- Email: [email protected]
P1-183 Three-dimensional structure of an autonomously folded domain of human amyloid-β precursor protein
S148
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Poster Session P I : Molecular Mechanisms of Neurodegeneration - ~-Amyloidosis T H R E E - D I M E N S I O N A L S T R U C T U R E O F AN AUTONOMOUSLY FOLDED DOMAIN OF HUMAN AMYLOID-[3 P R E C U R S O R P R O T E I N
Irina Dulubova*, Angela Ho, Iryna Huryeva, Thomas C. Stidhof, Josep Rizo. UT Southwestern Medical Center, Dallas, TX, USA. Contact e-mail: Irina.Dulubova@ UTSouthwestern.edu
Background: Production of amyloid-[~ peptide (A[~) through proteolytic processing of Amyloid-~ Precursor Protein (APP) by [3- and y-secretases is known to play a major role in the pathogenesis of Alzheimer's disease. APP is a ubiquitously expressed membrane glycoprotein with a large NH2-terminal extracellular sequence, a single transmembrane region, and a short COOH-terminal intracellular fragment (AICD). The extracellular sequence of APP includes a large conserved region, the CAPPD (for Central APP Domain), whose structure and function are unknown. The CAPPD is adjacent to the [3-secretase cleavage site on APP, making this region an attractive target for potential regulation of the cleavage. A recent study (Ho&Siidhof, PNAS, in press) has reported that F-spondin, a secreted signaling molecule implicated in neuronal development and repair, binds to the CAPPD, and that this binding inhibits [3-secretase cleavage of APP in transfected cells. Objective: To analyze the structural properties of the CAPPD and to evaluate its interaction with F-spondin as a first step towards examining the biological function of this region. Methods: We have analyzed recombinant proteins encompassing the entire CAPPD of human APP by partial proteolysis, and examined a stable domain contained within the CAPPD by multidimensional nuclear magnetic resonance (NMR) spectroscopy. Results: We found an autonomously folded domain that comprises the C-terminal half of the CAPPD and have determined its three-dimensional structure by NMR spectroscopy. The structure revealed that the domain is composed of four a-helices. We are now using this structural information to design mutants that might modulate F-spondin binding, and are currently studying the effect of the mutations on F-spondin binding and APP processing. Conclusions: The extracellular sequence of APP contains a large conserved region, the CAPPD, which in tum includes an autonomously folded domain. The three-dimensional structure of this domain at atomic resolution now allows the generation of structure-based mutants to test the significance of this region in vivo. We anticipate that our structural studies will provide new insights into APP function and into the mechanisms of regulation of APP processing.
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A 50 BASE E L E M E N T I M M E D I A T E L Y DISTAL T O T H E S T O P C O D O N C O N T R O L S APP MRNA STABILITY
Pamela R. Westmark*, James S. Malter. University of Wisconsin, Madison, WI, USA. Contact e-mail: [email protected]
Background: Alzheimer's disease (AD) is characterized by the overproduction and deposition of [3-amyloid, a 39-42 amino acid peptide that is cleaved from the amyloid precursor protein (APP). Understanding of the molecular regulation of APP mRNA and protein levels is vital to understanding [3-amyloid accumulation and deposition in AD. APP and downstream [3-amyloid production are dependent on steady state levels of APP mRNA. APP mRNA degradation, abundance, and downstream APP synthesis are controlled by at least two 31-untranslated region (UTR) regulatory elements. The 50 base stop codon element (50sce) acts as an APP mRNA stabilizer and opposes the action of a 29 base element, which destabilizes APP mRNA. Both elements function through mRNA binding protein interactions. Objective: By mutating the 50sce, we attempted to characterize and inhibit the 50sce cis acting element adjacent to the stop codon. Methods: We sequentially mutated 5 nucleotides at a time in the 50sce and screened the resulting RNA fragments for mRNA binding interactions. Then, APP-WT and mutant cDNAs were co-transfected into B103 cells and the amounts of APP, APP mRNA and stability determined. Results: Mutations at positions 2257 to 2261, 2267 to 2271, 2287 to 2291, 2292 to 2296, 2257 to 2261/2267 to 2271, and 2257 to 2261/2287 to 2291, led to increased in vitro binding, while the reverse was seen with changes at positions 2262 to 2266, 2277 to 2281, 2297 to 2301, 2262 to 2266/2277
to 2281, 2262 to 2266/2297 to 2301, 2277 to 2281/2282 to 2286, and 2277 to 2281/2297 to 2301. The expression levels of transfected APP mutant mRNAs and protein correlated with protein binding affinities, which was consistent with the hypothesis that the latter binds to and stabilizes the former. Co-transfection of WT and mutant APP cDNAs also demonstrated modulation of WT mRNA content and APP synthesis. Conclusions: These data suggested that modest sequence changes could lead to significant alterations in mRNA-protein interactions and stability. Destabilization of APP mRNA by antagonization of the 50sce element could provide a novel mechanism to pharmacologically manipulate APP and [3-amyloid production.
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ANALYSIS O F S I N G L E A L Z H E I M E R P L A Q U E C O R E S BY L A S E R - C A P T U R E M I C R O S C O P Y A N D N A N O - E L E C T R O S P R A Y f r A N D E M MASS SPECTROMETRY
Lars O. Tjemherg*, Nenad Bogdanovic, Birgitta Axelsson, Bengt Winhlad, Jan N~slund. Karolinska Institutet, Huddinge, Sweden. Contact e-mail: Lars. Tjernberg @neurotec.ki.se
Background: Polymerization of the 40-42 residue amyloid [3-peptide (A[3) into amyloid plaques is a central event in Alzheimer's disease (AD) pathogenesis. It has been speculated that other proteins are of importance in the polymerization process and co-deposit with AI3 in the plaque cores. Identifying these proteins and preventing them from interacting with A[3 could thus be a possible therapeutic approach for prevention and treatment of AD. Objective(s): The aim of this study was to develop a highly selective and sensitive method for the analysis of amyloid plaque cores. Methods: Post mortem brain was homogenized and centrifuged in buffer containing protease inhibitor cocktail. The resulting pellet was washed and centrifuged in 30% sucrose. The pellet was washed with SDS and filtered through a 40 Ixm nylon mesh. The filtrate was further purified by velocity sedimentation centrifugation. Plaque-enriched fractions were pooled and dispensed on a glass slide. The glass slide was placed in laser microscope and selected plaques were catapulted into a tube cap by a laser pulse. The plaques were dissolved in formic acid, lyophilized and digested with trypsin. The samples were desalted and concentrated before injection onto a reversed phase capillary column coupled online to an ion-trap mass spectrometer. Mass spectra (MS) and MS/MS spectra were automatically recorded. The MASCOT software was used for matching MS/MS spectra to sequences deposited in the NCBI protein database. Results: The use of laser catapulting as the final step for isolation of plaque cores resulted in a preparation free from contaminating aggregates. One single plaque was sufficient for the unbiased identification of the major plaque component, AlL Interestingly, even when 100 plaques were catapulted and analyzed, no other protein than A[3 could be identified. Given that the total recovery of potential plaque core proteins are similar as for A[~, no other protein is present in the plaque cores at levels above 1% as compared to AlL Conclusions: Laser pressure catapulting in combination with LC-MS/MS enables highly selective and sensitive analysis of plaque cores. We suggest that this method can be used for the analysis of aggregates and inclusion bodies found in other neurodegenerative disorders
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U N B I A S E D C O M P A R A T I V E STUDY O F T H E A L Z H E I M E R ' S A M Y L O I D P E P T I D E S W I T H PC-12 CELLS
David A. Bateman*, Avi Chakrabartty. University of Toronto, Toronto, ON, Canada. Contact e-mail: [email protected]
Background: Alzheimer's disease is linked to the formation of amyloid fibrils, which has been shown to be catalyzed by the release of Alzheimer's peptides Abeta40 and Abeta42. These peptides are proteolytic cleavage products from the amyloid precursor protein and are forty and forty-two amino acids in length. The peptides start to deposit in brains as plasma membrane-bound diffuse plaques and have been shown to specifically interact with certain phospholipids and gangliosides. Current methods utilize
•
Poster Session P I : Molecular Mechanisms of Neurodegeneration - ~-Amyloidosis T H R E E - D I M E N S I O N A L S T R U C T U R E O F AN AUTONOMOUSLY FOLDED DOMAIN OF HUMAN AMYLOID-[3 P R E C U R S O R P R O T E I N
Irina Dulubova*, Angela Ho, Iryna Huryeva, Thomas C. Stidhof, Josep Rizo. UT Southwestern Medical Center, Dallas, TX, USA. Contact e-mail: Irina.Dulubova@ UTSouthwestern.edu
Background: Production of amyloid-[~ peptide (A[~) through proteolytic processing of Amyloid-~ Precursor Protein (APP) by [3- and y-secretases is known to play a major role in the pathogenesis of Alzheimer's disease. APP is a ubiquitously expressed membrane glycoprotein with a large NH2-terminal extracellular sequence, a single transmembrane region, and a short COOH-terminal intracellular fragment (AICD). The extracellular sequence of APP includes a large conserved region, the CAPPD (for Central APP Domain), whose structure and function are unknown. The CAPPD is adjacent to the [3-secretase cleavage site on APP, making this region an attractive target for potential regulation of the cleavage. A recent study (Ho&Siidhof, PNAS, in press) has reported that F-spondin, a secreted signaling molecule implicated in neuronal development and repair, binds to the CAPPD, and that this binding inhibits [3-secretase cleavage of APP in transfected cells. Objective: To analyze the structural properties of the CAPPD and to evaluate its interaction with F-spondin as a first step towards examining the biological function of this region. Methods: We have analyzed recombinant proteins encompassing the entire CAPPD of human APP by partial proteolysis, and examined a stable domain contained within the CAPPD by multidimensional nuclear magnetic resonance (NMR) spectroscopy. Results: We found an autonomously folded domain that comprises the C-terminal half of the CAPPD and have determined its three-dimensional structure by NMR spectroscopy. The structure revealed that the domain is composed of four a-helices. We are now using this structural information to design mutants that might modulate F-spondin binding, and are currently studying the effect of the mutations on F-spondin binding and APP processing. Conclusions: The extracellular sequence of APP contains a large conserved region, the CAPPD, which in tum includes an autonomously folded domain. The three-dimensional structure of this domain at atomic resolution now allows the generation of structure-based mutants to test the significance of this region in vivo. We anticipate that our structural studies will provide new insights into APP function and into the mechanisms of regulation of APP processing.
~
A 50 BASE E L E M E N T I M M E D I A T E L Y DISTAL T O T H E S T O P C O D O N C O N T R O L S APP MRNA STABILITY
Pamela R. Westmark*, James S. Malter. University of Wisconsin, Madison, WI, USA. Contact e-mail: [email protected]
Background: Alzheimer's disease (AD) is characterized by the overproduction and deposition of [3-amyloid, a 39-42 amino acid peptide that is cleaved from the amyloid precursor protein (APP). Understanding of the molecular regulation of APP mRNA and protein levels is vital to understanding [3-amyloid accumulation and deposition in AD. APP and downstream [3-amyloid production are dependent on steady state levels of APP mRNA. APP mRNA degradation, abundance, and downstream APP synthesis are controlled by at least two 31-untranslated region (UTR) regulatory elements. The 50 base stop codon element (50sce) acts as an APP mRNA stabilizer and opposes the action of a 29 base element, which destabilizes APP mRNA. Both elements function through mRNA binding protein interactions. Objective: By mutating the 50sce, we attempted to characterize and inhibit the 50sce cis acting element adjacent to the stop codon. Methods: We sequentially mutated 5 nucleotides at a time in the 50sce and screened the resulting RNA fragments for mRNA binding interactions. Then, APP-WT and mutant cDNAs were co-transfected into B103 cells and the amounts of APP, APP mRNA and stability determined. Results: Mutations at positions 2257 to 2261, 2267 to 2271, 2287 to 2291, 2292 to 2296, 2257 to 2261/2267 to 2271, and 2257 to 2261/2287 to 2291, led to increased in vitro binding, while the reverse was seen with changes at positions 2262 to 2266, 2277 to 2281, 2297 to 2301, 2262 to 2266/2277
to 2281, 2262 to 2266/2297 to 2301, 2277 to 2281/2282 to 2286, and 2277 to 2281/2297 to 2301. The expression levels of transfected APP mutant mRNAs and protein correlated with protein binding affinities, which was consistent with the hypothesis that the latter binds to and stabilizes the former. Co-transfection of WT and mutant APP cDNAs also demonstrated modulation of WT mRNA content and APP synthesis. Conclusions: These data suggested that modest sequence changes could lead to significant alterations in mRNA-protein interactions and stability. Destabilization of APP mRNA by antagonization of the 50sce element could provide a novel mechanism to pharmacologically manipulate APP and [3-amyloid production.
•
ANALYSIS O F S I N G L E A L Z H E I M E R P L A Q U E C O R E S BY L A S E R - C A P T U R E M I C R O S C O P Y A N D N A N O - E L E C T R O S P R A Y f r A N D E M MASS SPECTROMETRY
Lars O. Tjemherg*, Nenad Bogdanovic, Birgitta Axelsson, Bengt Winhlad, Jan N~slund. Karolinska Institutet, Huddinge, Sweden. Contact e-mail: Lars. Tjernberg @neurotec.ki.se
Background: Polymerization of the 40-42 residue amyloid [3-peptide (A[3) into amyloid plaques is a central event in Alzheimer's disease (AD) pathogenesis. It has been speculated that other proteins are of importance in the polymerization process and co-deposit with AI3 in the plaque cores. Identifying these proteins and preventing them from interacting with A[3 could thus be a possible therapeutic approach for prevention and treatment of AD. Objective(s): The aim of this study was to develop a highly selective and sensitive method for the analysis of amyloid plaque cores. Methods: Post mortem brain was homogenized and centrifuged in buffer containing protease inhibitor cocktail. The resulting pellet was washed and centrifuged in 30% sucrose. The pellet was washed with SDS and filtered through a 40 Ixm nylon mesh. The filtrate was further purified by velocity sedimentation centrifugation. Plaque-enriched fractions were pooled and dispensed on a glass slide. The glass slide was placed in laser microscope and selected plaques were catapulted into a tube cap by a laser pulse. The plaques were dissolved in formic acid, lyophilized and digested with trypsin. The samples were desalted and concentrated before injection onto a reversed phase capillary column coupled online to an ion-trap mass spectrometer. Mass spectra (MS) and MS/MS spectra were automatically recorded. The MASCOT software was used for matching MS/MS spectra to sequences deposited in the NCBI protein database. Results: The use of laser catapulting as the final step for isolation of plaque cores resulted in a preparation free from contaminating aggregates. One single plaque was sufficient for the unbiased identification of the major plaque component, AlL Interestingly, even when 100 plaques were catapulted and analyzed, no other protein than A[3 could be identified. Given that the total recovery of potential plaque core proteins are similar as for A[~, no other protein is present in the plaque cores at levels above 1% as compared to AlL Conclusions: Laser pressure catapulting in combination with LC-MS/MS enables highly selective and sensitive analysis of plaque cores. We suggest that this method can be used for the analysis of aggregates and inclusion bodies found in other neurodegenerative disorders
•
U N B I A S E D C O M P A R A T I V E STUDY O F T H E A L Z H E I M E R ' S A M Y L O I D P E P T I D E S W I T H PC-12 CELLS
David A. Bateman*, Avi Chakrabartty. University of Toronto, Toronto, ON, Canada. Contact e-mail: [email protected]
Background: Alzheimer's disease is linked to the formation of amyloid fibrils, which has been shown to be catalyzed by the release of Alzheimer's peptides Abeta40 and Abeta42. These peptides are proteolytic cleavage products from the amyloid precursor protein and are forty and forty-two amino acids in length. The peptides start to deposit in brains as plasma membrane-bound diffuse plaques and have been shown to specifically interact with certain phospholipids and gangliosides. Current methods utilize