The overall goals of this grant proposal is to determine role(s) of lens proteinases, their cognate inhibitors and proteolytic products of crystallins in the formation of high mo. wt. (HMW) proteins during the development of senile cataract. In this study, human lenses of different ages and cataractous lenses will be utilized. The following four specific aims will be pursued: (1) Regulation of proteolysis in lens: The regulation of proteolysis in the lens at the enzyme and protein substrate levels will be determined, i.e. by searching for zymogens and/or proteinase-inhibitor complexes of two lens proteinases, i.e.a 25 kDa and a membrane proteinase, and examining the effect of oxidation on endogenous proteinase inhibitors and the preferential proteolysis of native vs. oxidatively modified crystallins by endogenous proteinases. (2) Origin of degraded polypeptides from crystallins: The identity of """"""""parent crystallins"""""""" from which the three in vivo found degraded polypeptides of human lenses, i.e. 5.5 kDa, 9 kDa and 15 kDa, originated will be established by comparing their amino acid sequences. Lens crystallins will be proteolyzed in vitro with the two lens proteinases (25 kDa and a membrane proteinase) and proteolysis products compared with similar polypeptides found in vivo for their Mr's and partial N-terminal amino acid sequences. In addition, the origin of the degraded polypeptides from crystallins by a non-enzymatic reaction (Fenton reaction) will also be examined. (3) Cross-linking of degraded polypeptides to form HMW proteins: The mechanism of cross-linking to form HMW proteins in vitro by degraded polypeptides per se or between these polypeptides and individual crystallins will be determined. (4) Search for and quantitation of cross-linked products of degraded polypeptides in HMW and water insoluble proteins of normal aging and cataractous lenses. Antibodies raised against the specific cross-linked degraded polypeptides will be utilized to identify and quantitate the levels of aggregates of these polypeptides by Western blot analysis and a radioimmunoassay method respectively. The above studies will show: (i) how the lens proteolytic process is regulated in vivo; (ii) whether the degradation of crystallins in vivo is enzymatic or non-enzymatic, and (iii) if the degraded polypeptides cross-link per se and with individual crystallins to form the HMW proteins that exist in increasing levels in aging and cataractous lenses.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY006400-04A3
Application #
3262420
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1985-09-01
Project End
1994-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
4
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Missouri Lions Eye Research Foundation
Department
Type
DUNS #
City
Columbia
State
MO
Country
United States
Zip Code
65201
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