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.     

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 forms a homodimer that is labeled by an active site-directed gamma-secretase inhibitor

Presenilin (PS) is the presumptive catalytic component of the intramembrane aspartyl protease gamma-secretase complex. Recently a family of presenilin homologs was identified. One member of this family, signal peptide peptidase (SPP), has been shown to be a protease, which supports the hypothesis that PS and presenilin homologs are related intramembrane-cleaving aspartyl proteases. SPP has been reported as a glycoprotein of approximately 45 kDa. Our initial characterization of SPP isolated from human brain and cell lines demonstrated that SPP is primarily present as an SDS-stable approximately 95-kDa protein on Western blots. Upon heating or treatment of this approximately 95-kDa SPP band with acid, a approximately 45-kDa band could be resolved. Co-purification of two different epitope-tagged forms of SPP from a stably transfected cell line expressing both tagged versions demonstrated that the approximately 95-kDa band is a homodimer of SPP. Pulse-chase metabolic labeling studies demonstrated that the SPP homodimer assembles rapidly and is metabolically stable. In a glycerol velocity gradient, SPP sedimented from approximately 100-200 kDa. Significantly the SPP homodimer was specifically labeled by an active site-directed photoaffinity probe (III-63) for PS, indicating that the active sites of SPP and PS/gamma-secretase are similar and providing strong evidence that the homodimer is functionally active. Collectively these data suggest that SPP exists in vivo as a functional dimer.     

Regulated intramembrane proteolysis of Bri2 (Itm2b) by ADAM10 and SPPL2a/SPPL2b

Presenilin, the catalytic component of the gamma-secretase complex, type IV prepilin peptidases, and signal peptide peptidase (SPP) are the founding members of the family of intramembrane-cleaving GXGD aspartyl proteases. SPP-like (SPPL) proteases, such as SPPL2a, SPPL2b, SPPL2c, and SPPL3, also belong to the GXGD family. In contrast to gamma-secretase, for which numerous substrates have been identified, very few in vivo substrates are known for SPP and SPPLs. Here we demonstrate that Bri2 (Itm2b), a type II-oriented transmembrane protein associated with familial British and Danish dementia, undergoes regulated intramembrane proteolysis. In addition to the previously described ectodomain processing by furin and related proteases, we now describe that the Bri2 protein, similar to gamma-secretase substrates, undergoes an additional cleavage by ADAM10 in its ectodomain. This cleavage releases a soluble variant of Bri2, the BRICHOS domain, which is secreted into the extracellular space. Upon this shedding event, a membrane-bound Bri2 N-terminal fragment remains, which undergoes intramembrane proteolysis to produce an intracellular domain as well as a secreted low molecular weight C-terminal peptide. By expressing all known SPP/SPPL family members as well as their loss of function variants, we demonstrate that selectively SPPL2a and SPPL2b mediate the intramembrane cleavage, whereas neither SPP nor SPPL3 is capable of processing the Bri2 N-terminal fragment.     

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.     

Gamma-secretase inhibition

Development of Alzheimer's disease (AD) pathology appears to be causally related to age-dependent changes in the metabolism of the amyloid-beta peptide (A beta), leading to its enhanced aggregation and deposition. gamma-Secretase is a crucial enzyme for the generation of A beta from the amyloid-beta precursor protein and thus represents a valid potential therapeutic target for the treatment or prevention of AD. Enzyme activity has been shown to be dependent on the expression of presenilins and the identification of inhibitors containing transition-state analogue mimics, together with mutagenesis and knockout studies, confirms that presenilins may provide at least a component of the catalytic site for this putative aspartyl protease. Considerable effort has been expended to identify compounds which specifically reduce gamma-secretase activity in the central nervous system, and those with the appropriate properties are being utilized in on-going proof-of-concept studies in animals and humans, to determine the extent and duration of gamma-secretase inhibition required to elicit therapeutic benefits. gamma-Secretase-mediated substrate cleavage appears to fall into the category of 'regulated intramembrane proteolysis'. By virtue of its mechanistic similarities, the effects of gamma-secretase inhibitors on proteolysis and signalling through other substrates, such as Notch, has to be determined carefully, since this is likely to impact on the clinically safe dose of these compounds.     

A gamma-secretase-like intramembrane cleavage of TNFalpha by the GxGD aspartyl protease SPPL2b

Gamma-secretase and signal peptide peptidase (SPP) are unusual GxGD aspartyl proteases, which mediate intramembrane proteolysis. In addition to SPP, a family of SPP-like proteins (SPPLs) of unknown function has been identified. We demonstrate that SPPL2b utilizes multiple intramembrane cleavages to liberate the intracellular domain of tumor necrosis factor alpha (TNFalpha) into the cytosol and the carboxy-terminal counterpart into the extracellular space. These findings suggest common principles for regulated intramembrane proteolysis by GxGD aspartyl proteases.     

C-terminal fragment of presenilin is the molecular target of a dipeptidic gamma-secretase-specific inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester)

Gamma-secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1 and, Pen-2 that is responsible for the intramembrane proteolysis of various type I transmembrane proteins, including amyloid beta-precursor protein and Notch. The direct labeling of PS polypeptides by transition-state analogue gamma-secretase inhibitors suggested that PS represents the catalytic center of gamma-secretase. Here we show that one of the major gamma-secretase inhibitors of dipeptidic type, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), targets the C-terminal fragment of PS, especially the transmembrane domain 7 or more C-terminal region, by designing and synthesizing DAP-BpB (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-(S)-phenylglycine-4-(4-(8-biotinamido)octylamino)benzoyl)benzyl)methylamide), a photoactivable DAPT derivative. We also found that DAP-BpB selectively binds to the high molecular weight gamma-secretase complex in an activity-dependent manner. Photolabeling of PS by DAP-BpB is completely blocked by DAPT or its structural relatives (e.g. Compound E) as well as by arylsulfonamides. In contrast, transition-state analogue inhibitor L-685,458 or alpha-helical peptidic inhibitor attenuated the photolabeling of PS1 only at higher concentrations. These data illustrate the DAPT binding site as a novel functional domain within the PS C-terminal fragment that is distinct from the catalytic site or the substrate binding site.     

Intra- or intercomplex binding to the gamma-secretase enzyme. A model to differentiate inhibitor classes

Gamma-secretase is one of the critical enzymes required for the generation of amyloid-beta peptides from the beta-amyloid precursor protein. Because amyloid-beta peptides are generally accepted to play a key role in Alzheimer disease, gamma-secretase inhibition holds the promise for a disease-modifying therapy for this neurodegenerative condition. Although recent progress has enhanced the understanding of the biology and composition of the gamma-secretase enzyme complex, less information is available on the actual interaction of various inhibitor classes with the enzyme. Here we show that the two principal classes of inhibitor described in the scientific and patent literature, aspartyl protease transition state analogue and small molecule non-transition state inhibitors, display fundamental differences in the way they interact with the enzyme. Taking advantage of a gamma-secretase enzyme overexpressing cellular system and different radiolabeled gamma-secretase inhibitors, we observed that the maximal binding of non-transition state gamma-secretase inhibitors accounts only for half the number of catalytic sites of the recombinant enzyme complex. This characteristic stoichiometry can be best accommodated with a model whereby the non-transition state inhibitors bind to a unique site at the interface of a dimeric enzyme. Subsequent competition studies confirm that this site appears to be targeted by the main classes of small molecule gamma-secretase inhibitor. In contrast, the non-steroidal anti-inflammatory drug gamma-secretase modulator sulindac sulfide displayed noncompetitive antagonism for all types of inhibitor. This finding suggests that non-steroidal anti-inflammatory drug-type gamma-secretase modulators target an alternative site on the enzyme, thereby changing the conformation of the binding sites for gamma-secretase inhibitors.     

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.     

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.     

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.     

Foamy Virus Envelope Protein Is a Substrate for Signal Peptide Peptidase-like 3 (SPPL3)

Signal peptide peptidase (SPP), its homologs, the SPP-like proteases SPPL2a/b/c and SPPL3, as well as presenilin, the catalytic subunit of the γ-secretase complex, are intramembrane-cleaving aspartyl proteases of the GxGD type. In this study, we identified the 18-kDa leader peptide (LP18) of the foamy virus envelope protein (FVenv) as a new substrate for intramembrane proteolysis by human SPPL3 and SPPL2a/b. In contrast to SPPL2a/b and γ-secretase, which require substrates with an ectodomain shorter than 60 amino acids for efficient intramembrane proteolysis, SPPL3 cleaves mutant FVenv lacking the proprotein convertase cleavage site necessary for the prior shedding. Moreover, the cleavage product of FVenv generated by SPPL3 serves as a new substrate for consecutive intramembrane cleavage by SPPL2a/b. Thus, human SPPL3 is the first GxGD-type aspartyl protease shown to be capable of acting like a sheddase, similar to members of the rhomboid family, which belong to the class of intramembrane-cleaving serine proteases.     

Conserved residues within the putative active site of gamma-secretase differentially influence enzyme activity and inhibitor binding

Gamma-secretase performs the final processing step in the generation of amyloid-beta (Abeta) peptides, which are believed to be causative for Alzheimer's disease. Presenilins (PS) are required for gamma-secretase activity and the presence of two essential intramembranous aspartates (D257 and D385) has implicated this region as the putative catalytic centre of an aspartyl protease. The presence of several key hydrogen-bonding residues around the active site of classical aspartyl proteases led us to investigate the role of both the critical aspartates and two nearby conserved hydrogen bond donors in PS1. Generation of cell lines stably overexpressing the D257E, D385E, Y256F and Y389F engineered mutations has enabled us to determine their role in enzyme catalysis and binding of a transition state analogue gamma-secretase inhibitor. Here we report that replacement of either tyrosine residue alters gamma-secretase cleavage specificity, resulting in an increase in the production of the more pathogenic Abeta42 peptide in both cells and membranous enzyme preparations, without affecting inhibitor binding. In contrast, replacement of either of the aspartate residues precludes inhibitor binding in addition to inactivation of the enzyme. Together, these data further incriminate the region around the intramembranous aspartates as the active site of the enzyme, targeted by transition state analogue inhibitors, and highlight the roles of individual residues.     

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.     

Mechanism of gamma-secretase cleavage activation: is gamma-secretase regulated through autoinhibition involving the presenilin-1 exon 9 loop?

Maturation of gamma-secretase requires an endoproteolytic cleavage in presenilin-1 (PS1) within a peptide loop encoded by exon 9 of the corresponding gene. Deletion of the loop has been demonstrated to cause familial Alzheimer's disease. A synthetic peptide corresponding to the loop sequence was found to inhibit gamma-secretase in a cell-free enzymatic assay with an IC(50) of 2.1 microM, a value similar to the K(m) (3.5 microM) for the substrate C100. Truncation at either end, single amino acid substitutions at certain residues, sequence reversal, or randomization reduced its potency. Similar results were also observed in a cell-based assay using HEK293 cells expressing APP. In contrast to small-molecule gamma-secretase inhibitors, kinetic inhibition studies demonstrated competitive inhibition of gamma-secretase by the exon 9 peptide. Consistent with this finding, inhibitor cross-competition kinetics indicated noncompetitive binding between the exon 9 peptide and L685458, a transition-state analogue presumably binding at the catalytic site, and ligand competition binding experiments revealed no competition between L685458 and the exon 9 peptide. These data are consistent with the proposed gamma-secretase mechanism involving separate substrate-binding and catalytic sites and binding of the exon 9 peptide at the substrate-binding site, but not the catalytic site of gamma-secretase. NMR analyses demonstrated the presence of a loop structure with a beta-turn in the middle of the exon 9 peptide and a loose alpha-helical conformation for the rest of the peptide. Such a structure supports the hypothesis that this exon 9 peptide can adopt a distinct conformation, one that is compact enough to occupy the putative substrate-binding site without necessarily interfering with binding of small molecule inhibitors at other sites on gamma-secretase. We hypothesize that gamma-secretase cleavage activation may be a result of a cleavage-induced conformational change that relieves the inhibitory effect of the intact exon 9 loop occupying the substrate-binding site on the immature enzyme. It is possible that the DeltaE9 mutation causes Alzheimer's disease because cleavage activation of gamma-secretase is no longer necessary, alleviating constraints on Abeta formation.     

Are presenilins intramembrane-cleaving proteases? Implications for the molecular mechanism of Alzheimer's disease.

The amyloid-beta protein (Abeta) is strongly implicated in the pathogenesis of Alzheimer's disease. The final step in the production of Abeta from the amyloid precursor protein (APP) is proteolysis by the unidentified gamma-secretases. This cleavage event is unusual in that it apparently occurs within the transmembrane region of the substrate. Studies with substrate-based inhibitors together with molecular modeling and mutagenesis of the gamma-secretase cleavage site of APP suggest that gamma-secretases are aspartyl proteases that catalyze a novel intramembranous proteolysis. This proteolysis requires the presenilins, proteins with eight transmembrane domains that are mutated in most cases of autosomal dominant familial Alzheimer's disease. Two conserved transmembrane aspartates in presenilins are essential for gamma-secretase activity, suggesting that presenilins themselves are gamma-secretases. Moreover, presenilins also mediate the apparently intramembranous cleavage of the Notch receptor, an event critical for Notch signaling and embryonic development. Thus, if presenilins are gamma-secretases, then they are also likely the proteases that cleave Notch within its transmembrane domain. Another protease, S2P, involved in the processing of the sterol regulatory element binding protein, is also a multipass integral membrane protein which cleaves within or very close to the transmembrane region of its substrate. Thus, presenilins and S2P appear to be members of a new type of polytopic protease with an intramembranous active site.     

Impas 1 possesses endoproteolytic activity against multipass membrane protein substrate cleaving the presenilin 1 holoprotein

Presenilins (PS1 and PS2) are supposed to be unusual aspartic proteases and components of the gamma-secretase complex regulating cleavage of type I proteins. Multiple mutations in PS1 are a major cause of familial early-onset Alzheimer's disease (AD). We and others recently identified PS-related families of proteins (IMPAS/PSH/signal peptide peptidases (SPP)). The functions of these proteins are yet to be determined. We found that intramembrane protease-associated or intramembrane protease aspartic protein Impas 1 (IMP1)/SPP induces intramembranous cleavage of PS1 holoprotein in cultured cells coexpressing these proteins. Mutations in evolutionary invariant sites in hIMP1 or specific gamma-secretase inhibitors abolish the hIMP1-mediated endoproteolysis of PS1. In contrast, neither AD-like mutations in hIMP1 nor in PS1 substrate abridge the PS1 cleavage. The data suggest that IMP1 is a bi-aspartic polytopic protease capable of cleaving transmembrane precursor proteins. These data, to our knowledge, are a first observation that a multipass transmembrane protein or the integral protease per se may be a primary substrate for an intramembranous proteolysis.     

Targeting presenilin-type aspartic protease signal peptide peptidase with gamma-secretase inhibitors

Presenilin is implicated in the pathogenesis of Alzheimer's disease. It is thought to constitute the catalytic subunit of the gamma-secretase complex that catalyzes intramembrane cleavage of beta-amyloid precursor protein, the last step in the generation of amyloidogenic Abeta peptides. The latter are major constituents of amyloid plaques in the brain of Alzheimer's disease patients. Inhibitors of gamma-secretase are considered potential therapeutics for the treatment of this disease because they prevent production of Abeta peptides. Recently, we discovered a family of presenilin-type aspartic proteases. The founding member, signal peptide peptidase, catalyzes intramembrane cleavage of distinct signal peptides in the endoplasmic reticulum membrane of animals. In humans, the protease plays a crucial role in the immune system. Moreover, it is exploited by the hepatitis C virus for the processing of the structural components of the virion and hence is an attractive target for anti-infective intervention. Signal peptide peptidase and presenilin share identical active site motifs and both catalyze intramembrane proteolysis. These common features let us speculate that gamma-secretase inhibitors directed against presenilin may also inhibit signal peptide peptidase. Here we demonstrate that some of the most potent known gamma-secretase inhibitors efficiently inhibit signal peptide peptidase. However, we found compounds that showed higher specificity for one or the other protease. Our findings highlight the possibility of developing selective inhibitors aimed at reducing Abeta generation without affecting other intramembrane-cleaving aspartic proteases.