During aging, an increasing proportion of total lens proteins becomes water insoluble (WI) either due to aggregation and/or cross-linking. The increased sizes of cross-linked multimers of crystallins become so large that they finally become water insoluble and cause lens opacity during age-related (senile) cataract development. Among the variety of post-translational modifications, deamidation and truncations of crystallins are identified as the most abundant during aging in human lenses. Therefore, these modifications playa major role in age-related aggregation and cross-linking of crystallins, and in tum, are significant causative factors in age-related cataract development. Our studies have shown that ~A3-crystallin exists as an activable proteinase in the lens, and the active enzyme is capable of proteolyzing aA-, aB-, yC- and yD-crystallins. Further, our studies demonstrated that ~A3 proteinase is inhibition by aA- and aB-crystallins. Based on these results, we have hypothesized that ~A3-proteinase activity is regulated in vivo by aA- and aB-crystallins as inhibitors, and the activated ~A3-proteinase proteolyzes a-, ~- and y-crystallins. The crystallin fragments per se aggregate and/or undergo post-translational modifications such as deamidation. The unmodified and modified crystallin fragments aggregate and cross-link with intact crystallins to first form the water soluble-high molecular weight (WS-HMW) proteins, where its components cross-link and become water insoluble. To test the above hypothesis, the proposed studies will be focused to answer the following two questions: (1) Which polypeptide (amino acids) forms the ~A3 proteinase active site, and how is the proteinase activity inhibited by aA- and aB-crystallins? (2) What are the roles of crystallin fragments and/or deamidated crystallins in aggregation and cross-linking processes of crystallins in vivo? To answer the first question, we will determine the ~A3-proteinase active site in the regions of the motifs III and IV, the proteinase-induced proteolysis of a-, ~- and y-crystallins in vivo, and the inhibition mechanism of ~A3-proteinase by aA and aB-crystallins. To answer the second question, we will determine whether the fragments of a-, ~- and y-crystallins are post-translationally modified in vivo during aging and cataract development, the mechanism of complex formation between crystallin fragments and deamidated crystallins, and effects of deamidation of Asn(s) in aA- and aB-crystallins on lens transparency using transgenic mouse models. Because human lenses will be used in these studies, the findings will be relevant in elucidation of in vivo properties of ~A3-proteinase, its regulation by aA- and aB-crystallins as inhibitors, the ~A3- proteinase-induced proteolysis of crystallins, and potential roles of protelyzed crystallin fragments and their deamidated species in aggregation and cross-linking process during development of opacity in aging human lenses. PHS
A cataract is a clouding of the lens in the eye, and most cataracts are related to aging. By age 80, more than half of all Americans either have a cataract or have had cataract surgery (National Eye Institute of NIH-Resource guide). The risk of cataract increases as one gets older, and the other risk factors for cataract include: diabetes, smoking and alcohol use, and prolonged exposure to sunlight. Although intraocular lens (made of silicone or plastic) implant following cataract surgery restores vision, the surgery is traumatic and expensive at an older age. Women have a significantly higher age-adjusted prevalence of cataract than men in this country. The total direct medical cost for cataract extraction alone is presently 6.8 billion dollars, which is estimated to substantially increase in future with the aging population in the USA. This cost was 5.5 billions in 1991, which could be substantially reduced if cataract development is delayed or prevented. Our studies will be focused to answer the following two questions: (1) What are molecular properties and functions of ~A3-crystallin proteinase in vivo? (2) What is the molecular mechanism of aggregation and crosslinking of truncated and/or deamidated crystallins in vivo? Our proposal is to determine the potential role of crystallin fragments and post-translationally derived deamidated crystallins as causative factors in the mechanism age-related cataract development. The above studies will provide answers to the central question regarding the mechanism of development of lens opacity in human. PHS 398/
