Hsp70s are ubiquitous and highly conserved molecular chaperones that play multiple essential roles in maintaining cellular protein homeostasis through assisting in protein folding, assembly, degradation, and transportation across membrane. The fundamental importance of maintaining protein homeostasis inevitably links Hsp70s with many destructive human diseases, most notably cancers and neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Thus, elucidating the structural and biochemical properties of Hsp70s will not only advance our understanding of the basic molecular mechanism of Hsp70-assited protein folding, but also provide crucial insights regarding how to target Hsp70s for therapeutic interventions in treating cancers and neurodegenerative disorders. Hsp70s have three key biochemical activities that are at the heart of the chaperone activity: ATPase, peptide substrate binding, and ATP-induced allosteric coupling. In spite of extensive efforts, the very basic mechanism of Hsp70-assisted protein folding is still ill-defined due to the lack of in-depth understanding o two key biochemical activities: 1) ATP-induced allosteric coupling is central to Hsp70s' chaperone activity~ however, all previous studies had failed to reveal the molecular mechanism. 2) The well-established single peptide binding site on Hsp70s has made it difficult to explain the efficient chaperone activity. Could there be additional peptide binding sites on Hsp70s that account for the high efficiency? Thus, the overall objective of this proposal is to analyze these two key biochemical activities in order to dissect the basic mechanisms of Hsp70 chaperone function. Recently, we solved the first crystal structure of an intact Hsp70 in the allosteric active state, and discovered a novel peptide substrate binding site on Hsp70s. Based on these original discoveries, we propose the following two Specific Aims: 1) elucidate the molecular mechanism of the ATP-driven allosteric coupling in Hsp70s, and 2) characterize a novel peptide binding site on Hsp70s and investigate its role in protein folding. To achieve our goal, we use a multidisciplinary approach combining X-ray crystallography, biochemistry, NMR, EPR, computational chemistry, and yeast and E.coli genetics. We expect that successful completion of this proposal will help us realize our long-term goal, which is to establish a thoroug mechanism understanding of the very basic mechanism of Hsp70 chaperone activity in protein folding.

Public Health Relevance

The ubiquitous molecular chaperone Hsp70s play multiple essential roles in maintaining cellular protein homeostasis, imbalance of which leads to many devastating human diseases, including cancers and neurodegenerative diseases. In this proposal, we aim to understand the basic molecular mechanism of Hsp70s' chaperone activity in maintaining protein homeostasis, and thereby provide crucial insights on how to target them for treating cancers and neurodegenerative diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098592-05
Application #
9279173
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Maas, Stefan
Project Start
2013-09-01
Project End
2018-05-31
Budget Start
2017-06-01
Budget End
2018-05-31
Support Year
5
Fiscal Year
2017
Total Cost
$260,775
Indirect Cost
$89,775
Name
Virginia Commonwealth University
Department
Physiology
Type
Schools of Medicine
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
Idikuda, Vinay; Gao, Weihua; Grant, Khade et al. (2018) Singlet oxygen modification abolishes voltage-dependent inactivation of the sea urchin spHCN channel. J Gen Physiol 150:1273-1286
Zhu, Mengye; Idikuda, Vinay Kumar; Wang, Jianbing et al. (2018) Shank3-deficient thalamocortical neurons show HCN channelopathy and alterations in intrinsic electrical properties. J Physiol 596:1259-1276
Yang, Jiao; Zong, Yinong; Su, Jiayue et al. (2017) Conformation transitions of the polypeptide-binding pocket support an active substrate release from Hsp70s. Nat Commun 8:1201
Liu, Qingdai; Li, Hongtao; Yang, Ying et al. (2017) A disulfide-bonded DnaK dimer is maintained in an ATP-bound state. Cell Stress Chaperones 22:201-212
Yang, Jiao; Zhou, Lei; Liu, Qinglian (2017) Data on the optimizations of expression and purification of human BiP/GRP78 protein in Escherichia coli. Data Brief 10:525-530
Liu, Chang; Xie, Changan; Grant, Khade et al. (2016) Patch-clamp fluorometry-based channel counting to determine HCN channel conductance. J Gen Physiol 148:65-76
Liu, Qinglian; Craig, Elizabeth A (2016) Molecular biology: Mature proteins braced by a chaperone. Nature 539:361-362
Sarbeng, Evans Boateng; Liu, Qingdai; Tian, Xueli et al. (2015) A functional DnaK dimer is essential for the efficient interaction with Hsp40 heat shock protein. J Biol Chem 290:8849-62
Yang, Jiao; Nune, Melesse; Zong, Yinong et al. (2015) Close and Allosteric Opening of the Polypeptide-Binding Site in a Human Hsp70 Chaperone BiP. Structure 23:2191-2203
Fong, Jerry J; Sreedhara, Karthik; Deng, Liwen et al. (2015) Immunomodulatory activity of extracellular Hsp70 mediated via paired receptors Siglec-5 and Siglec-14. EMBO J 34:2775-88

Showing the most recent 10 out of 13 publications