Gamma-D crystallin gene (CRYGD) mutation causes autosomal dominant congenital cerulean cataracts

Congenital cataracts are a major cause of bilateral visual impairment in childhood. We mapped the gene responsible for autosomal congenital cerulean cataracts to chromosome 2q33-35 in a four generation family of Moroccan descent. The maximum lod score (7.19 at recombination fraction theta=0) was obtained for marker D2S2208 near the g-crystallin gene (CRYG) cluster. Sequencing of the coding regions of the CRYGA, B, C, and D genes showed the presence of a heterozygous C>A transversion in exon 2 of CRYGD that is associated with cataracts in this family. This mutation resulted in a proline to threonine substitution at amino acid 23 of the protein in the first of the four Greek key motifs that characterise this protein. We show that although the x ray crystallography modelling does not indicate any change of the backbone conformation, the mutation affects a region of the Greek key motif that is important for determining the topology of this protein fold. Our data suggest strongly that the proline to threonine substitution may alter the protein folding or decrease the thermodynamic stability or solubility of the protein. Furthermore, this is the first report of a mutation in this gene resulting in autosomal dominant congenital cerulean cataracts

UV-radiation Induced Disruption of Dry-Cavities in Human γD-crystallin Results in Decreased Stability and Faster Unfolding

Age-onset cataracts are believed to be expedited by the accumulation of UV-damaged human γD-crystallins in the eye lens. Here we show with molecular dynamics simulations that the stability of γD-crystallin is greatly reduced by the conversion of tryptophan to kynurenine due to UV-radiation, consistent with previous experimental evidences. Furthermore, our atomic-detailed results reveal that kynurenine attracts more waters and other polar sidechains due to its additional amino and carbonyl groups on the damaged tryptophan sidechain, thus breaching the integrity of nearby dry center regions formed by the two Greek key motifs in each domain. The damaged tryptophan residues cause large fluctuations in the Tyr-Trp-Tyr sandwich-like hydrophobic clusters, which in turn break crucial hydrogen-bonds bridging two β-strands in the Greek key motifs at the “tyrosine corner”. Our findings may provide new insights for understanding of the molecular mechanism of the initial stages of UV-induced cataractogenesis.International Business Machines Corporation (IBM Blue Gene Program

Carbon turnover in the water-soluble protein of the adult human lens.

PurposeHuman eye lenses contain cells that persist from embryonic development. These unique, highly specialized fiber cells located at the core (nucleus) of the lens undergo pseudo-apoptosis to become devoid of cell nuclei and most organelles. Ostensibly lacking in protein transcriptional capabilities, it is currently believed that these nuclear fiber cells owe their extreme longevity to the perseverance of highly stable and densely packed crystallin proteins. Maintaining the structural and functional integrity of lenticular proteins is necessary to sustain cellular transparency and proper vision, yet the means by which the lens actually copes with a lifetime of oxidative stress, seemingly without any capacity for protein turnover and repair, is not completely understood. Although many years of research have been predicated upon the assumption that there is no protein turnover or renewal in nuclear fiber cells, we investigated whether or not different protein fractions possess protein of different ages by using the (14)C bomb pulse.MethodsAdult human lenses were concentrically dissected by gently removing the cell layers in water or shaving to the nucleus with a curved micrometer-controlled blade. The cells were lysed, and the proteins were separated into water-soluble and water-insoluble fractions. The small molecules were removed using 3 kDa spin filters. The (14)C/C was measured in paired protein fractions by accelerator mass spectrometry, and an average age for the material within the sample was assigned using the (14)C bomb pulse.ResultsThe water-insoluble fractions possessed (14)C/C ratios consistent with the age of the cells. In all cases, the water-soluble fractions contained carbon that was younger than the paired water-insoluble fraction.ConclusionsAs the first direct evidence of carbon turnover in protein from adult human nuclear fiber cells, this discovery supports the emerging view of the lens nucleus as a dynamic system capable of maintaining homeostasis in part due to intricate protein transport mechanisms and possibly protein repair. This finding implies that the lens plays an active role in the aversion of age-related nuclear (ARN) cataract

The Thermal Structural Transition of α-Crystallin Inhibits the Heat Induced Self-Aggregation

-crystallin, the major constituent of human lens, is a member of the heat-shock proteins family and it is known to have a quaternary structural transition at . The presence of calcium ions and/or temperature changes induce supramolecular self-aggregation, a process of relevance in the cataractogenesis. Here we investigate the potential effect of the bovine -crystallin's structural transition on the self-aggregation process. Along all the temperatures investigated, aggregation proceeds by forming intermediate molecular assemblies that successively aggregate in clusters. The final morphology of the aggregates, above and below , is similar, but the aggregation kinetics are completely different. The size of the intermediate molecular assemblies, and their repulsive energy barrier show a marked increase while crossing . Our results highlight the key role of heat modified form of -crystallin in protecting from aggregation and preserving the transparency of the lens under hyperthermic conditions

Insights into the structure and aggregation of lens crystallins and other aggregation-prone proteins

Cataract is the world's leading cause of blindness. The destabilization, partial unfolding, and aggregation of lens crystallin proteins cause the loss of lens transparency (opacification) and cataract formation. Numerous congenital mutations and age-related changes to the long-lived alpha-, beta- and gamma-crystallins are associated with cataract and their study has provided insight into the molecular basis of this disease. In this thesis, alpha- and gamma-crystallin isoforms have been characterised under crowded and oxidative conditions, respectively. Finally, the conformational heterogeneity of model proteins was studied by capillary electrophoresis as a prelude to such studies on the more complex crystallins. Chapter 2 details the structural characterisation of the disulfide-linked gammaS-crystallin dimer, an oxidative product in the aging lens. X-ray crystallography revealed an intermolecular disulfide bond from C24-C24' and two intramolecular disulfides, one in each subunit, between C22 and C26. Small angle X-ray scattering confirmed the extended in-solution biological assembly in lieu of a compact state. It was demonstrated that the disulfide-linked dimer was stable at glutathione concentrations akin to those in aged and catractous lenses. The dimer had a higher aggregation propensity compared to the monomeric form owing to uncooperative domain unfolding. These findings provide novel insight into the contributions of oxidative modification to the formation of age-related cataract. Chapter 3 describes the impacts that a highly crowded environment comparable to the eye lens has on the structure and function of the molecular chaperone alphaB-crystallin. Macromolecular crowding using Ficoll 400 induces significant destabilisation, unfolding, an increase in size/oligomeric state, and a loss of chaperone function leading to kinetically distinct amorphous and fibrillar aggregation. These results are recapitulated in-principle using the biologically relevant crowding agent bovine gamma-crystallin. Aggregation is prevented by the lens partner protein alphaA-crystallin at physiologically relevant ratios through an increase in the alphaA/alphaB-crystallin complex stability. These results complement multiple dilute in vitro and in vivo studies and provide support for therapeutic approaches prevent and reverse cataract via alpha-crystallin stabilisation. Chapter 4 investigates capillary electrophoresis as a method for studying the conformational heterogeneity of a protein. Bovine serum albumin (BSA), yeast alcohol dehydrogenase (YADH), and bovine alpha-lactalbumin (BLA) were used to assess the application of this method towards various conformational aspects in comparison to SEC-MALS. The method distinguished between BSA oligomers and two different monomer populations, multiple YADH monomer and tetramer conformations, and apo- and holo-BLA. The 'dispersity of electrophoretic mobilities' allowed a relative comparison of the levels of conformational heterogeneity between unrelated proteins. This enables for better interpretation of the heterogeneity of more complex proteins such as post-translationally modified crystallins in vivo and oligomeric alpha-crystallin. Overall, this thesis provides new insights into the molecular basis for post-translational and environmental changes in alpha- and gamma-crystallins that cause cataract