The conversion of soluble proteins into amyloid fibers is a feature of a number of clinical disorders including Alzheimer's, Huntington's and type II diabetes. In each medical disorder, the precursor protein has distinct primary and tertiary structure. However, the resultant fibers are remarkably similar at the histological and ultrastructural level. Fiber formation kinetics are similar to crystallization in that there exists a prolonged lag phase in which fiber is undetectable. This is followed by a cooperative transition to the fibrous state. Interestingly, both the fibrous state, and the intermediate states sampled during the lag phase have been identified as cytotoxic. Central to all these disorders, therefore, is the need to identify the molecular basis of conformational change. The overall goal of this proposal is to determine the molecular basis for amyloid conversion in two medically relevant systems. First, islet amyloid polypeptide(IAPP), a 37 residue peptide hormone that is cosecreted with insulin by the b-cells of the pancreas. In type II diabetics, it deposits as amyloid resulting in b-cell death. Second, renal diseases which necessitate treatment by dialysis result in the deposition of b-2 microglobulin (b2m) in the joints giving rise to a variety of skeletal pathologies. In both of these systems, it is wild-type, unmodified forms of the protein which aggregate. Our approach is to identify plausible changes in the in vivo environment of theses proteins and to determine the molecular impact of these changes on the folding and fibrillogenesis of these systems. In lAPP, our group recently reported the existence of an obligate intermediate and two nucleation processes. The first major aim is to determine the conformation and oligomeric changes associated with these phenomena. Furthermore, we have also determined that fibrillogenesis of lAPP is significantly perturbed by both insulin and the lipid bilayer of the secretory granule. As both of these components are perturbed in diabetics, we will determine the molecular basis for these effects. In Beta2m, our group recently discovered a novel interaction between b2m and Cu(ll) which can uniquely give rise to the nucleation of amyloid fibers under conditions present during hemodialysis therapy. Our second major aim is to determine the structural and energetic basis for divalent induced amyloidosis.
Our aims will be met by combined use of mutagenesis, optical, NMR and deuterium exchange techniques to elucidate the perturbation of protein folding and fibrillogenesis pathways.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054899-08
Application #
7163797
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Sechi, Salvatore
Project Start
1999-08-15
Project End
2008-01-10
Budget Start
2007-01-01
Budget End
2008-01-10
Support Year
8
Fiscal Year
2007
Total Cost
$298,777
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Wolfe, Leslie S; Calabrese, Matthew F; Nath, Abhinav et al. (2010) Protein-induced photophysical changes to the amyloid indicator dye thioflavin T. Proc Natl Acad Sci U S A 107:16863-8
Williamson, Jessica A; Loria, J Patrick; Miranker, Andrew D (2009) Helix stabilization precedes aqueous and bilayer-catalyzed fiber formation in islet amyloid polypeptide. J Mol Biol 393:383-96
Calabrese, Matthew F; Miranker, Andrew D (2009) Metal binding sheds light on mechanisms of amyloid assembly. Prion 3:1-4
Ruschak, Amy M; Miranker, Andrew D (2009) The role of prefibrillar structures in the assembly of a peptide amyloid. J Mol Biol 393:214-26
Blaho, Dorottya V; Miranker, Andrew D (2009) Delineating the conformational elements responsible for Cu(2+)-induced oligomerization of beta-2 microglobulin. Biochemistry 48:6610-7
Hebda, James A; Miranker, Andrew D (2009) The interplay of catalysis and toxicity by amyloid intermediates on lipid bilayers: insights from type II diabetes. Annu Rev Biophys 38:125-52
Calabrese, Matthew F; Eakin, Catherine M; Wang, Jimin M et al. (2008) A regulatable switch mediates self-association in an immunoglobulin fold. Nat Struct Mol Biol 15:965-71
Calabrese, Matthew F; Eakin, Catherine M; Wang, Jimin M et al. (2008) A regulatable switch mediates self-association in an immunoglobulin fold. Nat Struct Mol Biol :
Jaswal, Sheila S; Miranker, Andrew D (2007) Scope and utility of hydrogen exchange as a tool for mapping landscapes. Protein Sci 16:2378-90
Ruschak, Amy M; Miranker, Andrew D (2007) Fiber-dependent amyloid formation as catalysis of an existing reaction pathway. Proc Natl Acad Sci U S A 104:12341-6

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