The long term goal of this proposal is to understand at a molecular level the structural and kinetic factors which cause islet amyloid polypeptide (IAPP) to form fibrils. IAPP forms fibrils in the endocrine pancreas of type II diabetics contributing greatly to the pathogenesis of this disease. Curiously, IAPP isolated from amyloid deposits in afflicted patients is indistinguishable from that of healthy individuals. Clearly a perturbation of the interactions of IAPP with other physiological factors has taken place. Candidates for the factors which affect fibrillogenesis in this system include the cosecreted peptides (C-peptide, insulin, and proinsulin), changes in ionic conditions upon exocytosis, circulating serum amyloid polypeptide and extracellular proteoglycans. Our interest in this system stems both from its role in diabetes and as a model system for the study of protein conformational changes which give rise to amyloid fibrils. The first major aim of this work is to determine the intra-and inter-molecular interactions of soluble IAPP oligomers. Initially, we will focus on methods to determine the number, size and interconversion rates of these species. We will subsequently extend hydrogen deuterium exchange pulse labeling methods monitored by NMR and mass spectrometry to determine the residues involved in structure and the kinetics of their formation. This will provide valuable insights into the conformational transitions which give rise to fibrils. Elucidation of the interactions of IAPP with other granule components known to affect the in vitro formation of fibrils will then be undertaken. The second major aim of this proposal is to determine the molecular interactions of in vitro generated fibrils. Our structural investigations will use deuterium or methylene diradical labeling techniques to elucidate those residues which participate in fibril structure. Localization of label will be performed using fragmentation techniques followed by analysis with mass spectrometry. It is our objective to distinguish interactions within fibril monomers, between fibril monomers, between fibrils as they coil into higher order structures, and between fibrils and physiological factors which are universally found in amyloid plaques. We will also determine and characterize the soluble oligomeric states which nucleate fibril formation and elongate existing fibrils. Given our understanding of the conformations and oligomeric states of soluble IAPP determined in our first aim, we will be able to correlate fibril formation kinetics with the populations of different soluble states of IAPP.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054899-03
Application #
6381374
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Laughlin, Maren R
Project Start
1999-08-15
Project End
2003-07-31
Budget Start
2001-08-01
Budget End
2002-07-31
Support Year
3
Fiscal Year
2001
Total Cost
$207,050
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
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

Showing the most recent 10 out of 23 publications