Cataract, a major cause of blindness in the world, develops as a result of age-related modifications and aggregation of the eye lens proteins. Alpha-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 ?- and ?-crystallins during an in vitro chaperone assay at 37?C. Identify the interaction sites in a-? and a-? 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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