The ultimate goal of this project is to understand the components of fibrillogenesis pathway structurally, focusing on [3-amyloid (A[3) of Alzheimer's Disease (AD), The specific aims of this proposal are as follows: 1, To examine, by solid-state NMR, AfJl-40 fibrils seeded by brain amyloid from patients who died with Alzheimer's Disease, i,e"""""""" fibrils seeded by ex mortuo brain amyloid, A[31-40 can adopt more than one stable conformation in fibrils, and these conformations can be passed on to progeny fibrils, But which of the known conformations, if any, is adopted by A[31-40 in the brains of patients with AD? To address this question, we purified brain amyloid from AD patients, and used this material to seed fibril formation by labeled synthetic A[31-40, Exciting new data, recently published (PNAS 106:7443, 2009), indicate that brainseeded fibrils have a novel structure, distinct from either of the known A[31-40 fibril structures, To address the question of fibril heterogeneity and disease phenotype, we will also compare ex mortuo seeded fibrils formed using material from different anatomic locations of the brain, and from different patients, 2, To examine mutations of the AfJ peptide occurring adjacent to the """"""""bend region"""""""", Of the point mutations (associated with familial AD) within the A[3 sequence itself, most are at positions 21-23, adjacent to the bend region that is essential for association of the two [3-sheets of wild-type A[3 in fibrils, We have now shown by solid-state NMR and other data (Tycko et aI., Biochemistry 48: 6072, 2009) that fibrils of the Iowa mutant, D23N-A[3 have an unprecedented structure for full length AI3 fibrils, antiparallel l3-sheets, In addition, new data on the Japanese mutation, L E22-Af3 indicate that, contrary to published reports, it readily and abundantly makes j3-sheet fibrils, and new solid-state NMR data (unpublished) show that these, too, deviate from the structure of wild type Aj3 fibrils. In the revised proposal, we focus on these two exemplary mutant forms of Aj3, which have in common the loss of an anionic residue and very rapid fibril formation, though there are instructive differences as well. For this aim, we will: A) Examine the C-terminall3-sheet region of D23N-AI3 (not previous studied, but currently in progress);8) Examine both the N- and C-terminall3 sheet regions of ~E22-Aj3 (already in progress). C) We have recently shown that fibrils of both of these mutant peptides can cross-seed wild-type AI3. Accordingly, we will examine wild type A131-40 fibrils made by cross-seeding, using D23N-AI3 and ~E22-AI3 fibrils as seeds. In all of these cases, results will be compared with data obtained previously and new data on brain-seeded AI3 fibrils to determine whether the wild type peptide is also capable of adopting structural forms similar to those of the mutant peptides. The materials for these studies are already in place (e.g., brains from individuals dying with AD and controls), and new, streamlined methods of solid-state NMR make this project entirely feasible in the two year time frame.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS042852-06A2
Application #
7782452
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Sutherland, Margaret L
Project Start
2001-12-01
Project End
2011-08-31
Budget Start
2009-09-30
Budget End
2010-08-31
Support Year
6
Fiscal Year
2009
Total Cost
$353,194
Indirect Cost
Name
University of Chicago
Department
Pathology
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Scherpelz, Kathryn P; Lu, Jun-Xia; Tycko, Robert et al. (2016) Preparation of Amyloid Fibrils Seeded from Brain and Meninges. Methods Mol Biol 1345:299-312
Lu, Jun-Xia; Qiang, Wei; Yau, Wai-Ming et al. (2013) Molecular structure of ?-amyloid fibrils in Alzheimer's disease brain tissue. Cell 154:1257-68
Cloe, Adam L; Orgel, Joseph P R O; Sachleben, Joseph R et al. (2011) The Japanese mutant A? (?E22-A?(1-39)) forms fibrils instantaneously, with low-thioflavin T fluorescence: seeding of wild-type A?(1-40) into atypical fibrils by ?E22-A?(1-39). Biochemistry 50:2026-39
Bockhorn, Jessica J; Lazar, Kristi L; Gasser, Adam J et al. (2010) Novel semisynthetic method for generating full length beta-amyloid peptides. Biopolymers 94:511-20
Lanning, Jennifer D; Hawk, Andrew J; Derryberry, Johnmark et al. (2010) Chaperone-like N-methyl peptide inhibitors of polyglutamine aggregation. Biochemistry 49:7108-18
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Chopra, Neeraj; Gan, Wenxun; Schreiber, Hans et al. (2009) Versatile cyclic templates for assembly of axially oriented ligands. Bioconjug Chem 20:231-40
Paravastu, Anant K; Qahwash, Isam; Leapman, Richard D et al. (2009) Seeded growth of beta-amyloid fibrils from Alzheimer's brain-derived fibrils produces a distinct fibril structure. Proc Natl Acad Sci U S A 106:7443-8
Tycko, Robert; Sciarretta, Kimberly L; Orgel, Joseph P R O et al. (2009) Evidence for novel beta-sheet structures in Iowa mutant beta-amyloid fibrils. Biochemistry 48:6072-84
Carnemolla, Ronald; Ren, Xuefeng; Biswas, Tapan K et al. (2008) The specific amino acid sequence between helices 7 and 8 influences the binding specificity of human apolipoprotein A-I for high density lipoprotein (HDL) subclasses: a potential for HDL preferential generation. J Biol Chem 283:15779-88

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