Abstract
Cataract, opacity of the eye lens, is the leading cause of visual impairment worldwide. The crucial pathogenic factors that cause cataract are misfolding and aggregation of crystallin protein. βB1‐crystallin, which is the most abundant water‐soluble protein in mammalian lens, is essential for lens transparency. A previous study identified the missense mutation βB1‐S93R being responsible for congenital cataract. However, the exact pathogenic mechanism causing cataract remains unclear. The S93 residue, which is located at the first Greek‐key motif of βB1‐crystallin, is highly conserved, and its substitution to Arginine severely impaired hydrogen bonds and structural conformation, which were evaluated via Molecular Dynamic Simulation. The βB1‐S93R was also found to be prone to aggregation in both human cell lines and Escherichia coli. Then, we isolated the βB1‐S93R variant from inclusion bodies by protein renaturation. The βB1-S93R mutation exposed more hydrophobic residues, and the looser structural mutation was prone to aggregation. Furthermore, the S93R mutation reduced the structural stability of βB1-crystallin when incubated at physiological temperature and made it more sensitive to environmental stress, such as UV irradiation or oxidative stress. We also constructed a βB1-S93R cellular model and discovered that βB1-S93R was more sensitive to environmental stress, causing not only aggregate formation but also cellular apoptosis and impaired cellular viability. All of the results indicated that lower solubility and structural stability, sensitivity to environmental stress, vulnerability to aggregation, and impaired cellular viability of βB1-S93R might be involved in cataract development.
Highlights
Protein misfolding and aggregation are associated with many pathological disorders in humans, such as Neurodegenerative diseases, Cataract, and Type II diabetes (Dobson et al, 2001; Konarkowska et al, 2006; Andersen, 2006)
From the dimeric structure of the βB1-wild type (WT), S93 is located in the first Greek key motif and structurally close to the second Greek key motif (Figure 1B), implying that this site probably plays an important role in maintaining structural stability of βB1–crystallin
The western blot and the densitometric analyses showed that the WT protein mainly existed in the supernatant, while a considerable amount of S93R protein appeared in the precipitate (Figure 2D)
Summary
Protein misfolding and aggregation are associated with many pathological disorders in humans, such as Neurodegenerative diseases, Cataract, and Type II diabetes (Dobson et al, 2001; Konarkowska et al, 2006; Andersen, 2006). Protein might misfold and form insoluble aggregates under pathological conditions (Yang and Gruebele, 2003; Alam et al, 2017; Finkelstein, 2018). Cataract, which is characterized by opacity of the eye lens, is a prevalent disease associated with protein aggregation and the leading cause of blindness and visual impairment worldwide (Asbell et al, 2020). Misfolding and aggregation of different lens crystallins, which account for about 90% of the water soluble proteins of the lens, are considered the major pathogenic factors responsible for all types of cataracts (Luo et al, 2021). Mutations in various genes have been linked to both age-related and congenital cataracts, and among the more than 100 genes reported in congenital cataracts, crystallin mutations account for almost half of the total number of mutations, most of their pathogenetic mechanisms causing cataracts remain unclear (Li et al, 2020)
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