The British journal of ophthalmology · 2023
Heteromeric formation with βA3 protects the low thermal stability of βB1-L116P.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Xu Jingjie, Zhang Ying, Liu Jian, Hu Lidan, Luo Chenqi, Yao Ke, Chen Xiangjun
Аннотация
BACKGROUND/AIMS: Congenital cataract is the leading cause of visual disability and blindness in childhood. βB1-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 βB1-L116P.
METHODS: Protein isolation, size-exclusion chromatography, spectroscopy, Uncle stability screens and molecular dynamics simulations were used to assess βA3- and βB1-crystallin thermal stability, structural properties and heteromer formation.
RESULTS: Cells that overexpressed βB1-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, βA3 had a relative protective effect on βB1-L116P after heteromers were formed, although βA3 was relatively unstable and was usually protected by basic β-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 βB1-crystallin thermal stability in the presence of the cataract-related L116P mutation contributes significantly to congenital cataract formation. Moreover, its formation of heteromers with βA3 protects against the low thermal stability of βB1-L116P.
METHODS: Protein isolation, size-exclusion chromatography, spectroscopy, Uncle stability screens and molecular dynamics simulations were used to assess βA3- and βB1-crystallin thermal stability, structural properties and heteromer formation.
RESULTS: Cells that overexpressed βB1-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, βA3 had a relative protective effect on βB1-L116P after heteromers were formed, although βA3 was relatively unstable and was usually protected by basic β-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 βB1-crystallin thermal stability in the presence of the cataract-related L116P mutation contributes significantly to congenital cataract formation. Moreover, its formation of heteromers with βA3 protects against the low thermal stability of βB1-L116P.