Proteinase Inhbitors & Crystallin Fragments in Cataract

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DESCRIPTION (provided by applicant): During aging, an increasing proportion of total lens proteins becomes water insoluble (Wl), either due to aggregation or cross-linking, and their further cross-linking into covalent multimers is believed to cause opacity during age-related (senile) cataract development. Various post-translational modifications of crystallins are described in the literature as causative factors for cross-linking mechanism, but their relative roles remain undefined. Because the senile cataract development is a slow process and sometimes takes years, few specific modifications might act as triggers to accelerate the cross-linking mechanism and cause lens opacity. Our results show that modified crystallin fragments play an active role in the crystallin cross-linking process. To understand such a role of crystallin fragments, one must determine their origin, post-translational modifications and cross-linking mechanism in order to implicate them as a causative factor. Based on our results, we have hypothesized that beta A3/Al-crystallin exhibits proteinase activity and the activity is regulated in vivo due to its existence in an inactive state, inhibition by alpha crystallin, and also at the crystailin substrate level. On activation, the beta A3/A1-crystallin proteinase proteolyses crystallins, and crystallin fragments undergo post-translational modifications and cross-link per se and with two beaded filament proteins (pahkinin and filensin) to form covalent multimers. To test the above hypothesis, the proposed studies will be focused to answer the following four questions: (A) What is the molecular mechanism of activation of an Arg-bond hydrolyzing proteinase activity associated with beta A3/A1-crystallin? (B) How is the beta A3/A1-crystallin proteinase activity regulated in vivo? (C) What is the covalent cross-linking mechanism of post-translationally modified crystallin fragments with beaded filaments proteins (phakinin and filensin) during aging and cataract development? D) What are the potential role of newly discovered posttranslational modifications (i.e., deamidation of N to D residue, oxidation of W and M residues, and conversion of serine to dehydroalanine, and formylation of H residue) in crystallin fragments in the cross-linking mechanism with beaded filament proteins? The above studies will provide answers to the central question of how opacity develops during age-related cataract development. Because human lenses will be used in these studies, the findings will be relevant in elucidating the role of beta A3/A1-crystallin proteinase in the proteolysis of crystallins, their regulation in vivo, and, in particular, the mechanism of cross-linking of crystallin fragments per se and with pakinin and filensin.