- Email: [email protected]
P4-281 Characterization of the cleavage specificity of Alzheimer's disease γ-secretase identified by valine-scanning mutagenesis of the transmembrane domain of the Notch-1 protein
Poster Session t)4: Molecular Mechanisms of Neurodegeneration - Presenilins and Conclusions: In APH-l-transfected cells, APH-laS and APH-laL were expressed as ~22 kDa and ~24 kDa proteins, respectively. APH-lb appeared as a major ~ 25 kDa band and a minor ~26.5 kDa band; the latter may be a phosphorylated form. Interestingly, bands with twice the molecular sizes of APH-la and APHI-b were clearly observed, suggesting that APH-1 proteins form SDS-stable dimers. In addition, we obtained data implying that APH-1 proteins interact with themselves to increase their expression levels. The expression of APH-1 proteins may be influenced by their interaction.
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PHYSICAL INTERACTION BETWEEN BACE AND PRESENILIN: IS THAT FUNCTIONAL?
Georges Levesque*, Sebastien Hebert, Valerie Bourdages, Justin Dupuis, Chantal Godin. Laval University, Quebec, PQ, Canada. Contact e-mail:
Georges.Levesque @crsfa, ulaval.ca Background: Both 13- and y- secretases cleave amyloid beta precursor protein (APP) to produce 13-amyloid peptides. In Alzheimer's disease (AD) brain, these peptides are deposited in amyloid plaques and considered by many as the principal cause of dementia. The identity of 13-secretase has been shown m be the transmembrane aspartic protease, beta-site APP cleaving enzyme 1 (BACE1). Presenilin-1 (PS1), a polytopic muhitransmembrane protein, appears to constitute the catalytic component of y-secretase. We have recently demonstrated an interaction between BACE1 and wildtype PS-1 thus suggesting a direct, intimate collaboration in 13-amyloid peptide production. Objective: To study the functional role of this physical interaction. Methods: Yeast two-hybrid, cell transfections and western blotting were used to investigate the functional BACE/PS1 interaction. Results: We report that immature BACE1 preferentially interacts with full-length (holoprotein) PS1 at an endogenous level. Various AD and non-AD associated PS1 mutations do not abrogate binding with BACEI. Preliminary data suggest that immature BACE1 protein levels regulate PS 1 endoproteolysis. This downregulation effect on PS 1 was abolished with the D257A and D385A dominant negative mutants in PS1 -/- fibroblast cells. Conclusion: our results suggest that BACE1 binding to PS1 may lead to altered PS 1 metabolism and possibly, 13-amyloid production.
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CHARACTERIZATION OF THE CLEAVAGE SPECIFICITY OF ALZHEIMER'S DISEASE )'-SECRETASE IDENTIFIED BY VALINEoSCANNING MUTAGENESIS OF THE TRANSMEMBRANE DOMAIN OF THE NOTCH-1 PROTEIN
Hisashi Tanii*. Mie University, Faculty of Medicine, Dapartment of
Psychiatry, Mie, Japan. Contact e-mail: [email protected] Background: Familial AD (FAD)-associated presenilin forming -secretase complex mediates dual transmembraneous cleavage of APP (gamma-40/42 and 49) as well as Notch protein ($3 and $4). These cleavages liberate amyloid 13 into the extracelhilar space and AICD(APP intracellular cytoplasmic domain) translocate to nucleus, The cleavage of Notch-1 releases polypeptide Notch 13and also liberates NICD (Notch intracellular cytoplasmic domain) cut from $3 site. Objective(s): Here we noticed that APP has an amino acid valine just before and after garnma-secretase cleavage site of APP (gamma-40/42 and 49) and NICD is generated by cleavage at $3 between Gly1743 and Val1744 of Notch-l, suggesting that valine has some roles in gamma-secretase cleavage. The newly identified cleavage site of Notch-1 ($4) was identified in the center of four sequential alanine residues between Ala1731 and Ala1732 which has no valine. Methods: In order to investigate the effect of valine upon $4 cleavage site, we stably transfected human embryonic kidney 293 (K293) cells with F-NEXT (an N-terminally FLAG-tagged N deltaE variant) and mutated F-NEXT (residue 1726--1733 were replaced by valine). Results: Although each mutant generates various secreted species of Notch 13, $4 site is the major site for all these valine-variants. Valine mutation close to $4 site affects the generations of some Notch 13 species and the difference of cleavage activity was also observed. These amino acid substitutions sometimes influence distant site
$555
which resemble to the features of some kinds of APP mutation. All these valine-mntants have presenilin-dependent cleavage because the generation of Notch species was inhibited in cells expressing PS 1 D385N. Conclusions: Our results demonstrate mutation effects of valine upon gamma-secretase cleavage of Notch- 1
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ASSOCIATION AND LOCALIZATION OF NICASTRIN INFLUENCES THE ACTIVITY OF THE G-SECRETASE COMPLEX
Vanessa A. Morals* 1,2, Ryan R. Fortna 2, Adam S. Crystal 2, D.S. Pijak 2 , Tong Li 3, Philip C. Wong 3, J. Costa 1, Virginia M.-Y. Lee 2, Robert W. Doms 2. l Instituto de Tecnologia Quimica e Biologica/Instituto de
Biologia Experimental e Tecnologica, Oeiras, Portugal; 2Department of Microbiology, University of Pennsylvania School of Medicine/Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, Philadelphia, PA 19104, USA,"3John Hopkins University, School of Medicine, Baltimore, MD, USA. Contact e-mail: vmorais @itqb. unl.pt Background: Alzheimer's Disease (AD) is a progressive neurodegenerative disorder which is characterized by the accumulation of senile plaques, predominantly composed of amyloid-13 (A13)peptide. A13peptide is generated by the proteolytic processing of amyloid [3 precursor protein (APP) through sequential cleavages by the 13- and y-secretases. The y-secretase complex contains four noncovalently associated protein components that are essential for its function: Presenifin (PS), Nicastrin (NCT), Aphl and Pen2. NCT is a type I transmembrane protein containing multiple glycosylation sites and that appears to stabilize PS and be critical for trafficking of PS to the cell surface. Conversely, PS is required for maturation and trafficking of NCT. Objective(s): In this study, we address the function of chimeric proteins between human NCT (hNCT) and Caenorhabditis elegans NCT (ceNCT). Methods: To address the function of these chimeric proteins four different functional assays were performed in NCT - t - cells: 1) detection of secreted A13 through ELISA; 2) production of NICD through a Notch cleavage assay; 3) detection of PS1-CTF through a PS1-FL cleavage assay and 4) cleavage of C99 detected by a Luciferase reporter assay. Results: Recently, through the production of chimeric proteins between hNCT and ceNCT, we have identified a region that confers interaction of hNCT with other y-secretase components. The substitution of amino acids 620-721 in ceNCT with the corresponding hNCT region (chimera eeNCT-h(620"72t)) confers interaction of the former with the human y-secretase complex components. This region is composed of 50 amino acids adjacent to the TMD, the TMD and the intracellular C-terminal domain. We addressed the function of these chimeric proteins using four different functional assays, and observed that NCT - / - ceils were capable of producing NICD and A13 only in the presence of transfected hNCT. No chimeric protein was capable of recovering this phenotype, including the chimera ceNCT-h(62°721) that has the identified region required for association. Conclusions: These studies indicated that although ceNCT-h(620-721) associates with the y-secretase complex it does not form an active complex. To further elucidate which determinants of hNCT are required for function of NCT within the complex, additional chimeras should be constructed.
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ENDOPROTEOLYSIS OF PRESENILIN-1 IS AN INTRAMOLECULAR AUTOCATALYTIC EVENT
Anne L. Brunkan*, Maribel Martinez, Alison M. Goate. Washington
University School of Medicine, St. Louis, MO, USA. Contact e-mail: abrunkan @icarus, wustl, edu Background: Mutation in the presenilins (PS1, PS2) or the amyloid precursor protein (APP) account fur all known causes of familial Alzheimer's disease (FAD). APP is cleaved in a presenilin-dependent manner to form the A13fragments that accumulate in plaques in AD brains. The signaling protein Notch is also cleaved in a presenilin-dependent fashion to form NICD. PS contain the active site for the y-secretase complex that accomplishes this proteolysis in the presence of required co-factors Nicastrin, APH-1, and
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PHYSICAL INTERACTION BETWEEN BACE AND PRESENILIN: IS THAT FUNCTIONAL?
Georges Levesque*, Sebastien Hebert, Valerie Bourdages, Justin Dupuis, Chantal Godin. Laval University, Quebec, PQ, Canada. Contact e-mail:
Georges.Levesque @crsfa, ulaval.ca Background: Both 13- and y- secretases cleave amyloid beta precursor protein (APP) to produce 13-amyloid peptides. In Alzheimer's disease (AD) brain, these peptides are deposited in amyloid plaques and considered by many as the principal cause of dementia. The identity of 13-secretase has been shown m be the transmembrane aspartic protease, beta-site APP cleaving enzyme 1 (BACE1). Presenilin-1 (PS1), a polytopic muhitransmembrane protein, appears to constitute the catalytic component of y-secretase. We have recently demonstrated an interaction between BACE1 and wildtype PS-1 thus suggesting a direct, intimate collaboration in 13-amyloid peptide production. Objective: To study the functional role of this physical interaction. Methods: Yeast two-hybrid, cell transfections and western blotting were used to investigate the functional BACE/PS1 interaction. Results: We report that immature BACE1 preferentially interacts with full-length (holoprotein) PS1 at an endogenous level. Various AD and non-AD associated PS1 mutations do not abrogate binding with BACEI. Preliminary data suggest that immature BACE1 protein levels regulate PS 1 endoproteolysis. This downregulation effect on PS 1 was abolished with the D257A and D385A dominant negative mutants in PS1 -/- fibroblast cells. Conclusion: our results suggest that BACE1 binding to PS1 may lead to altered PS 1 metabolism and possibly, 13-amyloid production.
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CHARACTERIZATION OF THE CLEAVAGE SPECIFICITY OF ALZHEIMER'S DISEASE )'-SECRETASE IDENTIFIED BY VALINEoSCANNING MUTAGENESIS OF THE TRANSMEMBRANE DOMAIN OF THE NOTCH-1 PROTEIN
Hisashi Tanii*. Mie University, Faculty of Medicine, Dapartment of
Psychiatry, Mie, Japan. Contact e-mail: [email protected] Background: Familial AD (FAD)-associated presenilin forming -secretase complex mediates dual transmembraneous cleavage of APP (gamma-40/42 and 49) as well as Notch protein ($3 and $4). These cleavages liberate amyloid 13 into the extracelhilar space and AICD(APP intracellular cytoplasmic domain) translocate to nucleus, The cleavage of Notch-1 releases polypeptide Notch 13and also liberates NICD (Notch intracellular cytoplasmic domain) cut from $3 site. Objective(s): Here we noticed that APP has an amino acid valine just before and after garnma-secretase cleavage site of APP (gamma-40/42 and 49) and NICD is generated by cleavage at $3 between Gly1743 and Val1744 of Notch-l, suggesting that valine has some roles in gamma-secretase cleavage. The newly identified cleavage site of Notch-1 ($4) was identified in the center of four sequential alanine residues between Ala1731 and Ala1732 which has no valine. Methods: In order to investigate the effect of valine upon $4 cleavage site, we stably transfected human embryonic kidney 293 (K293) cells with F-NEXT (an N-terminally FLAG-tagged N deltaE variant) and mutated F-NEXT (residue 1726--1733 were replaced by valine). Results: Although each mutant generates various secreted species of Notch 13, $4 site is the major site for all these valine-variants. Valine mutation close to $4 site affects the generations of some Notch 13 species and the difference of cleavage activity was also observed. These amino acid substitutions sometimes influence distant site
$555
which resemble to the features of some kinds of APP mutation. All these valine-mntants have presenilin-dependent cleavage because the generation of Notch species was inhibited in cells expressing PS 1 D385N. Conclusions: Our results demonstrate mutation effects of valine upon gamma-secretase cleavage of Notch- 1
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ASSOCIATION AND LOCALIZATION OF NICASTRIN INFLUENCES THE ACTIVITY OF THE G-SECRETASE COMPLEX
Vanessa A. Morals* 1,2, Ryan R. Fortna 2, Adam S. Crystal 2, D.S. Pijak 2 , Tong Li 3, Philip C. Wong 3, J. Costa 1, Virginia M.-Y. Lee 2, Robert W. Doms 2. l Instituto de Tecnologia Quimica e Biologica/Instituto de
Biologia Experimental e Tecnologica, Oeiras, Portugal; 2Department of Microbiology, University of Pennsylvania School of Medicine/Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, Philadelphia, PA 19104, USA,"3John Hopkins University, School of Medicine, Baltimore, MD, USA. Contact e-mail: vmorais @itqb. unl.pt Background: Alzheimer's Disease (AD) is a progressive neurodegenerative disorder which is characterized by the accumulation of senile plaques, predominantly composed of amyloid-13 (A13)peptide. A13peptide is generated by the proteolytic processing of amyloid [3 precursor protein (APP) through sequential cleavages by the 13- and y-secretases. The y-secretase complex contains four noncovalently associated protein components that are essential for its function: Presenifin (PS), Nicastrin (NCT), Aphl and Pen2. NCT is a type I transmembrane protein containing multiple glycosylation sites and that appears to stabilize PS and be critical for trafficking of PS to the cell surface. Conversely, PS is required for maturation and trafficking of NCT. Objective(s): In this study, we address the function of chimeric proteins between human NCT (hNCT) and Caenorhabditis elegans NCT (ceNCT). Methods: To address the function of these chimeric proteins four different functional assays were performed in NCT - t - cells: 1) detection of secreted A13 through ELISA; 2) production of NICD through a Notch cleavage assay; 3) detection of PS1-CTF through a PS1-FL cleavage assay and 4) cleavage of C99 detected by a Luciferase reporter assay. Results: Recently, through the production of chimeric proteins between hNCT and ceNCT, we have identified a region that confers interaction of hNCT with other y-secretase components. The substitution of amino acids 620-721 in ceNCT with the corresponding hNCT region (chimera eeNCT-h(620"72t)) confers interaction of the former with the human y-secretase complex components. This region is composed of 50 amino acids adjacent to the TMD, the TMD and the intracellular C-terminal domain. We addressed the function of these chimeric proteins using four different functional assays, and observed that NCT - / - ceils were capable of producing NICD and A13 only in the presence of transfected hNCT. No chimeric protein was capable of recovering this phenotype, including the chimera ceNCT-h(62°721) that has the identified region required for association. Conclusions: These studies indicated that although ceNCT-h(620-721) associates with the y-secretase complex it does not form an active complex. To further elucidate which determinants of hNCT are required for function of NCT within the complex, additional chimeras should be constructed.
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ENDOPROTEOLYSIS OF PRESENILIN-1 IS AN INTRAMOLECULAR AUTOCATALYTIC EVENT
Anne L. Brunkan*, Maribel Martinez, Alison M. Goate. Washington
University School of Medicine, St. Louis, MO, USA. Contact e-mail: abrunkan @icarus, wustl, edu Background: Mutation in the presenilins (PS1, PS2) or the amyloid precursor protein (APP) account fur all known causes of familial Alzheimer's disease (FAD). APP is cleaved in a presenilin-dependent manner to form the A13fragments that accumulate in plaques in AD brains. The signaling protein Notch is also cleaved in a presenilin-dependent fashion to form NICD. PS contain the active site for the y-secretase complex that accomplishes this proteolysis in the presence of required co-factors Nicastrin, APH-1, and