Date of Award

1-1-2021

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Chemistry

Content Description

1 online resource (xv, 130 pages) : color illustrations.

Dissertation/Thesis Chair

Jayanti Pande

Committee Members

Alexander Shekhtman, Li Niu, Ting Wang, Sho-Ya Wang

Keywords

lens crystallins, nuclear cataract, Cataract, Crystalline lens, Mutation (Biology), Proteins

Subject Categories

Biochemistry | Chemistry

Abstract

Cataract, or clouding of the ocular lens, among the most common types of eye diseases, is the leading cause of blindness worldwide. With the opacity or clouding of the lens, light incident on the lens is scattered rather than being transmitted and is thus prevented from focusing on the retina. The lens becomes cataractous due to a large number of reasons, among which aging and genetic mutations are two of the most common factors. Clouding of the center of the lens or nuclear opacity, is the most frequently observed type of age-onset cataract, as well as inherited, congenital cataract [1, 2]. The nuclear region is the oldest part of the lens and is particularly rich in the γ-crystallin proteins, which are expressed at very high concentrations (up to 400 mg/mL) in the mammalian eye lens [3]. Among the cataract-associated protein modifications, a significant number occur in the γ-crystallin family. Based on our previous work, we know that genetic mutations and post-translational modifications (PTMs) of the γ-crystallins alter molecular interactions and lead to either (a) self-association or (b) association with other crystallins [4, 5, 6, 7]. Such changes reduce protein solubility and result in the formation of a variety of condensed-phases, which are responsible for the light scattering and opacity, thus leading to cataract. The main objective of this dissertation is to determine the molecular mechanisms by which missense mutations in γ-crystallins lead to cataract formation in a model system in vitro. The strategy involves the expression of native and mutant crystallins in E. coli and the comparison of their physicochemical properties in solution using a number of biophysical and biochemical methods. The focus here is on the investigations of three cataract-associated mutations of the γ-crystallins, (i) the Tyr66Asn (Y66N) mutation and (ii) the Val41Met (V41M) mutation, both of which occur in human γS-crystallin (HGS), and (iii) the Ser10Arg (S10R) mutation in mouse γB-crystallin (MGB). Y66N is a missense mutation in HGS, associated with dominant infantile cataracts [8]. This mutant protein is believed to translocate to the cell membrane, although HGS, like other crystallins, is a highly water-soluble cytosolic protein. The mutation is located in the tyrosine corner of the N terminal domain (N-td) of HGS, which is suspected to be one of the protein-folding nucleation sites [9]. Although the mutant protein has been well characterized, there is no molecular mechanism presented as to how this mutant leads to the observed opacity [8]. We found that the Y66N mutant had two forms - a soluble, and an insoluble form - when expressed at physiological temperature in vivo (in E. coli). By performing spectroscopic and molecular dynamics (MD) simulations, we revealed the importance of the tyrosine corner in protein stability and the folding process. We thereby provided insights into the protein folding defect arising from the Tyr to Asn mutation, which leads to the formation of the condensed phase of Y66N and lens opacity. We are the first to show and propose that the compromised solubility of Y66N could be the result of a protein-folding defect. The S10R mutation in MGB was reported to be associated with nuclear cataract, which could apparently be reversed by the over-expression of α3 connexin (Cx46) [10, 11]. Here again, the molecular basis for the aggregation due to the mutation and the subsequent changes were not apparent. We show that, even though the mutation does not involve a cysteine, some cysteine residues in the mutant protein are activated leading to the enhanced formation of intermolecular disulfide-crosslinked protein aggregates in the S10R mutant under oxidizing conditions. We suggest that once these deposits accumulate, they inhibit glutathione transport which results in the oxidative stress-mediated downstream changes. Overexpression of connexin 46 – which transports reduced glutathione (GSH) into the nucleus [12, 13] – suppresses aggregation. We thus provide an important missing link to explain cataractogenic changes due to this mutation. We also examined the V41M mutation in HGS, associated with hereditary cataracts [14]. Vendra et al [15] showed that V41M had lower thermal and chemical stability, and enhanced surface hydrophobicity compared to the wild type (WT) protein, which can increase its aggregation propensity. Previous work from this laboratory [5] identified the structural differences between V41M and HGS at the residue-level using NMR spectroscopy and showed that the mutant displayed significant perturbations near the two adjacent β-strands of the first and the second “Greek-key” motif and thus created hydrophobic surface patches in the N-td. Specifically, we noted that the S-pi interaction brought about by the introduction of a Met residue in place of Val might be the key determinant. In this dissertation, we describe further details of such an interaction, which affects the protein surface, and facilitates the binding of ANS (8-anilinonaphthalene sulfonate) – a probe of hydrophobic surface. Further, using the temperature-dependence of the heterologous expression in E. coli, we demonstrate how this structural perturbation in the mutant differentiates the mutant from the WT protein in vivo. We propose that the formation of insoluble (aggregated) form of the mutant observed in E. coli, mimics the aggregation of the mutant in the lens, which causes opacity and cataract. The work in this dissertation provides, for the first time, a detailed mechanistic understanding of how three cataract-associated, missense mutants in the γ-crystallins generate light-scattering elements in highly soluble proteins. This work thus expands the knowledge base of the growing variety of molecular mechanisms underlying cataract-associated missense mutations in the γ-crystallins.

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