The "wear and tear" of lens aging is recorded at the molecular level through accumulated modifications of the crystallins in its fiber cells. This slow environmental perturbation in the absence of protein turn-over leads to a progressive loss in crystallins stability and solubility and alters protein-protein interactions, all of which compromise lens transparency and refractivity. Roughly 35% of the fiber cell weight consists of the molecular chaperone, a-crystallin, a protein-stability sensor that binds aggregation-prone proteins. Attractive interactions between a-crystallin and destabilized ?- and ?-crystallins are a central facet of lens aging. The exhaustion of chaperone capacity is hypothesized to be a central catalyst for age-related cataract. Furthermore, the earliest stage of age-related nuclear cataract is temporally correlated with the appearance of disulfide cross-linked aggregates of crystallins. The long term goal of this grant is to develop an understanding of the interrelationships between crystallins stability, chaperone structure, affinity and capacity, and molecular crowding in the process of lens aging and the development of cataract. In the next funding period, we will test two hypotheses regarding the molecular mechanisms of age-related and hereditary cataracts.
Aims 1 and 2 will undertake a systematic analysis of the energetic threshold required to trigger binding of destabilized ?- and ?-crystallins to a-crystallin and determine whether chaperone-driven interactions lead to formation of disulfide cross-links through the entropic advantage afforded by complex formation. Critical to this endeavor is the use of an innovative label-free approach, backscattering interferometry, which allows characterization of these interactions in real time. We will also test the hypothesis that A-crystallin cysteine mutants linked to congenital cataract lead to a compressed aging process from the perspective of titration of a-crystallin capacity and formation of aggregates. Based on preliminary data, complex formation by these mutants is driven by their increased affinity and capacity rather than by substrate destabilization and results in the formation of disulfide cross-links.
In aim 3, state of the art structural tools will be employed to provide snapshots of a chaperone activated state and its complex with the substrate. These studies will complement the mechanistic insight of aims 1 and 2 by identifying sequence and structural motifs involved in binding, and defining the basis of activation by oligomer expansion of small heat-shock proteins. Cataract formation is a leading cause of blindness worldwide and affects 20 million adults in the United States. In the most common type, human nuclear cataract, protein aggregation and disulfide cross-linking are major molecular events. The development of a mechanistic perspective on these molecular transformations is of fundamental biochemical importance and may well have an impact on the development of intervention and therapeutic strategies.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY012018-15
Application #
8212087
Study Section
Special Emphasis Panel (ZRG1-BDCN-F (02))
Program Officer
Araj, Houmam H
Project Start
1998-02-01
Project End
2013-02-28
Budget Start
2012-02-01
Budget End
2013-02-28
Support Year
15
Fiscal Year
2012
Total Cost
$495,898
Indirect Cost
$168,864
Name
Vanderbilt University Medical Center
Department
Physiology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
McDonald, Ezelle T; Bortolus, Marco; Koteiche, Hanane A et al. (2012) Sequence, structure, and dynamic determinants of Hsp27 (HspB1) equilibrium dissociation are encoded by the N-terminal domain. Biochemistry 51:1257-68
Latham, Joey C; Stein, Richard A; Bornhop, Darryl J et al. (2009) Free-solution label-free detection of alpha-crystallin chaperone interactions by back-scattering interferometry. Anal Chem 81:1865-71
McHaourab, Hassane S; Godar, Jared A; Stewart, Phoebe L (2009) Structure and mechanism of protein stability sensors: chaperone activity of small heat shock proteins. Biochemistry 48:3828-37
Koteiche, Hanane A; Kumar, M Satish; McHaourab, Hassane S (2007) Analysis of betaB1-crystallin unfolding equilibrium by spin and fluorescence labeling: evidence of a dimeric intermediate. FEBS Lett 581:1933-8
McHaourab, Hassane S; Kumar, M Satish; Koteiche, Hanane A (2007) Specificity of alphaA-crystallin binding to destabilized mutants of betaB1-crystallin. FEBS Lett 581:1939-43
Zou, Ping; Surendhran, Kavitha; Mchaourab, Hassane S (2007) Distance measurements by fluorescence energy homotransfer: evaluation in T4 lysozyme and correlation with dipolar coupling between spin labels. Biophys J 92:L27-9
Shi, Jian; Koteiche, Hanane A; McHaourab, Hassane S et al. (2006) Cryoelectron microscopy and EPR analysis of engineered symmetric and polydisperse Hsp16.5 assemblies reveals determinants of polydispersity and substrate binding. J Biol Chem 281:40420-8
Shashidharamurthy, R; Koteiche, Hanane A; Dong, Jinhui et al. (2005) Mechanism of chaperone function in small heat shock proteins: dissociation of the HSP27 oligomer is required for recognition and binding of destabilized T4 lysozyme. J Biol Chem 280:5281-9
Sathish, Hasige A; Koteiche, Hanane A; McHaourab, Hassane S (2004) Binding of destabilized betaB2-crystallin mutants to alpha-crystallin: the role of a folding intermediate. J Biol Chem 279:16425-32