Conserved tryptophan residues are critical for the structure and the stability of β/γ-crystallin in the lenses of vertebrates. During aging, in which the lenses are continuously exposed to ultraviolet irradiation and other environmental stresses, oxidation of tryptophan residues in β/γ-crystallin is triggered and impacts the lens proteins to varying degrees. Kynurenine derivatives, formed by oxidation of tryptophan, accumulate, resulting in destabilization and insolubilization of β/γ-crystallin, which correlates with age-related cataract formation. To understand the contribution of tryptophan modification on the structure and stability of human βB2-crystallin, five tryptophan residues were mutated to phenylalanine considering its similarity in structure and hydrophilicity to kynurenine. Among all mutants, W59F and W151F altered the stability and homo-oligomerization of βB2-crystallin-W59F promoted tetramerization whereas W151F blocked oligomerization. Most W59F dimers transformed into tetramer in a month, and the separated dimer and tetramer of W59F demonstrated different structures and hydrophobicity, implying that the biochemical properties of βB2-crystallin vary over time. By using SAXS, we found that the dimer of βB2-crystallin in solution resembled the lattice βB1-crystallin dimer (face-en-face), whereas the tetramer of βB2-crystallin in solution resembled its lattice tetramer (domain-swapped). Our results suggest that homo-oligomerization of βB2-crystallin includes potential inter-subunit reactions, such as dissociation, unfolding, and re-formation of the dimers into a tetramer in solution. The W>F mutants are useful in studying different folding states of βB2-crystallin in lens.

BACKGROUND: Congenital cataracts, which can arise due to a combination of factors like environmental influences and genetic predisposition, significantly impact children\'s visual health globally. The occurrence rate of congenital cataracts varies from 0. 63 to 9.74 per 10,000 births. There are 7.4 instances per 10,000 children, with the highest occurrence seen in Asia. Symptoms of the disease include clouding of the lens and visual impairment. Timely identification of the condition plays a crucial role in the management and outlook of pediatric patients.
OBJECTIVE: This investigation aimed to discover causative mutations in four separate Chinese family lineages.
METHODS: The detailed clinical data and family history of four Chinese families with autosomal dominant congenital cataracts were carefully documented. Examination of the Whole Exome Sequencing was utilized to identify the genetic anomalies present in the familial cases. Subsequent validation of the identified mutations was carried out using PCR and Sanger sequencing. Following this, various computational predictive programs were utilized to evaluate how the mutations impact the structure and function of the protein.
RESULTS: The sequencing results reveal four potential disease-causing mutations: c.436G > A (p.V146M) of CRYBB2 Family 1, c.26G > T (p.R9I) of GJA3 in family 2, c.227G > A (p.R76H) of GJA8 in family 3, c.-168G > T of FTL in family 4. Among them, the causative mutation in Family GJA3 is novel, and Family FTL is a rare cataract syndrome. These familial mutations showed complete co-segregation with the affected individuals, with no presence in unaffected family members or the 100 controls. Several bioinformatic prediction tools also support the likely pathogenicity of these mutations.
CONCLUSIONS: Our findings expand the mutational and phenotypic spectrum of genes associated with congenital cataracts and provide clues to the pathogenesis of congenital cataracts. These data also demonstrate the importance of NGS technology for the molecular diagnosis of congenital cataract patients.

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.

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.
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.
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.
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.

Congenital cataract, a common disease with lens opacification, causes blindness in the newborn worldwide and is mainly caused by abnormal aggregation of crystallin. As the main structural protein in the mammalian lens, βB1-crystallin has an important role in the maintenance of lens transparency. Recently, the L116P mutation in βB1-CRY was found in a Chinese family with congenital nuclear cataracts, while its underlying pathogenic mechanism remains unclear. In the current study, the βB1 wild-type protein was purified, and the mutated form, βB1-L116P, was examined for examining the effect on structural stability and susceptibility against environmental stresses. Our results reveal low solubility and structural stability of βB1-L116P at physiological temperature, which markedly impaired the protein structure and the oligomerization of βB1-crystallin. Under guanidine hydrochloride-induced denaturing conditions, βB1-L116P mutation perturbed the protein unfolding process, making it prone to amyloid fibrils aggregation. More importantly, the L116P mutation increased susceptibility of βB1-crystallin against UV radiation. βB1-L116P overexpression led to the formation of more serious intracellular aggresomes under UV radiation or oxidative stress. Furthermore, the βB1-L116P mutation increased the sensitivity to the proteolysis process. These results indicate that the low structural stability, susceptibility to amyloid fibrils aggregation, and protease degradation of βB1-L116P may contribute to cataract development and associated symptoms.

Expression of β-crystallin B2 (CRYβB2) is elevated in African American (AA) breast tumors. The underlying mechanisms of CRYβB2-induced malignancy and the association of CRYβB2 protein expression with survival have not yet been described. Here, we report that the expression of CRYβB2 in breast cancer cells increases stemness, growth, and metastasis. Transcriptomics data revealed that CRYβB2 upregulates genes that are functionally associated with unfolded protein response, oxidative phosphorylation, and DNA repair, while down-regulating genes related to apoptosis. CRYβB2 in tumors promotes de-differentiation, an increase in mesenchymal markers and cancer-associated fibroblasts, and enlargement of nucleoli. Proteome microarrays identified a direct interaction between CRYβB2 and the nucleolar protein, nucleolin. CRYβB2 induces nucleolin, leading to the activation of AKT and EGFR signaling. CRISPR studies revealed a dependency on nucleolin for the pro-tumorigenic effects of CRYβB2. Triple-negative breast cancer (TNBC) xenografts with upregulated CRYβB2 are distinctively sensitive to the nucleolin aptamer, AS-1411. Lastly, in AA patients, higher levels of nucleolar CRYβB2 in primary TNBC correlates with decreased survival. In summary, CRYβB2 is upregulated in breast tumors of AA patients and induces oncogenic alterations consistent with an aggressive cancer phenotype. CRYβB2 increases sensitivity to nucleolin inhibitors and may promote breast cancer disparity.

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.

Adequate knowledge of protein conformations is crucial for understanding their function and their association properties with other proteins. The cataract disease is correlated with conformational changes in key proteins called crystallins. These changes are due to mutations or post-translational modifications that may lead to protein unfolding, and thus the formation of aggregate states. Human βB2-crystallin (HβB2C) is found in high proportion in the eye lens, and its mutations are related to some cataracts. HβB2C also associates into dimers, tetramers, and other higher-order supramolecular complexes. However, it is the only protein of the βγ-crystallin family that has been found in an extended conformation. Therefore, we hypothesize that the extended conformation is not energetically favourable and that HβB2C may adopt a closed (completely folded) conformation, similar to the other members of the βγ-crystallin family. To corroborate this hypothesis, we performed extensive molecular dynamics simulations of HβB2C in its monomeric and dimeric conformations, using all-atom and coarse-grained scales. We employed Markov state model (MSM) analysis to characterize the conformational and kinetically relevant states in the folding process of monomeric HβB2C. The MSM analysis clearly shows that HβB2C adopts a completely folded structure, and this conformation is the most kinetically and energetically favourable one. In contrast, the extended conformations are kinetically unstable and energetically unfavourable. Our MSM analysis also reveals a key metastable state, which is particularly interesting because it is from this state that the folded state is reached. The folded state is stabilized by the formation of two salt bridges between the residue-pairs E74-R187 and R97-E166 and the two hydrophobic residue-pairs V59-L164 and V72-V151. Furthermore, free energy surface (FES) analysis revealed that the HβB2C dimer with both monomers in a closed conformation (face-en-face dimer) is energetically more stable than the domain-swapped dimer (crystallographic structure). The results presented in this report shed light on the molecular details of the folding mechanism of HβB2C in an aqueous environment and may contribute to interpreting different experimental findings. Finally, a detailed knowledge of HβB2C folding may be key to the rational design of potential molecules to treat cataract disease.

β/γ-Crystallins, the main structural protein in human lenses, have highly stable structure for keeping the lens transparent. Their mutations have been linked to cataracts. In this study, we identified 10 new mutations of β/γ-crystallins in lens proteomic dataset of cataract patients using bioinformatics tools. Of these, two double mutants, S175G/H181Q of βΒ2-crystallin and P24S/S31G of γD-crystallin, were found mutations occurred in the largest loop linking the distant β-sheets in the Greek key motif. We selected these double mutants for identifying the properties of these mutations, employing biochemical assay, the identification of protein modifications with nanoUPLC-ESI-TOF tandem MS and examining their structural dynamics with hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that both double mutations decrease protein stability and induce the aggregation of β/γ-crystallin, possibly causing cataracts. This finding suggests that both the double mutants can serve as biomarkers of cataracts.