Lens cataracts, interfering with normal vision, affect 50% of the American population over age 75. Human gammaD-crystallin (HgammaD-Crys) and Human gammaS-crystallin (HgammaS-Crys) are monomeric two-domain major proteins of the human eye lens and significant components of mature-onset cataracts. Mature-onset cataracts are thought to be an aggregated state of modified, damaged, or partially unfolded states of lens crystallins. This proposal treats mature-onset cataract as a protein deposition disease, and focuses on in vitro unfolding, refolding and aggregation experiments with purified proteins, to find in vitro models for the precursors to lens protein aggregation and/or chaperone recognition, in situ. For other protein deposition diseases such systems have been key steps toward targeted searches for therapeutic agents inhibiting pathological deposition. Purified recombinant HgammaD-Crys can be refolded in vitro at 37 degrees C and neutral pH in the absence of cellular factors. Formation of high molecular weight fibrils competes with productive refolding. A partially unfolded species with the C-terminal domain folded, and the N-terminal domain unfolded, has been identified as an intermediate in the unfolding and refolding pathway, and is a candidate for the precursor in fibril formation. HgammaS-Crys also refolds in vitro. Though HgammaS molecules do not self-aggregate during refolding, they are recruited into the HgammaD fibrils when refolding with both proteins present. These co-aggregation reactions of partially folded intermediates - under conditions of productive refolding - provide in vitro models for lens opacity. In this proposal, we use a set of HgammaD mutant proteins, including triple-tryptophan replacements, to a) deepen understanding of the molecular basis of the extraordinary stability of the crystallins in their solution state, b) to map the pathway of the partially folded HgammaD and HgammaS crystallins polymerizing into fibrillar and other aggregated states, with emphasis on identification of putative nucleating and propagating species, and c) to determine whether partially folded species or their fibrillar state formed in vitro are substrates of the alpha-crystallins, and whether they are generated in vitro by photo-oxidative stress, thought to be acting on the crystallins within the lens.
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