Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2011. / Cataloged from PDF version of thesis. [Alpha] and [gamma] in title on title page appear as Greek symbols. Vita. / Includes bibliographical references (p. 148-168). / Cataract, the leading cause of blindness worldwide, is the opacification of the eye lens. In.age-related cataract, as well as roughly one half of congenital cataract cases, aggregation or precipitation of crystallin proteins results in the formation of large structures that scatter light, preventing the pinpoint focusing on the retina normally accomplished by the lens. The human eye lens is composed of fiber cells packed with crystallins up to 450 mg/ml. Human yD-crystallin (HyD-Crys) is a monomeric, two-domain protein, predominantly localized to the lens nucleus. Both domains of this long-lived protein have double Greek key p-sheet folds with well-packed hydrophobic cores. Three mutations resulting in amino acid substitutions in the y-crystallin buried cores-two in the N-terminal domain (N-td), and one in the C-terminal domain (C-td)-cause earlyonset cataract in mice. It has not been possible to identify the aggregating precursors within lens tissues, and the question persists as to the nature of structure and stability changes in the crystallins leading to their increased propensity for aggregation and cataract formation within the lens environment. To compare in vivo cataract-forming phenotypes with in vitro unfolding and aggregation of y-crystallins, mouse mutant substitutions were introduced into HyD-Crys. WT HyD-Crys unfolds in vitro through a three-state pathway, exhibiting an intermediate with the N-td unfolded, and the C-td native-like. L5S and V75D also displayed three-state unfolding pathways, with the first transition, unfolding of the N-td, shifted to significantly lower denaturant concentrations. 190F was globally destabilized and the overall unfolding transition was shifted to lower denaturant concentrations. During thermal denaturation, the mutant proteins exhibited lowered thermal stability compared with WT. Kinetic unfolding experiments further confirmed this destabilization. The cataract phenotype of increased protein aggregation could be a direct property of the mutation, or could reflect an inability to be recognized by the a-crystallin chaperone. We have therefore carried out experiments comparing the interactions of WT HyD-Crys, V75D and 190F with a major component of the lenticular chaperone system, the small heat shock protein human aB-crystallin. Suppression levels of the aggregation that competes with the refolding pathway after dilution from GdnHCl were indistinguishable between WT and mutant proteins. However, dramatic differences among the mutants were observed under different conditions, including physiological conditions in the absence of denaturant and partially destabilizing acidic conditions such as those that may exist in the vicinity of degrading lysosomes in maturing lens fiber cells. In particular, incubation under physiological conditions highlighted differential interactions between the lens chaperone and the mutant HyD-Crys proteins themselves. Destabilized precursors to the cataractous state may populate various non-native structural conformations that, although aggregation-prone, elude recognition by acrystallin and efficiently continue along the aggregation pathway. Coupled with the observation of different unfolding pathways for the mutant crystallins, these results support the existence of multiple pathways for cataract formation in which partially unfolded species are differentially recognized by the passive lens chaperone system. While these observations were made utilizing destabilized mutant proteins, the same mechanisms may underlie the formation of age-related cataractous aggregates induced in vivo by covalent modification of the lens proteins. / by Kate L. Moreau. / Ph.D.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/62781 |
| Date | January 2011 |
| Creators | Moreau, Kate L. (Kate Lauren) |
| Contributors | Jonathan A. King., Massachusetts Institute of Technology. Dept. of Biology., Massachusetts Institute of Technology. Dept. of Biology. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 192 p., application/pdf |
| Rights | M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission., http://dspace.mit.edu/handle/1721.1/7582 |
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