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Structural and biochemical analysis of the heparin-induced E1 dimer of the amyloid precursor protein
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Developing Topics
TABLE 1 Parent-Offspring Concordance in the ADC Registry. All AD1
Father-Son (%) Father-Daughter (%) Mother-Son (%) Mother-Daughter (%) 1 2 3
65 Onset AD2 (LOAD)
60 Onset AD3 (EOAD)
Concordant
Discordant
Concordant
Discordant
Concordant
Discordant
374 (17.1) 358 (12.2) 738 (32.9) 870 (28.7)
1814 2572 1501 2166
248 (15.1) 239 (10.3) 516 (30.4) 636 (26.5)
1390 2082 1182 1765
13 (4.3) 17 (4.8) 23 (7.4) 30 (8.1)
290 338 287 341
Concordant pairs with any age of onset. Concordant pairs who both have onset 65 years. Concordant pairs who both have onset 60 years.
but has had a profound impact on our understanding of the disease. There is a widespread misperception that dominant mutations in amyloid precursor protein, presenilin 1, and presenilin 2 cause most cases of EOAD even though they explain less than 10% of all EOAD cases. Here we set out to determine the genetic contribution to the remaining w90% of EOAD cases. Methods: A liability threshold model of disease was used to estimate heritability of EOAD and late-onset AD (LOAD) using concordance for AD among parent-offspring pairs. Individuals with probable AD and detailed parental history (n ¼ 5,370) were identified in a research registry of 32 United States Alzheimer’s Disease Centers. Results: For LOAD (n ¼ 4,302), we found sex-specific parent-offspring concordance that ranged from w10-30% resulting in a heritability of 69.8% (95% CI: 64.6-75.0%) and equal heritability for both sexes regardless of parental gender. For EOAD (n ¼ 702), we found that the parent-offspring concordance is ¼ 10% and EOAD heritability is 92-100% for all likely values of EOAD prevalence. Conclusions: We confirm LOAD is a highly polygenic disease. By contrast, the data for EOAD suggest it is an almost entirely genetically based disease, and the low rate of concordance and sex-specific pattern observed lead us to reject the hypotheses that EOAD is a purely dominant, mitochondrial, X-linked, or polygenic disorder. The most likely explanation of the data is that w90% of EOAD cases are due to autosomal recessive causes. P4-344
PRO-APOPTOTIC FUNCTION OF PIN1-MEDIATED NOTCH1 ACTIVATION
Sang-Ha Baik, Sungkyunkwan University, Suwon, South Korea. Background: The peptidyl-prolyl cis/trans isomerase (PPIase) Pin1 regulates factors involved in the control of cell growth and apoptosis. Notch1 is a transmembrane receptor crucial for neuronal development, neurogenesis, and neuronal death. Proteolytic cleavage of Notch1 by g-secretase results in liberation of the intracellular domain of Notch1 (NICD), which translocates into the nucleus and regulates gene expression. Methods: Here, we demonstrate that Pin1 stimulates Notch1 activation leading to an increased production of Notch1 intracellular domain (NICD) by gamma-secretase. Results: Overexpression of Pin1 enhances gamma-secretase activity and NICD production. Conversely, down-regulation of Pin1 using siRNA reduces gamma-secretase activity and NICD production. Pin1 overexpressed cells were vulnerable to glucose deprivation or chemical hypoxia induced cell death. The incidence of glucose deprivation or hypoxia-induced cell death was diminished in Pin1(-/-) mouse embryonic fibroblast cells or neuronal cells transfected with Pin1 siRNA. Conclusions: These results suggest novel functions of Pin1 in Notch1 activation and Notch1-mediated cell death. P4-344
STRUCTURAL AND BIOCHEMICAL ANALYSIS OF THE HEPARIN-INDUCED E1 DIMER OF THE AMYLOID PRECURSOR PROTEIN
Sven Dahms, Sandra Hoefgen, Dirk Roeser, Manuel Than, Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI) Jena, Jena, Germany. Background: The amyloid precursor protein (APP) is the key player in Alzheimer’s disease pathology because imbalanced proteolytic processing by the alpha-, beta and gamma-secretases leads to excessive production of neurotoxic Abeta peptide species. On the other hand, especially the N-terminal E1 domain, comprising the growth factor like domain (GFLD) and the copper binding do-
main has been implicated in physiological, neurotrophic functions of APP. In order to gain a structural understanding of the processes underlining the physiological action of APP we investigated the structure, function and biochemistry of the complete E1 domain. Methods: To analyze the structural properties facilitating APP function, we investigated the E1-domain by means of X-ray crystallography. The recombinant protein was expressed in E. coli and purified in a three-step chromatography procedure. We confirmed the functional features implicated by the structure via limited proteolysis, analytical size exclusion chromatography, static light scattering, ITC and molecular modeling. Results: The crystal structure of the APP-E1 domain was solved and refined to 2.7 A showing a so far unknown interaction of the subdomains. GFLD and CuBD interact via an evolutionary conserved interface in a pH dependent manner. Dimerization of the E1-domain is induced by heparin in a pH dependent manner. Based on a dimeric assembly found in the E1-domain crystals we modeled the heparin-[APP-E1]2 in agreement to our biochemical data. Conclusions: Heparin induced dimerization of the E1 domain provides a structural explanation for the ability of full length APP to dimerize and mediate cell adhesion, a prerequisite for neurotrophic action of the protein. The sub-domains interact tightly, forming a closed structural and hence a functional entity. Heparin induced dimerization as well as inter-domain interaction is altered upon pH shift from 7.4 to 5.7 giving rise for a pH dependent function of APP dependent on the sub-cellular localization. P4-345
DEVELOPMENT OF A NOVEL PROTEOMIC APPROACH FOR THE UNBIASED IDENTIFICATION OF NITRATED PROTEINS IN ALZHEIMER’S DISEASE BRAINS
Tal Nuriel, Emily Mercer, Yuliang Ma, Steven Gross, Weill Cornell Medical College, New York, New York, United States. Background: Nitration of tyrosine and tryptophan residues in proteins is commonly observed in the setting of neurodegenerative diseases such as Alzheimer’s disease. It has been proposed that this protein modification contributes to disease pathogenesis by inhibiting or modulating the activity of vital cellular proteins. To more fully understand the pathological effect of protein nitration on the progression of Alzheimer’s disease, it is important to broadly identify the proteins that undergo nitration in vivo, as well as the specific amino acid sites that get modified. Methods: To date, unbiased identification of nitrated proteins has primarily employed 2D-gel electrophoresis followed by Western Blotting with an anti-nitrotyrosine antibody. This method, however, suffers from limited coverage and infrequent discovery of the specific modification sites. To overcome these shortcomings, we have developed a solidphase, chemical-capture approach for unbiased and high-throughput discovery of nitrotyrosine and nitrotryptophan sites in proteins. This approach relies on a detailed modification strategy for the selective attachment of an agarose bead at sites of nitration, allowing for the purification of nitrated proteins, followed by trypsinolysis and subsequent identification of peptides and nitration sites by nanoflow LC-MS/MS. Results: We have demonstrated the validity of this method using nitrated BSA and peroxynitrite-treated rat brain homogenates. Furthermore, we have successfully identified endogenous protein nitration sites in the brains of transgenic mice containing an Alzheimer’s-like phenotype. Conclusions: These discoveries, along with future applications of this method, will advance our understanding of the role of protein nitration in Alzheimer’s disease, as well as other neurodegenerative disorders.
Developing Topics
TABLE 1 Parent-Offspring Concordance in the ADC Registry. All AD1
Father-Son (%) Father-Daughter (%) Mother-Son (%) Mother-Daughter (%) 1 2 3
65 Onset AD2 (LOAD)
60 Onset AD3 (EOAD)
Concordant
Discordant
Concordant
Discordant
Concordant
Discordant
374 (17.1) 358 (12.2) 738 (32.9) 870 (28.7)
1814 2572 1501 2166
248 (15.1) 239 (10.3) 516 (30.4) 636 (26.5)
1390 2082 1182 1765
13 (4.3) 17 (4.8) 23 (7.4) 30 (8.1)
290 338 287 341
Concordant pairs with any age of onset. Concordant pairs who both have onset 65 years. Concordant pairs who both have onset 60 years.
but has had a profound impact on our understanding of the disease. There is a widespread misperception that dominant mutations in amyloid precursor protein, presenilin 1, and presenilin 2 cause most cases of EOAD even though they explain less than 10% of all EOAD cases. Here we set out to determine the genetic contribution to the remaining w90% of EOAD cases. Methods: A liability threshold model of disease was used to estimate heritability of EOAD and late-onset AD (LOAD) using concordance for AD among parent-offspring pairs. Individuals with probable AD and detailed parental history (n ¼ 5,370) were identified in a research registry of 32 United States Alzheimer’s Disease Centers. Results: For LOAD (n ¼ 4,302), we found sex-specific parent-offspring concordance that ranged from w10-30% resulting in a heritability of 69.8% (95% CI: 64.6-75.0%) and equal heritability for both sexes regardless of parental gender. For EOAD (n ¼ 702), we found that the parent-offspring concordance is ¼ 10% and EOAD heritability is 92-100% for all likely values of EOAD prevalence. Conclusions: We confirm LOAD is a highly polygenic disease. By contrast, the data for EOAD suggest it is an almost entirely genetically based disease, and the low rate of concordance and sex-specific pattern observed lead us to reject the hypotheses that EOAD is a purely dominant, mitochondrial, X-linked, or polygenic disorder. The most likely explanation of the data is that w90% of EOAD cases are due to autosomal recessive causes. P4-344
PRO-APOPTOTIC FUNCTION OF PIN1-MEDIATED NOTCH1 ACTIVATION
Sang-Ha Baik, Sungkyunkwan University, Suwon, South Korea. Background: The peptidyl-prolyl cis/trans isomerase (PPIase) Pin1 regulates factors involved in the control of cell growth and apoptosis. Notch1 is a transmembrane receptor crucial for neuronal development, neurogenesis, and neuronal death. Proteolytic cleavage of Notch1 by g-secretase results in liberation of the intracellular domain of Notch1 (NICD), which translocates into the nucleus and regulates gene expression. Methods: Here, we demonstrate that Pin1 stimulates Notch1 activation leading to an increased production of Notch1 intracellular domain (NICD) by gamma-secretase. Results: Overexpression of Pin1 enhances gamma-secretase activity and NICD production. Conversely, down-regulation of Pin1 using siRNA reduces gamma-secretase activity and NICD production. Pin1 overexpressed cells were vulnerable to glucose deprivation or chemical hypoxia induced cell death. The incidence of glucose deprivation or hypoxia-induced cell death was diminished in Pin1(-/-) mouse embryonic fibroblast cells or neuronal cells transfected with Pin1 siRNA. Conclusions: These results suggest novel functions of Pin1 in Notch1 activation and Notch1-mediated cell death. P4-344
STRUCTURAL AND BIOCHEMICAL ANALYSIS OF THE HEPARIN-INDUCED E1 DIMER OF THE AMYLOID PRECURSOR PROTEIN
Sven Dahms, Sandra Hoefgen, Dirk Roeser, Manuel Than, Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI) Jena, Jena, Germany. Background: The amyloid precursor protein (APP) is the key player in Alzheimer’s disease pathology because imbalanced proteolytic processing by the alpha-, beta and gamma-secretases leads to excessive production of neurotoxic Abeta peptide species. On the other hand, especially the N-terminal E1 domain, comprising the growth factor like domain (GFLD) and the copper binding do-
main has been implicated in physiological, neurotrophic functions of APP. In order to gain a structural understanding of the processes underlining the physiological action of APP we investigated the structure, function and biochemistry of the complete E1 domain. Methods: To analyze the structural properties facilitating APP function, we investigated the E1-domain by means of X-ray crystallography. The recombinant protein was expressed in E. coli and purified in a three-step chromatography procedure. We confirmed the functional features implicated by the structure via limited proteolysis, analytical size exclusion chromatography, static light scattering, ITC and molecular modeling. Results: The crystal structure of the APP-E1 domain was solved and refined to 2.7 A showing a so far unknown interaction of the subdomains. GFLD and CuBD interact via an evolutionary conserved interface in a pH dependent manner. Dimerization of the E1-domain is induced by heparin in a pH dependent manner. Based on a dimeric assembly found in the E1-domain crystals we modeled the heparin-[APP-E1]2 in agreement to our biochemical data. Conclusions: Heparin induced dimerization of the E1 domain provides a structural explanation for the ability of full length APP to dimerize and mediate cell adhesion, a prerequisite for neurotrophic action of the protein. The sub-domains interact tightly, forming a closed structural and hence a functional entity. Heparin induced dimerization as well as inter-domain interaction is altered upon pH shift from 7.4 to 5.7 giving rise for a pH dependent function of APP dependent on the sub-cellular localization. P4-345
DEVELOPMENT OF A NOVEL PROTEOMIC APPROACH FOR THE UNBIASED IDENTIFICATION OF NITRATED PROTEINS IN ALZHEIMER’S DISEASE BRAINS
Tal Nuriel, Emily Mercer, Yuliang Ma, Steven Gross, Weill Cornell Medical College, New York, New York, United States. Background: Nitration of tyrosine and tryptophan residues in proteins is commonly observed in the setting of neurodegenerative diseases such as Alzheimer’s disease. It has been proposed that this protein modification contributes to disease pathogenesis by inhibiting or modulating the activity of vital cellular proteins. To more fully understand the pathological effect of protein nitration on the progression of Alzheimer’s disease, it is important to broadly identify the proteins that undergo nitration in vivo, as well as the specific amino acid sites that get modified. Methods: To date, unbiased identification of nitrated proteins has primarily employed 2D-gel electrophoresis followed by Western Blotting with an anti-nitrotyrosine antibody. This method, however, suffers from limited coverage and infrequent discovery of the specific modification sites. To overcome these shortcomings, we have developed a solidphase, chemical-capture approach for unbiased and high-throughput discovery of nitrotyrosine and nitrotryptophan sites in proteins. This approach relies on a detailed modification strategy for the selective attachment of an agarose bead at sites of nitration, allowing for the purification of nitrated proteins, followed by trypsinolysis and subsequent identification of peptides and nitration sites by nanoflow LC-MS/MS. Results: We have demonstrated the validity of this method using nitrated BSA and peroxynitrite-treated rat brain homogenates. Furthermore, we have successfully identified endogenous protein nitration sites in the brains of transgenic mice containing an Alzheimer’s-like phenotype. Conclusions: These discoveries, along with future applications of this method, will advance our understanding of the role of protein nitration in Alzheimer’s disease, as well as other neurodegenerative disorders.