The objective of this application is to understand how novel ATPase modulators of Hsp70 change the fate of its pathologically important client protein, tau. The central hypothesis states that ATPase modulation mediates the fate of tau by affecting which co-chaperones associate with Hsp70. Completion of this work will define the Hsp70 complexes that fate tau for refolding and degradation. Consequently, new and innovative ways to pharmacologically intervene in Alzheimer's disease (AD) and other tauopathies may be identified. The following aims will test the central hypothesis.
Aim 1 : Identify the co-chaperone composition of complexes induced by Hsp70 ATPase modulators in a cell- based model. The working hypothesis states that Hsp70 ATPase stimulators recruit a co- chaperone which promotes the accumulation of tau. Conversely, Inhibitors recruit a co-chaperone which promotes proteosomal degradation of tau. This hypothesis will be tested by performing an immunoprecipitatlon of tau, in both treated and untreated cells, followed by mass spectrometry.
Aim 2 : Determine the effect of ATPase modulators on IHsp70 binding interactions with co- chaperones and client protein tau. The working model postulates that Hsp70 ATPase modulation changes the binding affinity of certain co-chaperones for the Hsp70-tau complex. Surface plasmon resonance and an enzyme-linked immunosorbent assay will be used to test the effect of ATPase modulators on the formation of binary and ternary complexes between Hsp70, tau, and co-chaperones.
Aim 3 : Understand the structural basis for the effect of ATPase modulation on Hsp70, tau, co- chaperone complex formation. We predict that ATPase modulators cause Hsp70 to predominantly populate specific nucleotide bound conformations. Previous work, using partial proteolysis, has shown that distinctive cleavage patterns define the ATP and ADP conformations of Hsp70. Thus, this technique will be used to probe the structure of Hsp70 after treatment. Site-directed mutagenesis will also be used to define binding surfaces and allosteric networks affected by ATPase modulators. Relevance: More than five million Americans suffer from Alzheimer's disease (AD). In AD, misfolded proteins build up in the brain causing severe and irreversible memory loss. By therapeutically targeting a chaperone factor involved in the folding and degradation of misfolded proteins we may be able to take advantage of the body's own intrinsic system to treat AD.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
5F30AG035464-03
Application #
8196724
Study Section
Special Emphasis Panel (ZRG1-F03B-H (20))
Program Officer
Wise, Bradley C
Project Start
2009-12-01
Project End
2012-06-30
Budget Start
2011-12-01
Budget End
2012-06-30
Support Year
3
Fiscal Year
2012
Total Cost
$24,732
Indirect Cost
Name
University of Michigan Ann Arbor
Department
None
Type
Organized Research Units
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Thompson, Andrea D; Bernard, Steffen M; Skiniotis, Georgios et al. (2012) Visualization and functional analysis of the oligomeric states of Escherichia coli heat shock protein 70 (Hsp70/DnaK). Cell Stress Chaperones 17:313-27
Chang, Lyra; Thompson, Andrea D; Ung, Peter et al. (2010) Mutagenesis reveals the complex relationships between ATPase rate and the chaperone activities of Escherichia coli heat shock protein 70 (Hsp70/DnaK). J Biol Chem 285:21282-91