Cataract, a major cause of blindness in the world, develops as a result of age-related modifications and aggregation of the eye lens proteins. a-Crystallin accounts for nearly 40% of the adult lens proteins but its structure-function is yet to be fully understood. The chaperone-like activity of a-crystallin is believed to play a central role in maintaining lens transparency. We propose the following specific aims to increase our understanding of chaperone function of a-crystallin and its subunit organization to meet our long-term goal of understanding structure-function of a-crystallin. 1) Confirm that residues 70-88 in aA-crystallin and residues 73-92 in aB-crystallin are the major chaperone sites. Determine the amino acid sequences (binding site) in aB-crystallin that contribute to the enhanced hydrophobicity and chaperone function at 37?C following deletion of 54-61 sequence. 2) Identify the a-crystallin binding site(s) in p- and y-crystallins during an in vitro chaperone assay at 37?C. Identify the interaction sites in a-p and a-y complexes in human lens high-molecular-weight aggregates with the use of novel cross- linkers and mass spectrometric analysis. 3) Determine the directional preference and orientation of ADH peptide (YSGVCHTDLHAWHGDWPLPVK) during its interaction with aA- crystallin by site-directed fluorescence labeling and quenching studies. 4) Identify and characterize the aB-aB-;aB-aA- and aA-aA- crystallin interaction sites using cysteine scanning mutagenesis and chemical modification. We plan to accomplish these specific aims by site-directed mutagenesis studies and the use of novel cross-linkers and mass spectrometric methods. Understanding the structure of a-crystallin and its mechanisms of its action, including its interaction with other lens proteins, is likely to provide us better tools to delay or prevent cataractogenesis.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Anterior Eye Disease Study Section (AED)
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Araj, Houmam H
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University of Missouri-Columbia
Schools of Medicine
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Santhoshkumar, Puttur; Karmakar, Srabani; Sharma, Krishna K (2016) Structural and functional consequences of chaperone site deletion in ?A-crystallin. Biochim Biophys Acta 1864:1529-38
Raju, Murugesan; Santhoshkumar, Puttur; Sharma, K Krishna (2012) ?A-Crystallin-derived mini-chaperone modulates stability and function of cataract causing ?AG98R-crystallin. PLoS One 7:e44077
Santhoshkumar, Puttur; Raju, Murugesan; Sharma, K Krishna (2011) ?A-crystallin peptide SDRDKFVIFLDVKHF accumulating in aging lens impairs the function of ?-crystallin and induces lens protein aggregation. PLoS One 6:e19291
Raju, Murugesan; Santhoshkumar, Puttur; Henzl, T Michael et al. (2011) Identification and characterization of a copper-binding site in ýýA-crystallin. Free Radic Biol Med 50:1429-36
Raju, Murugesan; Santhoshkumar, Puttur; Sharma, K Krishna (2011) Cataract-causing ?AG98R-crystallin mutant dissociates into monomers having chaperone activity. Mol Vis 17:7-15
Sharma, K Krishna; Santhoshkumar, Puttur (2009) Lens aging: effects of crystallins. Biochim Biophys Acta 1790:1095-108
Santhoshkumar, Puttur; Murugesan, Raju; Sharma, K Krishna (2009) Deletion of (54)FLRAPSWF(61) residues decreases the oligomeric size and enhances the chaperone function of alphaB-crystallin. Biochemistry 48:5066-73
Santhoshkumar, Puttur; Udupa, Padmanabha; Murugesan, Raju et al. (2008) Significance of interactions of low molecular weight crystallin fragments in lens aging and cataract formation. J Biol Chem 283:8477-85
Murugesan, Raju; Santhoshkumar, Puttur; Sharma, K Krishna (2008) Role of alphaBI5 and alphaBT162 residues in subunit interaction during oligomerization of alphaB-crystallin. Mol Vis 14:1835-44
Rao, Guruprasad; Santhoshkumar, Puttur; Sharma, K Krishna (2008) Anti-chaperone betaA3/A1(102-117) peptide interacting sites in human alphaB-crystallin. Mol Vis 14:666-74

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