Crystallin Research Articles

Decrease of alpha-crystallin A by miR-325-3p in retinal cells under blue light exposure

Exposure to blue light can lead to retinal degeneration, causing adverse effects on eye health. Although the loss of retinal cells due to blue light exposure has been observed, the precise molecular mechanisms underlying this process remain poorly understood. In this study, we investigate the role of alpha crystallin A (CRYAA) in neuro-retinal degeneration and their regulation by blue light. We observed significant apoptotic cell death in both the retina of rats and the cultured neuro-retinal cells. The expressions of Cryaa mRNA and protein were significantly down-regulated in the retina exposed to blue light. We identified that miR-325-3p reduces Cryaa mRNA and protein by binding to its 3'-untranslated region (UTR). Up-regulation of miR-325-3p destabilized Cryaa mRNA and suppresses CRYAA, whereas down-regulation of miR-325-3p increased both expressions. Blue light-induced neuro-retinal cell death was alleviated by CRYAA overexpression. These results highlight the critical role of Cryaa mRNA and miR-325-3p molecular axis in blue light-induced retinal degeneration. Consequently, targeting CRYAA and miR-325-3p presents a potential strategy for protecting against blue light-induced retinal degeneration.

The Aqueous Stem Bark Extract of Alstonia boonei Exhibits Anticataract Activity in Sprague Dawley Rat.

In Africa, Alstonia boonei is used folklorically for the management of the multitude of conditions including cataract, which accounts for 50% of cases of blindness in the region. The current study set out to probe the traditional use of the aqueous extract of Alstonia boonei stem bark (ABE) as an anticataract remedy using Sprague Dawley rat models. We investigated the probable phytochemical constituents in the extract, in vitro antioxidant potential, and its in vitro aldose reductase inhibition. For the anticataract investigations, diabetic cataract was induced using galactose in 3-week-old Sprague Dawley rats, and age-related cataract was induced by the administration of sodium selenite to 10-day-old rat pups. Cataract scores in both models were determined after treatment with 30, 100, and 300 mgkg-1 doses of ABE and 10 mlkg-1 of distilled water. Lens glutathione, total lens protein, soluble lens proteins (alpha-A) crystallin, and aquaporin 0 levels in the enucleated lens homogenates were determined. Changes in lens to body weight were also determined with histopathological analysis done on the lenses in the selenite-induced cataract model. The presence of alkaloids, tannins, flavonoids, glycosides, and triterpenoids was identified in the extract. The extract inhibited aldose reductase activity with IC50 of 92.30 μgml-1. The 30, 100, and 300 mgkg-1ABE-treated rats recorded significantly (p < 0.05) reduced cataract scores indicating a delay in cataractogenesis in galactose-induced cataract and in selenite-induced cataractogenesis as well. Markers of lens transparency such as AQP0, alpha-A crystallin, and total lens proteins and lens glutathione levels were significantly (p < 0.05) preserved. In conclusion, this study establishes the anticataract potential of the aqueous stem bark extract of Alstonia boonei in Sprague Dawley rat models.

Anticataract Effect of the Aqueous Extract of the Flowers of Aspilia africana in Murine Model of Diabetic and Age-Related Cataracts.

The use of Aspilia africana in traditional medicine for the management of ocular diseases has been reported in India and some indigenous communities of Africa. The aim of this study was to investigate the aqueous extract of the flowers of A. africana (AAE) as an anticataract remedy using murine models of diabetic and senile cataracts. Preliminary phytochemical screening of the extract, in vitro antioxidant assays, and in vitro aldose reductase inhibitory activity were performed. For anticataract investigations of the extracts, diabetic cataract was induced by galactose administration in 3-week-old Sprague Dawley rats. The evaluation of experimentally induced age-related cataract was performed by administering sodium selenite to 10-day-old rat pups. The phytochemical analysis revealed the presence of alkaloids, tannins, flavonoids, glycosides, and saponins. In vitro aldose reductase inhibitory property of the extract on rat lenses revealed that the AAE inhibited the enzyme activity with IC50 of 12.12 µg/ml. For the anticataract investigations, 30, 100, and 300 mg·kg-1AAE-treated rats recorded significantly low (p ≤ 0.0001) cataract scores compared to the negative control rats, indicating a delay in cataractogenesis from the second week of treatment in the galactose-induced cataractogenesis. Similarly, the treatment with AAE caused a significant reduction (p ≤ 0.0001) in cataract scores compared to the negative control rats in the selenite-induced cataractogenesis. Markers of lens transparency, such as aquaporin 0, alpha-A crystallin, and total lens proteins and lens glutathione levels, were significantly preserved (p ≤ 0.05-0.0001) in each cataract model after AAE treatment. The study established the anticataract potential of the aqueous extract of flowers of A. africana in murine models, hence giving scientific credence to its folkloric use in the management of cataract.

Damage resistance of gamma crystallins: Weathering heat, UV, and ionizing radiation.

The γ-crystallins are structural and refractive proteins of the vertebrate lens. They belong to the βγ-crystallin superfamily, which is characterized by an exceptionally stable double-Greek key fold. In the context of the lens, these proteins must remain stable and soluble for the lifetime of the organism. Recent work in my group focuses on understanding how γ-crystallins maintain functionality in the face of damage caused by aging and oxidation. I will present results on the impact of heat, UV light, and ionizing radiation on human γS-crystallin and cataract-associated variants. We find that the γ-crystallins are not particularly resistant to damage; on the contrary, they appear to be selected for the ability to remain soluble even when the protein surface is significantly modified and transient unfolding intermediates are populated. These have been investigated using a variety of biophysical techniques, including circular dichroism, mass spectrometry, fluorescence, and NMR spectroscopy. In particular, mass spectrometry measurements on irradiated samples show evidence of both deamidation and oxidative damage, while high-resolution NMR data allow us to locate protein regions and specific residues that are involved in conformational changes, altered dynamics, and protein-protein contacts. We will discuss proposed aggregation mechanisms mediated by different types of protein modification, as well as the relevance of these findings to human cataract disease.

Study of the migration of Fasciola hepatica juveniles across the intestinal barrier of the host by quantitative proteomics in an ex vivo model.

Fasciola hepatica is a trematode parasite that infects animals and humans causing fasciolosis, a worldwide-distributed disease responsible for important economic losses and health problems. This disease is of growing public health concern since parasite isolates resistant to the current treatment (triclabendazole) have increasingly been described. F. hepatica infects its vertebrate host after ingestion of the encysted parasite (metacercariae), which are found in the water or attached to plants. Upon ingestion, newly excysted juveniles of F. hepatica (FhNEJ) emerge in the intestinal lumen and cross the intestinal barrier, reach the peritoneum and migrate to the biliary ducts, where adult worms fully develop. Despite the efforts made to develop new therapeutic and preventive tools, to date, protection against F. hepatica obtained in different animal models is far from optimal. Early events of host-FhNEJ interactions are of paramount importance for the infection progress in fasciolosis, especially those occurring at the host-parasite interface. Nevertheless, studies of FhNEJ responses to the changing host environment encountered during migration across host tissues are still scarce. Here, we set-up an ex vivo model coupled with quantitative SWATH-MS proteomics to study early host-parasite interaction events in fasciolosis. After comparing tegument and somatic fractions from control parasites and FhNEJ that managed to cross a mouse intestinal section ex vivo, a set of parasite proteins whose expression was statistically different were found. These included upregulation of cathepsins L3 and L4, proteolytic inhibitor Fh serpin 2, and a number of molecules linked with nutrient uptake and metabolism, including histone H4, H2A and H2B, low density lipoprotein receptor, tetraspanin, fatty acid binding protein a and glutathione-S-transferase. Downregulated proteins in FhNEJ after gut passage were more numerous than the upregulated ones, and included the heath shock proteins HSP90 and alpha crystallin, amongst others. This study brings new insights into early host-parasite interactions in fasciolosis and sheds light on the proteomic changes in FhNEJ triggered upon excystment and intestinal wall crossing, which could serve to define new targets for the prevention and treatment of this widespread parasitic disease.

Whole genome assembly of the armored loricariid catfish Ancistrus triradiatus highlights herbivory signatures

The catfish Ancistrus triradiatus belongs to the species-rich family Loricariidae. Loricariids display remarkable traits such as herbivory, a benthic lifestyle, the absence of scales but the presence of dermal bony plates. They are exported as ornamental fish worldwide, with escaped fishes becoming a threat locally. Although genetic and phylogenetic studies are continuously increasing and developmental genetic investigations are underway, no genome assembly has been formally proposed for Loricariidae yet. We report a high-quality genome assembly of Ancistrus triradiatus using long and short reads, and a newly assembled transcriptome. The genome assembly is composed of 9530 scaffolds, including 85.6% of ray-finned fish BUSCOs, and 26,885 predicted protein-coding genes. The genomic GC content is higher than in other catfishes, reflecting the higher metabolism associated with herbivory. The examination of the SCPP gene family indicates that the genes presumably triggering scale loss when absent, are present in the scaleless A. triradiatus, questioning their explanatory role. The analysis of the opsin gene repertoire revealed that gene losses associated to the nocturnal lifestyle of catfishes were not entirely found in A. triradiatus, as the UV-sensitive opsin 5 is present. Finally, most gene family expansions were related to immunity except the gamma crystallin gene family which controls pupil shape and sub-aquatic vision. Thus, the genome of A. triradiatus reveals that fish herbivory may be related to the photic zone habitat, conditions metabolism, photoreception and visual functions. This genome is the first for the catfish suborder Loricarioidei and will serve as backbone for future genetic, developmental and conservation studies.

Three Novel Mutations of Microphthalmos Identified in Two Chinese Families.

Genetic alterations are a major cause of microphthalmos, while novel-related genes and mutations in microphthalmos have rarely been explored. To identify the underlying genetic defect responsible for microphthalmos eyes in two three-generation Chinese families, we screened 425 genes involved in common inherited non-syndromic eye diseases with next-generation sequencing-based target capture sequencing of the two probands of two three-generation Chinese families diagnosed with microphthalmos. Variants were filtered and analyzed to identify possible disease-causing variants before Sanger sequencing validation. We enrolled two families with microphthalmos (Family 1: microphthalmos with congenital ocular coloboma and Family 2: simple microphthalmos). Two novel heterozygous mutations, Peroxidasin (PXDN) c.3165C>T (p.Pro1055Pro) and PXDN c.2640C>G (p.Arg880Arg), were found in Family 1, and Crystallin Beta B2 (CRYBB2) c.481G>A (p.Gly161Arg) was found in Family 2, but none of the mutations were found in the unaffected individuals, who were phenotypically normal. Multiple orthologous sequence alignment (MSA) revealed that the CRYBB2 p.Gly161Arg mutation was a deleterious effect mutation. In conclusion, the three novel mutations found in our study extend our current understanding of the genetic basis of microphthalmos and provide early pre-symptomatic diagnosis and emphasize the significance of genetic diagnosis of microphthalmos.

Analysis of Global and Residue‐Specific yB Crystallin Protein Dynamics via NMR Spectroscopy and Dynamic Light Scattering

The proteins that comprise the water‐soluble interior of the optic lens are referred to as crystallin proteins, of which three major categories have been identified: alpha (a), beta (b), and gamma (y). Gamma and beta crystallin proteins have been observed to undergo aggregation and phase‐separation over time, which can lead to the formation of cataracts, while alpha crystallin limits this formation by removing deformed protein aggregates from solution. Here, we describe our studies on bovine yB crystallin, which is the homologue to human yD crystallin. To better understand the intermolecular interactions between yB crystallins, we overexpressed the 15N‐labeled recombinant protein in Escherichia coli, purified it via column chromatography, and studied the effect of solution conditions on protein dynamics using NMR spectroscopy and dynamic light scattering techniques. Prior research from our group employed T1‐T2 NMR experiments to probe the effect of temperature and protein concentration on global protein dynamics, while more recent 1H‐15N HSQC experiments allowed us to visualize the amide proton peaks for yB crystallin protein at various concentrations. We observed small peak shifts for select residues as the protein concentration increased, which we hypothesized were due to increased intermolecular interactions between yB crystallin molecules. Analysis of these NMR spectra, combined with dynamic light scattering data on the same samples, provide further insight into both global dynamics as well as residue‐specific interactions.

Approximation for dielectric geometry modified screened electrostatic gamma crystallin interactions

We provide a useful and rapid approximation to dielectric-geometry modified, screened electrostatic interactions that is adapted to the case of a liquid-liquid phase separating protein, bovine gamma-B crystallin, for which extensive experimental and theoretical work has been published. We calibrate this model with use of experimental light scattering data on second virial coefficients, and then apply this approximation to study a space of approximately 150,000 relative orientations of neighboring pairs of gamma crystallins, at various separation distances.

Patterns of T and B cell responses to Mycobacterium tuberculosis membrane-associated antigens and their relationship with disease activity in rheumatoid arthritis patients with latent tuberculosis infection.

This study was aimed at exploring whether latent tuberculosis infection (LTBI) contributes to the pathogenesis of immune-mediated inflammatory diseases in a TB endemic setting. We screened 198 rheumatoid arthritis (RA) patients with tuberculin skin test (TST) and studied 61 (median DAS28-ESR = 6.3) who were positive. Whole blood T cell proliferative responses to Mycobacterium tuberculosis (Mtb) membrane (MtM) antigens, including the latency-induced protein alpha crystallin (Acr), were determined by flow cytometry using Ki67 expression as the marker for nuclear proliferation. Serum antibody levels were determined by ELISA. Follow-up investigations (at 3-6, 9-12 and 15-18 months after baseline) were performed in 41 patients who were classified empirically as 'high' (HR-T/HR-B) or 'low' (LR-T/LR-B) responders based on their dynamic T cell or antibody responses. Significant correlations were seen between baseline T cell responses to MtM and Acr, and between IgG, IgA and IgM antibody responses to MtM. However, no correlation was seen between T and B cell responses. At all time points during the follow-up, T cell responses to both antigens (except for MtM at one point) were significantly higher in HR-T (n = 25) than LR-T (n = 16) patients. Levels of IgA and IgM (but not IgG) antibodies to MtM were also significantly higher in HR-B (n = 13) than LR-B (n = 28) at all time points. Importantly, HR-T patients exhibited significantly higher baseline and follow-up DAS28 scores than LR-T. Ten (of 61) patients had a history of TB and developed RA 6 years (median) after contracting TB. Three new TB cases (1 from TST-positive and 2 from TST-negative groups) emerged during the follow-up. Our results suggest that persistently elevated T cell responses to Mtb antigens may contribute to disease activity in RA.

The aging mouse lens transcriptome

Age is a major risk factor for cataract (ARC). However, the influence of aging on the lens transcriptome is under studied. Lens epithelial (LEC) and fiber cells (LFC) were isolated from young (3 month old) and aged (24 month old) C57BL/6J mice, and the transcriptome elucidated via RNAseq. EdgeR estimated differential gene expression in pairwise contrasts, and Advaita's Ipathway guide and custom R scripts were used to evaluate the potential biological significance of differentially expressed genes (DEGs). This analysis revealed age-dependent decreases in lens differentiation marker expression in both LECs and LFCs, with gamma crystallin transcripts downregulating nearly 50 fold in aged LFCs. The expression of the transcription factors Hsf4 and Maf, which are known to activate lens fiber cell preferred genes, are downregulated, while FoxE3, which represses gamma crystallin expression, is upregulated in aged fibers. Aged LECs upregulate genes controlling the immune response, complement pathways, and cellular stress responses, including glutathione peroxidase 3 (Gpx3). Aged LFCs exhibit broad changes in the expression of genes regulating cell communication, and upregulate genes involved in antigen processing/presentation and cholesterol metabolism, while changes in the expression of mitochondrial respiratory chain genes are consistent with mitochondrial stress, including upregulation of NDufa4l2, which encodes an alternate electron transport chain protein. However, age did not profoundly affect the response of LECs to injury as both young and aged LECs upregulate inflammatory gene signatures at 24h post injury to similar extents. These RNAseq profiles provide a rich data set that can be mined to understand the genetic regulation of lens aging and how this impinges on the pathophysiology of age related cataract.

Elucidating the Electrostatic Interactions of Bovine Gamma‐B Crystallin via Site‐Directed Mutagenesis

The proteins that comprise the water-soluble interior of the optic lens are referred to as crystallin proteins, of which three major categories have been identified: alpha (a), beta (b), and gamma (y). Gamma and beta crystallin proteins have been observed to aggregate with time, which can lead to the formation of cataracts, while alpha crystallin limits this formation by removing deformed protein aggregates from solution. Our studies specifically involve the use of bovine yB crystallin, which is the homologue to human yD crystallin. Prior research from our group has generated computer models of the electrostatic interactions between human yD crystallin molecules to identify the specific residues involved in gamma-gamma and gamma-alpha interactions. Here, we describe our work with a site-directed mutant of bovine yB crystallin (D107A), which we propose to govern inter-protein interactions, and more specifically, gamma-alpha interactions. We seek to compare mutant and wild-type proteins using NMR spectroscopy and dynamic light scattering to better understand how individual residues dictate gamma-gamma and gamma-alpha interactions.

Abstract B116: Evaluating the ability of clinically approved chemotherapeutics to decrease race-associated breast cancer mortality disparities via targeting the tumor-promoting effect of Crystallin Beta B2

Abstract In the United States, breast cancer (BC) remains the second leading cause of cancer death in women. The unequal burden in the mortality of BC in African American (AA) women compared to Caucasian American (CA) women goes without full explanation. When socioeconomic factors such as education and income are accounted for, this mortality disparity persists. Therefore, to acquire an understanding of how to end BC disparities, it is crucial to examine the underlying cellular and molecular mechanisms driving this disproportionate outcome. Studies on gene expression analyses have identified a particular gene, CRYβB2, with higher expression in AA breast tumors. Expression of CRYβB2 was also found to be increased in studies comparing AA and CA colorectal, renal, glioblastoma, and prostate cancers, and has been linked with decreased survival. Although the precise mechanisms of elevated CRYβB2 expression remain unknown, overexpression is significantly associated with BC tumor promotion. In view of this, we investigated the biological effects of CRYβB2 in basal-like, triple negative breast cancer (TNBC) cell lines. CRYβB2 expression was manipulated via CRIPSR/Cas9-mediated knockout and lentiviral overexpression systems. Our preliminary data show enhanced proliferation and invasion in 3D cultures with cells overexpressing CRYβB2 compared to controls, suggesting two mechanisms by which CRYβB2-overexpression may enhance tumor promotion and promote poor patient outcome. To identify drugs that may specifically target cells overexpressing CRYβB2, we subjected one of our CRYβB2 model systems (SUM159 cells overexpressing CRYβB2, with CRYβB2 knocked out, and the parental SUM159 cells with low CRYβB2 expression) to increasing doses of 125 clinically approved chemotherapeutics. High content live cell imaging was used to evaluate the effects of the drugs by measuring 3D spheroid BC cell growth, and IC50 were generated to determine whether cells with altered CRYβB2 expression were more or less sensitive to particular classes of drugs. Drugs identified with increased cytotoxicity to cells overexpressing CRYβB2 were confirmed using two additional TNBC models with altered CRYβB2 levels. Additional in vitro mechanistic studies evaluating the effect of the drugs on proliferation, cytotoxicity and invasion of CRYβB2-overexpressing were executed in 3D cultures with live cell imaging. Drugs that significantly affect the proliferation or invasion of CRYβB2-overexpressing cells will be evaluated in future studies for efficacy and toxicity in conjunction with other chemotherapeutics via in vivo AA-specific xenografts and PDX models. This information will be used to explore the potential use of these chemotherapeutics for neoadjuvant or adjuvant therapy in BC patients with tumors that overexpress CRYβB2. The identification of clinically approved drugs that inhibit CRYβB2 function may quickly be applied in the clinic to increase survival rates until more effective and less toxic, targeted-therapies are developed. Citation Format: Portia L. Andrews, Denise K. Reaves, Megan E. Martin, Michael Tarpley, Kevin P. Williams, Jodie M. Fleming. Evaluating the ability of clinically approved chemotherapeutics to decrease race-associated breast cancer mortality disparities via targeting the tumor-promoting effect of Crystallin Beta B2 [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B116.

Transcriptome profiling of differentiated lenses through RNA sequencing

Objective: To gain insight into the transcriptional landscape including mRNA, long non-coding RNA (lncRNA), and circular RNA (circRNA) of the differentiated lens. Methods: Experiment research. The total RNAs of the differentiated lenses were extracted and purified. Total RNAs of 16-week, 23-week, and 25-week differentiated lenses were then sequenced using Illumina HiSeq 2500, and analyzed using bioinformatics tools. The top expressed and differentially expressed mRNAs and lncRNAs were screened. The expressions of overlap genes among the 16-week, 23-week, and 25-week lenses were analyzed by Venn diagram. The expression tendency of lens-specific genes was obtained and verified with real-time polymerase chain reaction. Results: A total of 67 518 311 mapped reads were obtained from differentiated lenses at 16 weeks, 99 440 160 at 23 weeks, and 67 262 320 at 25 weeks. The gene overlap expression analysis showed 740 of the top 1 000 highly expressed mRNAs, 170 of the top 300 highly expressed lncRNAs, and 69 of the top 100 highly expressed circRNAs overlapping expressed in lenses at 16, 23, and 25 weeks, respectively. Lens specific gene expression analysis revealed that the expression of crystallin (CRY) AA, CRYGA, CRYGB, CRYGC, CRYGD, CRYGEP, and CRYGS was upregulated, while the expression of gap junction (GJ) A3 and GJA8 was downregulated with the differentiation of lenses. Conclusion: The lens transcriptome profile shows that more than half of the high expressed mRNA, lncRNA and circRNA at different differentiation stages are overlapping expressed, and all of them have high expression of lens specific protein genes, such as CRY, GJ etc. (Chin J Ophthalmol, 2020, 56: 356-363).

Aggregation of Gamma S-Crystallin Mediated by UV Light and Divalent Metal Cations

Aggregation of Gamma S-Crystallin Mediated by UV Light and Divalent Metal Cations

Buried Tryptophans Contributing to the High Kinetic Stability of the Long-lived Gamma Crystallins and their Oxidative Damage Opening the Pathway to the Aggregated State Associated with Cataracts

Buried Tryptophans Contributing to the High Kinetic Stability of the Long-lived Gamma Crystallins and their Oxidative Damage Opening the Pathway to the Aggregated State Associated with Cataracts

Immune responses to Mycobacterium tuberculosis membrane-associated antigens including alpha crystallin can potentially discriminate between latent infection and active tuberculosis disease.

Changes in expression of membrane antigens may accompany the transition of Mycobacterium tuberculosis (Mtb) from 'dormant' to 'active' states. We have determined whether antibody and T cell responses to Mtb membrane (MtM)-associated antigens, especially the latency-induced protein alpha crystallin (Acr), can discriminate between latent tuberculosis infection (LTBI) and active TB (ATB) disease. Study subjects comprised a previously described cohort of healthcare workers (HCWs, n = 43) and smear-positive ATB patients (n = 10). HCWs were further categorized as occupational contacts (OC, n = 30), household contacts of TB (HC, n = 8) and cured TB (CTB, n = 5). Levels (ΔOD) of serum antibody isotypes (IgG, IgA and IgM) were determined by ELISA and blood T cell proliferative responses were determined by flow cytometry using Ki67 protein as marker for DNA synthesis. Antibodies to MtM and Acr were predominantly IgG and their levels in HCWs and ATB did not differ significantly. However, HCWs showed a significantly higher level of anti-MtM IgM and a significantly lower level of anti-Acr IgA antibodies than the ATB patients. Also, a larger proportion of HCWs showed a high (>1) ΔODAcr/ΔODMtM ratio for IgG. HCWs also showed a higher, though not significantly different from ATB, avidity of anti-MtM (IgG) antibodies. A higher proportion of HCWs (35% of OC, 62.5% of HC and 20% of CTB), compared with ATB (10%) showed a positive T cell response to Acr along with significant difference (P <0.05) between HC and ATB. A significant correlation (r = 0.60, P <0.0001) was noted between T cell responses of HCWs towards Acr and MtM (reported earlier by us) and both responses tended to decline with rising exposure to the infection. Even so, positive responses to Acr (38.5%) were significantly lower than to MtM (92%). Neither antibody nor T cell responses to either antigen appeared affected by BCG vaccination or reactivity to tuberculin. Results of the study suggest that the levels of IgM antibodies to MtM, IgA antibodies to Acr and proliferative T cell responses to both the antigens can potentially discriminate between LTBI and active TB disease. They also underscore the necessity of SOPs for antibody assays.

In silico analysis and high-risk pathogenic phenotype predictions of non-synonymous single nucleotide polymorphisms in human Crystallin beta A4 gene associated with congenital cataract.

In order to provide a cost-effective method to narrow down the number of pathogenic Crystallin beta A4 (CRYBA4) non-synonymous single nucleotide polymorphisms (nsSNPs), we collected nsSNP information of the CRYBA4 gene from SNP databases and literature, predicting the pathogenicity and possible changes of protein properties and structures using multiple bioinformatics tools. The nsSNP data of the CRYBA4 gene were collected from 4 databases and published literature. According to 12 criteria, six bioinformatics tools were chosen to predict the pathogenicity. I-Mutant 2.0, Mupro and INPS online tools were used to analyze the effects of amino acid substitution on protein stability by calculating the value of ΔΔG. ConSurf, SOPMA, GETAREA and HOPE online tools were used to predict the evolutionary conservation of amino acids, solvent accessible surface areas, and the physical and chemical properties and changes of protein structure. All 157 CRYBA4 nsSNPs were analyzed. Forty-four CRYBA4 high-risk pathogenic nsSNPs (predicted to be pathogenic by all six software tools) were detected out of the 157 CRYBA4 nsSNPs, four of which (c.283C>T, p.R95W; c.449T>A, p.V150D; c.475G>A, p.G159R; c.575G>C, p.R192P) should be focused on because of their high potential pathogenicity and possibility of changing protein properties. Thirty high-risk nsSNPs were predicted to cause a decrease of protein stability. Twenty-nine high-risk nsSNPs occurred in evolutionary conserved positions. Twenty-two high-risk nsSNPs occurred in the core of the protein. It is predicted that these high-risk pathogenic nsSNPs can cause changes in the physical and chemical properties of amino acids, resulting in structural changes of proteins and changes in the interactions between domains and other molecules, thus affecting the function of proteins. This study provides important reference value when narrowing down the number of pathogenic CRYBA4 nsSNPs and studying the pathogenesis of congenital cataracts. By using this method, we can easily find 44 high-risk pathogenic nsSNPs out of 157 CRYBA4 nsSNPs.

Study on iTRAQ-based differential expression protein analysis in nuclear cataract lens with different axial length

Objective To identify and verify differentially expressed proteins in lens of nuclear cataract eyes with different axial lengths. Methods Forty-eight nuclear cataract patients with the hardness grade Ⅲ to Ⅳ were enrolled from January 2016 to December 2017 in the east area of Xining city.The lens nucleus was obtained during extracapsular cataract extraction (ECCE) surgery.The samples were divided into six groups according to age and axial length, with 8 samples in each group.Seven normal transparent human lens nuclei were obtained from donors during corneal transplantation surgery and served as normal control.Water-soluble, water-insoluble and water-insoluble-urea-soluble protein fractions were extracted separately and merged to analyze by using 8-plex isobaric tags for relative and absolute quantitation (iTRAQ) labeling combined with 2D-LC/MS/MS.The data were processed with ProteinPilot software.Two technical replicates of iTRAQ analyses were carried out.Up-regulated and down-regulated proteins were selected in two replicates as candidate biomarkers.Twenty lens nuclei from 20 nulear cataract patients were divided into long axis young age group, normal axis young age group, long axis old age group and normal axis old age group.Western blot was used to confirm the most different proteins.This study protocol was approved by the Ethics Committee of TangDu Hospital of Air Force Medical University (TDLL2015046). Written consent was obtained from each participant. Results Eighty proteins were identified.Six proteins were selected as differentially-expressed proteins.Sorbitol dehydrogenase (SDH) was elevated whereas beta-crystallin B2 (CRYBB2) was decreased in the long axis groups.The relative expression of SDH protein in the long axis low age group was 1.204±0.142, which was significantly higher than 0.711±0.072 in the normal axis low age group, the relative expression level of SDH in the long axis old age group was 1.092±0.334, which was significantly higher than 0.617±0.089 in the normal axis old age group, the difference was statistically significant (all at P<0.05). Conclusions The expression levels of SDH in the nuclear cataract were elevated with long axial length.SDH may be involved in the pathogenesis of axial myopia-associated nuclear cataract. Key words: Axial lengths, eye; Human lens, crystalline; Nuclear cataract; Isotope labeling; Proteomics

Elucidating the Intermolecular Interactions of a Cataract‐Causing Protein: γB Crystallin

Gamma crystallin proteins can be found in the eye lens, helping to maintain transparency through their intermolecular interactions. Because crystallins remain in the lens after their initial synthesis, mutations caused by prolonged exposure to environmental stresses can have permanent consequences, including aggregation and liquid‐liquid phase separation of the crystallins. These structural deviations can cause clouding of the lens, a condition known as cataracts. With the use of light scattering and nuclear magnetic resonance (NMR) spectroscopy, we aim to study the detailed interactions between crystallins. We have expressed and isolated recombinant bovine Gamma B crystallin protein in Escherichia coli and performed T1/T2 NMR experiments to measure the longitudinal and transverse relaxation times of the protein. These experiments allowed us to analyze the crystallins in terms of the attractive forces between molecules and how those forces change with the manipulation of environmental conditions (temperature, pH). Analysis of our current data suggests higher rates of intermolecular interaction at higher concentrations of crystallins and lower temperatures.Support or Funding InformationRIT and NIH Grant EY018249This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.