these inhibitors is virtually negated by the fact that γ-secretase is an essential part of the Notch signaling pathway, rendering the compounds unacceptably toxic upon chronic exposure. However, these compounds have served as useful chemical tools for biological investigations. In contrast, γ-secretase modulators continue to be of keen interest as possible AD therapeutics. These modulators either shift Aβ production to shorter, less pathogenic peptides or inhibit the proteolysis of APP selectively compared to that of Notch. The various chemical types of inhibitors and modulators will be discussed, along with their use as probes for basic biology and their potential as AD therapeutics.
protease; amyloid; active site; docking site; allosteric sites; chemical probes
Membrane protein biogenesis requires the coordinated movement of hydrophobic transmembrane domains (TMD) from the cytosolic vestibule of the Sec61 channel into the lipid bilayer. Molecular insight into TMD integration has been hampered by the difficulty of characterizing intermediates during this intrinsically dynamic process. In this study, we show that cotransin, a substrate-selective Sec61 inhibitor, traps nascent TMDs in the cytosolic vestibule, permitting detailed interrogation of an early pre-integration intermediate. Site-specific crosslinking revealed the pre-integrated TMD docked to Sec61 near the cytosolic tip of the lateral gate. Escape from cotransin-arrest depends not only on cotransin concentration, but also on the biophysical properties of the TMD. Genetic selection of cotransin-resistant cancer cells uncovered multiple mutations clustered near the lumenal plug of Sec61α, thus revealing cotransin’s likely site of action. Our results suggest that TMD/lateral gate interactions facilitate TMD transfer into the membrane, a process that is allosterically modulated by cotransin binding to the plug.
Cells are surrounded by a plasma membrane that acts like a barrier around the cell—keeping the cell’s boundaries distinct from surrounding cells and helping to regulate the contents of the cell. This plasma membrane is made up mostly of two layers of fatty molecules, and is also studded with proteins. Some of these membrane proteins act as channels that allow nutrients and other chemicals to enter and leave the cell, while others allow the cell to communicate with other cells and the outside environment.
Like all proteins, membrane proteins are chains of amino acids that are linked together by a molecular machine called a ribosome. The ribosomes that make membrane proteins are located on the outside of a membrane-enclosed compartment within the cell called the endoplasmic reticulum. To eventually become embedded within a membrane, a new protein must—at the same time as it is being built—enter a channel within the membrane of the endoplasmic reticulum. The newly synthesized protein chain enters this channel, called Sec61, via an entrance near the ribosome and then threads its way toward the inside of the endoplasmic reticulum. However, there is also a ‘side-gate’ in Sec61 that allows specific segments the new protein to escape the channel and become embedded within the membrane. From here, the membrane protein can be trafficked to other destinations within the cell, including the plasma membrane. However, how the newly forming protein chain passes through the side-gate of Sec61 is not well understood.
Now MacKinnon, Paavilainen et al. have used a small molecule called cotransin—which is known to interfere with the passage of proteins through Sec61—to observe the interactions between the Sec61 channel and the new protein. Cotransin appears to trap the new protein chain within the Sec61 channel by essentially ‘locking’ the side-gate.
MacKinnon, Paavilainen et al. observed that the trapped protein interacts with the inside of the channel at the end closest to the ribosome—which is the likely location of the side-gate. In contrast, cotransin likely binds at the other end of the channel, to a piece of Sec61 that serves to plug the exit into the endoplasmic reticulum; and this plug is directly connected to the side-gate. By preventing the plug from moving out of the way, cotransin can somehow stop the new protein from passing through the side-gate. However, MacKinnon, Paavilainen et al. did find that some membrane proteins with certain physical and chemical properties could get through the gate, despite the presence of cotransin. The next challenge is to resolve exactly how interactions between cotransin and the Sec61 plug can block the escape of new proteins into the membrane.
cotransin; transmembrane domain; endoplasmic reticulum; human
The 4 kDa amyloid β-peptide (Aβ) is strongly implicated the pathogenesis of Alzheimer’s disease (AD), and this peptide is cut out of the amyloid β-protein precursor (APP) by the sequential action of β- and γ-secretases. γ-Secretase is a membrane-embedded protease complex that cleaves the transmembrane region of APP to produce Aβ, and this protease is a top target for developing AD therapeutics. A number of inhibitors of the γ-secretase complex have been identified, including peptidomimetics that block the active site, helical peptides that interact with the initial substrate docking site, and other less peptide-like, more drug-like compounds. To date, one γ-secretase inhibitor has advanced into late-phase clinical trials for the treatment of AD, but serious concerns remain. The γ-secretase complex cleaves a number of other substrates, and γ-secretase inhibitors cause in vivo toxicities by blocking proteolysis of one essential substrate, the Notch receptor. Thus, compounds that modulate γ-secretase, rather than inhibit it, to selectively alter Aβ production without hindering signal transduction from the Notch receptor would be more ideal. Such modulators have been discovered and advanced, with one compound in late-phase clinical trials, renewing interest in γ-secretase as a therapeutic target.
Secondary active transporters from several protein families share a core of two five-helix inverted repeats that has become known as the LeuT fold. The known high-resolution protein structures with this fold were analyzed by structural superposition of the core transmembrane domains (TMDs). Three angle parameters derived from the mean TMD axes correlate with accessibility of the central binding site from the outside or inside. Structural transitions between distinct conformations were analyzed for four proteins in terms of changes in relative TMD arrangement and in internal conformation of TMDs. Collectively moving groups of TMDs were found to be correlated in the covariance matrix of elastic network models. The main features of the structural transitions can be reproduced with the 5 % slowest normal modes of anisotropic elastic network models. These results support the rocking bundle model for the major conformational change between the outward- and inward-facing states of the protein and point to an important role for the independently moving last TMDs of each repeat in occluding access to the central binding site. Occlusion is also supported by flexing of some individual TMDs in the collectively moving bundle and hash motifs.
Electronic supplementary material
The online version of this article (doi:10.1007/s00249-012-0802-z) contains supplementary material, which is available to authorized users.
Membrane proteins; Secondary active transport; Elastic network models; Protein structure; Protein dynamics
γ-Secretase cleaves the carboxyl-terminal fragment (βCTF) of APP not only in the middle of the transmembrane domain (γ-cleavage), but also at sites close to the membrane/cytoplasm boundary (ε-cleavage), to produce the amyloid β protein (Aβ) and the APP intracellular domain (AICD), respectively. The AICD49–99 and AICD50–99 species were identified as counterparts of the long Aβ species Aβ48 and Aβ49, respectively. We found that Aβ40 and AICD50–99 were the predominant species in cells expressing wild-type APP and presenilin, whereas the production of Aβ42 and AICD49–99 was enhanced in cells expressing familial Alzheimer's disease mutants of APP and presenilin. These long Aβ species were identified in cell lysates and mouse brain extracts, which suggests that ε-cleavage is the first cleavage of βCTF to produce Aβ by γ-secretase. Here, we review the progress of research on the mechanism underlying the proteolysis of the APP transmembrane domain based on tri- and tetrapeptide release.
γ-Secretase is an unusual protease with an intramembrane catalytic site that cleaves many type I membrane proteins, including the amyloid β-protein (Aβ) precursor (APP) and the Notch receptor. Genetic and biochemical studies have identified four membrane proteins as components of γ-secretase: heterodimeric presenilin composed of its N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2. Here we demonstrated that certain compounds, including protein kinase inhibitors and their derivatives, act directly on purified γ-secretase to selectively block cleavage of APP- but not Notch-based substrates. Moreover, ATP activated the generation of the APP intracellular domain and Aβ, but not the generation of the Notch intracellular domain by the purified protease complex, and was a direct competitor of the APP-selective inhibitors, as were other nucleotides. In accord, purified γ-secretase bound specifically to an ATP-linked resin. Finally, a photoactivable ATP analog specifically labeled presenilin 1-C-terminal fragments in purified γ-secretase preparations; the labeling was blocked by ATP itself and APP-selective γ-secretase inhibitors. We concluded that a nucleotide-binding site exists within γ-secretase, and certain compounds that bind to this site can specifically modulate the generation of Aβ while sparing Notch. Drugs targeting the γ-secretase nucleotide-binding site represent an attractive strategy for safely treating Alzheimer disease.
Several familial Alzheimer disease (FAD) mutations within the transmembrane region of the amyloid precursor protein (APP) increase the Aβ42/40 ratio without increasing total Aβ production. In the present study, we analyzed the impact of FAD mutations and γ-secretase modulators (GSMs) that alter the Aβ42/40 ratio on APP C-terminus (CT) positioning relative to the membrane, reasoning that changes in the alignment of the APP intramembranous domain and presenilin 1 (PS1) may impact the PS1/γ-secretase cleavage site on APP.
By using a Förster resonance energy transfer (FRET)-based technique, fluorescent lifetime imaging microscopy (FLIM), we show that Aβ42/40 ratio-modulating factors which target either APP substrate or PS1/γ-secretase affect proximity of the APP-CT to the membrane and change PS1 conformation.
Thus, we propose that there is a reciprocal relationship between APP-CT positioning relative to the membrane and PS1 conformation, suggesting that factors that modulate either APP positioning in the membrane or PS1 conformation could be exploited therapeutically.
Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a “finger” domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an “open” conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a “closed” conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins.
γ-secretase is a large ubiquitously expressed protease complex composed of four core subunits: presenilin, Aph1, PEN-2, and nicastrin. The function of γ-secretase in the cells is to proteolytically cleave various proteins within their transmembrane domains. Presenilin and Aph1 occur as alternative variants belonging to mutually exclusive γ-secretase complexes and providing the complexes with heterogeneous biochemical and physiological properties. γ-secretase is proposed to have a role in the development and progression of cancer and γ-secretase inhibitors are intensively studied for their probable anti-tumor effects in various types of cancer models. Here, we for the first time determined mRNA expression levels of presenilin-1, presenilin-2, Aph1a, Aph1b, PEN-2, and nicastrin in a set of breast cancer tissue samples (N = 55) by quantitative real-time PCR in order to clarify the clinical significance of the expression of different γ-secretase complex components in breast cancer. We found a high positive correlation between the subunit expression levels implying a common regulation of transcription. Our univariate Kaplan-Meier survival analyses established low expression level of γ-secretase complex as a risk factor for breast cancer specific mortality. The tumors expressing low levels of γ-secretase complex were characterized by high histopathological tumor grade, low or no expression of estrogen and progesterone receptors and consequently high probability to fall into the class of triple negative breast cancer tumors. These results may provide novel tools to further categorize breast cancer tumors, especially the highly aggressive and poorly treatable breast cancer type of triple negative cases, and suggest a significant role for γ-secretase in breast cancer.
The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues1–4. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis5–7, and mutations in presenilin are associated with Alzheimer's disease8–10. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6Å resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related11,12. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.
Presenilin 1(PS1) is the catalytic subunit of γ-secretase, the enzyme responsible for the Aβ C-terminal cleavage site, which results in the production of Aβ peptides of various lengths. Production of longer forms of the Aβ peptide occur in patients with autosomal dominant Alzheimer disease (AD) due to mutations in presenilin. Many modulators of γ-secretase function have been described. We hypothesize that these modulators act by a common mechanism by allosterically modifying the structure of presenilin.
To test this hypothesis we generated a genetically encoded GFP-PS1-RFP (G-PS1-R) FRET probe that allows monitoring of the conformation of the PS1 molecule in its native environment in live cells. We show that G-PS1-R can be incorporated into the γ-secretase complex, reconstituting its activity in PS1/2 deficient cells. Using Förster resonance energy transfer (FRET)-based approaches we show that various pharmacological and genetic manipulations that target either γ-secretase components (PS1, Pen2, Aph1) or γ-secretase substrate (amyloid precursor protein, APP) and are known to change Aβ42 production are associated with a consistent conformational change in PS1.
These results strongly support the hypothesis that allosteric changes in PS1 conformation underlie changes in the Aβ42/40 ratio. Direct measurement of physiological and pathological changes in the conformation of PS1/γ-secretase may provide insight into molecular mechanism of Aβ42 generation, which could be exploited therapeutically.
The amyloid-β peptide (Aβ), implicated in the pathogenesis of Alzheimer's disease (AD), is produced through sequential proteolysis of the Aβ precursor protein (APP) by β- and γ-secretases. Thus, blocking either of these two proteases, directly or indirectly, is potentially worthwhile toward developing AD therapeutics. β-Secretase is a membrane-tethered pepsin-like aspartyl protease suitable for structure-based design, whereas γ-secretase is an unusual, heterotetrameric membrane-embedded aspartyl protease. While γ-secretase inhibitors entered clinical trials first due to their superior pharmacological properties (for example, brain penetration) over β-secretase inhibitors, it has since become clear that γ-secretase inhibitors can cause mechanism-based toxicities owing to interference with the proteolysis of another γ-secretase substrate, the Notch receptor. Strategies for targeting Aβ production at the γ-secretase level without blocking Notch signalling will be discussed. Other strategies utilizing cell-based screening have led to the identification of novel Aβ lowering agents that likewise leave Notch proteolysis intact. The mechanism by which these agents lower Aβ is unknown, but these compounds may ultimately reveal new targets for AD therapeutics.
The presenilins are transmembrane proteins that, as part of a large protein complex, regulate the cleavage of other transmembrane proteins, notably the receptor Notch and the β-amyloid precursor protein. Mutations in presenilin genes increase the production of neurotoxic forms of the amyloid β peptide and contribute to 20-50% of early-onset cases of inherited Alzheimer's disease.
The presenilins are evolutionarily conserved transmembrane proteins that regulate cleavage of certain other proteins in their transmembrane domains. The clinical significance of this regulation is shown by the contribution of presenilin mutations to 20-50% of early-onset cases of inherited Alzheimer's disease. Although the precise molecular mechanism underlying presenilin function or dysfunction remains elusive, presenilins are thought to be part of a complex of proteins that has 'γ-secretase cleavage' activity, which is clearly central in the pathogenesis of Alzheimer's disease. Mutations in presenilins increase the production of the longer isoforms of amyloid β peptide, which are neurotoxic and prone to self-aggregation. Biochemical studies indicate that the presenilins do not act alone but operate within large heteromeric protein complexes, whose components and enzymatic core are the subject of much study and controversy; one essential component is nicastrin. The presenilin primary sequence is remarkably well conserved in eukaryotes, suggesting some functional conservation; indeed, defects caused by mutations in the nemotode presenilin homolog can be rescued by human presenilin.
The presenilin/γ-secretase complex, an unusual intramembrane aspartyl protease, plays an essential role in cellular signaling and membrane protein turnover. Its ability to liberate numerous intracellular signaling proteins from the membrane and also mediate the secretion of amyloid-β protein (Aβ) has made modulation of γ-secretase activity a therapeutic goal for cancer and Alzheimer disease. Although the proteolysis of the prototypical substrates Notch and β-amyloid precursor protein (APP) has been intensely studied, the full spectrum of substrates and the determinants that make a transmembrane protein a substrate remain unclear. Using an unbiased approach to substrate identification, we surveyed the proteome of a human cell line for targets of γ-secretase and found a relatively small population of new substrates, all of which are type I transmembrane proteins but have diverse biological roles. By comparing these substrates to type I proteins not regulated by γ-secretase, we determined that besides a short ectodomain, γ-secretase requires permissive transmembrane and cytoplasmic domains to bind and cleave its substrates. In addition, we provide evidence for at least two mechanisms that can target a substrate for γ cleavage: one in which a substrate with a short ectodomain is directly cleaved independent of sheddase association, and a second where a substrate requires ectodomain shedding to instruct subsequent γ-secretase processing. These findings expand our understanding of the mechanisms of substrate selection as well as the diverse cellular processes to which γ-secretase contributes.
All cells face the challenge of removing transmembrane proteins from the lipid bilayer for the purpose of signaling or degradation. One molecular solution to this problem is the multiprotein enzyme complex γ-secretase, which is able to hydrolyze several known transmembrane proteins within the hydrophobic lipid environment. Due to its central role in the pathogenesis of Alzheimer disease, modulation of γ-secretase activity has become a therapeutic goal. However, the number and diversity of proteins that can be cleaved by this protease remain unknown, and the attributes that target these proteins to γ-secretase are unclear. In this study, we used an unbiased approach to substrate identification and surveyed the proteome for targets of γ-secretase. Of the thousands of proteins detectable, only a relative few were substrates of γ-secretase, all of which were type I transmembrane proteins. In addition to validating several of these novel substrates, we compared them to other proteins that we identified as nonsubstrates and determined that there are specific domains that can activate or inhibit γ-secretase processing. These findings should advance our understanding of the many cellular processes regulated by γ-secretase and may offer insights into how γ-secretase can be exploited for therapeutic purposes.
Using an unbiased quantitative proteomics approach, novel substrate targets for the protease γ-secretase are identified and analyzed to determine which domains enable their cleavage.
The γ-secretase complex, consisting of presenilin, nicastrin, presenilin enhancer-2 (PEN-2), and anterior pharynx defective-1 (APH-1) cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. The regulatory mechanisms governing the multistep assembly of this “proteasome of the membrane” are unknown. We characterize a new interaction partner of nicastrin, the retrieval receptor Rer1p. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 substantially increased binding of nicastrin to Rer1p, demonstrating the competitive nature of these interactions. Moreover, Rer1p expression levels control the formation of γ-secretase subcomplexes and, concomitantly, total cellular γ-secretase activity. We identify Rer1p as a novel limiting factor that negatively regulates γ-secretase complex assembly by competing with APH-1 during active recycling between the endoplasmic reticulum (ER) and Golgi. We conclude that total cellular γ-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value.