Cataract, a major cause of visual impairment worldwide, is the opacification of the eye’s crystalline lens due to aggregation of the crystallin proteins. The research reported here is aimed at investigating the aggregating behavior of γ-crystallin proteins in various incubation conditions. Thioflavin T binding assay, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, intrinsic (tryptophan) fluorescence spectroscopy, light scattering, and electron microscopy were used for structural characterization. Molecular dynamics simulations and bioinformatics prediction were performed to gain insights into the γD-crystallin mechanisms of fibrillogenesis. We first demonstrated that, except at pH 7.0 and 37°C, the aggregation of γD-crystallin was observed to be augmented upon incubation, as revealed by turbidity measurements. Next, the types of aggregates (fibrillar or non-fibrillar aggregates) formed under different incubation conditions were identified. We found that, while a variety of non-fibrillar, granular species were detected in the sample incubated under pH 7.0, the fibrillogenesis of human γD-crystallin could be induced by acidic pH (pH 2.0). In addition, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and intrinsic fluorescence spectroscopy were used to characterize the structural and conformational features in different incubation conditions. Our results suggested that incubation under acidic condition led to a considerable change in the secondary structure and an enhancement in solvent-exposure of the hydrophobic regions of human γD-crystallin. Finally, molecular dynamics simulations and bioinformatics prediction were performed to better explain the differences between the structures and/or conformations of the human γD-crystallin samples and to reveal potential key protein region involved in the varied aggregation behavior. Bioinformatics analyses revealed that the initiation of amyloid formation of human γD-crystallin may be associated with a region within the C-terminal domain. We believe the results from this research may contribute to a better understanding of the possible mechanisms underlying the pathogenesis of senile nuclear cataract.
The current research is aimed at exploring the inhibitory effect of glutathione on fibril formation of an important four disulfide bond-containing whey protein, α-lactalbumin. Through numerous spectroscopic techniques and transmission electron microscopy, we found that the inhibition of amyloid formation of α-lactalbumin was dependent on the glutathione concentration and fibrillation was significantly attenuated in the presence of 5 mM glutathione. Moreover, the data from the measurements using 4,4′-dithiodipyridine reagent revealed that the treatment of α-lactalbumin with glutathione led to the exposure of sulfhydryl groups. Also, the observed inhibition of α-lactalbumin by glutathione was correlated with the reduction of disulfide bridges of protein. The results presented here suggest that the addition of food compatible reducing agent/dietary supplement such as glutathione would be useful for preventing the formation of milk protein fibrillar aggregates. The presence of these resulting aggregates can then, in turn, be used to modulate the key properties of food products such as protein beverage and yogurt.
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