Small heat shock proteins (sHSPs) are intimately linked to cell survival under conditions of stress. sHSPs act at an early stage of the stress response, by recognizing partly unfolded proteins to inhibit formation of potentially toxic aggregates. Failed sHSP function is associated with cataract, cardiac myopathies, motor neuropathies, and neurodegenerative disease. The archetypal human sHSP, ?B-crystallin ("?B"), is a major protein of the eye lens and along with another related sHSP, ?A-crystallin, is largely responsible for maintaining lens transparency throughout one's lifetime. Despite their critical importance to human health, understanding of the mechanisms by which sHSPs perform their functions remains rudimentary. Information on sHSP structure has been limited by the fact that the proteins exist as large, dynamic, polydisperse oligomeric assemblies. A structure of the ~600 kDa ?B oligomer has been solved using a combination of data from solid- state NMR, small-angle x-ray scattering, and electron microscopy, providing insights into the assembly of the oligomer. This continuing project seeks answers to three overarching questions: How do inherited mutations in sHSPs affect their structure and consequently, their function? How is sHSP structure and function modulated by changes in cellular conditions? and How do sHSPs recognize and bind client proteins? We propose to apply approaches that combine data from solution-state and solid-state NMR, small-angle x-ray scattering, single particle electron microscopy, and tandem mass spectrometry used to determine the structure of the most widely known ?B mutant, R120G ?B, involved in cataracts and cardiomyopathies (Aim 1). A mutant of ?B that represents the activated state associated with acidosis conditions will be investigated in Aim 2 to uncover the molecular mechanism of sHSP activation by pH. ?B/client protein interactions will be studied in Aim 3, using peptides and a model client protein to define the determinants of sHSP client recognition. The proposed studies build on the burgeoning progress towards understanding the structural biology of ?B and the important family of sHSPs afforded by developments of techniques capable of studying large heterogenous species.

Public Health Relevance

Small heat shock proteins (sHSPs) are a cell's first line of defense against potentially dire consequences of stress conditions that include ischemia, hypoxia, and acidosis. Inherited mutations in sHSP genes are associated with disorders that affect a variety of tissues and organs, especially the eye lens where they cause cataracts and the heart, where they cause myopathies. We propose to study two human sHSPs,??B-crystallin and HSP27, to learn how their structures and functions are modulated in response to stress conditions and how mutations alter these properties.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY017370-07
Application #
8584288
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Araj, Houmam H
Project Start
2006-04-01
Project End
2015-11-30
Budget Start
2014-02-01
Budget End
2014-11-30
Support Year
7
Fiscal Year
2014
Total Cost
$403,605
Indirect Cost
$122,625
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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Carlson, Anne E; Rosenbaum, Joel C; Brelidze, Tinatin I et al. (2013) Flavonoid regulation of HCN2 channels. J Biol Chem 288:33136-45
Jehle, Stefan; Vollmar, Breanna S; Bardiaux, Benjamin et al. (2011) N-terminal domain of alphaB-crystallin provides a conformational switch for multimerization and structural heterogeneity. Proc Natl Acad Sci U S A 108:6409-14
Jehle, Stefan; Rajagopal, Ponni; Bardiaux, Benjamin et al. (2010) Solid-state NMR and SAXS studies provide a structural basis for the activation of alphaB-crystallin oligomers. Nat Struct Mol Biol 17:1037-42
Jehle, Stefan; van Rossum, Barth; Stout, Joseph R et al. (2009) alphaB-crystallin: a hybrid solid-state/solution-state NMR investigation reveals structural aspects of the heterogeneous oligomer. J Mol Biol 385:1481-97