The Experts below are selected from a list of 9765 Experts worldwide ranked by ideXlab platform
Michael S. Wolfe - One of the best experts on this subject based on the ideXlab platform.
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Cryoelectron microscopy structure of purified Gamma-Secretase at 12 A resolution.
Journal of molecular biology, 2008Co-Authors: Pamela Osenkowski, Patrick C. Fraering, Michael S. Wolfe, Lorène Aeschbach, Dennis J SelkoeAbstract:Gamma-Secretase, an integral membrane protein complex, catalyzes the intramembrane cleavage of the beta-amyloid precursor protein (APP) during the neuronal production of the amyloid beta-peptide. As such, the protease has emerged as a key target for developing agents to treat and prevent Alzheimer's disease. Existing biochemical studies conflict on the oligomeric assembly state of the protease complex, and its detailed structure is not known. Here, we report that purified active human Gamma-Secretase in digitonin has a total molecular mass of approximately 230 kDa when measured by scanning transmission electron microscopy. This result supports a complex that is monomeric for each of the four component proteins. We further report the three-dimensional structure of the Gamma-Secretase complex at 12 A resolution as obtained by cryoelectron microscopy and single-particle image reconstruction. The structure reveals several domains on the extracellular side, three solvent-accessible low-density cavities, and a potential substrate-binding surface groove in the transmembrane region of the complex.
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Gamma-Secretase inhibition and modulation for Alzheimer's disease.
Current Alzheimer research, 2008Co-Authors: Michael S. WolfeAbstract:Gamma-Secretase is a multi-protein complex that proteolyzes the transmembrane region of the amyloid beta-peptide (Abeta) precursor (APP), producing the Abeta peptide implicated in the pathogenesis of Alzheimer's disease (AD). This protease has been a top target for AD, and various inhibitors have been identified, including transition-state analogue inhibitors that interact with the active site, helical peptides that interact with the initial substrate docking site, and other less peptide-like, more drug-like compounds. Although one Gamma-Secretase inhibitor has advanced into late-phase clinical trials, concerns about inhibiting this protease remain. The protease complex cleaves a number of other substrates, and in vivo toxicities observed with Gamma-Secretase inhibitors are apparently due to blocking one particularly important substrate, the Notch receptor. Thus, the potential of Gamma-Secretase as therapeutic target likely depends on the ability to selectively inhibit Abeta production without hindering Notch proteolysis (i.e., modulation rather than inhibition). The discovery of Gamma-Secretase modulators has revived Gamma-Secretase as an attractive target and has so far resulted in one compound in late-phase clinical trials. The identification of other modulators in a variety of structural classes raise the hope that more promising agents will soon be in the pipeline.
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Gamma-Secretase: structure, function, and modulation for Alzheimer's disease.
Current topics in medicinal chemistry, 2008Co-Authors: Michael S. WolfeAbstract:Abstract Gamma-Secretase proteolyzes a variety of membrane-associated fragments derived from type I integral membrane proteins, including the amyloid beta-protein precursor, involved in Alzheimer's disease, and the Notch receptor, critical for cellular differentiation. This protease is composed of four integral membrane proteins: presenilin, nicastrin, Aph-1 and Pen-2. Assembly of these four components leads to presenilin autoproteolysis into two subunits, each of which contributes one aspartate to the active site of an aspartyl protease. The protease contains an initial docking site for substrate, where it binds prior to passing between the two presenilin subunits to the internal water-containing active site. The extracellular region of nicastrin also interacts with the N-terminus of the substrate as an essential step in substrate recognition and processing. Modulation of APP processing without interfering with Notch signaling is an important therapeutic goal, and allosteric sites on the protease allow such selective modulation. A better structural and mechanistic understanding of Gamma-Secretase should ultimately allow structure-based design of more potent and selective modulators.
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Detergent-dependent dissociation of active Gamma-Secretase reveals an interaction between Pen-2 and PS1-NTF and offers a model for subunit organization within the complex.
Biochemistry, 2004Co-Authors: Patrick C. Fraering, Matthew J. Lavoie, Beth L. Ostaszewski, W. Taylor Kimberly, Dennis J Selkoe, Michael S. WolfeAbstract:Gamma-Secretase is a member of a new class of proteases with an intramembrane catalytic site and cleaves numerous type I membrane proteins, including the amyloid beta-protein precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of Gamma-Secretase: a heterodimeric form of presenilin (PS), composed of its N- and C-terminal fragments (PS-NTF and PS-CTF, respectively), a highly glycosylated, mature form of nicastrin (NCT), Aph-1, and Pen-2. However, it is unclear how these components interact physically with each other and assemble into functional complexes. We and others recently found that Aph-1 interacts with a less glycosylated, immature form of nicastrin as an intermediate toward full assembly of Gamma-Secretase. Here we show that (1) the detergent dodecyl beta-d-maltoside (DDM) mediates the dissociation and inactivation of active Gamma-Secretase in a concentration-dependent manner, (2) DDM-dependent dissociation of the active Gamma-Secretase complex generates two major inactive complexes (Pen-2-PS1-NTF and mNCT-Aph-1) and two minor inactive complexes (mNCT-Aph1-PS1-CTF and PS1-NTF-PS1-CTF), and (3) Pen-2 can also associate with the PS holoprotein in complexes devoid of NCT and Aph-1. Taken together, our results demonstrate that Pen-2 interacts with PS-NTF within active Gamma-Secretase and offer a model for how the components of active Gamma-Secretase interact physically with each other.
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identity and function of Gamma Secretase
Journal of Neuroscience Research, 2003Co-Authors: Taylor W Kimberly, Michael S. WolfeAbstract:Gamma-Secretase catalyzes intramembrane proteolysis of various type I membrane proteins, including the amyloid-beta precursor protein and the Notch receptor. Despite its importance in the pathogenesis of Alzheimer's disease and to normal development, this protease has eluded identification until only very recently. Four membrane proteins are now known to be members of the protease complex: presenilin, nicastrin, aph-1, and pen-2. Recent findings suggest that these four proteins are sufficient to reconstitute the active Gamma-Secretase complex and that together they mediate the cell surface signaling of a variety of receptors via intramembrane proteolysis.
Dennis J Selkoe - One of the best experts on this subject based on the ideXlab platform.
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Cryoelectron microscopy structure of purified Gamma-Secretase at 12 A resolution.
Journal of molecular biology, 2008Co-Authors: Pamela Osenkowski, Patrick C. Fraering, Michael S. Wolfe, Lorène Aeschbach, Dennis J SelkoeAbstract:Gamma-Secretase, an integral membrane protein complex, catalyzes the intramembrane cleavage of the beta-amyloid precursor protein (APP) during the neuronal production of the amyloid beta-peptide. As such, the protease has emerged as a key target for developing agents to treat and prevent Alzheimer's disease. Existing biochemical studies conflict on the oligomeric assembly state of the protease complex, and its detailed structure is not known. Here, we report that purified active human Gamma-Secretase in digitonin has a total molecular mass of approximately 230 kDa when measured by scanning transmission electron microscopy. This result supports a complex that is monomeric for each of the four component proteins. We further report the three-dimensional structure of the Gamma-Secretase complex at 12 A resolution as obtained by cryoelectron microscopy and single-particle image reconstruction. The structure reveals several domains on the extracellular side, three solvent-accessible low-density cavities, and a potential substrate-binding surface groove in the transmembrane region of the complex.
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Detergent-dependent dissociation of active Gamma-Secretase reveals an interaction between Pen-2 and PS1-NTF and offers a model for subunit organization within the complex.
Biochemistry, 2004Co-Authors: Patrick C. Fraering, Matthew J. Lavoie, Beth L. Ostaszewski, W. Taylor Kimberly, Dennis J Selkoe, Michael S. WolfeAbstract:Gamma-Secretase is a member of a new class of proteases with an intramembrane catalytic site and cleaves numerous type I membrane proteins, including the amyloid beta-protein precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of Gamma-Secretase: a heterodimeric form of presenilin (PS), composed of its N- and C-terminal fragments (PS-NTF and PS-CTF, respectively), a highly glycosylated, mature form of nicastrin (NCT), Aph-1, and Pen-2. However, it is unclear how these components interact physically with each other and assemble into functional complexes. We and others recently found that Aph-1 interacts with a less glycosylated, immature form of nicastrin as an intermediate toward full assembly of Gamma-Secretase. Here we show that (1) the detergent dodecyl beta-d-maltoside (DDM) mediates the dissociation and inactivation of active Gamma-Secretase in a concentration-dependent manner, (2) DDM-dependent dissociation of the active Gamma-Secretase complex generates two major inactive complexes (Pen-2-PS1-NTF and mNCT-Aph-1) and two minor inactive complexes (mNCT-Aph1-PS1-CTF and PS1-NTF-PS1-CTF), and (3) Pen-2 can also associate with the PS holoprotein in complexes devoid of NCT and Aph-1. Taken together, our results demonstrate that Pen-2 interacts with PS-NTF within active Gamma-Secretase and offer a model for how the components of active Gamma-Secretase interact physically with each other.
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Notch and the amyloid precursor protein are cleaved by similar Gamma-Secretase(s).
Biochemistry, 2003Co-Authors: W. Taylor Kimberly, Beth L. Ostaszewski, Dennis J Selkoe, Thekla S Diehl, William P Esler, Jun Gao, Michael S. WolfeAbstract:Gamma-Secretase is an intramembrane-cleaving protease whose substrates include Notch and the amyloid precursor protein (APP). On the basis of initial genetic and pharmacologic data, the Gamma-Secretase activity responsible for cleavage of both proteins appears to be identical. However, apparent differences in the cleavage site and in sequence specificity raise questions about the degree of similarity between Notch and APP Gamma-like proteolysis. In an effort to resolve this issue directly, we established an in vitro Gamma-Secretase activity assay that cleaves both APP- and Notch-based substrates, C100Flag and N100Flag. Analysis with specific Gamma-Secretase inhibitors, dominant-negative Gamma-Secretase preparations, and antibody co-immunoprecipitations all demonstrated identical cleavage of these substrates. Most importantly, we found that these substrates prevented cleavage of each other, indicating that the same Gamma-Secretase complex can cleave either protein. Finally, we provide evidence that both substrates are cut at two distinct regions in the transmembrane domain. These data resolve some of the apparent conflicts and strongly indicate that Notch and APP are proteolyzed by the same enzyme(s).
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FAD mutations in presenilin-1 or amyloid precursor protein decrease the efficacy of a Gamma-Secretase inhibitor: evidence for direct involvement of PS1 in the Gamma-Secretase cleavage complex.
Neurobiology of disease, 2000Co-Authors: W Xia, Beth L. Ostaszewski, W T Kimberly, T Rahmati, C L Moore, M S Wolfe, Dennis J SelkoeAbstract:To investigate the mechanism of regulation of Ass production by familial Alzheimer's disease (FAD)-linked presenilin 1 (PS1), we used a cell-free system that allows de novo Ass generation to examine whether PS1 participates directly in the Gamma-Secretase reaction. Optimal Ass generation in vitro was achieved at mildly acidic pH and could be inhibited by the aspartyl protease inhibitor pepstatin A, consistent with the suggestion that Gamma-Secretase is an aspartyl protease. Dominant negative mutations of the critical transmembrane aspartates in PS1 or full deletion of PS1 did not alter the maturation of APP in the secretory pathway. Instead, PS1 had a direct effect on the inhibition of Ass production by a designed peptidomimetic inhibitor: the inhibition was significantly less effective in cells expressing FAD-causing mutations in either APP or PS1 than in cells expressing the wild-type proteins. Taken together, these findings suggest that PS1 participates physically in a complex with APP during the Gamma-Secretase cleavage event.
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Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and Gamma-Secretase activity.
Nature, 1999Co-Authors: Michael S. Wolfe, Beth L. Ostaszewski, W. Taylor Kimberly, Weiming Xia, Thekla S Diehl, Dennis J SelkoeAbstract:Accumulation of the amyloid-beta protein (Abeta) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimer's disease. The final step in the generation of Abeta from the beta-amyloid precursor protein is an apparently intramembranous proteolysis by the elusive Gamma-Secretase(s). The most common cause of familial Alzheimer's disease is mutation of the genes encoding presenilins 1 and 2, which alters Gamma-Secretase activity to increase the production of the highly amyloidogenic Abeta42 isoform. Moreover, deletion of presenilin-1 in mice greatly reduces Gamma-Secretase activity, indicating that presenilin-1 mediates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate residues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Abeta production and increases the amounts of the carboxy-terminal fragments of beta-amyloid precursor protein that are the substrates of Gamma-Secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp --> Ala mutations also prevented the normal endoproteolysis of presenilin-1 in the TM6 --> TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimer's disease and which does not require this cleavage, the Asp 385 --> Ala mutation still inhibited Gamma-Secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-1 endoproteolysis and Gamma-Secretase activity, and suggest that presenilin 1 is either a unique diaspartyl cofactor for Gamma-Secretase or is itself Gamma-Secretase, an autoactivated intramembranous aspartyl protease.
Taisuke Tomita - One of the best experts on this subject based on the ideXlab platform.
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Alzheimer's disease treatment by inhibition/modulation of the Gamma-Secretase activity
Rinsho shinkeigaku = Clinical neurology, 2009Co-Authors: Taisuke TomitaAbstract:Several lines of evidence indicate that the production and deposition of amyloid-beta peptides (Abeta) contribute to the etiology of Alzheimer's disease. Inhibition or modulation of Gamma-Secretase, that is a responsible enzyme for the Abeta production, is one of the plausible therapeutics for Alzheimer's disease. However, the Gamma-Secretase is an unusual aspartic protease that cleaves the scissile bond within the transmembrane domain of several membrane protein including APP and Notch receptor. Thus, development of drugs that regulate the production of Abeta without affecting the Notch signaling is now demanding. Extensive drug screening and development allow that some Secretase inhibitors and modulators have advanced into late-phase clinical trials, whereas the molecular mechanisms of Notch-sparing effect by these compounds effect still remain unknown. Identification of the molecular targets and mechanisms of these compounds using chemical biological approaches is currently underway. This review focuses on the recent development of inhibitors/modulators and provides a direction for the effective treatment of AD through inhibition/modulation of the Gamma-Secretase activity.
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Targeting the structure and function relationships of the Gamma-Secretase for the development of Alzheimer's disease
Rinsho shinkeigaku = Clinical neurology, 2008Co-Authors: Taisuke TomitaAbstract:Genetic and biological studies provide evidence that the production and deposition of amyloid-beta peptides (Abeta) contribute to the etiology of Alzheimer's disease. Thus, beta- and Gamma-Secretases, that are involved in the Abeta generation, are plausible molecular targets for Alzheimer's disease treatment. Gamma-Secretase is an unusual aspartic protease that cleaves the scissile bond within the transmembrane domain. This unusual enzyme is composed of a high molecular weight membrane protein complex containing presenilin, nicastrin, Aph-1 and Pen-2. Drugs that regulate the production of Abeta by inhibiting or modulating Gamma-Secretase activity could provide a disease-modifying effect on AD, although recent studies suggest that Gamma-Secretase plays important roles in cellular signaling including Notch pathway. Thus, understanding the molecular mechanism whereby Gamma-Secretase recognizes and cleaves its substrate is a critical issue for the development of compounds that specifically regulate Abeta-generating Gamma-Secretase activity. I will review our structural studies on the Gamma-Secretase complex, and envision the direction for developing effective and selective Gamma-Secretase inhibitors as therapeutics for AD.
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At the frontline of Alzheimer's disease treatment: Gamma-Secretase inhibitor/modulator mechanism.
Naunyn-Schmiedeberg's archives of pharmacology, 2007Co-Authors: Taisuke TomitaAbstract:Genetic and biological studies provide evidence that the production and deposition of amyloid-beta peptides (Abeta) contribute to the etiology of Alzheimer's disease. beta- and Gamma-Secretases, which are responsible for the generation of Abeta, are plausible molecular targets for Alzheimer's disease treatment. Gamma-Secretase is an unusual aspartic protease that cleaves the scissile bond within the transmembrane domain. This unusual enzyme is composed of a high molecular weight membrane protein complex containing presenilin, nicastrin, Aph-1 and Pen-2. Drugs that regulate the production of Abeta by inhibiting or modulating Gamma-Secretase activity could provide a disease-modifying effect on Alzheimer's disease, although recent studies suggest that Gamma-Secretase plays important roles in cellular signaling including Notch. Thus, understanding the molecular mechanism whereby Gamma-Secretase recognizes and cleaves its substrate is a critical issue for the development of compounds that specifically regulate Abeta-generating Gamma-Secretase activity. This review focuses on the structure and function relationship of Gamma-Secretase complex and the mode of action of the Gamma-Secretase inhibitors.
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divergent synthesis of multifunctional molecular probes to elucidate the enzyme specificity of dipeptidic Gamma Secretase inhibitors
ACS Chemical Biology, 2007Co-Authors: Haruhiko Fuwa, Hiroyuki Miyashita, Yasuko Takahashi, Yu Konno, Naoto Watanabe, Makoto Sasaki, Hideaki Natsugari, Toshiyuki Kan, Tohru Fukuyama, Taisuke TomitaAbstract:Divergent synthesis of multifunctional molecular probes based on caprolactam-derived dipeptidic Gamma-Secretase inhibitors (GSIs), Compound E (CE) and LY411575 analogue (DBZ), was efficiently accomplished by means of Cu(I)-catalyzed azide/alkyne fusion reaction. Photoaffinity labeling experiments using these derivatives coupled to photoactivatable and biotin moieties provided direct evidence that the molecular targets of CE and DBZ are the N-terminal fragment of presenilin 1 within the Gamma-Secretase complex. Moreover, these photoprobes directly targeted signal peptide peptidase. These data suggest that the divergent synthesis of molecular probes has been successfully applied to characterize the interaction of GSIs with their molecular targets and define the structural requirements for inhibitor binding to intramembrane-cleaving proteases.
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Three-dimensional structure of the Gamma-Secretase complex.
Biochemical and biophysical research communications, 2006Co-Authors: Toshihiko Ogura, Kazuhiro Mio, Ikuo Hayashi, Hiroyuki Miyashita, Rie Fukuda, Raphael Kopan, Tatsuhiko Kodama, Takao Hamakubo, Takeshi Iwastubo, Taisuke TomitaAbstract:Gamma-Secretase belongs to an atypical class of aspartic proteases that hydrolyzes peptide bonds within the transmembrane domain of substrates, including amyloid-beta precursor protein and Notch. Gamma-Secretase is comprised of presenilin, nicastrin, APH-1, and PEN-2 which form a large multimeric membrane protein complex, the three-dimensional structure of which is unknown. To gain insight into the structure of this complex enzyme, we purified functional Gamma-Secretase complex reconstituted in Sf9 cells and analyzed it using negative stain electron microscopy and 3D reconstruction techniques. Analysis of 2341 negatively stained particle images resulted in the three-dimensional representation of Gamma-Secretase at a resolution of 48 angstroms. The structure occupies a volume of 560 x 320 x 240 angstroms and resembles a flat heart comprised of two oppositely faced, dimpled domains. A low density space containing multiple pores resides between the domains. Some of the dimples in the putative transmembrane region may house the catalytic site. The large dimensions are consistent with the observation that Gamma-Secretase activity resides within a high molecular weight complex.
Klaus Elenius - One of the best experts on this subject based on the ideXlab platform.
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Gamma-Secretase-dependent signaling of receptor tyrosine kinases
Oncogene, 2019Co-Authors: Johannes A.m. Merilahti, Klaus EleniusAbstract:Human genome harbors 55 receptor tyrosine kinases (RTK). At least half of the RTKs have been reported to be cleaved by Gamma-Secretase-mediated regulated intramembrane proteolysis. The two-step process involves releasing the RTK ectodomain to the extracellular space by proteolytic cleavage called shedding, followed by cleavage in the RTK transmembrane domain by the Gamma-Secretase complex resulting in release of a soluble RTK intracellular domain. This intracellular domain, including the tyrosine kinase domain, can in turn translocate to various cellular compartments, such as the nucleus or proteasome. The soluble intracellular domain may interact with transcriptional regulators and other proteins to induce specific effects on cell survival, proliferation, and differentiation, establishing an additional signaling mode for the cleavable RTKs. On the other hand, the same process can facilitate RTK turnover and proteasomal degradation. In this review we focus on the regulation of RTK shedding and Gamma-Secretase cleavage, as well as signaling promoted by the soluble RTK ICDs. In addition, therapeutic implications of increased knowledge on RTK cleavage on cancer drug development are discussed.
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Genome-wide screen of Gamma-Secretase-mediated intramembrane cleavage of receptor tyrosine kinases.
Molecular biology of the cell, 2017Co-Authors: Johannes A.m. Merilahti, Veera K. Ojala, Anna M. Knittle, Arto T. Pulliainen, Klaus EleniusAbstract:Receptor tyrosine kinases (RTKs) have been demonstrated to signal via regulated intramembrane proteolysis, in which ectodomain shedding and subsequent intramembrane cleavage by Gamma-Secretase leads to release of a soluble intracellular receptor fragment with functional activity. For most RTKs, however, it is unknown whether they can exploit this new signaling mechanism. Here we used a system-wide screen to address the frequency of susceptibility to Gamma-Secretase cleavage among human RTKs. The screen covering 45 of the 55 human RTKs identified 12 new as well as all nine previously published Gamma-Secretase substrates. We biochemically validated the screen by demonstrating that the release of a soluble intracellular fragment from endogenous AXL was dependent on the sheddase disintegrin and metalloprotease 10 (ADAM10) and the Gamma-Secretase component presenilin-1. Functional analysis of the cleavable RTKs indicated that proliferation promoted by overexpression of the TAM family members AXL or TYRO3 depends on Gamma-Secretase cleavage. Taken together, these data indicate that Gamma-Secretase-mediated cleavage provides an additional signaling mechanism for numerous human RTKs.
Frederic Checler - One of the best experts on this subject based on the ideXlab platform.
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p53 is regulated by and regulates members of the Gamma-Secretase complex.
Neurodegenerative Diseases, 2010Co-Authors: Frederic Checler, Raphaelle Pardossi-piquard, Julie Dunys, Cristine Alves Da CostaAbstract:Amyloid beta-peptides is the generic term for a set of hydrophobic peptides that accumulate in Alzheimer's disease (AD)-affected brains. These amyloid-beta peptide fragments are mainly generated by an enzymatic machinery referred to as Gamma-Secretase complex that is built up by the association of four distinct proteins, namely presenilin 1 (PS1) or PS2, nicastrin, Aph-1 and Pen-2. AD is also characterized by exacerbated cell death that appears linked to the tumor suppressor p53. Interestingly, all members of the Gamma-Secretase complex control p53-dependent cell death. On the other hand, p53 appears to be able to regulate directly or indirectly the expression and transcription of PS1, PS2 and Pen-2. This review will focus on the functional cross-talk between the members of the Gamma-Secretase complex and p53 and will discuss the putative implication of this oncogene in AD pathology.
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TMP21 Transmembrane Domain Regulates {Gamma}-Secretase Cleavage.
Journal of Biological Chemistry, 2009Co-Authors: Raphaelle Pardossi-piquard, Christopher Bohm, Fusheng Chen, Soshi Kanemoto, Frederic Checler, Gerold Schmitt-ulms, Peter St George-hyslop, Paul E FraserAbstract:TMP21 has been shown to be associated with the Gamma-Secretase complex and can specifically regulate Gamma-cleavage without affecting epsilon-mediated proteolysis. To explore the basis of this activity, TMP21 modulation of Gamma-Secretase activity was investigated independent of epsilon-cleavage using an APPepsilon construct which lacks the AICD domain. The APPepsilon construct behaves similar to the full-length precursor protein with respect to alpha- and beta-cleavages and is able to undergo normal Gamma-processing. Co-expression of APPepsilon and TMP21 resulted in the accumulation of membrane-embedded higher molecular weight Abeta-positive fragments consistent with an inhibition of Gamma-Secretase cleavage. The APPepsilon system was used to examine the functional domains of TMP21 through the investigation of a series of TMP21-p24a chimera proteins. It was found that chimeras containing the transmembrane domain bound to the Gamma-Secretase complex and could decrease Gamma-Secretase proteolytic processing. This was confirmed though investigation of a synthetic peptide corresponding to the TMP21 transmembrane helix. The isolated TMP21 TM peptide but not the homologous p24a domain was able to reduce Abeta production in a dose-dependent fashion. These observations suggest that the TMP21 transmembrane domain promotes its association with the presenilin complex that results in decreased Gamma-cleavage activity.
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Pharmacological evidences for DFK167-sensitive presenilin-independent Gamma-Secretase-like activity.
Journal of Neurochemistry, 2009Co-Authors: Jean Sevalle, Erwan Ayral, Jean-françois Hernandez, Jean Martinez, Frederic CheclerAbstract:Amyloid-beta (Abeta) peptides production is thought to be a key event in the neurodegenerative process ultimately leading to Alzheimer's disease (AD) pathology. A bulk of studies concur to propose that the C-terminal moiety of Abeta is released from its precursor beta-amyloid precursor protein by a high molecular weight enzymatic complex referred to as Gamma-Secretase, that is composed of at least, nicastrin (NCT), Aph-1, Pen-2, and presenilins (PS) 1 or 2. They are thought to harbor the Gamma-Secretase catalytic activity. However, several lines of evidence suggest that additional Gamma-Secretase-like activities could potentially contribute to Abeta production. By means of a quenched fluorimetric substrate (JMV2660) mimicking the beta-amyloid precursor protein sequence targeted by Gamma-Secretase, we first show that as expected, this probe allows monitoring of an activity detectable in several cell systems including the neuronal cell line telencephalon specific murine neurons (TSM1). This activity is reduced by DFK167, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), and LY68458, three inhibitors known to functionally interact with PS. Interestingly, JMV2660 but not the unrelated peptide JMV2692, inhibits Abeta production in an in vitroGamma-Secretase assay as expected from a putative substrate competitor. This activity is enhanced by PS1 and PS2 mutations known to be responsible for familial forms of AD and reduced by aspartyl mutations inactivating PS or in cells devoid of PS or NCT. However, we clearly establish that residual JMV2660-hydrolysing activity could be recovered in PS- and NCT-deficient fibroblasts and that this activity remained inhibited by DFK167. Overall, our study describes the presence of a proteolytic activity displaying Gamma-Secretase-like properties but independent of PS and still blocked by DFK167, suggesting that the PS-dependent complex could not be the unique Gamma-Secretase activity responsible for Abeta production and delineates PS-independent Gamma-Secretase activity as a potential additional therapeutic target to fight AD pathology.
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p53-dependent control of cell death by nicastrin: lack of requirement for presenilin-dependent Gamma-Secretase complex.
Journal of Neurochemistry, 2009Co-Authors: Raphaelle Pardossi-piquard, Frederic Checler, Peter St George-hyslop, Julie Dunys, Emilie Giaime, Marie-victoire Guillot-sestier, Cristine Alves Da CostaAbstract:Nicastrin (NCT) is a component of the presenilin (PS)-dependent Gamma-Secretase complexes that liberate amyloid beta-peptides from the beta-Amyloid Precursor Protein. Several lines of evidence indicate that the members of these complexes could also contribute to the control of cell death. Here we show that over-expression of NCT increases the viability of human embryonic kidney (HEK293) cells and decreases staurosporine (STS)- and thapsigargin (TPS)-induced caspase-3 activation in various cell lines from human and neuronal origins by Akt-dependent pathway. NCT lowers p53 expression, transcriptional activity and promoter transactivation and reduces p53 phosphorylation. NCT-associated protection against STS-stimulated cell death was completely abolished by p53 deficiency. Conversely, the depletion of NCT drastically enhances STS-induced caspase-3 activation and p53 pathway and favored p53 nuclear translocation. We examined whether NCT protective function depends on PS-dependent Gamma-Secretase activity. First, a 29-amino acid deletion known to reduce NCT-dependent amyloid beta-peptide production did not affect NCT-associated protective phenotype. Second, NCT still reduces STS-induced caspase-3 activation in fibroblasts lacking PS1 and PS2. Third, the Gamma-Secretase inhibitor DFK167 did not affect NCT-mediated reduction of p53 activity. Altogether, our study indicates that NCT controls cell death via phosphoinositide 3-kinase/Akt and p53-dependent pathways and that this function remains independent of the activity and molecular integrity of the Gamma-Secretase complexes.
