Proteinase Inhbitors & Crystallin Fragments in Cataract

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During aging, an increasing proportion of total lens proteins becomes water insoluble (Wl), either due to aggregation and/or cross-linking. A further cross-linking of these species into covalent multimers is believed to cause opacity during age-related(senile) cataract development.A variety of post-translational modifications of crystallins are described in the literature as causativefactors for cross-linking mechanism, but their relative roles remain unclear. Because the senile cataract development is a slow process and sometimes takes years, few specific modifications might act as triggersto acceleratethe cross-linking mechanism and cause lens opacity. Our results show that modified crystallinfragments 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 implicatethem as a causativefactor. Based on our results, we have hypothesized that bA3/A1 -crystallin contains proteinaseactivity, but the activity is regulated in vivo because the enzyme activity needed activation, and the active enzyme is inhibited by a-crystallin. The bA3/A1-crystallin proteinase proteolyses a-, b- and g-crystallins, and the crystallin fragments undergo post-translationalmodifications, leading to their cross-linking per se and with phakinin and filensin (lens beaded filament proteins) to form covalent multimers. These covalent multimers cause lens opacity. To test the above hypothesis,the proposed studies will be focused to answer the following three major questions: (A) What is the molecular mechanism of activation of an Arg-bond hydrolyzing proteinase activity of bA3/ A1-crystallin? (B) How is the b A3/A1-crystaltin proteinase activity regulated in vivo? (C) What is the covalent cross- linking mechanismof post-translationallymodified crystallin fragments per se and with phakinin and filensin? The above studieswill provide an answerto 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 b A3/A1-crystal!in proteinase in the proteolysis of crystallins, their regulation in vivo, and in particular the mechanismof cross-linkingof crystallinfragments per se and with phakinin and filensin.