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.     

Presenilin-1 affects trafficking and processing of betaAPP and is targeted in a complex with nicastrin to the plasma membrane

Amyloid beta-peptide (Abeta) is generated by the consecutive cleavages of beta- and gamma-secretase. The intramembraneous gamma-secretase cleavage critically depends on the activity of presenilins (PS1 and PS2). Although there is evidence that PSs are aspartyl proteases with gamma-secretase activity, it remains controversial whether their subcellular localization overlaps with the cellular sites of Abeta production. We now demonstrate that biologically active GFP-tagged PS1 as well as endogenous PS1 are targeted to the plasma membrane (PM) of living cells. On the way to the PM, PS1 binds to nicastrin (Nct), an essential component of the gamma-secretase complex. This complex is targeted through the secretory pathway where PS1-bound Nct becomes endoglycosidase H resistant. Moreover, surface-biotinylated Nct can be coimmunoprecipitated with PS1 antibodies, demonstrating that this complex is located to cellular sites with gamma-secretase activity. Inactivating PS1 or PS2 function by mutagenesis of one of the critical aspartate residues or by gamma-secretase inhibitors results in delayed reinternalization of the beta-amyloid precursor protein and its accumulation at the cell surface. Our data suggest that PS is targeted as a biologically active complex with Nct through the secretory pathway to the cell surface and suggest a dual function of PS in gamma-secretase processing and in trafficking.     

A presenilin-independent aspartyl protease prefers the gamma-42 site cleavage

beta-Amyloid peptides (Abeta40 and Abeta42) are the major constituents of amyloid plaques, which are one of the hallmarks of Alzheimer's disease (AD). The Abeta is derived from sequential cleavages of amyloid precursor protein (APP) by beta- and gamma-secretases. gamma-Secretase consists of at least four proteins where presenilins (PS1 and PS2 or PS) are the catalytic subunit involved in the gamma-site cleavage of APP. Secretion of both Abeta40 and Abeta42 is significantly reduced in PS1 knock-out cells and completely abolished in cells deficient for both PS1 and PS2. Consequently, both the PS proteins play essential roles in the production of the secretory of Abeta from cells. Recent studies in primary neurons, however, suggest that PSs are not required for intracellular Abeta42 accumulation; thus the intracellular Abeta42 appears to be generated in a PS-independent manner. Here we present the first biochemical evidence indicating that Abeta, especially Abeta42, can be generated in the absence of PS based on an in vitrogamma-secretase assay employing membranes prepared from PS-deficient Blastocyst-derived (BD) cells. This PS-independent gamma-secretase (PSIG) activity is sensitive to the changes in pH and displays an optimal activity at pH 6.0. Pepstatin A is a potent inhibitor for this proteolytic activity with IC50 of 1.2 nm and 0.4 nm for Abeta40 and Abeta42 generation, respectively. These results indicate that these PS-independent gamma-site cleavages are mediated by an aspartyl protease. More importantly, the PSIG activity displays a distinct preference in mediating the 42-site cleavage over the 40-site cleavage, thereby generating Abeta42 as the predominant product.     

PEN-2 enhances gamma-cleavage after presenilin heterodimer formation

The presenilin (PS) complex, including PS, nicastrin, APH-1 and PEN-2, is essential for gamma-secretase activity, which is required for amyloid beta-protein (Abeta) generation. However, the precise individual roles of the three cofactors in the PS complex in Abeta generation remain to be clarified. Here, to distinguish the roles of PS cofactors in gamma-secretase activity from those in PS endoproteolysis, we investigated their roles in the gamma-secretase activity reconstituted by the coexpression of PS N- and C-terminal fragments (NTF and CTF) in PS-null cells. We demonstrate that the coexpression of PS1 NTF and CTF forms the heterodimer and restores Abeta generation in PS-null cells. The generation of Abeta was saturable at a certain expression level of PS1 NTF/CTF, while the overexpression of PEN-2 alone resulted in a further increase in Abeta generation. Although PEN-2 did not enhance PS1 NTF/CTF heterodimer formation, PEN-2 expression reduced the IC50 of a specific gamma-secretase inhibitor, a transition state analogue, for Abeta generation, suggesting that PEN-2 expression enhances the affinity or the accessibility of the substrate to the catalytic site. Thus, our results strongly suggest that PEN-2 is not only an essential component of the gamma-secretase complex but also an enhancer of gamma-cleavage after PS heterodimer formation.     

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.     

Presenilin function and gamma-secretase activity

Alzheimer's disease (AD) is the most common form of dementia and is characterized pathologically by the accumulation of beta-amyloid (Abeta) plaques and neurofibrillary tangles in the brain. Genetic studies of AD first highlighted the importance of the presenilins (PS). Subsequent functional studies have demonstrated that PS form the catalytic subunit of the gamma-secretase complex that produces the Abeta peptide, confirming the central role of PS in AD biology. Here, we review the studies that have characterized PS function in the gamma-secretase complex in Caenorhabditis elegans, mice and in in vitro cell culture systems, including studies of PS structure, PS interactions with substrates and other gamma-secretase complex members, and the evidence supporting the hypothesis that PS are aspartyl proteases that are active in intramembranous proteolysis. A thorough knowledge of the mechanism of PS cleavage in the context of the gamma-secretase complex will further our understanding of the molecular mechanisms that cause AD, and may allow the development of therapeutics that can alter Abeta production and modify the risk for AD.     

Intramembrane proteolysis of GXGD-type aspartyl proteases is slowed by a familial Alzheimer disease-like mutation

More than 150 familial Alzheimer disease (FAD)-associated missense mutations in presenilins (PS1 and PS2), the catalytic subunit of the gamma-secretase complex, cause aberrant amyloid beta-peptide (Abeta) production, by increasing the relative production of the highly amyloidogenic 42-amino acid variant. The molecular mechanism behind this pathological activity is unclear, and different possibilities ranging from a gain of function to a loss of function have been discussed. gamma-Secretase, signal peptide peptidase (SPP) and SPP-like proteases (SPPLs) belong to the same family of GXGD-type intramembrane cleaving aspartyl proteases and share several functional similarities. We have introduced the FAD-associated PS1 G384A mutation, which occurs within the highly conserved GXGD motif of PS1 right next to the catalytically critical aspartate residue, into the corresponding GXGD motif of the signal peptide peptidase-like 2b (SPPL2b). Compared with wild-type SPPL2b, mutant SPPL2b slowed intramembrane proteolysis of tumor necrosis factor alpha and caused a relative increase of longer intracellular cleavage products. Because the N termini of the secreted counterparts remain unchanged, the mutation selectively affects the liberation of the intracellular processing products. In vitro experiments demonstrate that the apparent accumulation of longer intracellular cleavage products is the result of slowed sequential intramembrane cleavage. The longer cleavage products are still converted to shorter peptides, however only after prolonged incubation time. This suggests that FAD-associated PS mutation may also result in reduced intramembrane cleavage of beta-amyloid precursor protein (betaAPP). Indeed, in vitro experiments demonstrate slowed intramembrane proteolysis by gamma-secretase containing PS1 with the G384A mutation. As compared with wild-type PS1, the mutation selectively slowed Abeta40 production, whereas Abeta42 generation remained unaffected. Thus, the PS1 G384A mutation causes a selective loss of function by slowing the processing pathway leading to the benign Abeta40.     

Familial Alzheimer disease-linked presenilin 1 variants enhance production of both Abeta 1-40 and Abeta 1-42 peptides that are only partially sensitive to a potent aspartyl protease transition state inhibitor of "gamma-secretase"

Presenilin 1 (PS1) plays an essential role in intramembranous "gamma-secretase" processing of several type I membrane proteins, including the beta-amyloid precursor proteins (APP) and Notch1. In this report, we examine the activity of two familial Alzheimer's disease-linked PS1 variants on the production of secreted Abeta peptides and the effects of L-685,458, a potent gamma-secretase inhibitor, on inhibition of Abeta peptides from cells expressing these PS1 variants. We now report that PS1 variants enhance the production and secretion of both Abeta1-42 and Abeta1-40 peptides. More surprisingly, whereas the IC(50) for inhibition of Abeta1-40 peptide production from cells expressing wild-type PS1 is approximately 1.5 microm, cells expressing the PS1deltaE9 mutant PS1 exhibit an IC(50) of approximately 4 microm. Immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry reveal that the levels of Abeta1-43 peptides are elevated in medium of PS1deltaE9 cells treated with higher concentrations of inhibitor. The differential effects of wild-type and mutant PS1 on gamma-secretase production of Abeta peptides and the disparity in sensitivity of these peptides to a potent gamma-secretase suggest that PS may be necessary, but not sufficient, to catalyze hydrolysis at the scissile bonds that generate the termini of Abeta1-40 and Abeta1-42 peptides.     

FAD-linked mutations in presenilin 1 alter the length of Abeta peptides derived from betaAPP transmembrane domain mutants

gamma-Secretase is an enzymatic activity responsible for the final cleavage of the amyloid precursor protein leading to the production of the amyloid beta-peptide (Abeta). gamma-Secretase is likely an aspartyl protease, since its activity can be inhibited by both pepstatin and active-site directed aspartyl protease inhibitors. Recent work has indicated that presenilins 1 and 2 may actually be the gamma-secretase enzymes. Presenilin (PS) mutations, which lead to an increase in the production of a longer form of Abeta, are also the most common cause of familial Alzheimer's disease (FAD). Therefore, in an attempt to better characterize the substrate preferences of gamma-secretase, we performed experiments to determine how FAD-linked mutations in PS1 would affect the generation of Abeta peptides from full length precursor substrates that we have previously demonstrated to be proteolytically cleaved at alternative sites and/or by enzymatic activities that are pharmacologically distinct. Presenilin mutations increased the production of Abeta peptides from sites distal to the primary cleavage site ('longer' peptides) and in several cases also decreased production of 'shorter' peptides. These results support a model in which the FAD-linked mutants subtly alter the conformation of the gamma-secretase complex to favor the production of long Abeta.     

Transition-state analogue gamma-secretase inhibitors stabilize a 900 kDa presenilin/nicastrin complex

Gamma-secretase mediates the final step, which generates Alzheimer's disease Abeta amyloid protein, by cleaving the transmembrane domain of the amyloid-beta protein precursor. Four gene products, presenilin, nicastrin, APH-1, and PEN-2, are required for gamma-secretase activity that is contained within a high molecular mass complex. To further characterize gamma-secretase, we probed membranes from human neuroblastoma SH-SY5Y cells with gamma-secretase inhibitor biotin derivatives of L-685,458, pepstatin A, and the difluoro alcohol 1-Bt. These inhibitor derivatives bound and precipitated PS1 fragments from membrane CHAPSO extracts. Analysis of PS1 complexes by blue native gel electrophoresis and western blotting indicated that the CHAPSO extracts contained complexes of approximately 900, 500, and 400 kDa. With this technique, derivatives of the three inhibitors were detected only in association with the 900 kDa species. Size-exclusion chromatography showed that 13% of PS1 immunoreactivity extracted with CHAPSO was comprised within a >or=900 kDa species with the remaining eluting in fractions of 669-250 kDa but that most enzymatic activity was associated with the 900 kDa fractions. After treatment with L-685,458 inhibitor, 49% PS1 immunoreactivity was eluted in the 900 kDa fraction, supporting evidence that the inhibitor stabilized this complex. Subcellular fractionation of SH-SY5Y cells indicated that the 900 kDa complex was formed as PS1 and NCT matured through the secretory pathway and that enzymatic activity correlated with complex maturation. From these observations, we propose a model for the structure of active gamma-secretase that would consist of dimerization of 400-500 kDa subunits and be consistent with the apparent molecular mass of the complex.