Congenital cataracts account for approximately 5-20% of childhood blindness worldwide and 22-30% of childhood blindness in developing countries. Genetic disorders are the primary cause of congenital cataracts. In this work, we investigated the underlying molecular mechanism of G149V point missense mutation in βB2-crystallin, which was first identified in a three-generation Chinese family with two affected members diagnosed with congenital cataracts. Spectroscopic experiments were performed to determine the structural differences between the wild type (WT) and the G149V mutant of βB2-crystallin. The results showed that the G149V mutation significantly changed the secondary and tertiary structure of βB2-crystallin. The polarity of the tryptophan microenvironment and the hydrophobicity of the mutant protein increased. The G149V mutation made the protein structure loose and the interaction between oligomers was reduced, which decreased the stability of the protein. Furthermore, we compared βB2-crystallin WT and the G149V mutant with their biophysical properties under environmental stress. We found that the G149V mutation makes βB2-crystallin more sensitive to environmental stresses (oxidative stress, UV irradiation, and heat shock) and more likely to aggregate and form precipitation. These features might be important to the pathogenesis of βB2-crystallin G149V mutant related to congenital cataracts.

Crystallins, the major constituent proteins of mammalian lenses, are significant not only for the maintenance of eye lens stability, transparency, and refraction, but also fulfill various physiopathological functions in extraocular tissues. βB2-crystallin, for example, is a multifunctional protein expressed in the human retina, brain, testis, ovary, and multiple tumors. Mutations in the βB2 crystallin gene or denaturation of βB2-crystallin protein are associated with cataracts, ocular pathologies, and psychiatric disorders. A prominent role for βB2-crystallins in axonal growth and regeneration, as well as in dendritic outgrowth, has been demonstrated after optic nerve injury. Studies in βB2-crystallin-null mice revealed morphological and functional abnormalities in testis and ovaries, indicating βB2-crystallin contributes to male and female fertility in mice. Interestingly, although pathogenic significance remains obscure, several studies identified a clear correlation between βB2 crystallin expression and the prognosis of patients with breast cancer, colorectal cancer, prostate cancer, renal cell carcinoma, and glioblastoma in the African American population. This review summarizes the physiological and pathological functions of βB2-crystallin in the eye and other organs and tissues and discusses findings related to the expression and potential role of βB2-crystallin in tumors.

Studies have established that congenital cataract is the major cause of blindness in children across the globe. The β-crystallin protein family is the richest and most soluble structural protein in the lens. Their solubility and stability are essential in maintaining lens transparency. In this study, we identified a novel βB2 mutation W151R in a rare progressive cortical congenital cataract family and explored its pathogenesis using purified protein and mutant related cataract-cell models. Due to its low solubility and poor structural stability, the βB2 W151R mutation was prone to aggregation. Moreover, the W151R mutation enhanced the exposure of the hydrophobic side chains in the fourth Greek Key motif, which were readily degraded by trypsin. However, upon the administration of lanosterol, the negative effect of the W151R mutation was reversed. Therefore, lanosterol is a potential therapeutic option for cataracts.

β/γ-Crystallins are predominant structural proteins in vertebrate lens with unique properties of extremely high solubility, long-term stability and resistance to UV damage. Four conserved Trp residues in β/γ-crystallins account for UV absorbance and thereafter fluorescence quenching to avoid photodamage. Herein we found that βB2-crystallin Trp fluorescence was greatly enhanced by the introduction of an extra unquenched Trp fluorophore by cataract-associated mutations S31W and R145W. Both mutations impaired oligomerization, decreased stability and promote thermal aggregation, while S31W was more deleterious. S31W accelerated βB2-crystallin aggregation under UV damaging conditions, whereas R145W delayed. These observations suggested that the introduction of an extra Trp fluorophore had complicated effects on βB2-crystallin stability and aggregation against various stresses. Our findings highlight that the number of Trp fluorophores in β/γ-crystallin is evolutionarily optimized to exquisitely perform their structural roles in the lens.

βΒ2-crystallin (CRYBB2) is expressed at an increased level in the postnatal lens cortex and is associated with cataracts. Improved understanding of the underlying biology of cataracts is likely to be critical for the development of early detection strategies and new therapeutics. The present study aimed to identify long non-coding RNAs (lncRNAs) and mRNAs associated with CRYBB2 knockdown (KO)-induced cataracts. RNAs from 3 non-treated mice and 3 CRYBB2 KO mice were analyzed using the Affymetrix GeneChip Mouse Gene 2.0 ST array. A total of 149 lncRNAs and 803 mRNAs were identified to have upregulated expression, including Snora73b, Klk1b22 and Rnu3a, while the expression levels of 180 lncRNAs and 732 mRNAs were downregulated in CRYBB2 KO mice, including Snord82, Snhg9 and Foxn3. This lncRNA and mRNA expression profile of mice with CRYBB2 KO provides a basis for studying the genetic mechanisms of cataract progression.

Crystallins are a major family of proteins located within the lens of the eye. Cataracts are thought to be due to the formation of insoluble fibrillar aggregates, which are largely composed of proteins from the crystallin family. Today the only cataract treatment that exists is surgery and this can be difficult to access for individuals in the developing world. Development of novel pharmacotherapeutic approaches for the treatment of cataract rests on the specific targeting of these structures. βB2-crystallin, a member of β-crystallin family, is a large component of the crystallin proteins within the lens, and as such was used to form model fibrils in vitro. Peptides were identified, using phage display techniques, that bound to these fibrils with high affinity. Fibrillation of recombinantly expressed human βB2-crystallin was performed in 10% (v/v) trifluoroethanol (TFE) solution (pH 2.0) at various temperatures, and its amyloid-like structure was confirmed using Thioflavin-T (ThT) assay, transmission electron microscopy (TEM), and X-ray fiber diffraction (XRFD) analysis. Affinity of identified phage-displayed peptides were analyzed using enzyme-linked immunosorbent assay (ELISA). Specific binding of a cyclic peptide (CKQFKDTTC) showed the highest affinity, which was confirmed using a competitive inhibition assay.

β/γ-Crystallins, the predominant structural proteins in vertebrate lens with lifelong stability to maintain lens transparency, share a high similarity in their primary sequences and tertiary structures. Four conserved Trp residues have been shown to be important to γ-crystallin structure, stability and protection against UV irradiation, whereas their roles in β-crystallins remain elusive. Herein we found that two congenital cataract-causing mutations, W59C and W151C, dramatically decreased βB2-crystallin solubility and stability against thermal and guanidine hydrochloride-induced denaturation. The two mutated proteins were prone to form aggregates when irradiated by UV light in the tubes or exogenously expressed in the cells. Although W59 and W151 are structurally identical in β/γ-crystallin domains, substituting them by Cys led to dissimilar influences on βB2-crystallin stability. Our results suggested that the conserved Trp residues might play a more crucial role in the correct folding and structural integrity of β-crystallin domains than in γ-crystallins.

βγ-Crystallins are long-lived eye lens proteins that are crucial for lens transparency and refractive power. Each βγ-crystallin comprises two homologous domains, which are connected by a short linker. γ-Crystallins are monomeric, while β-crystallins crystallize as dimers and multimers. In the crystal, human βB2-crystallin is a domain-swapped dimer while the N-terminally truncated βB1-crystallin forms a face-en-face dimer. Combining and integrating data from multi-angle light scattering, nuclear magnetic resonance, and small-angle X-ray scattering of full-length and terminally truncated human βB2-crystallin in solution, we show that both these βB2-crystallin proteins are dimeric, possess C2 symmetry, and are more compact than domain-swapped dimers. Importantly, no inter-molecular paramagnetic relaxation enhancement effects compatible with domain swapping were detected. Our collective experimental results unambiguously demonstrate that, in solution, human βB2-crystallin is not domain swapped and exhibits a face-en-face dimer structure similar to the crystal structure of truncated βB1-crystallin.

Cataract is characterized by the formation of light-scattering protein aggregates in the lens. β/γ-Crystallins are the predominant structural proteins in the cytosol of lens fiber cells, and more than fifty β/γ-crystallin mutations have been linked to autosomal dominant congenital cataract. However, the structural role of these mutations in the formation of the core structures of amorphous aggregates or amyloid-like fibrils has not been elucidated yet. In this research, we studied the effects of the V187M and R188H mutations on the aggregation and fibrillization of βB2-crystallin during acid denaturation. The behavior of V187M was the same as the WT protein, suggesting that the residue at position 187 contributed little to the aggregation/fibrillization process. R188H promoted the formation of amorphous aggregates at pH above 3 and accelerated fibrillization at pH 3. The distinct behaviors of the mutants suggested that the residue at position 188 might play a regulatory role in βB2-crystallin aggregation/fibrillization but not reside in the core of the aggregates/fibrils.

Congenital cataract is the leading cause of childhood blindness worldwide. Investigations of the effects of inherited mutations on protein structure and function not only help us to understand the molecular mechanisms underlying congenital hereditary cataract, but also facilitate the study of complicated cataract and non-lens abnormities caused by lens-specific genes. In this research, we studied the effects of the V187M, V187E and R188H mutations on βB2-crystallin structure and stability using a combination of biophysical, cellular and molecular dynamic simulation analysis. Both V187 and R188 are located at the last strand of βB2-crystallin Greek-key motif 4. All of the three mutations promoted βB2-crystallin aggregation in vitro and at the cellular level. These three mutations affected βB2-crystallin quite differentially: V187M influenced the hydrophobic core of the C-terminal domain, V187E was a Greek-key motif breaker with the disruption of the backbone H-bonding network, while R188H perturbed the dynamic oligomeric equilibrium by dissociating the dimer and stabilizing the tetramer. Our results highlighted the importance of the last strand in the structural integrity, folding, assembly and stability of β-crystallins. More importantly, we proposed that the perturbation of the dynamic equilibrium between β-crystallin oligomers was an important mechanism of congenital hereditary cataract. The selective stabilization of one specific high-order oligomer by mutations might also be deleterious to the stability and folding of the β-crystalllin homomers and heteromers. The long-term structural stability and functional maintenance of β-crystallins are achieved by the precisely regulated oligomeric equilibrium.