The Effects of Age-Related Modifications on the Stability and Function of Human Eye Lens Crystallins
Creators: name: Ray, Nicholas John
Publisher: name: The Australian National University
Year: 2015
Subjects: Crystallin
Cataract
Aging
Post-translational modification
Protein
Deamidation
Eye
Eye lens

description: The crystallins are the primary proteins of the eye lens and are largely responsible for its high transparency, high refractive index and long-term stability. While the crystallin proteins are often able to remain stable for the lifetime of an individual, the lack of protein turnover in the lens means they are subject to a wide range of age-related, post-translational modifications. These modifications build up over the protein’s life and many are correlated with lens opacification and cataract-related blindness. This thesis details the investigation of several such age-related modifications, characterising their effect on crystallin protein structure, function and stability. Chapter 3 investigates the structures of several short peptides that are the product of truncations in crystallin proteins. The crystallins undergo spontaneous peptide cleavage events as the lens ages via several non-enzymatic pathways. One common such truncation pathway involves an N,O-acyl shift that can occur at the N-terminal side of serine residues. This rearrangement is responsible for the appearance of several short peptides in the lens as it ages, such as αA-crystallin 55-65 (LDSGISEVR), βA3-crystallin 200-215 (SHAQTSQIQSIRRIQQ) and γS-crystallin 167-178 (SPAVQSFRRIVE). In this study these three peptides were characterised structurally using NMR spectroscopy. Both βA3 200-215 and γS 167-178 are proposed to interact with the membrane of lens fibre cells, upon which they undergo a change in secondary structure. NMR spectroscopy was performed on both of these peptides under membrane-mimicking solvent conditions with the aim of characterising this structural change. Based on NMR secondary chemical shifts and NOE connectivities along their backbones, both peptides possess a region of secondary structure in their centre. Solution structures where generated using torsion angle dynamics simulation based on NMR-derived restraints, showing a short turn-like nascent helix in the γS-crystallin peptide and a longer region of well-defined α-helix in the βA3-crystallin peptide. The stable structures of these peptides under membrane-like conditions may permit peptide infiltration into the walls of lens fibre cells and contribute to age-related damage. The αA-crystallin 55-65 fragment was likewise structurally characterised using NMR spectroscopy. This peptide contains a major site of aspartic acid isomerisation at Asp58 and both L- and D- isoforms of aspartic and isoaspartic acid are common in aged lenses at this location. Structures for this protein fragment containing each of these isomers were generated from NMR-derived restraints. The native L-Asp containing peptide exhibited a region of turn-like structure in its central region and the presence of non-native aspartic acid isomers caused a loss of structure and an increase in disorder. It was also found that aspartic and isoaspartic acid residues are readily distinguishable via their characteristic NMR spectroscopic NOE patterns. Chapter 4 details the biophysical characterisation of deamidated γS-crystallin. Deamidation is a spontaneous chemical modification that converts Asn and Gln residues to Asp and Glu via a cyclic succinimide intermediate. The deamidation of Asn76 in human γS-crystallin to Asp is correlated with age-related cataract formation but is also common in healthy lenses. Biophysical characterisation was performed on wild type and N76D γS-crystallin using turbidity measurements to monitor aggregation, intrinsic fluorescence and circular dichroism spectroscopy to determine the folded state and NMR spectroscopy for identifying local changes in structure. Protein mass was determined using multi-angle light scattering and analytical ultracentrifugation methods. It was found that, relative to the wild type protein, deamidation at Asn76 in γS-crystallin causes an increase in the thermal stability and resistance to thermally induced aggregation alongside a decrease in stability to denaturants, a propensity to aggregate rapidly once destabilised and a tendency to form a dimer. The apparent increase in thermal stability upon deamidation is attributed to the formation of dimer, which prevents the unfolding of the inherently less stable monomer. Overall, it was found that deamidation of Asn76 causes a decrease in stability of γS-crystallin but this is offset by an increased tendency for dimer formation. Chapter 5 describes the structural and functional characterisation of deamidated αA-crystallin. Deamidation of Gln147 in human αA-crystallin to glutamic acid is common in aged lenses and may have a causative effect in cataract formation. The α-crystallins are small heat-shock proteins that act as molecular chaperones to prevent aggregation of proteins under stress and are the major defence against protein unfolding and aggregation in the lens. Deamidated αA-crystallin was structurally characterised using a variety of biophysical methods, including NMR, circular dichroism and intrinsic fluorescence spectroscopy, and the effect of deamidation on the protein's chaperone ability was determined for several aggregating model system. Deamidated αA-crystallin generally exhibited slightly reduced chaperone ability and a small loss of overall structure while also showing an increase in thermal stability and an increased tendency to form larger oligomers. It is likely that deamidation at Gln147 causes a loss of structure in the protein's central α-crystallin domain that leads to a slight loss of function but also increases the protein's tendency to form large oligomers. Such a loss of function could combine with other sources of age-related damage to the crystallins to contribute to lens opacification. Each of the age-related post-translational modifications examined in this thesis has a potentially negative influence on lens supramolecular protein structure and therefore contributes to cataract formation. However, none of these modifications results in a disproportionate loss of function or stability. It is likely that modifications such as these act cumulatively in the lens to generate overall instability. As modifications build up in the lens with age, the likelihood of damage resulting in protein unfolding and aggregation increases. Protein aggregates cause light scattering which contribute to lens opacification and hence cataract.