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Background/aims Congenital cataract is the leading cause of visual disability and blindness in childhood. ßB1-crystallin (CRYBB1) comprises about 1/10th of crystallin structural proteins, forming heteromers to maintain lens transparency. We previously reported a CRYBB1 mutation (c.347T>C, p.L116P) affecting 16 patients in a congenital nuclear cataract family. In this study, we investigate the underlying pathogenic mechanism of ßB1-L116P. Methods Protein isolation, size-exclusion chromatography, spectroscopy, Uncle stability screens and molecular dynamics simulations were used to assess ßA3- and ßB1-crystallin thermal stability, structural properties and heteromer formation. Results Cells that overexpressed ßB1-L116P tended to form aggregates and precipitations under heat-shock stress. Thermal denaturation and time-dependent turbidity experiments showed that thermal stability was significantly impaired. Moreover, protein instability appeared to increase with elevated concentrations detected by the Uncle system. Additionally, ßA3 had a relative protective effect on ßB1-L116P after heteromers were formed, although ßA3 was relatively unstable and was usually protected by basic ß-crystallins. Molecular dynamic simulations revealed that L116P mutation altered the hydrophobic residues at the surface around the mutant site, providing solvents more access to the internal and hydrophobic parts of the protein. Conclusions Decreased ßB1-crystallin thermal stability in the presence of the cataract-related L116P mutation contributes significantly to congenital cataract formation. Moreover, its formation of heteromers with ßA3 protects against the low thermal stability of ßB1-L116P. [ABSTRACT FROM AUTHOR]

Background Age-related cataract, which presents as a cloudy lens, is the primary cause of vision impairment worldwide and can cause more than 80% senile blindness. Previous studies mainly explored the profile of lens proteins at a low concentration because of technical limitations, which could not reflect physiological status. This study focuses on protein stability changes with ageing under physiological conditions using a novel equipment, Unchained Labs (Uncle), to evaluate protein thermal stability. Methods Samples were assessed through Unchained Labs, size-exclusion chromatography, western blot and biophysics approaches including the Thioflavin T, ultraviolet and internal fluorescence. Results With age, the melting temperature value shifted from 67.8°C in the young group to 64.2°C in the aged group. Meanwhile, crystallin may form more isomeric oligomers and easy to be degraded in aged lenses. The spectroscopic and size-exclusion chromatography results show a higher solubility after administrated with lanosterol under the environmental stress. Conclusion We are the first to explore rabbit lens protein stability changes with ageing using biophysical methods under physiological conditions, and this study can conclude that the structural stability and solubility of lens proteins decrease with ageing. Additionally, lanosterol could aid in resolving protein aggregation, making it a potential therapeutic option for cataracts. So, this study provides cataract models for anti-cataract drug developments [ABSTRACT FROM AUTHOR]