- Email: [email protected]
P4-293 The presenilins, chromosome missegregation, and Alzheimer's disease
$558
Poster Session P4: Molecular Mechanisms of Neurodegeneration - Presenilins
data suggest that 132 -CTFs are substrates for PS/y -secretase mediated cleavage. We also found that 132 -CTFs were generated from two distinct extacellular domain cleavage mediated by BACE1 and metalloproteases, respectively. We are currently performing site-directed mutagenesis studies to confirm the BACEl-dependent cleavage site of 132. We also show that, like BACEl-derived APP-C-terminal fragments (CTFs), over-expression of 132 -CTFs increase cellular sensitivity to apoptosis. Interestingly, t32 -CTF levels were markedly elevated in the brains of AD patients, closely correlating with increased levels of BACE1 (p < 0.01, n = 10 for AD cases, 11 cases for the age matched controls). These results suggest that abnormal processing of 132 as well as APP might be implicated in the pathogenesis of AD presumably owing to elevated BACE1 activity in AD brains. Possible pathological mechanisms of abnormal processing of 132 will be discussed.
~4~-~
CHARACTERIZATION OF PRESENILIN-1 ASSOCIATED PROTEINS
Guoqing Lin* 1, Xulun Zhang 1, Andres Shevchenko 2, Anna Shevchenko 2, Sangram Sisodia 1. 1 University of Chicago, Chicago, IL, USA; 2Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. Contact e-mail: [email protected]
Background: Presenilin-1 (PS1) is a polytopic membrane protein and mutant variants are linked with early-onset familial Alzheimer's disease (FAD). Together with Aphl, Nicastrin and Pen-2, PS1 promotes intramambranous, y-secretase processing of type-1 membrane proteins, including APP and Notch. Objective(s): We sought to identify additional proteins that may modulate PS1 function and y-secretase activity. Methods: a stable 293 cell line expressing TAP-tagged PSI was used for affinity purification of PSi-associated proteins and MALDI-MS was employed to identify these polypeptides. Results: Several PS 1 associated proteins have been identified, Structural comparisons indicated that some of these are involved in protein trafficking, and proteolytic activities. The levels of PS1-NTF, CTF and A13 are evaluated by overexpression and siRNA down regulation of these PS1 associated proteins. Conclusions: Our preliminary studies suggest that several PS1 associated proteins regulate PS1 and y-secretase activity, and present studies are focused on understanding the function of these novel regulators of PS1 function.
•
GAMMA-SECRETASE INHIBITORS STABILIZE A 900 KDA PRESENILIN COMPLEX: A TETRAMERIC MODEL FOR GAMMA-SECRETASE
Genevieve Evin*, Louise D. Cantefford, David E. Hoke, R,M. Damian Holsinger, Janetta G. Culvenor, Colin L. Masters. The University of Melbourne, Parkville, Australia. Contact e-mail: [email protected] y-Secretase carries out the ultimate proteolytic step in the generation of Alzheimer's disease A13 amyloid protein by cleaving the amyloid protein precursor within its transmembrane domain. The same enzyme activity is involved in processing membrane domains of a subset of type I integral proteins, including Notch. Four gene products, presenilin, nicastrin, APH-1, and PEN-2 are required for y-secretase activity that is contained within a high molecular weight complex. To further characterize y-secretase, we probed membranes from SH-SY5Y cells with y-secretase inhibitor biotin-derivatives. Derivatives of L-685,458, pepstatin and a difluoroalcohol transition-state analog (1-Bt) were found to interact tightly with presenilin 1 (PSI) C-terminal fragment and with a ~70 kDa species containing immunoreactivity for PS1 N-terminal region and for APH-1. Blue native gel electrophoresis followed by western blotting indicated that CHAPSO solubilized a PS1 complex of high molecular weight, that was broken down into 360 and 450 kDa species upon addition of dodecylmaitoside, and that Brij-35 extracted a 450 kDa PS1 complex. With this technique, derivatives of the three inhibitors were found to interact preferentially with the 900 kDa species, whatever detergent was used to extract PS 1 complexes, but an interaction with the 450 kDa species was also evident. Superose-6 size-exclusion chromatography showed that, after treatment with L-685,458, PS1 immunoreactivity from membrane Brij-extract was more condensed
in a fraction of 900 kDa, suggesting the inhibitor stabilized this high molecular weight complex. Pre-incubating Brij-35 membrane extracts with L-685,458 enhanced binding of the L-685,458 biotin-derivative, suggesting that the inhibitor stabilized a conformation favorable for binding of the biotinylated probe. From these observations, we propose a tetrameric model for the structure of y-secretase that would be consistent with the apparent molecular mass of the complex and would encompass two symmetrical active sites able to bind simultaneously two inhibitor molecules. (supported by the Australian NHMRC)
~ T H E PRESENILINS, CHROMOSOME MISSEGREGATION, AND ALZHEIMER'S DISEASE Debrah I. Boeras*, Antoneta Granic, Jaya Padmanabhan, Huntington Potter. USE College of Medicine, Tampa, FL, USA. Contact e-maiL" dboeras@hsc, usfedu
Background: Mutations in two genes, Presenilin 1 (PS1), and Presenilin 2 (PS2), have been shown to cause the majority of early-onset familial Alzheimer's Disease (FAD). The presenilins are found to be partly associated with the nuclear membrane and other cellular structures involved in cell division and chromosome segregation. We have previously demonstrated that fibroblasts from FAD patients with mutant presenilin genes, PS-1 or PS-2, show an increase in chromosome defects including trisomy 21, the chromosome abnormality that underlies Down syndrome and causes such individuals to develop AD pathology by age 30-40. (Geller and Potter, 1999) Furthermore, the APP gene, whose cleavage leads to amyloid formation, is located on chromosome 21. Objective(s): To further investigate the involvement of the presenilins in the cell cycle, and more importantly, in chromosome missegregation, we are using the following two experimental systems: 1) transfected ceils expressing either normal human presenilin genes or FAD mutant presenilin genes; 2) cells from mice expressing either normal or FAD mutant presenilins. Methods: We have subcloned the normal and FAD mutant PS genes into expression vectors and introduced them into mammalian cells with normal chromosome complements. Transfected cells expressing normal and mutant presenilins have been examined for changes in their karyotype by both standard metaphase chromosome analysis and by fluorescence in situ hybridization (FISH). Levels of PS gene expression in the transfected cells have been analyzed with real time PCR. Cell-specific immunocytochemistry and FISH with single copy mouse probes is being used to assess the level of aneuploidy in isolated primary neurons of presenifin transgenic mice. Results: Together these experiments indicate that the presenilins are involved in or affect cell cycle regulation and/or chromosome segregation and that the FAD mutations alter the presenilin function in the cell cycle.
~ - ]
MECHANISMS OF PRESENILIN REGULATION BY UBIQUILIN
Leann K. Massey* 1 Mervyn J. Monteiro 2. 1 University of Maryland, Baltimore, MD, USA," 2 University of Maryland Biotechnology Institute, Baltimore, MD, USA. Contact e-mail: lmassO01 @umaryland.edu Mutations in presenilin proteins (PS1 and PS2) lead to early onset Alzheimer's disease (AD) making the study of these proteins and the molecular interactions that regulate their levels important in characterizing AD. There are multiple forms of PS proteins in cells, the FL protein and their endoproteolytic fragments, termed the NH2-terminal fragment (NTF) and the COOH-terminal fragment (CTF). Currently, the NTF and CTF are proposed to be the active form of PS, residing in the y-secretase complex and cleaving APP to release A13.Thus, studying PS-interactors that modulate fragment levels could provide important information regarding y-secretase activity. Rudy Tanzi and co-workers recently used genetic screening to find new genes involved in AD and identified the ubiquilin-1 gene as one potential candidate. Prior to this, we demonstrated that the ubiquilin-1 protein, which contains ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, interacts both in vivo and in vitro with the large hydrophilic loop of full-length (FL) PS stabilizing it and perhaps acting as a molecular
Poster Session P4: Molecular Mechanisms of Neurodegeneration - Presenilins
data suggest that 132 -CTFs are substrates for PS/y -secretase mediated cleavage. We also found that 132 -CTFs were generated from two distinct extacellular domain cleavage mediated by BACE1 and metalloproteases, respectively. We are currently performing site-directed mutagenesis studies to confirm the BACEl-dependent cleavage site of 132. We also show that, like BACEl-derived APP-C-terminal fragments (CTFs), over-expression of 132 -CTFs increase cellular sensitivity to apoptosis. Interestingly, t32 -CTF levels were markedly elevated in the brains of AD patients, closely correlating with increased levels of BACE1 (p < 0.01, n = 10 for AD cases, 11 cases for the age matched controls). These results suggest that abnormal processing of 132 as well as APP might be implicated in the pathogenesis of AD presumably owing to elevated BACE1 activity in AD brains. Possible pathological mechanisms of abnormal processing of 132 will be discussed.
~4~-~
CHARACTERIZATION OF PRESENILIN-1 ASSOCIATED PROTEINS
Guoqing Lin* 1, Xulun Zhang 1, Andres Shevchenko 2, Anna Shevchenko 2, Sangram Sisodia 1. 1 University of Chicago, Chicago, IL, USA; 2Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. Contact e-mail: [email protected]
Background: Presenilin-1 (PS1) is a polytopic membrane protein and mutant variants are linked with early-onset familial Alzheimer's disease (FAD). Together with Aphl, Nicastrin and Pen-2, PS1 promotes intramambranous, y-secretase processing of type-1 membrane proteins, including APP and Notch. Objective(s): We sought to identify additional proteins that may modulate PS1 function and y-secretase activity. Methods: a stable 293 cell line expressing TAP-tagged PSI was used for affinity purification of PSi-associated proteins and MALDI-MS was employed to identify these polypeptides. Results: Several PS 1 associated proteins have been identified, Structural comparisons indicated that some of these are involved in protein trafficking, and proteolytic activities. The levels of PS1-NTF, CTF and A13 are evaluated by overexpression and siRNA down regulation of these PS1 associated proteins. Conclusions: Our preliminary studies suggest that several PS1 associated proteins regulate PS1 and y-secretase activity, and present studies are focused on understanding the function of these novel regulators of PS1 function.
•
GAMMA-SECRETASE INHIBITORS STABILIZE A 900 KDA PRESENILIN COMPLEX: A TETRAMERIC MODEL FOR GAMMA-SECRETASE
Genevieve Evin*, Louise D. Cantefford, David E. Hoke, R,M. Damian Holsinger, Janetta G. Culvenor, Colin L. Masters. The University of Melbourne, Parkville, Australia. Contact e-mail: [email protected] y-Secretase carries out the ultimate proteolytic step in the generation of Alzheimer's disease A13 amyloid protein by cleaving the amyloid protein precursor within its transmembrane domain. The same enzyme activity is involved in processing membrane domains of a subset of type I integral proteins, including Notch. Four gene products, presenilin, nicastrin, APH-1, and PEN-2 are required for y-secretase activity that is contained within a high molecular weight complex. To further characterize y-secretase, we probed membranes from SH-SY5Y cells with y-secretase inhibitor biotin-derivatives. Derivatives of L-685,458, pepstatin and a difluoroalcohol transition-state analog (1-Bt) were found to interact tightly with presenilin 1 (PSI) C-terminal fragment and with a ~70 kDa species containing immunoreactivity for PS1 N-terminal region and for APH-1. Blue native gel electrophoresis followed by western blotting indicated that CHAPSO solubilized a PS1 complex of high molecular weight, that was broken down into 360 and 450 kDa species upon addition of dodecylmaitoside, and that Brij-35 extracted a 450 kDa PS1 complex. With this technique, derivatives of the three inhibitors were found to interact preferentially with the 900 kDa species, whatever detergent was used to extract PS 1 complexes, but an interaction with the 450 kDa species was also evident. Superose-6 size-exclusion chromatography showed that, after treatment with L-685,458, PS1 immunoreactivity from membrane Brij-extract was more condensed
in a fraction of 900 kDa, suggesting the inhibitor stabilized this high molecular weight complex. Pre-incubating Brij-35 membrane extracts with L-685,458 enhanced binding of the L-685,458 biotin-derivative, suggesting that the inhibitor stabilized a conformation favorable for binding of the biotinylated probe. From these observations, we propose a tetrameric model for the structure of y-secretase that would be consistent with the apparent molecular mass of the complex and would encompass two symmetrical active sites able to bind simultaneously two inhibitor molecules. (supported by the Australian NHMRC)
~ T H E PRESENILINS, CHROMOSOME MISSEGREGATION, AND ALZHEIMER'S DISEASE Debrah I. Boeras*, Antoneta Granic, Jaya Padmanabhan, Huntington Potter. USE College of Medicine, Tampa, FL, USA. Contact e-maiL" dboeras@hsc, usfedu
Background: Mutations in two genes, Presenilin 1 (PS1), and Presenilin 2 (PS2), have been shown to cause the majority of early-onset familial Alzheimer's Disease (FAD). The presenilins are found to be partly associated with the nuclear membrane and other cellular structures involved in cell division and chromosome segregation. We have previously demonstrated that fibroblasts from FAD patients with mutant presenilin genes, PS-1 or PS-2, show an increase in chromosome defects including trisomy 21, the chromosome abnormality that underlies Down syndrome and causes such individuals to develop AD pathology by age 30-40. (Geller and Potter, 1999) Furthermore, the APP gene, whose cleavage leads to amyloid formation, is located on chromosome 21. Objective(s): To further investigate the involvement of the presenilins in the cell cycle, and more importantly, in chromosome missegregation, we are using the following two experimental systems: 1) transfected ceils expressing either normal human presenilin genes or FAD mutant presenilin genes; 2) cells from mice expressing either normal or FAD mutant presenilins. Methods: We have subcloned the normal and FAD mutant PS genes into expression vectors and introduced them into mammalian cells with normal chromosome complements. Transfected cells expressing normal and mutant presenilins have been examined for changes in their karyotype by both standard metaphase chromosome analysis and by fluorescence in situ hybridization (FISH). Levels of PS gene expression in the transfected cells have been analyzed with real time PCR. Cell-specific immunocytochemistry and FISH with single copy mouse probes is being used to assess the level of aneuploidy in isolated primary neurons of presenifin transgenic mice. Results: Together these experiments indicate that the presenilins are involved in or affect cell cycle regulation and/or chromosome segregation and that the FAD mutations alter the presenilin function in the cell cycle.
~ - ]
MECHANISMS OF PRESENILIN REGULATION BY UBIQUILIN
Leann K. Massey* 1 Mervyn J. Monteiro 2. 1 University of Maryland, Baltimore, MD, USA," 2 University of Maryland Biotechnology Institute, Baltimore, MD, USA. Contact e-mail: lmassO01 @umaryland.edu Mutations in presenilin proteins (PS1 and PS2) lead to early onset Alzheimer's disease (AD) making the study of these proteins and the molecular interactions that regulate their levels important in characterizing AD. There are multiple forms of PS proteins in cells, the FL protein and their endoproteolytic fragments, termed the NH2-terminal fragment (NTF) and the COOH-terminal fragment (CTF). Currently, the NTF and CTF are proposed to be the active form of PS, residing in the y-secretase complex and cleaving APP to release A13.Thus, studying PS-interactors that modulate fragment levels could provide important information regarding y-secretase activity. Rudy Tanzi and co-workers recently used genetic screening to find new genes involved in AD and identified the ubiquilin-1 gene as one potential candidate. Prior to this, we demonstrated that the ubiquilin-1 protein, which contains ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, interacts both in vivo and in vitro with the large hydrophilic loop of full-length (FL) PS stabilizing it and perhaps acting as a molecular