Modification of proteins play a pivotal role in changing a normal lens into a cataractous lens. Proteolytic cleavage of peptide bonds in crystallins by endogenous enzymes is probably one of the most frequent and important modifications. So far, information about these cleavages in crystallins is limited in terms of their enzymatic nature, regulation at the enzyme and substrate levels, effect on crystallin functions and secondary modifications in crystallin fragments. Based on preliminary results, the P.I. has hypothesized that the degradation of crystallins is regulated at both enzyme and substrate levels and the cleaved fragments undergo secondary modification such as glycation via the Mail lard reaction and therefore directly participate in the cross-linking of the lens proteins which is central to the development of lens opacity. To test this hypothesis, the following three specific aims will be pursued: (1) specific Aim #1: Determine the mechanism of autolytic activation of a membrane proteinase from a putative zymogen type activation of the BA31A1-crystallin proteinase by a detergent such as sodium deoxycholate. (2) specific Aim #2: Determine the role of the membrane proteinase and BA31A1-crystallin proteinase in the in vivo proteolysis of selected crystallins. (3) Specific Aim #3: Determine crosslinking of degraded polypeptides in general and specifically of a 9 kDa yD-crystallin fragment to form homologous (crystallin fragments alone) and heterologous (crystallin fragments plus native crystallins) multimers possibly via the glycation mechanism of the Maillard reaction. The above studies will address the questions regarding the in vivo regulation of crystallin degradation and the role of endogenous proteinases in this process. This study will also answer the questions whether the degraded polypeptides, on secondary modifications, acquire cross-linking properties and participate directly in protein cross-linking during the development of lens opacity. Since all of the proposed studies will be carried out in human lenses, the results will be directly relevant to the human senile cataract problem.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Visual Sciences A Study Section (VISA)
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Liberman, Ellen S
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University of Alabama Birmingham
Schools of Optometry/Ophthalmol
United States
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Srivastava, O P; Srivastava, K; Chaves, J M et al. (2017) Post-translationally modified human lens crystallin fragments show aggregation in vitro. Biochem Biophys Rep 10:94-131
Chaves, Jose M; Gupta, Ratna; Srivastava, Kiran et al. (2017) Human alpha A-crystallin missing N-terminal domain poorly complexes with filensin and phakinin. Biochem Biophys Res Commun 494:402-408
Hegde, Shylaja; Kesterson, Robert A; Srivastava, Om P (2016) CRY?A3/A1-Crystallin Knockout Develops Nuclear Cataract and Causes Impaired Lysosomal Cargo Clearance and Calpain Activation. PLoS One 11:e0149027
Tiwary, Ekta; Hegde, Shylaja; Purushotham, Sangeetha et al. (2015) Interaction of ?A3-Crystallin with Deamidated Mutants of ?A- and ?B-Crystallins. PLoS One 10:e0144621
Hegde, Shylaja M; Srivastava, Kiran; Tiwary, Ekta et al. (2014) Molecular mechanism of formation of cortical opacity in CRYAAN101D transgenic mice. Invest Ophthalmol Vis Sci 55:6398-408
Gupta, Ratna; Asomugha, Chinwe O; Srivastava, Om P (2011) The common modification in alphaA-crystallin in the lens, N101D, is associated with increased opacity in a mouse model. J Biol Chem 286:11579-92
Asomugha, C O; Gupta, R; Srivastava, O P (2010) Identification of crystallin modifications in the human lens cortex and nucleus using laser capture microdissection and CyDye labeling. Mol Vis 16:476-94
Gupta, R; Chen, J; Srivastava, O P (2010) A serine-type protease activity of human lens ?A3-crystallin is responsible for its autodegradation. Mol Vis 16:2242-52
Gupta, Ratna; Srivastava, Om P (2009) Identification of interaction sites between human betaA3- and alphaA/alphaB-crystallins by mammalian two-hybrid and fluorescence resonance energy transfer acceptor photobleaching methods. J Biol Chem 284:18481-92
Srivastava, K; Gupta, R; Chaves, J M et al. (2009) Truncated human betaB1-crystallin shows altered structural properties and interaction with human betaA3-crystallin. Biochemistry 48:7179-89