Early Proteomic Characteristics and Changes in the Optic Nerve Head, Optic Nerve, and Retina in a Rat Model of Ocular Hypertension.

The pathogenesis of glaucoma is still unknown. There are few studies on the dynamic change of tissue-specific and time-specific molecular pathophysiology caused by ocular hypertension (OHT). This study aimed to identify the early proteomic alterations in the retina, optic nerve head (ONH), and optic nerve (ON). After establishing a rat model of OHT, we harvested the tissues from control and glaucomatous eyes and analyzed the changes in protein expression using a multiplexed quantitative proteomics approach (TMT-MS3). Our study identified 6403 proteins after 1-day OHT and 4399 proteins after 7-days OHT in the retina, 5493 proteins after 1-day OHT and 4544 proteins after 7-days OHT in ONH, and 5455 proteins after 1-day OHT and 3835 proteins after 7-days OHT in the ON. Of these, 560 and 489 differential proteins were identified on day 1 and 7 after OHT in the retina, 428 and 761 differential proteins were identified on day 1 and 7 after OHT in the ONH, and 257 and 205 differential proteins on days 1 and 7 after OHT in the ON. Computational analysis on day 1 and 7 of OHT revealed that alpha-2 macroglobulin was upregulated across two time points and three tissues stably. The differentially expressed proteins between day 1 and 7 after OHT in the retina, ONH, and ON were associated with glutathione metabolism, mitochondrial dysfunction/oxidative phosphorylation, oxidative stress, microtubule, and crystallin. And the most significant change in retina are crystallins. We validated this proteomic result with the Western blot of crystallin proteins and found that upregulated on day 1 but recovered on day 7 after OHT, which are promising as therapeutic targets. These findings provide insights into the time- and region-order mechanisms that are specifically affected in the retina, ONH, and ON in response to elevated IOP during the early stages.

Cataract-causing Y204X mutation of crystallin protein CRYßB1 promotes its C-terminal degradation and higher-order oligomerization.

Crystallin proteins are a class of main structural proteins of the vertebrate eye lens, and their solubility and stability directly determine transparency and refractive power of the lens. Mutation in genes that encode these crystallin proteins is the most common cause for congenital cataracts. Despite extensive studies, the pathogenic and molecular mechanisms that effect congenital cataracts remain unclear. In this study, we identified a novel mutation in CRYBB1 from a congenital cataract family, and demonstrated that this mutation led to an early termination of mRNA translation, resulting in a 49-residue C-terminally truncated CRYßB1 protein. We show this mutant is susceptible to proteolysis, which allowed us to determine a 1.2-Å resolution crystal structure of CRYßB1 without the entire C-terminal domain. In this crystal lattice, we observed that two N-terminal domain monomers form a dimer that structurally resembles the WT monomer, but with different surface characteristics. Biochemical analyses and cell-based data also suggested that this mutant is significantly more liable to aggregate and degrade compared to WT CRYßB1. Taken together, our results provide an insight into the mechanism regarding how a mutant crystalin contributes to the development of congenital cataract possibly through alteration of inter-protein interactions that result in protein aggregation.

IgG Fc-binding protein positively regulates the assembly of pore-forming protein complex ßγ-CAT evolved to drive cell vesicular delivery and transport.

Cell membranes form barriers for molecule exchange between the cytosol and the extracellular environments. ßγ-CAT, a complex of pore-forming protein BmALP1 (two ßγ-crystallin domains with an aerolysin pore-forming domain) and the trefoil factor BmTFF3, has been identified in toad Bombina maxima. It plays pivotal roles, via inducing channel formation in various intracellular or extracellular vesicles, as well as in nutrient acquisition, maintaining water balance, and antigen presentation. Thus, such a protein machine should be tightly regulated. Indeed, BmALP3 (a paralog of BmALP1) oxidizes BmALP1 to form a water-soluble polymer, leading to dissociation of the ßγ-CAT complex and loss of biological activity. Here, we found that the B. maxima IgG Fc-binding protein (FCGBP), a well-conserved vertebrate mucin-like protein with unknown functions, acted as a positive regulator for ßγ-CAT complex assembly. The interactions among FCGBP, BmALP1, and BmTFF3 were revealed by co-immunoprecipitation assays. Interestingly, FCGBP reversed the inhibitory effect of BmALP3 on the ßγ-CAT complex. Furthermore, FCGBP reduced BmALP1 polymers and facilitated the assembly of ßγ-CAT with the biological pore-forming activity in the presence of BmTFF3. Our findings define the role of FCGBP in mediating the assembly of a pore-forming protein machine evolved to drive cell vesicular delivery and transport.

A crystallin mutant cataract with mineral deposits.

Connexin mutant mice develop cataracts containing calcium precipitates. To test whether pathologic mineralization is a general mechanism contributing to the disease, we characterized the lenses from a nonconnexin mutant mouse cataract model. By cosegregation of the phenotype with a satellite marker and genomic sequencing, we identified the mutant as a 5-bp duplication in the γC-crystallin gene (Crygcdup). Homozygous mice developed severe cataracts early, and heterozygous animals developed small cataracts later in life. Immunoblotting studies showed that the mutant lenses contained decreased levels of crystallins, connexin46, and connexin50 but increased levels of resident proteins of the nucleus, endoplasmic reticulum, and mitochondria. The reductions in fiber cell connexins were associated with a scarcity of gap junction punctae as detected by immunofluorescence and significant reductions in gap junction-mediated coupling between fiber cells in Crygcdup lenses. Particles that stained with the calcium deposit dye, Alizarin red, were abundant in the insoluble fraction from homozygous lenses but nearly absent in wild-type and heterozygous lens preparations. Whole-mount homozygous lenses were stained with Alizarin red in the cataract region. Mineralized material with a regional distribution similar to the cataract was detected in homozygous lenses (but not wild-type lenses) by micro-computed tomography. Attenuated total internal reflection Fourier-transform infrared microspectroscopy identified the mineral as apatite. These results are consistent with previous findings that loss of lens fiber cell gap junctional coupling leads to the formation of calcium precipitates. They also support the hypothesis that pathologic mineralization contributes to the formation of cataracts of different etiologies.

Transcriptome alterations in sf3b4-depleted zebrafish: Insights into cataract formation in retinitis pigmentosa model.

Posterior subcapsular cataract (PSC) frequently develops as a complication in patients with retinitis pigmentosa (RP). Despite numerous scientific investigations, the intricate pathomechanisms underlying cataract formation in individuals affected by RP remain elusive. Therefore, our study aims to elucidate the potential pathogenesis of cataracts in an RP model using splicing factor subunit 3b (sf3b4) mutant zebrafish. By analyzing our previously published transcriptome dataset, we identified that, in addition to RP, cataract was listed as the second condition in our transcriptomic analysis. Furthermore, we confirmed the presence of nucleus retention in the lens fiber cells, along with abnormal cytoskeleton expression in both the lens fiber cells and lens epithelial cells in sf3b4-depleted fish. Upon closer examination, we identified 20 differentially expressed genes (DEGs) that played a role in cataract formation, with 95 % of them related to the downregulation of structural lens proteins. Additionally, we also identified that among all the DEGs, 13 % were associated with fibrotic processes. It seems that the significant upregulation of inflammatory mediators, in conjunction with TGF-ß signaling, plays a central role in the cellular biology of PSC and posterior capsular opacification (PCO) in sf3b4 mutant fish. In summary, our study provides valuable insights into cataract formation in the RP model of sf3b4 mutants, highlighting its complexity driven by changes in structural lens proteins and increased cytokines/growth factors.

Mutations in alpha-B-crystallin cause autosomal dominant axonal Charcot-Marie-Tooth disease with congenital cataracts.

BACKGROUND AND PURPOSE: Mutations in the alpha-B-crystallin (CRYAB) gene have initially been associated with myofibrillar myopathy, dilated cardiomyopathy and cataracts. For the first time, peripheral neuropathy is reported here as a novel phenotype associated with CRYAB. METHODS: Whole-exome sequencing was performed in two unrelated families with genetically unsolved axonal Charcot-Marie-Tooth disease (CMT2), assessing clinical, neurophysiological and radiological features. RESULTS: The pathogenic CRYAB variant c.358A>G;p.Arg120Gly was segregated in all affected patients from two unrelated families. The disease presented as late onset CMT2 (onset over 40 years) with distal sensory and motor impairment and congenital cataracts. Muscle involvement was probably associated in cases showing mild axial and diaphragmatic weakness. In all cases, nerve conduction studies demonstrated the presence of an axonal sensorimotor neuropathy along with chronic neurogenic changes on needle examination. DISCUSSION: In cases with late onset autosomal dominant CMT2 and congenital cataracts, it is recommended that CRYAB is considered for genetic testing. The identification of CRYAB mutations causing CMT2 further supports a continuous spectrum of expressivity, from myopathic to neuropathic and mixed forms, of a growing number of genes involved in protein degradation and chaperone-assisted autophagy.

X-ray linear dichroic ptychography.

Biominerals such as seashells, coral skeletons, bone, and tooth enamel are optically anisotropic crystalline materials with unique nanoscale and microscale organization that translates into exceptional macroscopic mechanical properties, providing inspiration for engineering new and superior biomimetic structures. Using Seriatopora aculeata coral skeleton as a model, here, we experimentally demonstrate X-ray linear dichroic ptychography and map the c-axis orientations of the aragonite (CaCO3) crystals. Linear dichroic phase imaging at the oxygen K-edge energy shows strong polarization-dependent contrast and reveals the presence of both narrow (<35°) and wide (>35°) c-axis angular spread in the coral samples. These X-ray ptychography results are corroborated by four-dimensional (4D) scanning transmission electron microscopy (STEM) on the same samples. Evidence of co-oriented, but disconnected, corallite subdomains indicates jagged crystal boundaries consistent with formation by amorphous nanoparticle attachment. We expect that the combination of X-ray linear dichroic ptychography and 4D STEM could be an important multimodal tool to study nano-crystallites, interfaces, nucleation, and mineral growth of optically anisotropic materials at multiple length scales.

Cholesterol Content Regulates the Interaction of αA-, αB-, and α-Crystallin with the Model of Human Lens-Lipid Membranes.

α-Crystallin (αABc) is a major protein comprised of αA-crystallin (αAc) and αB-crystallin (αBc) that is found in the human eye lens and works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress. However, with age and cataract formation, the concentration of αABc in the eye lens cytoplasm decreases, with a corresponding increase in the membrane-bound αABc. This study uses the electron paramagnetic resonance (EPR) spin-labeling method to investigate the role of cholesterol (Chol) and Chol bilayer domains (CBDs) in the binding of αAc, αBc, and αABc to the Chol/model of human lens-lipid (Chol/MHLL) membranes. The maximum percentage of membrane surface occupied (MMSO) by αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trends: MMSO (αAc) > MMSO (αBc) ≈ MMSO (αABc), indicating that a higher amount of αAc binds to these membranes compared to αBc and αABc. However, with an increase in the Chol concentration in the Chol/MHLL membranes, the MMSO by αAc, αBc, and αABc decreases until it is completely diminished at a mixing ratio of 1.5. The Ka of αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trend: Ka (αBc) ≈ Ka (αABc) > Ka (αAc), but it was close to zero with the diminished binding at a Chol/MHLL mixing ratio of 1.5. The mobility near the membrane headgroup regions decreased with αAc, αBc, and αABc binding, and the Chol antagonized the capacity of the αAc, αBc, and αABc to decrease mobility near the headgroup regions. No significant change in membrane order near the headgroup regions was observed, with an increase in αAc, αBc, and αABc concentrations. Our results show that αAc, αBc, and αABc bind differently with Chol/MHLL membranes at mixing ratios of 0 and 0.5, decreasing the mobility and increasing hydrophobicity near the membrane headgroup region, likely forming the hydrophobic barrier for the passage of polar and ionic molecules, including antioxidants (glutathione), creating an oxidative environment inside the lens, leading to the development of cataracts. However, all binding was completely diminished at a mixing ratio of 1.5, indicating that high Chol and CBDs inhibit the binding of αAc, αBc, and αABc to membranes, preventing the formation of hydrophobic barriers and likely protecting against cataract formation.

Insights into the biochemical and biophysical mechanisms mediating the longevity of the transparent optics of the eye lens.

In the human eye, a transparent cornea and lens combine to form the "refracton" to focus images on the retina. This requires the refracton to have a high refractive index "n," mediated largely by extracellular collagen fibrils in the corneal stroma and the highly concentrated crystallin proteins in the cytoplasm of the lens fiber cells. Transparency is a result of short-range order in the spatial arrangement of corneal collagen fibrils and lens crystallins, generated in part by post-translational modifications (PTMs). However, while corneal collagen is remodeled continuously and replaced, lens crystallins are very long-lived and are not replaced and so accumulate PTMs over a lifetime. Eventually, a tipping point is reached when protein aggregation results in increased light scatter, inevitably leading to the iconic protein condensation-based disease, age-related cataract (ARC). Cataracts account for 50% of vision impairment worldwide, affecting far more people than other well-known protein aggregation-based diseases. However, because accumulation of crystallin PTMs begins before birth and long before ARC presents, we postulate that the lens protein PTMs contribute to a "cataractogenic load" that not only increases with age but also has protective effects on optical function by stabilizing lens crystallins until a tipping point is reached. In this review, we highlight decades of experimental findings that support the potential for PTMs to be protective during normal development. We hypothesize that ARC is preventable by protecting the biochemical and biophysical properties of lens proteins needed to maintain transparency, refraction, and optical function.

Copper Reductase Activity and Free Radical Chemistry by Cataract-Associated Human Lens γ-Crystallins.

Cataracts are caused by high-molecular-weight aggregates of human eye lens proteins that scatter light, causing lens opacity. Metal ions have emerged as important potential players in the etiology of cataract disease, as human lens γ-crystallins are susceptible to metal-induced aggregation. Here, the interaction of Cu2+ ions with γD-, γC-, and γS-crystallins, the three most abundant γ-crystallins in the lens, has been evaluated. Cu2+ ions induced non-amyloid aggregation in all three proteins. Solution turbidimetry, sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), circular dichroism, and differential scanning calorimetry showed that the mechanism for Cu-induced aggregation involves: (i) loss of ß-sheet structure in the N-terminal domain; (ii) decreased thermal and kinetic stability; (iii) formation of metal-bridged species; and (iv) formation of disulfide-bridged dimers. Isothermal titration calorimetry (ITC) revealed distinct Cu2+ binding affinities in the γ-crystallins. Electron paramagnetic resonance (EPR) revealed two distinct Cu2+ binding sites in each protein. Spin quantitation demonstrated the reduction of γ-crystallin-bound Cu2+ ions to Cu+ under aerobic conditions, while X-ray absorption spectroscopy (XAS) confirmed the presence of linear or trigonal Cu+ binding sites in γ-crystallins. Our EPR and XAS studies revealed that γ-crystallins' Cu2+ reductase activity yields a protein-based free radical that is likely a Tyr-based species in human γD-crystallin. This unique free radical chemistry carried out by distinct redox-active Cu sites in human lens γ-crystallins likely contributes to the mechanism of copper-induced aggregation. In the context of an aging human lens, γ-crystallins could act not only as structural proteins but also as key players for metal and redox homeostasis.

Novel cataract-causing variant c.177dupC in c-MAF regulates the expression of crystallin genes for cell apoptosis via a mitochondria-dependent pathway.

Congenital cataract (CC) is regarded as the most common hereditary ophthalmic disease in children. Mutations in CC-associated genes play important roles in CC formation, which provides the basis for molecular diagnosis and therapy. Among these CC-associated genes, v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (c-MAF) is considered an important transcription factor for eye and lens development. In this study, we recruited a three-generation Chinese Han family with CC. Gene sequencing revealed a novel duplication mutation in c-MAF (NM_005360.5: c.177dup) that caused frameshifting at residue 60 (p. M60fs) of c-MAF. Additionally, in the patient blood samples, the expression levels of related crystallin and noncrystallin genes confirmed that this novel duplication variant impaired the transactivation of c-MAF. Further functional analyses suggested that the c-MAF mutant induces the transcriptional inhibition of CRYAA and CRYGA and subsequently influences ME and G6PD expression levels, ultimately resulting in ROS generation and further leading to cell apoptosis via mitochondria-dependent pathways. In conclusion, we report a novel c-MAF heterozygous mutation that plays a vital role in CC formation in a Chinese family, broadening the genetic spectrum of CC.

Impact of α-crystallin protein loss on zebrafish lens development.

The α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataracts, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study, we used CRISPR gene editing to produce zebrafish lines with mutations in each of the three α-crystallin genes (cryaa, cryaba and cryabb) to prevent protein production. The absence of each α-crystallin protein was analyzed by mass spectrometry, and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larvae at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of αBa-crystallin produced no substantial lens defects. Our cryabb mutant produced a truncated αBb-crystallin protein and showed no substantial change in lens development. Mutation of each α-crystallin gene did not alter the mRNA levels of the remaining two, suggesting a lack of genetic compensation. These data suggest that αA-crystallin plays some role in lens development, but the range of phenotype severity in null mutants indicates its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels for these genes in lens epithelial and fiber cells through five days of development. Future experiments can explore the molecular mechanisms leading to lens defects in cryaa null mutants and the impact of αA-crystallin loss during zebrafish lens aging.

Optimization of aqueous extraction of antioxidants from Chrysanthemum (C. morifolium Ramat and C. indicum L.) flowers and evaluation of their protection from glycoxidation damage on human αA-crystallin.

Chrysanthemum tea is commonly consumed by Chinese consumers mainly due to the Chrysanthemum flower being a potential source of antioxidants. The current study investigates the effects of extraction time and temperature on Chrysanthemum flower aqueous extract (CFAE) antioxidant capacity, including Trolox equivalent antioxidant capacity (TEAC), ferrous iron-chelating activity, and superoxide radical scavenging capacity (SRSC) using a two-factor, three-level factorial design of the response surface method (RSM). The TEAC and SRSC of CFAE are higher at higher temperatures and longer times up to a certain point, and the highest TEAC and SRSC are achieved at a 100 °C extraction temperature for 45 min. The fructose induced-αA-crystallin (Cry) glycation model system was used to evaluate the effects of the CFAE on anti-glycoxidation activities. The antioxidant ingredients obtained from CFAE significantly impede the production of advanced glycation end products from protein glycoxidation products (dityrosine, kynurenine, and N'-methylkynurenine) in the glycation process of αA-Cry and exhibit strong anti-glycating activity. The glycation inhibitory effects of CFAE are concentration-dependent. C. indicum L. exhibits greater potential for preventing cataracts compared to C. morifolium Ramat CFAE's antioxidant and anti-glycation properties suggest its potential application as a natural ingredient in the development of agents to combat glycation.

The Relationship between Protein-Protein Interactions and Liquid-Liquid Phase Separation for Monoclonal Antibodies.

Being able to predict and control concentrated solution properties for solutions of monoclonal antibodies (mAbs) is critical for developing therapeutic formulations. At higher protein concentrations, undesirable solution properties include high viscosities, opalescence, particle formation, and precipitation. The overall aim of this work is to understand the relationship between commonly measured dilute solution parameters, the reduced osmotic second virial coefficient b22 and the diffusion interaction parameter kD and liquid-liquid phase separation, which occurs at higher protein concentrations. For globular proteins such as lysozyme or γB-crystallin, the location of the liquid-liquid coexistence curve is controlled by the net protein-protein interaction, which is related to b22. Because many mAbs undergo reversible self-association due to forming highly directional interactions, it is not known if b22 can be used as a reliable predictor for LLPS since increasing the anisotropy in the interaction potential causes phase separation to occur at much stonger net protein-protein attractions or lower values of b22. Here, we map the coexistence curves for three mAbs, referred to as COE-01, COE-07, and COE-19, in terms of b22 and kD values. The measurements are carried out at a low salt condition near the pI, where protein-protein interactions are expected to be anisotropic due to the presence of electrostatic attractions, and under salting-out conditions at high ammonium sulfate concentrations, which is expected to reduce the anisotropy by screening electrostatic interactions. We also show that deviations from a linear correlation between b22 and kD can be used as an indicator of reversible self-association. Each of the mAbs under salting-out conditions follows the correlation supporting the hypothesis that protein-protein interactions are nonspecific, while deviations from the correlation occur for COE-01 and COE-19 under low salt conditions indicating the mAbs undergo reversible self-association. For five out of the six conditions, the onset of phase separation, as reflected by the reduced virial coefficient at the critical point b22c occurs in a small window -1.6 > b22c > -2.3, which is similar to what has been observed for lysozyme and for bovine γB-crystallin. Under low salt conditions, b22c ≈ -5.1 for COE-19, which we previously showed to self-associate into small oligomers. Our findings suggest that under conditions where mAb interactions are weakly anisotropic, such as occur at high salt conditions, phase separation will begin to occur in a small window of b22. Deviations from the window can occur when mAbs undergo reversible self-association, although this is not always the case and likely depends upon whether or not highly directional interactions are passivated in the oligomer formation. We expect fitting LLPS measurements to simplified interaction models for mAbs will provide additional insight into the nature of the protein-protein interactions and guide their development for calculating concentrated solution properties.

Mimicking effects of cholesterol in lipid bilayer membranes by self-assembled amphiphilic block copolymers.

The effect of cholesterol on biological membranes is important in biochemistry. In this study, a polymer system is used to simulate the consequences of varying cholesterol content in membranes. The system consists of an AB-diblock copolymer, a hydrophilic homopolymer hA, and a hydrophobic rigid homopolymer C, corresponding to phospholipid, water, and cholesterol, respectively. The effect of the C-polymer content on the membrane is studied within the framework of a self-consistent field model. The results show that the liquid-crystal behavior of B and C has a great influence on the chemical potential of cholesterol in bilayer membranes. The effects of the interaction strength between components, characterized by the Flory-Huggins parameters and the Maier-Saupe parameter, were studied. Some consequences of adding a coil headgroup to the C-rod are presented. Results of our model are compared to experimental findings for cholesterol-containing lipid bilayer membranes.

ATCUN-like Copper Site in ßB2-Crystallin Plays a Protective Role in Cataract-Associated Aggregation.

Cataract is the leading cause of blindness worldwide, and it is caused by crystallin damage and aggregation. Senile cataractous lenses have relatively high levels of metals, while some metal ions can directly induce the aggregation of human γ-crystallins. Here, we evaluated the impact of divalent metal ions in the aggregation of human ßB2-crystallin, one of the most abundant crystallins in the lens. Turbidity assays showed that Pb2+, Hg2+, Cu2+, and Zn2+ ions induce the aggregation of ßB2-crystallin. Metal-induced aggregation is partially reverted by a chelating agent, indicating the formation of metal-bridged species. Our study focused on the mechanism of copper-induced aggregation of ßB2-crystallin, finding that it involves metal-bridging, disulfide-bridging, and loss of protein stability. Circular dichroism and electron paramagnetic resonance (EPR) revealed the presence of at least three Cu2+ binding sites in ßB2-crystallin, one of them with spectroscopic features typical for Cu2+ bound to an amino-terminal copper and nickel (ATCUN) binding motif, which is found in Cu transport proteins. The ATCUN-like Cu binding site is located at the unstructured N-terminus of ßB2-crystallin, and it could be modeled by a peptide with the first six residues in the protein sequence (NH2-ASDHQF-). Isothermal titration calorimetry indicates a nanomolar Cu2+ binding affinity for the ATCUN-like site. An N-truncated form of ßB2-crystallin is more susceptible to Cu-induced aggregation and is less thermally stable, indicating a protective role for the ATCUN-like site. EPR and X-ray absorption spectroscopy studies reveal the presence of a copper redox active site in ßB2-crystallin that is associated with metal-induced aggregation and formation of disulfide-bridged oligomers. Our study demonstrates metal-induced aggregation of ßB2-crystallin and the presence of putative copper binding sites in the protein. Whether the copper-transport ATCUN-like site in ßB2-crystallin plays a functional/protective role or constitutes a vestige from its evolution as a lens structural protein remains to be elucidated.

Butein Inhibits the Glycation of α-Crystallin: An Approach in Prevention of Retinopathy.

The aggregation of lens proteins induced by glycation is one of the key drivers of diabetic retinopathy and development of diabetic cataracts. Moreover, glycation also causes numerous alterations not only to the tertiary structure of lens proteins but also to serum proteins. There are also evidences of covalent crosslinking among lens crystallins resulting in development of cataract. In this article, the inhibitory potential of butein was tested against the glucose induced glycation and the aggregation α-crystallin (α-cry). The results showed that there was inhibition of advanced glycation products (78.28%) and early glycation products (86.30%) following the treatment of butein. Additionally, the presence of butein caused a significant improvement in the tested biochemical markers of glycation. The treatment with butein reduced the free lysine modification to 23.67%. The secondary and tertiary structural distortions of α-cry were also protected. The mechanism of inhibition further investigated at the molecular level using biophysical and computational techniques. The interaction data showed the butein exhibited strong affinity towards the α-cry. The binding event was entropically driven and energetically favourable. The Gibb's free energy of the interaction was found to be -5.99 to -7.17 kcal mol-1. The binding site of butein in α-cry was deciphered by molecular docking and the dynamics was studied using molecular dynamics (MD) simulations. The simulation data showed that butein formed stable complex with α-cry under physiological conditions. Most of the tested parameters from molecular simulations, such as secondary structure, was found to be stable. The data clearly show the potential of butein in inhibiting the glycation induced aggregation of α-cry and hence can be developed as useful inhibitor in the management of diabetic cataract and retinopathy.

Mutational analysis of CRYAA gene of cataract and investigating risk assessment factors responsible for eye diseases in district buner, KPK, Pakistan.

This research has been designed to analyze the risk factors of major eye diseases and the genetic alterations contributing to the manifestation of such disease. For this purpose, data was collected from 256 patients diagnosed by an ophthalmologist by using a specialized questionnaire. Blood samples were collected from 100 patients to perform a genetic investigation of cataracts. Whole genomic DNA was extracted from blood samples via the phenol-chloroform method. The purified DNA was used as the template for the amplification of about 400 bp fragments amplifying exons 1 and 2 of the CRYAA gene. The statistical analysis showed that 68% of individuals were blind due to cataracts. During molecular analysis, nucleotide sequences obtained have resulted in one silent mutation that occured at 20 positions in exon 2. It was replacing A>G which in turn substitutes the Lysine at position 70 for Arginine. It was interpreted by statistical analysis that this mutation did not result in a significant change in the CRYAA gene. In addition, protein analysis showed no significant changes in the structure of normal and mutated genes. At last, it is concluded that environmental risk factors play a major role in the studied diseases as compared to genetic factors. It is recommended to extend the study to a larger population to study all exons of the CRYAA gene as well as develop better estimates of the magnitude of the problems of visual loss and eye diseases in the Pakistani population.

Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation.

Lens transparency is established by abundant accumulation of crystallin proteins and loss of organelles in the fiber cells. It requires an efficient translation of lens messenger RNAs (mRNAs) to overcome the progressively reduced transcriptional activity that results from denucleation. Inappropriate regulation of this process impairs lens differentiation and causes cataract formation. However, the regulatory mechanism promoting protein synthesis from lens-expressed mRNAs remains unclear. Here we show that in zebrafish, the RNA-binding protein Rbm24 is critically required for the accumulation of crystallin proteins and terminal differentiation of lens fiber cells. In the developing lens, Rbm24 binds to a wide spectrum of lens-specific mRNAs through the RNA recognition motif and interacts with cytoplasmic polyadenylation element-binding protein (Cpeb1b) and cytoplasmic poly(A)-binding protein (Pabpc1l) through the C-terminal region. Loss of Rbm24 reduces the stability of a subset of lens mRNAs encoding heat shock proteins and shortens the poly(A) tail length of crystallin mRNAs encoding lens structural components, thereby preventing their translation into functional proteins. This severely impairs lens transparency and results in blindness. Consistent with its highly conserved expression in differentiating lens fiber cells, the findings suggest that vertebrate Rbm24 represents a key regulator of cytoplasmic polyadenylation and plays an essential role in the posttranscriptional control of lens development.

Refolding Increases the Chaperone-like Activity of αH-Crystallin and Reduces Its Hydrodynamic Diameter to That of α-Crystallin.

αH-Crystallin, a high molecular weight form of α-crystallin, is one of the major proteins in the lens nucleus. This high molecular weight aggregate (HMWA) plays an important role in the pathogenesis of cataracts. We have shown that the chaperone-like activity of HMWA is 40% of that of α-crystallin from the lens cortex. Refolding with urea significantly increased-up to 260%-the chaperone-like activity of α-crystallin and slightly reduced its hydrodynamic diameter (Dh). HMWA refolding resulted in an increase in chaperone-like activity up to 120% and a significant reduction of Dh of protein particles compared with that of α-crystallin. It was shown that the chaperone-like activity of HMWA, α-crystallin, and refolded α-crystallin but not refolded HMWA was strongly correlated with the denaturation enthalpy measured with differential scanning calorimetry (DSC). The DSC data demonstrated a significant increase in the native protein portion of refolded α-crystallin in comparison with authentic α-crystallin; however, the denaturation enthalpy of refolded HMWA was significantly decreased in comparison with authentic HMWA. The authors suggested that the increase in the chaperone-like activity of both α-crystallin and HMWA could be the result of the correction of misfolded proteins during renaturation and the rearrangement of protein supramolecular structures.