The molecular chaperones, Hsp70 and Hsp90 bind to unfolded proteins (e.g. clients) and recruit either "pro-folding" or "pro-degradation" co-chaperones, generating a series of distinct multi- protein complexes that either fold or degrade the bound protein. For some important clients, such as microtubule-binding protein tau (MAPT/tau), a key step in the "choice" to degrade seems to be the dynamic recruitment of the E3 ubiquitin conjugating ligase CHIP, which couples both Hsp70 and Hsp90 to the ubiquitin-proteasome system (UPS). However, little is known about the molecular events that govern CHIP assembly into the complex and we do not yet understand why some disease-associated proteins, such as hyper-phosphorylated tau, evade this process. Progress towards understanding how clients are loaded into the "pro-degradation" complex has been hindered by a lack of structural information, the dynamic nature of the protein-protein interactions and the difficulties of linking in vitro findings with cellular functions. We hypothesize that a rigorous ad comprehensive understanding of protein triage will emerge from a combination of chemical crosslinking-mass spectrometry (CXL-MS), cryo-electron microscopy (cryo-EM) and new chemical probes that trigger an acute switch to the "pro- degradation" complex. Guided by strong preliminary results, we will: (SA1) generate a complete CXL-MS signature of the Hsp70-CHIP-tau and Hsp90-CHIP-tau complexes and map their protein-protein contacts in vitro and in intact neuronal cells, (SA2) use these signatures, chemical probes and mass spectrometry to understand how the complexes form and how they recruit CHIP and other effectors of the UPS during active triage in the cytosol and (SA3) elucidate the macromolecular architecture of the chaperone complexes to, for the first time, link cellular observations with careful measurements of protein-protein contacts in vitro. This work is significant because it will reveal changes in th composition of macromolecular complexes that drive active protein triage and it is innovative because it links powerful in vitro approaches with new chemical probes and CXL-MS to address one of the key questions in cellular protein homeostasis. Moreover, the proposed work brings together a continuum of research expertise in structural, chemical and cellular approaches to the study of dynamic protein-protein interactions.

Public Health Relevance

Many serious and untreatable disorders, including Alzheimer's and Huntington's diseases are caused by defects in protein triage and the aberrant accumulation of misfolded proteins. To better treat these diseases, it is important to better understand the molecular mechanisms by which chaperones control protein homeostasis. EDITOR'S COMMENTS

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM109896-01
Application #
8668663
Study Section
Special Emphasis Panel (ZGM1-TRN-0 (MI))
Program Officer
Anderson, Vernon
Project Start
2014-06-01
Project End
2018-02-28
Budget Start
2014-06-01
Budget End
2015-02-28
Support Year
1
Fiscal Year
2014
Total Cost
$268,014
Indirect Cost
$88,355
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109