C-terminal PAL motif of presenilin and presenilin homologues required for normal active site conformation

The Alzheimer's disease-associated beta-amyloid peptide is produced through cleavage of amyloid precursor protein by beta-secretase and gamma-secretase. gamma-Secretase is a complex containing presenilin (PS) as the catalytic component and three essential cofactors: Nicastrin, anterior pharynx defective (APH-1) and presenilin enhancer-2 (PEN-2). PS and signal peptide peptidase (SPP) define a novel family of aspartyl proteases that cleave substrates within the transmembrane domain presumptively using two membrane-embedded aspartic acid residues for catalysis. Apart from the two aspartate-containing active site motifs, the only other region that is conserved between PS and SPP is a PAL sequence at the C-terminus. Although it has been well documented that this motif is essential for gamma-secretase activity, the mechanism underlying such a critical role is not understood. Here we show that mutations in this motif affect the conformation of the active site of gamma-secretase resulting in a complete loss of PS binding to a gamma-secretase transition state analog inhibitor, Merck C. Analogous mutations in SPP significantly inhibit its enzymatic activity. Furthermore, these mutations also abolish SPP binding to Merck C, indicating that SPP and gamma-secretase share a similar active site conformation, which is dependent on the PAL motif. Exploring the amino acid requirements within this motif reveals a very small side chain requirement, which is conserved during evolution. Together, these observations strongly support the hypothesis that the PAL motif contributes to the active site conformation of gamma-secretase and of SPP.     

Aspartyl protease inhibitor pepstatin binds to the presenilins of Alzheimer's disease.

Mutations in the presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease by altering gamma-secretase cleavage of the amyloid precursor protein, the last step in the generation of Abeta peptide. Ablation of presenilin (PS) genes, or mutation of two critical aspartates, abolishes gamma-secretase cleavage, suggesting that PS may be the gamma-secretases. Independently, inhibition experiments indicate that gamma-secretase is an aspartyl protease. To characterize the putative gamma-secretase activity associated with presenilins, lysates from human neuroblastoma SH-SY5Y and human brain homogenates were incubated with biotin derivatives of pepstatin, followed by immunoprecipitation of PS and associated proteins, and biotin detection by Western blotting. Precipitation with PS1 antibodies, directed to either N-terminal or loop regions, yielded the same 43 kDa band, of apparent molecular mass consistent with that of full-length PS1, although it may represent an aspartyl protease complexed with PS1. Incubation of cell lysates with pepstatin-biotin, followed by streptavidin precipitation and PS1 Western blotting, revealed PS1 fragments and full-length protein, indicating that pepstatin-biotin bound to both cleaved and uncleaved PS1. Binding could be competed by gamma-secretase inhibitor L-685,458 and could not be achieved with a PS1 mutant lacking the two transmembrane aspartates. Pepstatin-biotin was also shown to bind to PS2. PS1 was specifically absorbed to pepstatin-agarose, with an optimal pH of 6. Binding of pepstatin-biotin to PS1 from lymphocytes of a heterozygous carrier of pathologic exon 9 deletion was markedly decreased as compared to control lymphocytes, suggesting that this PS1 mutation altered the pepstatin binding site.     

Signal peptide peptidase: biochemical properties and modulation by nonsteroidal antiinflammatory drugs

Signal peptide peptidase (SPP) is an intramembrane aspartyl protease that cleaves remnant signal peptides after their release by signal peptidase. SPP contains active site motifs also found in presenilin, the catalytic component of the gamma-secretase complex of Alzheimer's disease. However, SPP has a membrane topology opposite that of presenilin, cleaves transmembrane substrates of opposite directionality, and does not require complexation with other proteins. Here we show that, upon isolation of membranes and solubilization with detergent, the biochemical characteristics of SPP are remarkably similar to gamma-secretase. The majority of the SPP-catalyzed cleavages occurred at a single site in a synthetic substrate based on the prolactin (Prl) signal sequence. However, as seen with cleavage of substrates by gamma-secretase, additional cuts at other minor sites are also observed. Like gamma-secretase, SPP is inhibited by helical peptidomimetics and apparently contains a substrate-binding site that is distinct from the active site. Surprisingly, certain nonsteroidal antiinflammatory drugs known to shift the site of proteolysis by gamma-secretase also alter the cleavage site of Prl by SPP. Together, these findings suggest that SPP and presenilin share certain biochemical properties, including a conserved drug-binding site for allosteric modulation of substrate proteolysis.     

Signal peptide peptidase and gamma-secretase share equivalent inhibitor binding pharmacology

The enzyme gamma-secretase has long been considered a potential pharmaceutical target for Alzheimer disease. Presenilin (the catalytic subunit of gamma-secretase) and signal peptide peptidase (SPP) are related transmembrane aspartyl proteases that cleave transmembrane substrates. SPP and gamma-secretase are pharmacologically similar in that they are targeted by many of the same small molecules, including transition state analogs, non-transition state inhibitors, and amyloid beta-peptide modulators. One difference between presenilin and SPP is that the proteolytic activity of presenilin functions only within a multisubunit complex, whereas SPP requires no additional protein cofactors for activity. In this study, gamma-secretase inhibitor radioligands were used to evaluate SPP and gamma-secretase inhibitor binding pharmacology. We found that the SPP enzyme exhibited distinct binding sites for transition state analogs, non-transition state inhibitors, and the nonsteroidal anti-inflammatory drug sulindac sulfide, analogous to those reported previously for gamma-secretase. In the course of this study, cultured cells were found to contain an abundance of SPP binding activity, most likely contributed by several of the SPP family proteins. The number of SPP binding sites was in excess of gamma-secretase binding sites, making it essential to use selective radioligands for evaluation of gamma-secretase binding under these conditions. This study provides further support for the idea that SPP is a useful model of inhibitory mechanisms and structure in the SPP/presenilin protein family.     

PEN-2 is an integral component of the gamma-secretase complex required for coordinated expression of presenilin and nicastrin

The Alzheimer disease-associated presenilin (PS) proteins apparently provide the active site of gamma-secretase, an unusual intramembrane-cleaving aspartyl protease. PSs principally occur as high molecular weight protein complexes that contain nicastrin (Nct) and additional so far unidentified components. Recently, PEN-2 has been implicated in gamma-secretase function. Here we identify PEN-2 as a critical component of PS1/gamma-secretase and PS2/gamma-secretase complexes. Strikingly, in the absence of PS1 and PS1/PS2, PEN-2 levels are strongly reduced. Similarly, PEN-2 levels are reduced upon RNA interference-mediated down-regulation of Nct. On the other side, down-regulation of PEN-2 by RNA interference is associated with reduced PS levels, impaired Nct maturation, and deficient gamma-secretase complex formation. We conclude that PEN-2 is an integral gamma-secretase complex component and that gamma-secretase complex components are expressed in a coordinated manner.     

Differential localization and identification of a critical aspartate suggest non-redundant proteolytic functions of the presenilin homologues SPPL2b and SPPL3

Signal peptide peptidase (SPP) is an unusual aspartyl protease that mediates clearance of signal peptides by proteolysis within the endoplasmic reticulum (ER). Like presenilins, which provide the proteolytically active subunit of the gamma-secretase complex, SPP contains a critical GXGD motif in its C-terminal catalytic center. Although SPP is known to be an aspartyl protease of the GXGD type, several presenilin homologues/SPP-like proteins (PSHs/SPPL) of unknown function have been identified by data base searches. We now investigated the subcellular localization and a putative proteolytic activity of PSHs/SPPLs in cultured cells and in an in vivo model. We demonstrate that SPPL2b is targeted through the secretory pathway to endosomes/lysosomes, whereas SPP and SPPL3 are restricted to the ER. As suggested by the differential subcellular localization of SPPL2b compared with SPP and SPPL3, we found distinct phenotypes upon antisense gripNA-mediated knockdown in zebrafish. spp and sppl3 knockdowns in zebrafish result in cell death within the central nervous system, whereas reduction of sppl2b expression causes erythrocyte accumulation in an enlarged caudal vein. Moreover, expression of D/A mutations of the putative C-terminal active sites of spp, sppl2, and sppl3 produced phenocopies of the respective knockdown phenotypes. Thus, our data suggest that all investigated PSHs/SPPLs are members of the novel family of GXGD aspartyl proteases. Furthermore, SPPL2b is shown to be the first member of the SPP/PSH/SPPL family that is not located within the ER but in endosomal/lysosomal vesicles.     

Presenilin endoproteolysis mediated by an aspartyl protease activity pharmacologically distinct from gamma-secretase

Presenilin (PS)-dependent gamma-secretase cleavage is the final proteolytic step in generating amyloid beta protein (A beta), a key peptide involved in the pathogenesis of Alzheimer's disease. PS undergoes endoproteolysis by an unidentified 'presenilinase' to generate the functional N-terminal and C-terminal fragment heterodimers (NTF/CTF) that may harbor the gamma-secretase active site. To better understand the relationship between presenilinase and gamma-secretase, we characterized the biochemical properties of presenilinase and compared them with those of gamma-secretase. Similar to gamma-secretase, presenilinase was most active at acidic pH 6.3. Aspartyl protease inhibitor pepstatin A blocked presenilinase activity with an IC50 of approximately 1 microM. Difluoroketone aspartyl protease transition state analogue MW167 was relatively selective for presenilinase (IC50 < 1 microM) over gamma-secretase (IC50-16 microM). Importantly, removing the transition state mimicking moiety simultaneously abolished both presenilinase and gamma-secretase inhibition, suggesting that presenilinase, like gamma-secretase, is an aspartyl protease. Interestingly, several of the most potent gamma-secretase inhibitors (IC50 = 0.3 or 20 nM) failed to block presenilinase activity. Although de novo generation of PS1 fragments coincided with production of A beta in vitro, blocking presenilinase activity without reducing pre-existing fragment levels permitted normal de novo generation of A beta and amyloid intracellular domain. Therefore, presenilinase has characteristics of an aspartyl protease, but this activity is distinct from gamma-secretase.     

Differential effects of inhibitors on the gamma-secretase complex. Mechanistic implications

Gamma-secretase is a protease complex of four integral membrane proteins, with presenilin (PS) as the apparent catalytic component, and this enzyme processes the transmembrane domains of a variety of substrates, including the amyloid beta-protein precursor and the Notch receptor. Here we explore the mechanisms of structurally diverse gamma-secretase inhibitors by examining their ability to displace an active site-directed photoprobe from PS heterodimers. Most gamma-secretase inhibitors, including a potent inhibitor of the PS-like signal peptide peptidase, blocked the photoprobe from binding to PS1, indicating that these compounds either bind directly to the active site or alter it through an allosteric interaction. Conversely, some reported inhibitors failed to displace this interaction, demonstrating that these compounds do not interfere with the protease by affecting its active site. Differential effects of the inhibitors with respect to photoprobe displacement and in cell-based and cell-free assays suggest that these compounds are important mechanistic tools for deciphering the workings of this intramembrane-cleaving protease complex and its similarity to other polytopic aspartyl proteases.     

A C-terminal region of signal peptide peptidase defines a functional domain for intramembrane aspartic protease catalysis

Intramembrane proteolysis is now firmly established as a prominent biological process, and structure elucidation is emerging as the new frontier in the understanding of these novel membrane-embedded enzymes. Reproducing this unusual hydrolysis within otherwise water-excluding transmembrane regions with purified proteins is a challenging prerequisite for such structural studies. Here we show the bacterial expression, purification, and reconstitution of proteolytically active signal peptide peptidase (SPP), a membrane-embedded enzyme in the presenilin family of aspartyl proteases. This finding formally proves that, unlike presenilin, SPP does not require any additional proteins for proteolysis. Surprisingly, the conserved C-terminal half of SPP is sufficient for proteolytic activity; purification and reconstitution of this engineered fragment of several SPP orthologues revealed that this region defines a functional domain for an intramembrane aspartyl protease. The discovery of minimal requirements for intramembrane proteolysis should facilitate mechanistic and structural analysis and help define general biochemical principles of hydrolysis in a hydrophobic environment.     

Gamma-secretase activity is associated with a conformational change of nicastrin

Gamma-secretase is a high molecular weight multicomponent protein complex with an unusual intramembrane-cleaving aspartyl protease activity. Gamma-secretase is intimately associated with Alzheimer disease because it catalyzes the proteolytic cleavage, which leads to the liberation of amyloid beta-peptide. At least presenilin (PS), Nicastrin (Nct), APH-1, and PEN-2 are constituents of the gamma-secretase complex, with PS apparently providing the active site of gamma-secretase. Expression of gamma-secretase complex components is tightly regulated, however little is known about the assembly of the complex. Here we demonstrate that Nct undergoes a major conformational change during the assembly of the gamma-secretase complex. The conformational change is directly associated with gamma-secretase function and involves the entire Nct ectodomain. Loss of function mutations generated by deletions failed to undergo the conformational change. Furthermore, the conformational alteration did not occur in the absence of PS. Our data thus suggest that gamma-secretase function critically depends on the structural "activation" of Nct.     

Vigilin interacts with signal peptide peptidase

ABSTRACT: BACKGROUND: Signal peptide peptidase (SPP), a member of the presenilin-like intra-membrane cleaving aspartyl protease family, migrates on Blue Native (BN) gels as 100 kDa, 200 kDa and 450 kDa species. SPP has recently been implicated in other non-proteolytic functions such as retro-translocation of MHC Class I molecules and binding of misfolded proteins in the endoplasmic reticulum (ER). These high molecular weight SPP complexes might contain additional proteins that regulate the proteolytic activity of SPP or support its non-catalytic functions. RESULTS: In this study, an unbiased iTRAQ-labeling mass spectrometry approach was used to identify SPP-interacting proteins. We found that vigilin, a ubiquitous multi-KH domain containing cytoplasmic protein involved in RNA binding and protein translation control, selectively enriched with SPP. Vigilin interacted with SPP and both proteins co-localized in restricted intracellular domains near the ER, biochemically co-fractionated and were part of the same 450 kDa complex on BN gels. However, vigilin does not alter the protease activity of SPP, suggesting that the SPP-vigilin interaction might be involved in the non-proteolytic functions of SPP. CONCLUSIONS: We have identified and validated vigilin as a novel interacting partner of SPP that could play an important role in the non-proteolytic functions of SPP. This data adds further weight to the idea that intramembrane-cleaving aspartyl proteases, such as presenilin and SPPs, could have other functions besides the proteolysis of short membrane stubs.     

Reconstitution of gamma-secretase activity

gamma-Secretase is a membrane protein complex with an unusual aspartyl protease activity that catalyses the regulated intramembranous cleavage of the beta-amyloid precursor protein (APP) to release the Alzheimer's disease (AD)-associated amyloid beta-peptide (Abeta) and the APP intracellular domain (AICD). Here we show the reconstitution of gamma-secretase activity in the yeast Saccharomyces cerevisiae, which lacks endogenous gamma-secretase activity. Reconstituted gamma-secretase activity depends on the presence of four complex components including presenilin (PS), nicastrin (Nct), APH-1 (refs 3-6) and PEN-2 (refs 4, 7), is associated with endoproteolysis of PS, and produces Abeta and AICD in vitro. Thus, the biological activity of gamma-secretase is reconstituted by the co-expression of human PS, Nct, APH-1 and PEN-2 in yeast.     

The presenilin proteins are components of multiple membrane-bound complexes that have different biological activities

Several lines of evidence have indicated that the presenilin proteins function within macromolecular complexes and are necessary for the regulated intramembranous proteolysis of certain type 1 transmembrane proteins, including the amyloid precursor protein, Notch, and p75. Data from multiple complementary experiments now suggest that there may be several distinct presenilin complexes. We show here that presenilin mutations and certain detergents affect the abundance and componentry of the presenilin complexes, and these structural effects correlate with their effects on gamma-secretase activity. Our data suggest that there are at least three complexes, including a approximately 150-kDa nicastrin-aph-1 complex (which is likely to be a precursor complex). There is a stable and abundant intermediate complex of approximately 440 kDa, which contains aph-1, pen-2, nicastrin, and PS1. However, it is the very low abundance, high mass (>/=670 kDa) heteromeric complexes that are associated with the highest gamma-secretase-specific activity.     

Distinct pharmacological effects of inhibitors of signal peptide peptidase and gamma-secretase

Signal peptide peptidase (SPP) and gamma-secretase are intramembrane aspartyl proteases that bear similar active site motifs but with opposite membrane topologies. Both proteases are inhibited by the same aspartyl protease transition-state analogue inhibitors, further evidence that these two enzymes have the same basic cleavage mechanism. Here we report that helical peptide inhibitors designed to mimic SPP substrates and interact with the SPP initial substrate-binding site (the "docking site") inhibit both SPP and gamma-secretase, but with submicromolar potency for SPP. SPP was labeled by helical peptide and transition-state analogue affinity probes but at distinct sites. Nonsteroidal anti-inflammatory drugs, which shift the site of proteolysis by SPP and gamma-secretase, did not affect the labeling of SPP or gamma-secretase by the helical peptide or transition-state analogue probes. On the other hand, another class of previously reported gamma-secretase modulators, naphthyl ketones, inhibited SPP activity as well as selective proteolysis by gamma-secretase. These naphthyl ketones significantly disrupted labeling of SPP by the helical peptide probe but did not block labeling of SPP by the transition-state analogue probe. With respect to gamma-secretase, the naphthyl ketone modulators allowed labeling by the transition-state analogue probe but not the helical peptide probe. Thus, the naphthyl ketones appear to alter the docking sites of both SPP and gamma-secretase. These results indicate that pharmacological effects of the four different classes of inhibitors (transition-state analogues, helical peptides, nonsteroidal anti-inflammatory drugs, and naphthyl ketones) are distinct from each other, and they reveal similarities and differences with how they affect SPP and gamma-secretase.     

Nicastrin expression in mouse peripheral tissues is not co-ordinated with presenilin and is high in muscle

Nicastrin was the first binding partner of presenilin (PS) shown to be a critical component of the presenilin/gamma-secretase complex essential in development and differentiation, and in generation of Alzheimer's disease Abeta amyloid peptide. To investigate the function of this glycoprotein, we compared nicastrin and presenilin protein expression in various mouse tissues. Western blot analysis of PS1, PS2 and nicastrin indicates their expression levels are not coordinated. In adult mouse, nicastrin is highly expressed in muscle membranes, whereas presenilin levels are very low. By Blue Native electrophoresis, a PS1 complex of 400 kDa was detected in lung, brain, thymus and heart; nicastrin was also detected as a 400-kDa complex in brain but in muscle it was detected with a complex mobility of 240 and 290 kDa, suggesting association with alternate protein complexes. Immunocytochemistry confirms strong intracellular expression of nicastrin in skeletal muscle and blood vessel smooth muscle. These findings suggest a function for nicastrin in muscle other than participation in the gamma-secretase complex.     

Presenilin 1 stabilizes the C-terminal fragment of the amyloid precursor protein independently of gamma-secretase activity

The cleavage of the transmembrane amyloid precursor protein (APP) by beta-secretase leaves the C-terminal fragment of APP, C99, anchored in the plasma membrane. C99 is subsequently processed by gamma-secretase, an unusual aspartyl protease activity largely dependent on presenilin (PS), generating the amyloid beta-peptide (Abeta) that accumulates in the brain of patients with Alzheimer's disease. It has been suggested that PS proteins are the catalytic core of this proteolytic activity, but a number of other proteins mandatory for gamma-secretase cleavage have also been discovered. The exact role of PS in the gamma-secretase activity remains a matter of debate, because cells devoid of PS still produce some forms of Abeta. Here, we used insect cells expressing C99 to demonstrate that the expression of presenilin 1 (PS1), which binds C99, not only increases the production of Abeta by these cells but also increases the intracellular levels of C99 to the same extent. Using pulse-chase experiments, we established that this results from an increased half-life of C99 in cells expressing PS1. In Chinese hamster ovary cells producing C99 from full-length human APP, similar results were observed. Finally, we show that a functional inhibitor of gamma-secretase does not alter the ability of PS1 to increase the intracellular levels of C99. This finding suggests that the binding of PS1 to C99 does not necessarily lead to its immediate cleavage by gamma-secretase, which could be a spatio-temporally regulated or an induced event, and provides biochemical evidence for the existence of a substrate-docking site on PS1.     

Two domains within the first putative transmembrane domain of presenilin 1 differentially influence presenilinase and gamma-secretase activity

Presenilins (PS) are thought to contain the active site for presenilinase endoproteolysis of PS and gamma-secretase cleavage of substrates. The structural requirements for PS incorporation into the gamma-secretase enzyme complex, complex stability and maturation, and appropriate presenilinase and gamma-secretase activity are poorly understood. We used rescue assays to identify sequences in transmembrane domain one (TM1) of PS1 required to support presenilinase and gamma-secretase activities. Swap mutations identified an N-terminal TM1 domain that is important for gamma-secretase activity only and a C-terminal TM1 domain that is essential for both presenilinase and gamma-secretase activities. Exchange of residues 95-98 of PS1 (sw95-98) completely abolishes both activities while the familial Alzheimer's disease mutation V96F significantly inhibits both activities. Reversion of residue 96 back to valine in the sw95-98 mutant rescues PS function, identifying V96 as the critical residue in this region. The TM1 mutants do not bind to an aspartyl protease transition state analog gamma-secretase inhibitor, indicating a conformational change induced by the mutations that abrogates catalytic activity. TM1 mutant PS1 molecules retain the ability to interact with gamma-secretase substrates and gamma-secretase complex members, although Nicastrin stability is decreased by the presence of these mutants. gamma-Secretase complexes that contain V96F mutant PS1 molecules display a partial loss of function for gamma-secretase that alters the ratio of amyloid-beta peptide species produced, leading to the amyloid-beta peptide aggregation that causes familial Alzheimer's disease.     

Co-expression of nicastrin and presenilin rescues a loss of function mutant of APH-1

gamma-Secretase is an intramembrane-cleaving aspartyl protease complex that mediates the final cleavage of beta-amyloid precursor protein to liberate the neurotoxic amyloid-beta peptide implicated in Alzheimer's disease. The four proteins presenilin (PS), nicastrin (NCT), APH-1, and PEN-2 are sufficient to reconstitute gamma-secretase activity in yeast. Although PS seems to contribute the catalytic core of the gamma-secretase complex, no distinct function could be attributed to the other components so far. In Caenorhabditis elegans, mutation of a glycine to an aspartic acid within a conserved GXXXG motif in the fourth transmembrane domain of APH-1 causes a loss of function phenotype. Surprisingly, we now found that the human homologue APH-1a carrying the equivalent mutation G122D is fully active in yeast co-expressing PS1, NCT, and PEN-2. To address this discrepancy, we expressed APH-1a G122D in HEK293 cells. As reported previously, overexpressed APH-1a G122D was not incorporated into the gamma-secretase complex. Separate overexpression of PS1, NCT, or PEN-2 together with APH-1a G122D allowed the formation of heterodimers lacking the other endogenous components. Only the combined overexpression of PS1 and NCT together with APH-1a G122D facilitated the formation of a fully active gamma-secretase complex. Under these conditions, APH-1a G122D supported the production of normal amounts of Abeta. We conclude that cooperative effects may stabilize a trim-eric complex of APH-1a G122D together with PS1 and NCT. Upon successful complex assembly, the GXXXG motif becomes dispensable for gamma-secretase activity.