- Email: [email protected]
P1-201 In search for APP-binding proteins
Poster Session P I : Molecular Mechanisms of Neurodegeneration - ~-Amyloidosis environment. Objective: To investigate the morphology and cytotoxicity of lipid/detergent induced Abeta oligomers. Methods: Circular dichroism spectroscopy was used to investigate the secondary structure of the detergent induced oligomers. Analytical ultraceutrifugation and platinum/carbon shadowing electron microscopy were used to examine the size and morphology of the aggregates. Thioflavin T and Congo Red binding assays were performed to study the amyloid characteristics of the detergent induced oligomers. A sufforhodamine B (SRB) assay was performed using PC12 cells to determine the cytotoxic properties of the Abeta aggregates. Results and Conclusions: To simulate a membrane environment, we have investigated Abeta association in the presence of various nondenaturing detergents. Using analytical ultracentfifugation, we find that the detergent promotes the instantaneous formation of large complexes of Abeta peptides that contain approximately 280 Abeta monomers; the peptide monomers adopt a folded beta-sheet structure within these complexes. Vesicles composed of phospholipids, gangliosides, and/or sphingomyelin prompt Abeta peptides to undergo similar structural transitions, suggesting that the Abeta complexes formed in detergent represent the memhrane-associated form of Abeta. Platinum/Carbon shadowing electron microscopy demonstrated that the detergent induces the formation of a monodispered sample of spherical particles and inhibits further association into amyloid fibrils. Furthermore, these membrane active spheroids were cytotoxic to cultured PC12 cells. Further characterization of these prefihriUar aggregates may lead to therapeutic strategies.
•
SOLUTION NMR STUDIES OF THE AB(1-40) AND AB(1-42) PEPTIDES ESTABLISH THAT THE MET35 OXIDATION STATE A F F E C T S THE MECHANISM OF AMYLOID F O R M A T I O N
Michael G. Zagorski* i, Liming Hou ~, Haiyan Shao 1, Yongbo Zhang 1, Nanda Menon 2. 1Case Western Reserve University, Cleveland, OH, USA; 2Recombinant Peptide Technologies, Inc., Athens, GA, USA. Contact e-mail: mxzl2 @cwru.edu Background: The pathogenesis of Alzheimer's disease is characterized by the aggregation and fibrillation of the 40-residue A13(1-40) and 42residue A1~(1-42) peptides into amyloid plaques. The structural changes associated with the conversion of monomeric A[3 peptide building blocks into multimeric fibrillar ~-strand aggregates remains unknown. Objectives: Recently, we established that oxidation of the methiohine-35 side-chain to the sulfoxide (Met35 red --+ Met35 °x) significantly impedes the rate of aggregation and fibrillation of the AI3 peptide. To explore this effect at greater resolution, we carefully compared the 1H, 15N, and 13C NMR chemical shifts of four A~3 peptides that had the Met35 reduced or oxidized (A13(140)Met35 red, A[g(1-40)Met35 °x, A[3(1-42)Met35 red, and AI3(1-42)Met35°x). The major advantage with the solution NMR technique is that it can provide site-specific structural information about the early events associated with aggregation and amyloid formation. Methods: Analytical ultracentfifugation, AFM, CD, and NMR. Results: With the use of a special disaggregation protocol, the highly aggregation prone AI3 peptides could be studied at higher, milli-molar concentrations (as required by NMR) in aqueous solution at neutral pH, remaining largely monomeric at 5°C as determined by sedimentation equilibrium studies. The NOE, amide-NH temperature coefficients, and chemical shift indices of the 1Hc~, 13Ce~ and I3C~, established that the four peptides are largely random, extended chain structures, with the Met35 °x reducing the propensity for [3-strand structure at two hydrophobic regions (Leu 17-Ala21 and Ile31-Va136), and turn- or bend-like structures at Asp7-Glul 1 and Phe20-Ser26. Additional NMR studies monitoting changes that occur during aging at 37°C established that, along with a gradual loss of signal/noise, the Met35 °x significantly hindered upfield chemical shift movements of the 2H NMR signals for the His6, Hisl3, and His 14 side-chains. Conclusions: The present NMR studies demonstrate that the Met35 red -+ Met35 °x conversion prevents aggregation by reducing both hydrophobic and electrostatic association, and that the AI3(1-40)Met35 red, Af3(1-40)Met35 °x, A~(1-42)Met35 rea, and AI3(1-42)Met35 °x peptides may associate differently, through specific, sharp changes in smacture during the initial stages of aggregation. The possible roles of the histidine side chains and salt bridge formation in A[3 seed formation are discussed.
~
I
N
S153
SEARCH FOR APP-BINDING PROTEINS
Carmela R. Abraham .1 , Sunyoung Oh 1, Aviva Ellenstein 1, Eric Berg 1, Peter O' Connor 1, Catherine Costello I , William E. Van Nostrand 2, Racbael Neve 3. l Boston University School of Medicine, Boston, MA, USA; 2SUNY at Stony Brook, Stony Brook, NY, USA; 3McLean Hospital, Belmont, MA, USA. Contact e-mail: [email protected] A key event in the pathogenesis of Alzheimer's Disease (AD) is the process of self-assembly of the A[3 peptide, which is generated from the proteolytic processing of Amyloid Precursor Protein (APP). Unfortunately, the physiological function of APP has been elusive. Yet, it is known that APP processing shares significant similarities with another type I transmemhrane protein, Notchl. Notch1 intracelhilar domain (NICD) is known to be involved in transcription via signaling pathway. It is plausible that APP C-terminal intracellular domain (AICD) might also play a role in signaling events. If this is true, APP should have potential ligands or binding partners to its extracellular domain. This work focuses on identification of proteins that bind to the extracellular domain of APP using a chemical cross -linking method to trap the APP-ligand complexes followed by mass spectrometric analyses to identify these complexes. Primary rat neuronal ceils over expressing human APP 751 by HSV infection were used for the crosslinking experiments. The cross-linked APP complexes were immunopurified using an APP antibody-agarose column. Cross-linked complexes were then separated by SDS-PAGE. A high molecular weight (~250kDa) band was detected by silver staining, excised from the get and digested with trypsin. The resultant peptides were extracted and analyzed using Matrix Assisted Laser Desorption/Ionization (MALDI)-Time-of-Flight (TOF) MS. Peptide masses from MALDI were searched against known protein sequences in available databases and several potential APP-binding proteins have been identified. Confirmation experiments using co-immunoprecipitation and double inununo-fluorescence following overexpression of these newly discovered proteins should enable identification of novel APP-interacting proteins and perhaps reveal the physiological function of APE Similarly to Notch1, modulation of these ligands may affect the proteolytic processing of APE
~ - ~
A M Y L O I D BETA (AB) AND A NON AB COMPONENT O F ALZHEIMER DISEASE AMYLOID (NAC) A F F E C T DIFFERENTLY BASIC AND R E C E P T O R S
MEDIATED NITRIC OXIDE SYNTHASE AND POLY(ADP-RIBOSE) POLYMERASE IN HIPPOCAMPUS Robert P. Strosznajder*, Agata Zambrzycka, Henryk Jesko, Joanna B. Strosznajder. Medical Research Centre Polish Academy of Sciences, Warsaw, Poland. Contact e-mail: [email protected] Amyloid beta (AB) and NAC peptides deposition leading to nerve endings degeneration during Alzheimer Disease (AD). AB depending on concentration and structure elicits neurotrophic or neurotoxic effect. NAC peptides fibrils could serve as a nucleus for the amyloid formation and have a strong tendency to form a beta sheet sa'ucmre. The molecular mechanism of these peptides neurotoxicity is not fully understood. The aim of this study was to investigate the role of NO in AB and NAC evoked macromolecules damage. Moreover, the activation of proapoptotic proteins by these peptides was investigated. The radiochemical and immunochemical methods were used in this study. This data indicated that AB 1-40/42 at 25 IzM and NAC 10 p,M activate free radicals formation and enhance oxidation of protein, lipids and DNA. Both peptldes by NO and other free radicals evoke DNA damage and enhance poly(ADP-ribose) polymerase (PARP) exclusively in hippocampus. Amyloid beta peptides AB 1-40/42 and its neurotoxic fragment AB25-35 stimulate caspase 3 activity in synaptic cytosol. NAC peptides release apoptotic inducing factor (AIF) from mitochondria and activate its translocation into nucleous indicated that this pathway is probably also involved in cell death. These peptides eliminate glutaminergic, NMDA receptor mediated signaling and also muscarirdc cholinergic receptor dependent signal transduction into PAR]?. Our last data indicated that PARP is the new nuclear target for these receptors signal transduction.
•
SOLUTION NMR STUDIES OF THE AB(1-40) AND AB(1-42) PEPTIDES ESTABLISH THAT THE MET35 OXIDATION STATE A F F E C T S THE MECHANISM OF AMYLOID F O R M A T I O N
Michael G. Zagorski* i, Liming Hou ~, Haiyan Shao 1, Yongbo Zhang 1, Nanda Menon 2. 1Case Western Reserve University, Cleveland, OH, USA; 2Recombinant Peptide Technologies, Inc., Athens, GA, USA. Contact e-mail: mxzl2 @cwru.edu Background: The pathogenesis of Alzheimer's disease is characterized by the aggregation and fibrillation of the 40-residue A13(1-40) and 42residue A1~(1-42) peptides into amyloid plaques. The structural changes associated with the conversion of monomeric A[3 peptide building blocks into multimeric fibrillar ~-strand aggregates remains unknown. Objectives: Recently, we established that oxidation of the methiohine-35 side-chain to the sulfoxide (Met35 red --+ Met35 °x) significantly impedes the rate of aggregation and fibrillation of the AI3 peptide. To explore this effect at greater resolution, we carefully compared the 1H, 15N, and 13C NMR chemical shifts of four A~3 peptides that had the Met35 reduced or oxidized (A13(140)Met35 red, A[g(1-40)Met35 °x, A[3(1-42)Met35 red, and AI3(1-42)Met35°x). The major advantage with the solution NMR technique is that it can provide site-specific structural information about the early events associated with aggregation and amyloid formation. Methods: Analytical ultracentfifugation, AFM, CD, and NMR. Results: With the use of a special disaggregation protocol, the highly aggregation prone AI3 peptides could be studied at higher, milli-molar concentrations (as required by NMR) in aqueous solution at neutral pH, remaining largely monomeric at 5°C as determined by sedimentation equilibrium studies. The NOE, amide-NH temperature coefficients, and chemical shift indices of the 1Hc~, 13Ce~ and I3C~, established that the four peptides are largely random, extended chain structures, with the Met35 °x reducing the propensity for [3-strand structure at two hydrophobic regions (Leu 17-Ala21 and Ile31-Va136), and turn- or bend-like structures at Asp7-Glul 1 and Phe20-Ser26. Additional NMR studies monitoting changes that occur during aging at 37°C established that, along with a gradual loss of signal/noise, the Met35 °x significantly hindered upfield chemical shift movements of the 2H NMR signals for the His6, Hisl3, and His 14 side-chains. Conclusions: The present NMR studies demonstrate that the Met35 red -+ Met35 °x conversion prevents aggregation by reducing both hydrophobic and electrostatic association, and that the AI3(1-40)Met35 red, Af3(1-40)Met35 °x, A~(1-42)Met35 rea, and AI3(1-42)Met35 °x peptides may associate differently, through specific, sharp changes in smacture during the initial stages of aggregation. The possible roles of the histidine side chains and salt bridge formation in A[3 seed formation are discussed.
~
I
N
S153
SEARCH FOR APP-BINDING PROTEINS
Carmela R. Abraham .1 , Sunyoung Oh 1, Aviva Ellenstein 1, Eric Berg 1, Peter O' Connor 1, Catherine Costello I , William E. Van Nostrand 2, Racbael Neve 3. l Boston University School of Medicine, Boston, MA, USA; 2SUNY at Stony Brook, Stony Brook, NY, USA; 3McLean Hospital, Belmont, MA, USA. Contact e-mail: [email protected] A key event in the pathogenesis of Alzheimer's Disease (AD) is the process of self-assembly of the A[3 peptide, which is generated from the proteolytic processing of Amyloid Precursor Protein (APP). Unfortunately, the physiological function of APP has been elusive. Yet, it is known that APP processing shares significant similarities with another type I transmemhrane protein, Notchl. Notch1 intracelhilar domain (NICD) is known to be involved in transcription via signaling pathway. It is plausible that APP C-terminal intracellular domain (AICD) might also play a role in signaling events. If this is true, APP should have potential ligands or binding partners to its extracellular domain. This work focuses on identification of proteins that bind to the extracellular domain of APP using a chemical cross -linking method to trap the APP-ligand complexes followed by mass spectrometric analyses to identify these complexes. Primary rat neuronal ceils over expressing human APP 751 by HSV infection were used for the crosslinking experiments. The cross-linked APP complexes were immunopurified using an APP antibody-agarose column. Cross-linked complexes were then separated by SDS-PAGE. A high molecular weight (~250kDa) band was detected by silver staining, excised from the get and digested with trypsin. The resultant peptides were extracted and analyzed using Matrix Assisted Laser Desorption/Ionization (MALDI)-Time-of-Flight (TOF) MS. Peptide masses from MALDI were searched against known protein sequences in available databases and several potential APP-binding proteins have been identified. Confirmation experiments using co-immunoprecipitation and double inununo-fluorescence following overexpression of these newly discovered proteins should enable identification of novel APP-interacting proteins and perhaps reveal the physiological function of APE Similarly to Notch1, modulation of these ligands may affect the proteolytic processing of APE
~ - ~
A M Y L O I D BETA (AB) AND A NON AB COMPONENT O F ALZHEIMER DISEASE AMYLOID (NAC) A F F E C T DIFFERENTLY BASIC AND R E C E P T O R S
MEDIATED NITRIC OXIDE SYNTHASE AND POLY(ADP-RIBOSE) POLYMERASE IN HIPPOCAMPUS Robert P. Strosznajder*, Agata Zambrzycka, Henryk Jesko, Joanna B. Strosznajder. Medical Research Centre Polish Academy of Sciences, Warsaw, Poland. Contact e-mail: [email protected] Amyloid beta (AB) and NAC peptides deposition leading to nerve endings degeneration during Alzheimer Disease (AD). AB depending on concentration and structure elicits neurotrophic or neurotoxic effect. NAC peptides fibrils could serve as a nucleus for the amyloid formation and have a strong tendency to form a beta sheet sa'ucmre. The molecular mechanism of these peptides neurotoxicity is not fully understood. The aim of this study was to investigate the role of NO in AB and NAC evoked macromolecules damage. Moreover, the activation of proapoptotic proteins by these peptides was investigated. The radiochemical and immunochemical methods were used in this study. This data indicated that AB 1-40/42 at 25 IzM and NAC 10 p,M activate free radicals formation and enhance oxidation of protein, lipids and DNA. Both peptldes by NO and other free radicals evoke DNA damage and enhance poly(ADP-ribose) polymerase (PARP) exclusively in hippocampus. Amyloid beta peptides AB 1-40/42 and its neurotoxic fragment AB25-35 stimulate caspase 3 activity in synaptic cytosol. NAC peptides release apoptotic inducing factor (AIF) from mitochondria and activate its translocation into nucleous indicated that this pathway is probably also involved in cell death. These peptides eliminate glutaminergic, NMDA receptor mediated signaling and also muscarirdc cholinergic receptor dependent signal transduction into PAR]?. Our last data indicated that PARP is the new nuclear target for these receptors signal transduction.