Presenilin 1 is linked with γ-secretase activity in the detergent solubilized state (original) (raw)
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Presenilin 1 is linked with gamma -secretase activity in the detergent solubilized state
Proceedings of the National Academy of Sciences, 2000
␥-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two A peptide isoforms, A40 and A42. Here we report the detergent solubilization and partial characterization of ␥-secretase. The activity of solubilized ␥-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the -secretase cleavage site. Cleavage of C100Flag by ␥-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the A40 or A42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the A40 and A42 termini. Recovery of catalytically competent, soluble ␥-secretase critically depends on the choice of detergent;
The many substrates of presenilin/γ-secretase
Journal of Alzheimer's disease : JAD, 2011
The Alzheimer's disease (AD)-associated amyloid-β protein precursor (AβPP) is cleaved by α-, β-, and presenilin (PS)/γ-secretases through sequential regulated proteolysis. These proteolytic events control the generation of the pathogenic amyloid-β (Aβ) peptide, which excessively accumulates in the brains of individuals afflicted by AD. A growing number of additional proteins cleaved by PS/γ-secretase continue to be discovered. Similarly to AβPP, most of these proteins are type-I transmembrane proteins involved in vital signaling functions regulating cell fate, adhesion, migration, neurite outgrowth, or synaptogenesis. All the identified proteins share common structural features, which are typical for their proteolysis. The consequences of the PS/γ-secretase-mediated cleavage on the function of many of these proteins are largely unknown. Here, we review the current literature on the proteolytic processing mediated by the versatile PS/γ-secretase complex. We begin by discussing th...
Biochemistry, 2011
γ-S ecretase is best known for its involvement in the proteolytic processing of amyloid precursor protein (APP) that generates β-amyloid peptide (Aβ), the major component in amyloid plaques that is a hallmark of Alzheimer's disease (AD) pathology (reviewed in ref 1). Processing of APP releases Aβ peptides of 37À42 residues (reviewed in refs 2À4). The longer forms, especially Aβ42, are more hydrophobic, prone to aggregation, and critical in the pathogenesis of AD. Blocking Aβ production with γ-secretase inhibitors (GSIs) is expected to slow or prevent the progression of AD and has thus become a major drug development effort in the past decade. In addition to APP, the substrates of γ-secretase include many single-transmembrane domain proteins (reviewed in ref 5), with the best characterized one being the cell surface receptor Notch. γ-Secretase processing of Notch generates the Notch intracellular domain (NICD), which is subsequently transported to the nucleus and acts as a transcription factor. Notch-mediated signaling activities are crucial for embryonic development as well as cell differentiation in adult animals. 6 Achieving a significant therapeutic window between Aβ reduction and side effects due to inhibition of Notch processing has been a major challenge for developing GSIs as a treatment for AD. γ-Secretase is a large membrane protein complex containing four essential components: presenilin (PS), aph-1, nicastrin (NCT), and pen-2. 2 PS is the catalytic subunit that contains the active site of the enzyme, but all components are essential as the absence of any of them leads to a complete loss of γ-secretase activity. Of the four components in the γ-secretase complex, both PS and aph-1 have two isoforms in humans. PS1 and PS2 are ∼60% homologous in sequence, and a similar degree of homology is found between aph-1a and aph-1b. The presence of different PS and aph-1 isoforms suggests the potential for heterogeneity of native γ-secretase such that there may be up to four different subtypes of γ-secretase complexes. The existence of two splicing variants of aph-1a (long and short forms) may further contribute to the heterogeneity of native γ-secretase. Little is known about the physiologic function of each individual γ-secretase complex. The biochemical characterization of the γ-secretase complex has been conducted mostly with the native enzyme from various cells and tissues, each of which likely contains a variable mixture of γ-secretase isoforms. The first generation of GSIs was developed using native enzyme preparations for screening and evaluation before the molecular identification of γ-secretase components. Understanding whether any of these compounds are selective for specific γ-secretase complexes may provide important insights into their in vivo efficacy and side effect
Presenilins and the γ-secretase: still a complex problem
Molecular Brain, 2010
The presenilins form part of a complex of membrane proteins that are involved in the proteolytic cleavage of cellsurface molecules. This article reviews the history of the discovery of the presenilins, their role in the pathogenesis of Alzheimer's disease and in the metabolism of the amyloid-β precursor protein. Unanswered questions about their biochemical mechanism of action and their effects on Ca 2+ homeostasis are examined.
Journal of Neurochemistry, 2003
Presenilin (PS)-dependent c-secretase cleavage is the final proteolytic step in generating amyloid b protein (Ab), 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 c-secretase active site. To better understand the relationship between presenilinase and c-secretase, we characterized the biochemical properties of presenilinase and compared them with those of c-secretase. Similar to c-secretase, presenilinase was most active at acidic pH 6.3. Aspartyl protease inhibitor pepstatin A blocked presenilinase activity with an IC 50 of 1 lM. Difluoroketone aspartyl protease transition state analogue MW167 was relatively selective for presenilinase (IC 50 < 1 lM) over c-secretase (IC 50 )16 lM). Importantly, removing the transition state mimicking moiety simultaneously abolished both presenilinase and c-secretase inhibition, suggesting that presenilinase, like c-secretase, is an aspartyl protease. Interestingly, several of the most potent c-secretase inhibitors (IC 50 ¼ 0.3 or 20 nM) failed to block presenilinase activity. Although de novo generation of PS1 fragments coincided with production of Ab in vitro, blocking presenilinase activity without reducing pre-existing fragment levels permitted normal de novo generation of Ab and amyloid intracellular domain. Therefore, presenilinase has characteristics of an aspartyl protease, but this activity is distinct from c-secretase.
2001
Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid  peptide (A). The A protein precursor (APP) is cleaved by -secretase to generate a C-terminal fragment, CTF, which in turn is cleaved by ␥-secretase to generate A. Alternative cleavage of the APP by ␣-secretase at A16/17 generates the C-terminal fragment, CTF␣. In addition to A, endoproteolytic cleavage of CTF␣ and CTF by ␥-secretase should yield a C-terminal fragment of 57-59 residues (CTF␥). However, CTF␥ 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 as well as an ϳ6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTF␥, and we describe an in vitro assay for ␥-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTF␥ fragment. Three compounds previously identified as ␥-secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2,2difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTF␥, providing additional evidence that the fragment arises from ␥-secretase cleavage. Consistent with reports that presenilins are the elusive ␥-secretases, subcellular fractionation studies showed that presenilin-1, CTF␣, and CTF are enriched in the CTF␥-generating fractions. The in vitro ␥-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for ␥-secretase inhibitors.
Transition-State Analogue γ-Secretase Inhibitors Stabilize a 900 kDa Presenilin/Nicastrin Complex †
Biochemistry, 2005
γ-Secretase mediates the final step, which generates Alzheimer's disease A amyloid protein, by cleaving the transmembrane domain of the amyloid-protein precursor. Four gene products, presenilin, nicastrin, APH-1, and PEN-2, are required for γ-secretase activity that is contained within a high molecular mass complex. To further characterize γ-secretase, we probed membranes from human neuroblastoma SH-SY5Y cells with γ-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 ∼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 g900 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 γ-secretase that would consist of dimerization of 400-500 kDa subunits and be consistent with the apparent molecular mass of the complex.
Biochemistry, 2004
Maturation of γ-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 γ-secretase in a cell-free enzymatic assay with an IC 50 of 2.1 µM, a value similar to the K m (3.5 µM) 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 cellbased assay using HEK293 cells expressing APP. In contrast to small-molecule γ-secretase inhibitors, kinetic inhibition studies demonstrated competitive inhibition of γ-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 γ-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 γ-secretase. NMR analyses demonstrated the presence of a loop structure with a -turn in the middle of the exon 9 peptide and a loose R-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 γ-secretase. We hypothesize that γ-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 ∆E9 mutation causes Alzheimer's disease because cleavage activation of γ-secretase is no longer necessary, alleviating constraints on A formation.
The Extreme C Terminus of Presenilin 1 Is Essential for γ-Secretase Complex Assembly and Activity
Journal of Biological Chemistry, 2004
The ␥-secretase complex catalyzes the cleavage of the amyloid precursor protein in its transmembrane domain resulting in the formation of the amyloid -peptide and the cytoplasmic APP intracellular domain. The active ␥-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 ␥-cleavage to occur. The PS proteins are themselves subjected to endoproteolytic cleavage resulting in the generation of an N-terminal and a Cterminal fragment that remain stably associated as a heterodimer. Here we investigated the effects of modifications on the C terminus of PS1 on PS1 endoproteolysis, ␥-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 ␥-secretase activity, PS1 endoproteolysis, and ␥-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 Cterminal 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. The formation and aggregation of the 40-42-residue amyloid -peptide (A) 1 in brain are implicated in Alzheimer's disease (AD) (1). Sequential proteolytic processing of the transmembrane amyloid precursor protein (APP) by -secretase and ␥-secretase generates the A peptide (2). ␥-Secretase cleavage
Specificity of presenilin-1- and presenilin-2-dependent γ-secretases towards substrate processing
Journal of Cellular and Molecular Medicine, 2017
The two presenilin-1 (PS1) and presenilin-2 (PS2) homologs are the catalytic core of the c-secretase complex, which has a major role in cell fate decision and Alzheimer's disease (AD) progression. Understanding the precise contribution of PS1-and PS2-dependent c-secretases to the production of b-amyloid peptide (Ab) from amyloid precursor protein (APP) remains an important challenge to design molecules efficiently modulating Ab release without affecting the processing of other c-secretase substrates. To that end, we studied PS1-and PS2-dependent substrate processing in murine cells lacking presenilins (PSs) (PS1KO, PS2KO or PS1-PS2 double-KO noted PSdKO) or stably re-expressing human PS1 or PS2 in an endogenous PS-null (PSdKO) background. We characterized the processing of APP and Notch on both endogenous and exogenous substrates, and we investigated the effect of pharmacological inhibitors targeting the PSs activity (DAPT and L-685,458). We found that murine PS1 c-secretase plays a predominant role in APP and Notch processing when compared to murine PS2 c-secretase. The inhibitors blocked more efficiently murine PS2-than murine PS1-dependent processing. Human PSs, especially human PS1, expression in a PSnull background efficiently restored APP and Notch processing. Strikingly, and contrary to the results obtained on murine PSs, pharmacological inhibitors appear to preferentially target human PS1-than human PS2-dependent c-secretase activity.