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.     

Presenilin-1 D257A and D385A mutants fail to cleave Notch in their endoproteolyzed forms, but only presenilin-1 D385A mutant can restore its gamma-secretase activity with the compensatory overexpression of normal C-terminal fragment

The enzyme gamma-secretase is involved in the cleavage of several type I membrane proteins, such as Notch 1 and amyloid precursor protein. Presenilin-1 (PS-1) is one of the critical integral membrane protein components of the gamma-secretase complex and is processed endoproteolytically, generating N- and C-terminal fragments (NTF and CTF, respectively). PS-1 is also known to incorporate into a high molecular weight complex by binding to other gamma-secretase components such as Nicastrin, Aph-1, and Pen-2. Mutations on PS-1 can alter the effects of gamma-secretase on its many substrates to different extents. Here, we showed that PS-1 mutants have a different activity for Notch cleavage, which depended on the PS-1 mutation site. We demonstrated that defective PS-1 mutants located in CTF, i.e. D385A and C410Y, could restore their gamma-secretase activities with the compensatory overexpression of wild type CTF or of minimal deleted CTF (amino acids 349-467). However, the defective PS-1 D257A mutant could not restore their gamma-secretase activities with the compensatory overexpression of wild type NTF. In comparison, both D257A NTF and D385A CTF could abolish the gamma-secretase activity of wild type and pathogenic PS-1 mutants. We also showed that PS-1 NTF but not CTF forms strong high molecular weight aggregates in SDS-PAGE. Taken together, results have shown that NTF and CTF integrate differently into high molecular weight aggregates and that PS-1 Asp-257 and Asp-385 have different accessibilities in their unendoproteolyzed conformation.     

Endoproteolysis of presenilin in vitro: inhibition by gamma-secretase inhibitors

The final proteolytic step to generate the amyloid beta-protein (Abeta) of Alzheimer's disease (AD) from beta-amyloid precursor protein (APP) is achieved by presenilin (PS)-dependent gamma-secretase cleavage. AD-causing mutations in PS1 and PS2 result in a selective and significant increase in production of the more amyloidogenic Abeta42 peptide. PS1 and PS2 undergo endoproteolysis by an unknown enzyme termed presenilinase to generate the functional complex of N- and C-terminal fragments (NTF/CTF). To investigate the endoproteolytic activity that generates active PS, we used a mammalian cell-free system that allows de novo human PS NTF and CTF generation. PS NTF and CTF generation in vitro was observed in endoplasmic reticulum (ER)-enriched fractions of membrane vesicles and to a lesser extent in Golgi/trans-Golgi-network (TGN)-enriched fractions. AD-causing mutations in PS1 and PS2 did not alter de novo generation of PS fragments. Removal of peripheral membrane-associated and cytosolic proteins did not prevent de novo generation of fragments, indicating that presenilinase activity corresponds to an integral membrane protein. Among several general inhibitors of different protease classes that blocked the presenilinase activity, pepstatin A was the most potent inhibitor. Screening available transition state analogue gamma-secretase inhibitors led to the identification of two compounds that were able to prevent the de novo generation of PS fragments, with an expected inhibition of Abeta generation. Our studies provide a biochemical approach to characterize and identify this elusive presenilinase.     

gamma-Secretase complexes containing N- and C-terminal fragments of different presenilin origin retain normal gamma-secretase activity

The gamma-secretase complex processes substrate proteins within membranes and consists of four proteins: presenilin (PS), nicastrin, Aph-1 and Pen-2. PS harbours the enzymatic activity of the complex, and there are two mammalian PS homologues: PS1 and PS2. PS undergoes endoproteolysis, generating the N- and C-terminal fragments, NTF and CTF, which represent the active species of PS. To characterize the functional similarity between complexes of various PS composition, we analysed PS1, PS2, and chimeric PS composed of the NTF from PS1 and CTF from PS2, or vice versa, in assembly and function of the gamma-secretase complex. Chimeric PSs, like PS1 and PS2, undergo normal endoproteolysis when introduced into cells devoid of endogenous PS. Furthermore, PS2 CTF can, at least partially, restore processing in a truncated PS1, which cannot undergo endoproteolysis. All PS forms enable maturation of nicastrin and cleave full length Notch receptors, indicating that both PS1 and PS2 are present at the cell surface. Finally, when co-introduced as separate molecules, NTF and CTF of different PS origin reconstitute gamma-secretase activity. In conclusion, these data show that endoproteolysis, NTF-CTF interactions, and the assembly and activity of gamma-secretase complexes are very conserved between PS1 and PS2.     

Smad mediates BMP-2-induced upregulation of FGF-evoked PC12 cell differentiation

To gain insight into presenilin-1 (PS1) structural aspects, we explored the structure–function relationship of its N- and C-terminal (NTF and CTF, respectively) complexes. We demonstrated that both NTF and CTF act as independent but inter-changing binding units capable of binding each other (NTF/CTF) or their homologues (NTF/NTF; CTF/CTF). The Alzheimer’s disease-associated PS1 mutations Y115H and M146L do not affect their ability to hetero- and/or homodimerize, thus conserving their basic integrity and function(s). These results suggest that PS1 associates intra-molecularly to form higher order complexes, which may be needed for endoproteolytic cleavage and/or γ-secretase-associated activity.     

Evidence that intramolecular associations between presenilin domains are obligatory for endoproteolytic processing.

Mutations in genes encoding presenilins (PS1 and PS2) cosegregate with the majority of early onset cases of familial Alzheimer's disease. PS1 and PS2 are polytopic membrane proteins that undergo endoproteolytic cleavage to generate stable NH2- and COOH-terminal derivatives (NTF and CTF, respectively). Several lines of evidence suggest that the endoproteolytic derivatives are likely the functional units of PS in vivo. In the present report, we examine the disposition of PS NTF and CTF assemblies in stable mouse N2a neuroblastoma cell lines expressing human PS polypeptides. We show that exogenous expression of PS1 NTFs neither assemble with endogenous CTF nor exhibit dominant negative inhibitory effects on the endogenous PS1 cleavage and the accumulation of derivatives. In cells co-expressing PS1 and PS2, PS1- and PS2-derived fragments do not form mixed assemblies. In contrast, cells expressing a chimeric PS1/PS2 polypeptide form stable PS1 NTF-PS2 CTF assemblies. Moreover, expression of chimeric PS1/PS2 polypeptides harboring a familial early onset AD-linked mutation (M146L) elevates the production of Abeta42 peptides. Our results provide evidence that assembly of structural domains contained within NH2- and COOH-terminal regions of PS occur prior to endoproteolytic cleavage.     

Chemical cross-linking provides a model of the gamma-secretase complex subunit architecture and evidence for close proximity of the C-terminal fragment of presenilin with APH-1

Gamma-secretase is an intramembrane cleaving aspartyl protease complex intimately implicated in Alzheimer disease pathogenesis. The protease is composed of the catalytic subunit presenilin (PS1 or PS2), the substrate receptor nicastrin (NCT), and two additional subunits, APH-1 (APH-1a, as long and short splice forms (APH-1aL, APH-1aS), or APH-1b) and PEN-2. Apart from the Alzheimer disease-associated beta-amyloid precursor protein, gamma-secretase has been shown to cleave a large number of other type I membrane proteins. Despite the progress in elucidating gamma-secretase function, basic questions concerning the precise organization of its subunits, their molecular interactions, and their exact stoichiometry in the complex are largely unresolved. Here we isolated endogenous human gamma-secretase from human embryonic kidney 293 cells and investigated the subunit architecture of the gamma-secretase complex formed by PS1, NCT, APH-1aL, and PEN-2 by chemical cross-linking. Using this approach, we provide evidence for the close neighborhood of the PS1 N- and C-terminal fragments (NTF and CTF, respectively), the PS1 NTF and PEN-2, the PS1 CTF and APH-1aL, and NCT and APH-1aL. We thus identify a previously unrecognized PS1 CTF/APH-1aL interaction, verify subunit interactions deduced previously from indirect approaches, and provide a model of the gamma-secretase complex subunit architecture. Finally, we further show that, like the PS1 CTF, the PS2 CTF also interacts with APH-1aL, and we provide evidence that these interactions also occur with the other APH-1 variants, suggesting similar subunit architectures of all gamma-secretase complexes.     

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.     

The extreme C terminus of presenilin 1 is essential for gamma-secretase complex assembly and activity

The gamma-secretase complex catalyzes the cleavage of the amyloid precursor protein in its transmembrane domain resulting in the formation of the amyloid beta-peptide and the cytoplasmic APP intracellular domain. The active gamma-secretase complex is composed of at least four subunits: presenilin (PS), nicastrin, Aph-1, and Pen-2, where the presence of all components is critically required for gamma-cleavage to occur. The PS proteins are themselves subjected to endoproteolytic cleavage resulting in the generation of an N-terminal and a C-terminal fragment that remain stably associated as a heterodimer. Here we investigated the effects of modifications on the C terminus of PS1 on PS1 endoproteolysis, gamma-secretase complex assembly, and activity in cells devoid of endogenous PS. We report that certain mutations and, in particular, deletions of the PS1 C terminus decrease gamma-secretase activity, PS1 endoproteolysis, and gamma-secretase complex formation. We demonstrate that the N- and C-terminal PS1 fragments can associate with each other in mutants having C-terminal truncations that cause loss of interaction with nicastrin and Aph-1. In addition, we show that the C-terminal fragment of PS1 alone can mediate interaction with nicastrin and Aph-1 in PS null cells expressing only the C-terminal fragment of PS1. Taken together, these data suggest that the PS1 N- and C-terminal fragment intermolecular interactions are independent of an association with nicastrin and Aph-1, and that nicastrin and Aph-1 interact with the C-terminal part of PS1 in the absence of an association with full-length PS1 or the N-terminal fragment.     

Glycogen synthase kinase-3beta regulates presenilin 1 C-terminal fragment levels

The majority of familial Alzheimer's disease cases have been attributed to mutations in the presenilin 1 (PS1) gene. PS1 is synthesized as an inactive holoprotein that undergoes endoproteolytic processing to generate a functional N- and C-terminal heterodimer (NTF and CTF, respectively). We identified a single residue in PS1, Ser(397), which regulates the CTF levels in a population of dimer that has a rapid turnover. This residue is part of a highly conserved glycogen synthase kinase-3beta (GSK-3beta) consensus phosphorylation site within the loop domain of PS1. Site-directed mutagenesis at the Ser(397) position increased levels of PS1 CTF but not NTF or holoprotein. Similar increases in only CTF levels were seen when cells expressing wild type PS1 were treated with lithium chloride, an inhibitor of GSK-3beta. Both wild type and PS1 S397A CTF displayed a biphasic turnover, reflecting rapidly degraded and stable populations. Rapid turnover was delayed for mutant PS1 S397A, causing increased CTF. These data demonstrate that PS1 NTF.CTF endoproteolytic fragments are generated in excess, that phosphorylation at Ser(397) by GSK-3beta regulates the discard of excess CTF, and that the disposal of surplus NTF is mediated by an independent mechanism. Overall, the results indicate that production of active NTF.CTF dimer is more complex than limited endoproteolysis of PS1 holoprotein and instead involves additional regulatory events.     

Pharmacological knock-down of the presenilin 1 heterodimer by a novel gamma -secretase inhibitor: implications for presenilin biology

Intramembranous cleavage of the beta-amyloid precursor protein by gamma-secretase is the final processing event generating amyloid-beta peptides, which are thought to be causative agents for Alzheimer's disease. Missense mutations in the presenilin genes co-segregate with early-onset Alzheimer's disease, and, recently, a close biochemical linkage between presenilins and the identity of gamma-secretase has been established. Here we describe for the first time that certain potent gamma-secretase inhibitors are able to interfere with the endoproteolytic processing of presenilin 1 (PS1). In addition, we identified a novel gamma-secretase inhibitor, [1S-benzyl-4R-[1-(5-cyclohexyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3(R,S)-ylcarbamoyl)-S-ethylcarbamoyl]-2R-hydroxy-5-phenyl-pentyl]-carbamic acid tert-butyl ester (CBAP), which not only physically interacts with PS1, but upon chronic treatment produces a "pharmacological knock-down" of PS1 fragments. This indicates that the observed accumulation of full-length PS1 is caused by a direct inhibition of its endoproteolysis. The subsequent use of CBAP as a biological tool to increase full-length PS1 levels in the absence of exogenous PS1 expression has provided evidence that wild-type PS1 endoproteolysis is not required either for PS1/gamma-secretase complex assembly or trafficking. Furthermore, in cell-based systems CBAP does not completely recapitulate PS1 loss-of-function phenotypes. Even though the beta-amyloid precursor protein cleavage and the S3 cleavage of the Notch receptor are inhibited by CBAP, an impairment of Trk receptor maturation was not observed.     

Carboxyl-terminal fragments of Alzheimer beta-amyloid precursor protein accumulate in restricted and unpredicted intracellular compartments in presenilin 1-deficient cells

Absence of functional presenilin 1 (PS1) protein leads to loss of gamma-secretase cleavage of the amyloid precursor protein (betaAPP), resulting in a dramatic reduction in amyloid beta peptide (Abeta) production and accumulation of alpha- or beta-secretase-cleaved COOH-terminal fragments of betaAPP (alpha- or beta-CTFs). The major COOH-terminal fragment (CTF) in brain was identified as betaAPP-CTF-(11-98), which is consistent with the observation that cultured neurons generate primarily Abeta-(11-40). In PS1(-/-) murine neurons and fibroblasts expressing the loss-of-function PS1(D385A) mutant, CTFs accumulated in the endoplasmic reticulum, Golgi, and lysosomes, but not late endosomes. There were some subtle differences in the subcellular distribution of CTFs in PS1(-/-) neurons as compared with PS1(D385A) mutant fibroblasts. However, there was no obvious redistribution of full-length betaAPP or of markers of other organelles in either mutant. Blockade of endoplasmic reticulum-to-Golgi trafficking indicated that in PS1(-/-) neurons (as in normal cells) trafficking of betaAPP to the Golgi compartment is necessary before alpha- and beta-secretase cleavages occur. Thus, although we cannot exclude a specific role for PS1 in trafficking of CTFs, these data argue against a major role in general protein trafficking. These results are more compatible with a role for PS1 either as the actual gamma-secretase catalytic activity or in other functions indirectly related to gamma-secretase catalysis (e.g. an activator of gamma-secretase, a substrate adaptor for gamma-secretase, or delivery of gamma-secretase to betaAPP-containing compartments).     

Positive and negative regulation of the gamma-secretase activity by nicastrin in a murine model

Nicastrin is a component of the gamma-secretase complex that has been shown to adhere to presenilin-1 (PS1), Notch, and APP. Here we demonstrate that Nicastrin-deficient mice showed a phenotype that is indistinguishable from PS1/PS2 double knock-out mice, whereas heterozygotes were healthy and viable. Fibroblasts derived from Nicastrin-deficient embryos were unable to generate amyloid beta-peptide and failed to release the intracellular domain of APP- or Notch1-Gal4-VP16 fusion proteins. Additionally, C- and N-terminal fragments of PS1 and the C-terminal fragments of PS2 were not detectable in Nicastrin-null fibroblasts, whereas full-length PS1 accumulated in null fibroblasts, indicating that Nicastrin is required for the endoproteolytic processing of presenilins. Interestingly, cells derived from Nicastrin heterozygotes produced relatively higher levels of amyloid beta-peptide whether the source was endogenous mouse or transfected human APP. These data demonstrate that Nicastrin is essential for the gamma-secretase cleavage of APP and Notch in mammalian cells and that Nicastrin has both positive and negative functions in the regulation of gamma-secretase activity.     

Nectin-1alpha,an immunoglobulin-like receptor involved in the formation of synapses,is a substrate for presenilin/gamma-secretase-like cleavage

Nectin-1 is a member of the immunoglobulin superfamily and a Ca(2+)-independent adherens junction protein involved in synapse formation. Here we show that nectin-1alpha undergoes intramembrane proteolytic processing analogous to that of the Alzheimer's disease amyloid precursor protein, mediated by a presenilin (PS)-dependent gamma-secretase-like activity. 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of Chinese hamster ovary cells activated a first proteolytic event, resulting in ectodomain shedding of nectin-1alpha. Subsequent cleavage of the remaining 26-kDa membrane-anchored C-terminal fragment (CTF) was inhibited independently by three specific gamma-secretase inhibitors and by expression of the dominant negative form of PS1. The PS/gamma-secretase-like cleavage product was detected in vivo following proteasome inhibitor treatment of cells. An in vitro gamma-secretase assay confirmed the generation of a 24-kDa nectin-1alpha intracellular domain, peripherally associated with the membrane fraction. We also found nectin-1alpha to interact with the N-terminal fragment of PS1. Finally, gamma-secretase inhibition resulted in beta-catenin release from cell junctions, concomitantly with the accumulation of the 26-kDa nectin-1alpha CTF, suggesting that high levels of nectin-1alpha CTF interfere with TPA-induced remodeling of cell-cell junctions. Our results are consistent with a previously reported role for PS/gamma-secretase in adherens junction function involving cleavage of cadherins. Similar to nectin-1, other members of the immunoglobulin superfamily involved in synapse formation may also serve as substrates for PS/gamma-secretase-like intramembrane proteolytic activity.     

Presenilin/gamma-secretase and alpha-secretase-like peptidases cleave human MHC Class I proteins

HLA (human leucocyte antigen)-A2 is an MHC Class I protein with primary functions in T-cell development and initi-ation of immune cell responses. MHC I proteins also play roles in intercellular adhesion, apoptosis, cell proliferation and neuronal plasticity. By utilizing a sequence comparison analysis, we recently identified HLA-A2 as a potential substrate for the Alzheimer's disease-associated PS1 (presenilin 1)/gamma-secretase. alpha-Secretase-like membrane metalloproteinases are responsible for an initial shedding event, partially mediated by ADAM (a disinteg-rin and metalloproteinase)-10. Accordingly, activation or inhibition of alpha-secretase-like membrane metalloproteinases directly modulated levels of a 14 kDa HLA-A2 CTF (C-terminal frag-ment) in CHO (Chinese-hamster ovary) cells. To show that the HLA-A2 CTF is subsequently cleaved by PS1/gamma-secretase, we re-duced its activity in cell lines stably expressing HLA-A2 and in Jurkat T-cells expressing endogenous MHC I. Treatment with specific PS1/gamma-secretase inhibitors or expression of a dominant-negative construct led to a significant accumulation of HLA-A2 CTFs. We also identified the PS1/gamma-secretase cleavage product of HLA-A2 CTF, termed HLA-A2 intracellular domain, in cell-free and cell-based experiments. In the absence of proteasome inhibitors, HLA-A2 intracellular domain underwent rapid degrad-ation. These data indicate that MHC I proteins undergo extra-cellular domain cleavage mediated by alpha-secretases and the cleavage product is subsequently cleaved by PS1/gamma-secretase.     

A novel gamma -secretase assay based on detection of the putative C-terminal fragment-gamma of amyloid beta protein precursor

Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid beta peptide (A beta). The A beta protein precursor (APP) is cleaved by beta-secretase to generate a C-terminal fragment, CTF beta, which in turn is cleaved by gamma-secretase to generate A beta. Alternative cleavage of the APP by alpha-secretase at A beta 16/17 generates the C-terminal fragment, CTFalpha. In addition to A beta, endoproteolytic cleavage of CTF alpha and CTF beta by gamma-secretase should yield a C-terminal fragment of 57-59 residues (CTF gamma). However, CTF gamma has not yet been reported in either brain or cell lysates, presumably due to its instability in vivo. We detected the in vitro generation of A beta as well as an approximately 6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTF gamma, and we describe an in vitro assay for gamma-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTF gamma fragment. Three compounds previously identified as gamma-secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2, 2-difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTF gamma, providing additional evidence that the fragment arises from gamma-secretase cleavage. Consistent with reports that presenilins are the elusive gamma-secretases, subcellular fractionation studies showed that presenilin-1, CTF alpha, and CTF beta are enriched in the CTF gamma-generating fractions. The in vitro gamma-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for gamma-secretase inhibitors.     

PEN-2 and APH-1 coordinately regulate proteolytic processing of presenilin 1

Presenilin (PS, PS1/PS2) complexes are known to be responsible for the intramembranous gamma-secretase cleavage of the beta-amyloid precursor protein and signaling receptor Notch. PS holoprotein undergoes endoproteolysis by an unknown enzymatic activity to generate NH(2)- and COOH-terminal fragments, a process that is required for the formation of the active and stable PS/-gamma-secretase complex. Biochemical and genetic studies have recently identified nicastrin, APH-1, and PEN-2 as essential cofactors that physically interact with PS1 and are necessary for the gamma-secretase activity. However, their precise function in regulating the PS complex and gamma-secretase activity remains unknown. Here, we demonstrate that endogenous PEN-2 preferentially interacts with PS1 holoprotein. Down-regulation of PEN-2 expression by small interfering RNA (siRNA) abolishes the endoproteolysis of PS1, whereas overexpression of PEN-2 promotes the production of PS1 fragments, indicating a critical role for PEN-2 in PS1 endoproteolysis. Interestingly, accumulation of full-length PS1 resulting from down-regulation of PEN-2 is alleviated by additional siRNA down-regulation of APH-1. Furthermore, overexpression of APH-1 facilitates PEN-2-mediated PS1 proteolysis, resulting in a significant increase in PS1 fragments. Our data reveal a direct role of PEN-2 in proteolytic cleavage of PS1 and a regulatory function of APH-1, in coordination with PEN-2, in the biogenesis of the PS1 complex.     

The N-terminus of presenilin-2 increases single channel activity of brain ryanodine receptors through direct protein-protein interaction

Presenilin-1 (PS1) and presenilin-2 (PS2) form the catalytic core in gamma-secretase complexes and mutations in these proteins result in aberrant cleavage of amyloid precursor protein leading to accumulation of the beta-amyloid in the brain of familial Alzheimer Disease patients. PS2 possesses a hydrophilic cytoplasmic N-terminal domain (PS2 NTF1-87) dispensable for gamma-secretase activity with physiological functions yet to be determined. The effects of this soluble 87 amino acid fragment of mouse PS2 on single channel activity of mouse brain ryanodine receptors (RyR) were determined. PS2 NTF1-87 application to the cytoplasmic side of the RyR significantly increased single channel activity by favoring higher sublevel openings. The Ca(2+) activation and desensitization ranges for RyRs were unchanged. We demonstrate facilitation of RyR gating by PS2 NTF1-87, which might represent a general mechanism of RyR regulation by presenilins potentially prone to be affected by mutations or external stimuli contributing to the development of neurodegenerative diseases.     

Functional domains in presenilin 1: the Tyr-288 residue controls gamma-secretase activity and endoproteolysis

Processing of the Alzheimer amyloid precursor protein (APP) into the amyloid beta-protein and the APP intracellular domain is a proteolysis event mediated by the gamma-secretase complex where presenilin (PS) proteins are key constituents. PS is subjected to an endoproteolytic cleavage, generating a stable heterodimer composed of an N-terminal and a C-terminal fragment. Here we aimed at further understanding the role of PS in endoproteolysis, in proteolytic processing of APP and Notch, and in assembly of the gamma-secretase complex. By using a truncation protocol and alanine scanning, we identified Tyr-288 in the PS1 N-terminal fragment as critical for PS-dependent intramembrane proteolysis. Further mutagenesis of the 288 site identified mutants differentially affecting endoproteolysis and gamma-secretase activity. The Y288F mutant was endoproteolyzed to the same extent as wild type PS but increased the amyloid beta-protein 42/40 ratio by approximately 75%. In contrast, the Y288N mutant was also endoproteolytically processed but was inactive in reconstituting gamma-secretase in PS null cells. The Y288D mutant was deficient in both endoproteolysis and gamma-secretase activity. All three mutant PS1 molecules were incorporated into gamma-secretase complexes and stabilized Pen-2 in PS null cells. Thus, mutations at Tyr-288 do not affect gamma-secretase complex assembly but can differentially control endoproteolysis and gamma-secretase activity.