Insight into Pathogenic Mechanism Underlying the Hereditary Cataract Caused by ßB2-G149V Mutation.

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 ßB2-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 ßB2-crystallin. The results showed that the G149V mutation significantly changed the secondary and tertiary structure of ßB2-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 ßB2-crystallin WT and the G149V mutant with their biophysical properties under environmental stress. We found that the G149V mutation makes ßB2-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 ßB2-crystallin G149V mutant related to congenital cataracts.

Heteromeric formation with ßA3 protects the low thermal stability of ßB1-L116P.

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.

Congenital cataract-causing mutation ßB1-L116P is prone to amyloid fibrils aggregation and protease degradation with low structural stability.

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, ßB1-crystallin has an important role in the maintenance of lens transparency. Recently, the L116P mutation in ßB1-CRY was found in a Chinese family with congenital nuclear cataracts, while its underlying pathogenic mechanism remains unclear. In the current study, the ßB1 wild-type protein was purified, and the mutated form, ßB1-L116P, was examined for examining the effect on structural stability and susceptibility against environmental stresses. Our results reveal low solubility and structural stability of ßB1-L116P at physiological temperature, which markedly impaired the protein structure and the oligomerization of ßB1-crystallin. Under guanidine hydrochloride-induced denaturing conditions, ßB1-L116P mutation perturbed the protein unfolding process, making it prone to amyloid fibrils aggregation. More importantly, the L116P mutation increased susceptibility of ßB1-crystallin against UV radiation. ßB1-L116P overexpression led to the formation of more serious intracellular aggresomes under UV radiation or oxidative stress. Furthermore, the ßB1-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 ßB1-L116P may contribute to cataract development and associated symptoms.

Novel Mutation in CRYBB3 Causing Pediatric Cataract and Microphthalmia.

Up to 25% of pediatric cataract cases are inherited, with half of the known mutant genes belonging to the crystallin family. Within these, crystallin beta B3 (CRYBB3) has the smallest number of reported variants. Clinical ophthalmological and genetic-dysmorphological evaluation were performed in three autosomal dominant family members with pediatric cataract and microphthalmia, as well as one unaffected family member. Peripheral blood was collected from all participating family members and next-generation sequencing was performed. Bioinformatics analysis revealed a novel missense variant c.467G>A/p.Gly156Glu in CRYBB3 in all family members with childhood cataract. This variant is classified as likely pathogenic by ACMG, and no previous descriptions of it were found in ClinVar, HGMD or Cat-Map. The only other mutation previously described in the fifth exon of CRYBB3 is a missense variant that causes a change in amino acid from the same 156th amino acid to arginine and has been associated with pediatric cataract and microphthalmia. To the best of our knowledge, this is the first time the c.467G>A/p.Gly156Glu variant is reported and the second time a mutation in CRYBB3 has been associated with microphthalmia.

CRYßB2 enhances tumorigenesis through upregulation of nucleolin in triple negative breast cancer.

Expression of ß-crystallin B2 (CRYßB2) is elevated in African American (AA) breast tumors. The underlying mechanisms of CRYßB2-induced malignancy and the association of CRYßB2 protein expression with survival have not yet been described. Here, we report that the expression of CRYßB2 in breast cancer cells increases stemness, growth, and metastasis. Transcriptomics data revealed that CRYßB2 upregulates genes that are functionally associated with unfolded protein response, oxidative phosphorylation, and DNA repair, while down-regulating genes related to apoptosis. CRYßB2 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 CRYßB2 and the nucleolar protein, nucleolin. CRYßB2 induces nucleolin, leading to the activation of AKT and EGFR signaling. CRISPR studies revealed a dependency on nucleolin for the pro-tumorigenic effects of CRYßB2. Triple-negative breast cancer (TNBC) xenografts with upregulated CRYßB2 are distinctively sensitive to the nucleolin aptamer, AS-1411. Lastly, in AA patients, higher levels of nucleolar CRYßB2 in primary TNBC correlates with decreased survival. In summary, CRYßB2 is upregulated in breast tumors of AA patients and induces oncogenic alterations consistent with an aggressive cancer phenotype. CRYßB2 increases sensitivity to nucleolin inhibitors and may promote breast cancer disparity.

A novel missense mutation of CRYBB1 causes congenital cataract in a Chinese family.

OBJECTIVE OF THE STUDY: To identify the pathogenic gene and mutation site of a Chinese family with congenital cataract. METHODS: Eight family members and 100 controls were employed, and targeted exome sequencing was used to identify the genetically pathogenic factor of the proband. RESULTS: Targeted next-generation sequencing identified a novel missense mutation c.209A>C (p.Q70P) of CRYBB1 gene in the family. Sanger sequencing results showed that this heterozygous mutation was a causative mutation, which was not found in unaffected family members and healthy controls. Bioinformatics predicts that the effect of this mutation on protein function is probably harmful. CONCLUSION: We demonstrate that c.209A>C of CRYBB1 gene is a pathogenic mutation in the family of congenital nuclear cataract in this study. This is the first report that this mutation leads to congenital nuclear cataract, which broadens the mutation spectrum of CRYBB1 gene in congenital nuclear cataract.

A novel CRYBB2 mutation causes autosomal dominant cataract: A report from a Chinese family.

PURPOSE: This study aimed to examine pathogenic mutation within one Chinese family of five-generations suffering from autosomal dominant cataract. METHODS: Next-generation sequencing and Sanger sequencing were used to find the pathogenic variants. RESULTS: A rare mutation, c.563G > A, in CRYBB2 gene was found in the proband that showed symptom of non-syndromic congenital autosomal dominant cataract. This mutation had been found in all affected individuals and in one healthy infant, but it did not exist between two individuals who did not develop such disease in that family, as well as in 100 healthy subjects who showed no relation with that family. Cataracts in this family varied with different severity of lens opacities and elongation of axial length. CONCLUSION: One missense mutation c.563G > A is reported in the CRYBB2 gene among one Chinese family suffering from early-onset cataract, and associated novel phenotypes are the elongation of axial length and the types of cataract. Our results expand the spectrum of associated phenotypes of CRYBB2 mutation.

ßB2 W151R mutant is prone to degradation, aggregation and exposes the hydrophobic side chains in the fourth Greek Key motif.

Studies have established that congenital cataract is the major cause of blindness in children across the globe. The ß-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 ßB2 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 ßB2 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.

Cataract-Associated New Mutants S175G/H181Q of ßΒ2-Crystallin and P24S/S31G of γD-Crystallin Are Involved in Protein Aggregation by Structural Changes.

ß/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 ß/γ-crystallins in lens proteomic dataset of cataract patients using bioinformatics tools. Of these, two double mutants, S175G/H181Q of ßΒ2-crystallin and P24S/S31G of γD-crystallin, were found mutations occurred in the largest loop linking the distant ß-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 ß/γ-crystallin, possibly causing cataracts. This finding suggests that both the double mutants can serve as biomarkers of cataracts.

A Novel Mutation p.S93R in CRYBB1 Associated with Dominant Congenital Cataract and Microphthalmia.

Purpose: To identify the pathogenetic mutations in a four-generation Chinese family with dominant congenital cataracts and microphthalmia.Methods: A four-generation Chinese family with dominant congenital cataracts were recruited. Genomic DNAs were collected from their peripheral blood leukocytes and subjected to whole exome sequencing. The genetic mutations were identified by bioinformatic analyses and verified by Sanger sequencing.Results: Whole exome sequencing revealed a c.279C>G point mutation in the CRYBB1 gene which was further verified by Sanger sequencing. The nucleotide replacement results in a novel mutation p.S93R in a conserved residue of ßB1 crystallin which is predicted to disrupt normal ßB1 structure and function.Conclusions: We identified a novel missense mutation p.S93R in CRYBB1 in a Chinese family with autosomal dominant congenital cataracts and microphthalmia. This serine residue is extremely conserved evolutionarily in more than 50 ßγ-crystallins of many species. These data will be very helpful to further understand the structural and functional features of crystallins.

A functional role for the cancer disparity-linked genes, CRYßB2 and CRYßB2P1, in the promotion of breast cancer.

BACKGROUND: In the USA, the breast cancer mortality rate is 41% higher for African-American women than non-Hispanic White women. While numerous gene expression studies have classified biological features that vary by race and may contribute to poorer outcomes, few studies have experimentally tested these associations. CRYßB2 gene expression has drawn particular interest because of its association with overall survival and African-American ethnicity in multiple cancers. Several reports indicate that overexpression of the CRYßB2 pseudogene, CRYßB2P1, and not CRYßB2 is linked with race and poor outcome. It remains unclear whether either or both genes are linked to breast cancer outcomes. This study investigates CRYßB2 and CRYßB2P1 expression in human breast cancers and breast cancer cell line models, with the goal of elucidating the mechanistic contribution of CRYßB2 and CRYßB2P1 to racial disparities. METHODS: Custom scripts for CRYßB2 or CRYßB2P1 were generated and used to identify reads that uniquely aligned to either gene. Gene expression according to race and tumor subtype were assessed using all available TCGA breast cancer RNA sequencing alignment samples (n = 1221). In addition, triple-negative breast cancer models engineered to have each gene overexpressed or knocked out were developed and evaluated by in vitro, biochemical, and in vivo assays to identify biological functions. RESULTS: We provide evidence that CRYßB2P1 is expressed at higher levels in breast tumors compared to CRYßB2, but only CRYßB2P1 is significantly increased in African-American tumors relative to White American tumors. We show that independent of CRYßB2, CRYßB2P1 enhances tumorigenesis in vivo via promoting cell proliferation. Our data also reveal that CRYßB2P1 may function as a non-coding RNA to regulate CRYßB2 expression. A key observation is that the combined overexpression of both genes was found to suppress cell growth. CRYßB2 overexpression in triple-negative breast cancers increases invasive cellular behaviors, tumor growth, IL6 production, immune cell chemoattraction, and the expression of metastasis-associated genes. These data underscore that both CRYßB2 and CRYßB2P1 promote tumor growth, but their mechanisms for tumor promotion are likely distinct. CONCLUSIONS: Our findings provide novel data emphasizing the need to distinguish and study the biological effects of both CRYßB2 and CRYßB2P1 as both genes independently promote tumor progression. Our data demonstrate novel molecular mechanisms of two understudied, disparity-linked molecules.

Molecular characterization of the human lens epithelium-derived cell line SRA01/04.

Cataract-associated gene discovery in human and animal models have informed on key aspects of human lens development, homeostasis and pathology. Additionally, in vitro models such as the culture of permanent human lens epithelium-derived cell lines (LECs) have also been utilized to understand the molecular biology of lens cells. However, these resources remain uncharacterized, specifically regarding their global gene expression and suitability to model lens cell biology. Therefore, we sought to molecularly characterize gene expression in the human LEC, SRA01/04, which is commonly used in lens studies. We first performed short tandem repeat (STR) analysis and validated SRA01/04 LEC for its human origin, as recommended by the eye research community. Next, we used Illumina HumanHT-12 v3.0 Expression BeadChip arrays to gain insights into the global gene expression profile of SRA01/04. Comparative analysis of SRA01/04 microarray data was performed using other resources such as the lens expression database iSyTE (integrated Systems Tool for Eye gene discovery), the cataract gene database Cat-Map and the published lens literature. This analysis showed that SRA01/04 significantly expresses >40% of the top iSyTE lens-enriched genes (313 out of 749) across different developmental stages. Further, SRA01/04 also significantly expresses ~53% (168 out of 318) of cataract-associated genes in Cat-Map. We also performed comparative gene expression analysis between SRA01/04 cells and the previously validated mouse LEC 21EM15. To gain insight into whether SRA01/04 reflects epithelial or fiber cell characteristics, we compared its gene expression profile to previously reported differentially expressed genes in isolated mouse lens epithelial and fiber cells. This analysis suggests that SRA01/04 has reduced expression of several fiber cell-enriched genes. In agreement with these findings, cell culture analysis demonstrates that SRA01/04 has reduced potential to initiate spontaneous lentoid body formation compared to 21EM15 cells. Next, to independently validate SRA01/04 microarray gene expression, we subjected several candidate genes to RT-PCR and RT-qPCR assays. This analysis demonstrates that SRA01/04 supports expression of many key genes associated with lens development and cataract, including CRYAB, CRYBB2, CRYGS, DKK3, EPHA2, ETV5, GJA1, HSPB1, INPPL1, ITGB1, PAX6, PVRL3, SFRP1, SPARC, TDRD7, and VIM, among others, and therefore can be relevant for understanding the mechanistic basis of these factors. At the same time, SRA01/04 cells do not exhibit robust expression of several genes known to be important to lens biology and cataract such as ALDH1A1, COL4A6, CP, CRYBA4, FOXE3, HMX1, HSF4, MAF, MEIS1, PITX3, PRX, SIX3, and TRPM3, among many others. Therefore, the present study offers a rich transcript-level resource for case-by-case evaluation of the potential advantages and limitations of SRA01/04 cells prior to their use in downstream investigations. In sum, these data show that the human LEC, SRA01/04, exhibits lens epithelial cell-like character reflected in the expression of several lens-enriched and cataract-associated genes, and therefore can be considered as a useful in vitro resource when combined with in vivo studies to gain insight into specific aspects of human lens epithelial cells.

Bidirectional Analysis of Cryba4-Crybb1 Nascent Transcription and Nuclear Accumulation of Crybb3 mRNAs in Lens Fibers.

Purpose: Crystallin gene expression during lens fiber cell differentiation is tightly spatially and temporally regulated. A significant fraction of mammalian genes is transcribed from adjacent promoters in opposite directions ("bidirectional" promoters). It is not known whether two proximal genes located on the same allele are simultaneously transcribed. Methods: Mouse lens transcriptome was analyzed for paired genes whose transcriptional start sites are separated by less than 5 kbp to identify coexpressed bidirectional promoter gene pairs. To probe these transcriptional mechanisms, nascent transcription of Cryba4, Crybb1, and Crybb3 genes from gene-rich part of chromosome 5 was visualized by RNA fluorescent in situ hybridizations (RNA FISH) in individual lens fiber cell nuclei. Results: Genome-wide lens transcriptome analysis by RNA-seq revealed that the Cryba4-Crybb1 pair has the highest Pearson correlation coefficient between their steady-state mRNA levels. Analysis of Cryba4 and Crybb1 nascent transcription revealed frequent simultaneous expression of both genes from the same allele. Nascent Crybb3 transcript visualization in "early" but not "late" differentiating lens fibers show nuclear accumulation of the spliced Crybb3 transcripts that was not affected in abnormal lens fiber cell nuclei depleted of chromatin remodeling enzyme Snf2h (Smarca5). Conclusions: The current study shows for the first time that two highly expressed lens crystallin genes, Cryba4 and Crybb1, can be simultaneously transcribed from adjacent bidirectional promoters and do not show nuclear accumulation. In contrast, spliced Crybb3 mRNAs transiently accumulate in early lens fiber cell nuclei. The gene pairs coexpressed during lens development showed significant enrichment in human "cataract" phenotype.

Crybb2 Mutations Consistently Affect Schizophrenia Endophenotypes in Mice.

As part of the ßγ-superfamily, ßB2-crystallin (CRYBB2) is an ocular structural protein in the lens, and mutation of the corresponding gene can cause cataracts. CRYBB2 also is expressed in non-lens tissue such as the adult mouse brain and is associated with neuropsychiatric disorders such as schizophrenia. Nevertheless, the robustness of this association as well as how CRYBB2 may contribute to disease-relevant phenotypes is unknown. To add further clarity to this issue, we performed a comprehensive analysis of behavioral and neurohistological alterations in mice with an allelic series of mutations in the C-terminal end of the Crybb2 gene. Behavioral phenotyping of these three ßB2-mutant lines Crybb2O377, Crybb2Philly, and Crybb2Aey2 included assessment of exploratory activity and anxiety-related behavior in the open field, sensorimotor gating measured by prepulse inhibition (PPI) of the acoustic startle reflex, cognitive performance measured by social discrimination, and spontaneous alternation in the Y-maze. In each mutant line, we also quantified the number of parvalbumin-positive (PV+) GABAergic interneurons in selected brain regions that express CRYBB2. While there were allele-specific differences in individual behaviors and affected brain areas, all three mutant lines exhibited consistent alterations in PPI that paralleled alterations in the PV+ cell number in the thalamic reticular nucleus (TRN). The direction of the PPI change mirrored that of the TRN PV+ cell number thereby suggesting a role for TRN PV+ cell number in modulating PPI. Moreover, as both altered PPI and PV+ cell number are schizophrenia-associated endophenotypes, our result implicates mutated Crybb2 in the development of this neuropsychiatric disorder.

Transgenic lysyl oxidase homolog 1 overexpression in the mouse eye results in the formation and release of protein aggregates.

Sequence variants in LOXL1 coding for the secreted enzyme lysyl oxidase homolog 1 (LOXL1) associate with pseudoexfoliation (PEX) syndrome, a condition that is characterized by the deposition of extracellular fibrillar PEX material in the anterior eye and other parts of the body. Since the specific role of LOXL1 in the pathogenesis of PEX is unclear, and an increase in its expression was reported for early stages of PEX syndrome, we generated and studied transgenic mice with ocular overexpression of its mouse ortholog Loxl1. The chicken ßB1-crystallin promoter was used to overexpress Loxl1 in the lenses of ßB1-crystallin-Loxl1 transgenic mice. Transgenic lenses contained high levels of the protein LOXL1 and its mRNA, which were both not detectable in lenses of wildtype littermates. In wildtype mice, immunoreactivity for LOXL1 was mainly seen extracellularly in region of the ciliary zonules. ßB1-crystallin-Loxl1 littermates showed an additional diffuse immunostaining in lens fibers and capsule, and in the inner limiting membrane and retina indicating secretion of soluble LOXL1 from transgenic lenses. In addition, lens fibers of transgenic animals contained multiple distinct spots of very intense LOXL1 immunoreactivity. By transmission electron microscopy, those spots correlated with electron-dense round or oval bodies of 20-50 nm in diameter which were localized in the rough endoplasmic reticulum and not seen in wildtype lenses. Immunogold electron microscopy confirmed that the electron-dense bodies contained LOXL1 indicating aggregation of insoluble LOXL1. Similar structures were seen in the extracellular lens capsule suggesting their secretion from lens fibers. Otherwise, no changes were seen between the eyes of ßB1-crystallin-Loxl1 mice and their wildtype littermates, neither by light microscopy and funduscopy of whole eyes, nor by scanning and quantitative transmission electron microscopy of ciliary epithelium and zonules. At one month of age, intraocular pressure was significantly higher in transgenic mice than in wildtype littermates. No differences in IOP were seen though at 2-5 months of age. We conclude that LOXL1 has a strong tendency to aggregate in the rER when expressed in vivo at high amounts. A similar scenario, involving intracellular aggregation of LOXL1 and secretion of LOXL1 aggregates into the extracellular space, may be involved in the early pathogenetic events in eyes of PEX patients.

A CRYBB2 mutation in a Taiwanese family with autosomal dominant cataract.

BACKGROUND/PURPOSE: To identify the underlying genetic cause of a Taiwanese family with autosomal dominant cerulean cataract. METHODS: A three-generation cerulean cataract family with 13 affected and 13 normal was identified. Whole exome sequencing, whole genome single nucleotide polymorphism genotyping and haplotype analysis, and fine mapping using polymorphic short tandem repeat markers were used to identify the causative gene mutation. RESULTS: Whole genome single nucleotide polymorphism genotyping and haplotype analysis mapped the candidate disease loci to chromosome 18 and chromosome 22. Polymorphic short tandem repeat markers further narrowed down the disease interval to chromosome 22 between markers D22S1174 and D22S1163. Whole exome sequencing was performed on selected individuals. Polymorphisms detected were filtered based on their genomic positions, allele frequency (<1%), and segregation within the pedigree. Affected individuals were found to be heterozygous carrying a C to T mutation on exon 6 of the CRYBB2 gene (with SNP ID: rs74315489). The mutation was predicted to produce a premature stop mutation Q155X. The mutation is co-segregation across the pedigree and the disease "T" allele was not detected in healthy members of the family and in additional 50 normal controls (100 chromosomes). Phylogenic protein alignment was also performed for the CRYBB2 gene across 68 species ranging from fishes, Sauropsida, Placentalia, carnivores, rodents, and primates with total 56 orthologous genes. The Q155 residue is 100% conserved across the evolutionary tree, indicating its crucial function. CONCLUSION: Here we identify the first Taiwanese cerulean cataract family carrying a CRYBB2_Q155X mutation.

Introduction of an extra tryptophan fluorophore by cataract-associating mutations destabilizes ßB2-crystallin and promotes aggregation.

ß/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 ß/γ-crystallins account for UV absorbance and thereafter fluorescence quenching to avoid photodamage. Herein we found that ßB2-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 ßB2-crystallin aggregation under UV damaging conditions, whereas R145W delayed. These observations suggested that the introduction of an extra Trp fluorophore had complicated effects on ßB2-crystallin stability and aggregation against various stresses. Our findings highlight that the number of Trp fluorophores in ß/γ-crystallin is evolutionarily optimized to exquisitely perform their structural roles in the lens.

Long Noncoding RNA CRYBG3 Blocks Cytokinesis by Directly Binding G-Actin.

The dynamic interchange between monomeric globular actin (G-actin) and polymeric filamentous actin filaments (F-actin) is fundamental and essential to many cellular processes, including cytokinesis and maintenance of genomic stability. Here, we report that the long noncoding RNA LNC CRYBG3 directly binds G-actin to inhibit its polymerization and formation of contractile rings, resulting in M-phase cell arrest. Knockdown of LNC CRYBG3 in tumor cells enhanced their malignant phenotypes. Nucleotide sequence 228-237 of the full-length LNC CRYBG3 and the ser14 domain of ß-actin is essential for their interaction, and mutation of either of these sites abrogated binding of LNC CRYBG3 to G-actin. Binding of LNC CRYBG3 to G-actin blocked nuclear localization of MAL, which consequently kept serum response factor (SRF) away from the promoter region of several immediate early genes, including JUNB and Arp3, which are necessary for cellular proliferation, tumor growth, adhesion, movement, and metastasis. These findings reveal a novel lncRNA-actin-MAL-SRF pathway and highlight LNC CRYBG3 as a means to block cytokinesis and to treat cancer by targeting the actin cytoskeleton.Significance: Identification of the long noncoding RNA LNC CRYBG3 as a mediator of microfilament disorganization marks it as a novel therapeutic antitumor strategy. Cancer Res; 78(16); 4563-72. ©2018 AACR.

Crybb2 associates with Tmsb4X and is crucial for dendrite morphogenesis.

Dendrite morphogenesis is a complex but well-orchestrated process. Various studies reported the involvement of alteration in dendrite morphology in different brain disorders, including neuropsychiatric disorders. Initially, ßB2-crystallin (gene symbol: Crybb2/CRYBB2) has been described as a structural protein of the ocular lens. Mutations of the corresponding gene, Crybb2, lead to cataract. Recent studies in mice suggested that mutations in Crybb2 cause alterations in hippocampal morphology and function, albeit its function in hippocampal neuron development remained elusive. In the current study, we found that Crybb2 contributes to dendritogenesis in vitro and in vivo. Furthermore, screening of previous data on differential expression-arrays, we found Tmsb4X up-regulated in Crybb2 mutants mouse brain. Additionally, Tmsb4X was co-expressed with Crybb2 at actin-enriched cell ruffles. Over-expression of Tmsb4X in cultured hippocampal neurons inhibited dendritogenesis, which phenocopied Crybb2 knock-down. The current study uncovers a new function of Crybb2 in brain development, especially in dendritogenesis, and the possible interplay partner Tmsb4X involved in this process.

[Analysis of disease-causing gene mutation in three Chinese families with congenital inherited cataract].

OBJECTIVE: To identify the disease-causing gene mutations in three Chinese pedigrees affected with congenital inherited cataract, in ordre to provide genetic counseling and prenatal diagnosis. METHODS: Using exons combined target region capture sequencing chip to screen the candidate disease-causing mutations, Sanger sequencing was used to confirm the disease-causing mutations. RESULTS: Family 1 was polymorphic cataract, family 2 was cerulean cataract, family 3 was coralliform cataract. The inheritance mode of the three pedigrees consisted with autosomal dominant inheritance. In family 1, a nonsense mutation of CRYßB2 gene c.463C>T in exon 6 result in a p.Q155X amino acid change. In family 2, a missense mutation of of CRYGD gene c.43C>T in exon 2 result in a p.R14C amino acid change. In family 3, a missense mutation of CRYGD gene c.70C>A in exon 2 result in a p.P23T amino aid change. No above-mentioned mutations were found in normal individuals. CONCLUSION: The nonsense mutation c.463C>T (p.Q155X) of CRYßB2 gene, the heterozygous mutations c.43C>T(p.R14C) of CRYGD gene and c.70C>A( p.P23T) of CRYGD gene was the disease-causing gene mutation in family 1, 2 and 3 respectively, our results provid genetic counseling and prenatal diagnosis for these three families.