Protein aggregation resulting from stress, disease or mutation poses a major threat to all cells. Damage due to protein aggregation is limited and repaired by a cellular protein quality control network consisting of chaperones and proteases. The proposed research investigates one component of this protein quality control network, the small heat shock proteins (sHSPs), a ubiquitous class of molecular chaperones. Expression and/or mutation of sHSPs are linked to multiple diseases of protein misfolding, including neurodegenerative diseases, myopathies and cataract. The mechanism of sHSP chaperone action and interaction with substrates, therefore, has wide-ranging implications for understanding cellular stress and disease processes, but remains poorly defined. Furthermore, sHSPs are an excellent model system for investigating the importance of protein dynamics in protein-protein recognition. We hypothesize that secondary, tertiary and quaternary dynamics underlie the effectiveness of sHSPs in recognizing and binding diverse denaturing proteins.
Aim 1 will use crosslinking and advanced mass spectrometry to define the nature of substrate sites bound by sHSPs and the organization of sHSP-substrate complexes formed in vitro. How the cellular environment impacts substrate recognition will then be tested in vivo in E. coli.
Aim 2 will determine how conformational flexibility of the sHSP N-terminal arm effects efficiency of sHSP substrate protection using iterations of molecular dynamics simulations and in vitro assays of the activity of mutant sHSPs. NMR will be used to obtain amino acid resolution of the dynamic interactions between sHSP and substrate. These complementary approaches will allow cross validation of results. sHSPs and substrates with known high resolution structures, or readily modeled by homology, will be used in Aims 1 and 2. The third and final Aim employs a model genetic system to test principles derived from the in vitro studies and to establish how sHSP-substrate interactions alter protein fate in the cell. In total, the proposed experiments will define not only how sHSPs recognize substrates and impact their metabolism, but also define new aspects of protein recognition and aggregate formation, which are critical to understanding many diseased states.

Public Health Relevance

All cells contain machinery to protect and repair proteins, the major workhorses of cells. Defects in this protein quality control network result in diseases of protein folding, including neurodegenerative disease, myopathies and cataract. Decline in this machinery is also associated with aging. The proposed research involves detailed biochemical studies of an important protein component of this protective network.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042762-20
Application #
8538401
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
1989-07-01
Project End
2015-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
20
Fiscal Year
2013
Total Cost
$308,708
Indirect Cost
$97,855
Name
University of Massachusetts Amherst
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003
Basha, Eman; Jones, Christopher; Blackwell, Anne E et al. (2013) An unusual dimeric small heat shock protein provides insight into the mechanism of this class of chaperones. J Mol Biol 425:1683-96
Basha, Eman; O'Neill, Heather; Vierling, Elizabeth (2012) Small heat shock proteins and ?-crystallins: dynamic proteins with flexible functions. Trends Biochem Sci 37:106-17
Basha, Eman; Jones, Christopher; Wysocki, Vicki et al. (2010) Mechanistic differences between two conserved classes of small heat shock proteins found in the plant cytosol. J Biol Chem 285:11489-97
Benesch, Justin L P; Aquilina, J Andrew; Baldwin, Andrew J et al. (2010) The quaternary organization and dynamics of the molecular chaperone HSP26 are thermally regulated. Chem Biol 17:1008-17
Jaya, Nomalie; Garcia, Victor; Vierling, Elizabeth (2009) Substrate binding site flexibility of the small heat shock protein molecular chaperones. Proc Natl Acad Sci U S A 106:15604-9
Painter, Alexander J; Jaya, Nomalie; Basha, Eman et al. (2008) Real-time monitoring of protein complexes reveals their quaternary organization and dynamics. Chem Biol 15:246-53
Basha, Eman; Friedrich, Kenneth L; Vierling, Elizabeth (2006) The N-terminal arm of small heat shock proteins is important for both chaperone activity and substrate specificity. J Biol Chem 281:39943-52
Giese, Kim C; Basha, Eman; Catague, Belmund Y et al. (2005) Evidence for an essential function of the N terminus of a small heat shock protein in vivo, independent of in vitro chaperone activity. Proc Natl Acad Sci U S A 102:18896-901
Giese, Kim C; Vierling, Elizabeth (2004) Mutants in a small heat shock protein that affect the oligomeric state. Analysis and allele-specific suppression. J Biol Chem 279:32674-83
Basha, Eman; Lee, Garrett J; Demeler, Borries et al. (2004) Chaperone activity of cytosolic small heat shock proteins from wheat. Eur J Biochem 271:1426-36

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