Towards the Identification and Characterization of Putative Adult Human Lens Epithelial Stem Cells.

The anterior lens epithelium has the ability to differentiate into lens fibres throughout its life. The present study aims to identify and functionally characterize the adult stem cells in the human lens epithelium. Whole mounts of lens epithelium from donor eyes (normal/cataract) were immunostained for SOX2, gap junction protein alpha 1 (GJA1), PAX6, α, ß and γ-crystallins, followed by a confocal analysis. The functional property of adult stem cells was analysed by their sphere forming ability using cultured lens epithelial cells from different zones. Based on marker expression, the lens epithelium was divided into four zones: the central zone, characterized by a small population of PAX6+, GJA1-, ß-crystallin- and γ-crystallin- cells; the germinative zone, characterized by PAX6+, GJA1+, ß-crystallin- and γ-crystallin-; the transitional zone, characterized by PAX6+, GJA1+, ß-crystallin+ and γ-crystallin-; and the equatorial zone, characterized by PAX6+/-, GJA1+, ß-crystallin+, and γ-crystallin+ cells. The putative lens epithelial stem cells identified as SOX2+ and GJA1 membrane expression negative cells were located only in the central zone (1.89 ± 0.84%). Compared to the other zones, a significant percentage of spheres were identified in the central zone (1.68 ± 1.04%), consistent with the location of the putative adult lens epithelial stem cells. In the cataractous lens, an absence of SOX2 expression and a significant reduction in sphere forming ability (0.33 ± 0.11%) were observed in the central zone. The above findings confirmed the presence of putative stem cells in the central zone of the adult human lens epithelium and indicated their probable association with cataract development.

FGF-2 Differentially Regulates Lens Epithelial Cell Behaviour during TGF-ß-Induced EMT.

Fibroblast growth factor (FGF) and transforming growth factor-beta (TGF-ß) can regulate and/or dysregulate lens epithelial cell (LEC) behaviour, including proliferation, fibre differentiation, and epithelial-mesenchymal transition (EMT). Earlier studies have investigated the crosstalk between FGF and TGF-ß in dictating lens cell fate, that appears to be dose dependent. Here, we tested the hypothesis that a fibre-differentiating dose of FGF differentially regulates the behaviour of lens epithelial cells undergoing TGF-ß-induced EMT. Postnatal 21-day-old rat lens epithelial explants were treated with a fibre-differentiating dose of FGF-2 (200 ng/mL) and/or TGF-ß2 (50 pg/mL) over a 7-day culture period. We compared central LECs (CLECs) and peripheral LECs (PLECs) using immunolabelling for changes in markers for EMT (α-SMA), lens fibre differentiation (ß-crystallin), epithelial cell adhesion (ß-catenin), and the cytoskeleton (alpha-tropomyosin), as well as Smad2/3- and MAPK/ERK1/2-signalling. Lens epithelial explants cotreated with FGF-2 and TGF-ß2 exhibited a differential response, with CLECs undergoing EMT while PLECs favoured more of a lens fibre differentiation response, compared to the TGF-ß-only-treated explants where all cells in the explants underwent EMT. The CLECs cotreated with FGF and TGF-ß immunolabelled for α-SMA, with minimal ß-crystallin, whereas the PLECs demonstrated strong ß-crystallin reactivity and little α-SMA. Interestingly, compared to the TGF-ß-only-treated explants, α-SMA was significantly decreased in the CLECs cotreated with FGF/TGF-ß. Smad-dependent and independent signalling was increased in the FGF-2/TGF-ß2 co-treated CLECs, that had a heightened number of cells with nuclear localisation of Smad2/3 compared to the PLECs, that in contrast had more pronounced ERK1/2-signalling over Smad2/3 activation. The current study has confirmed that FGF-2 is influential in differentially regulating the behaviour of LECs during TGF-ß-induced EMT, leading to a heterogenous cell population, typical of that observed in the development of post-surgical, posterior capsular opacification (PCO). This highlights the cooperative relationship between FGF and TGF-ß leading to lens pathology, providing a different perspective when considering preventative measures for controlling PCO.

Conformational stability of the deamidated and mutated human ßB2-crystallin.

Previous studies propose that genetic mutations and post-translational modifications in protein crystallins promote protein aggregation and are considered significant risk factors for cataract formation. The ßB2-crystallin (HßB2C) forms a high proportion of proteins in the human eye lens. Different congenital mutations and post-translational deamidations in ßB2-crystallin have been reported and linked to cataract formation. In this work, we employed extensive all-atom molecular dynamics simulations to evaluate the conformational stability of deamidated and mutated HßB2C. Our results show critical changes in the protein surface and its native contacts due to a modification in the conformational equilibrium of these proteins. The double deamidated (Q70E/Q162E) and single deamidated (Q70E) impact the well compact conformation of the HßB2C. These post-translational modifications allow the exposure of the protein hydrophobic interface, which lead to the exposure of electronegative residues. On the other hand, our mutational studies showed that the S143F mutation modifies the hydrogen-bond network of an antiparallel ß-sheet, unfolding the C-terminal domain. Interestingly, the chain termination mutation (Q155X) does not unfold the N-terminal domain. However, the resultant conformation is more compact and avoids the exposure of the hydrophobic interface. Our results provide valuable information about the first steps of HßB2C unfolding in the presence of deamidated amino acids that have been reported to appear during aging. The findings reported in this work are essential for the general knowledge of the initial steps in the cataract formation mechanism, which may be helpful for the further development of molecules with pharmacological potential against cataract disease.

Eye lens crystallin proteins inhibit the autocatalytic amyloid amplification nature of mature α-synuclein fibrils.

Parkinson´s disease is characterized by the accumulation of proteinaceous aggregates in Lewy bodies and Lewy Neurites. The main component found in such aggregates is α-synuclein. Here, we investigate how bovine eye lens crystallin proteins influence the aggregation kinetics of α-synuclein at mildly acidic pH (5.5) where the underlying aggregation mechanism of this protein is dominated by secondary nucleation of monomers on fibril surface providing an autocatalytic amyloid amplification process. Bovine α-, ßH- and γB-crystallins were found to display chaperone-like activity inhibiting α-synuclein aggregation. This effect was shown to be time-dependent, with early additions of α-crystallin capable of retarding and even inhibiting aggregation during the time frame of the experiment. The inhibitory nature of crystallins was further investigated using trap and seed kinetic experiments. We propose crystallins interact with mature α-synuclein fibrils, possibly binding along the surfaces and at fibril free ends, inhibiting both elongation and monomer-dependent secondary nucleation processes in a mechanism that may be generic to some chaperones that prevent the onset of protein misfolding related pathologies.

PNU282987 inhibits amyloid­ß aggregation by upregulating astrocytic endogenous αB­crystallin and HSP­70 via regulation of the α7AChR, PI3K/Akt/HSF­1 signaling axis.

Alzheimer's disease (AD) is a chronic and irreversible neurodegenerative disorder. Abnormal aggregation of the neurotoxic amyloid­ß (Aß) peptide is an early event in AD. The activation of astrocytic α7 nicotinic acetylcholine receptor (α7 nAChR) can inhibit Aß aggregation; thus, the molecular mechanism between α7 nAChR activation and Aß aggregation warrants further investigation. In the present study, Aß oligomer levels were assessed in astrocytic cell lysates after treatment with PNU282987 (a potent agonist of α7 nAChRs) or co­treatment with LY294002, a p­Akt inhibitor. The levels of heat shock factor­1 (HSF­1), heat shock protein 70 (HSP­70), and αB­crystallin (Cryab) in astrocytes treated with PNU282987 at various time­points or co­treated with methyllycaconitine (MLA), a selective α7 nAChR antagonist, as well as co­incubated with LY294002 were determined by western blotting. HSP­70 and Cryab levels were determined after HSF­1 knockdown (KD) in astrocytes. PNU282987 markedly inhibited Aß aggregation and upregulated HSF­1, Cryab, and HSP­70 in primary astrocytes, while the PNU282987­mediated neuroprotective effect was reversed by pre­treatment with MLA or LY294002. Moreover, the HSF­1 KD in astrocytes effectively decreased Cryab, but not HSP­70 expression. HSF­1 is necessary for the upregulation of Cryab expression, but not for that of HSP­70. HSF­1 and HSP­70 have a neuroprotective effect. Furthermore, the neuroprotective effect of PNU282987 against Aß aggregation was mediated by the canonical PI3K/Akt signaling pathway activation.

Aggregation of ß-crystallin through covalent binding to 1,2-naphthoquinone is rescued by α-crystallin chaperone.

Cataract induced by exposure to naphthalene is thought to mainly involve its metabolic activation, forming 1,2-naphthoquinone (1,2-NQ), which can modify proteins through chemical modifications. In the present study, we examined the effect of 1,2-NQ on aggregation of crystallins (cry) associated with cataract. Incubation of bovine ß-cry with 1,2-NQ caused covalent modification of ß-cry at Cys117 and Lys125 accompanied by reduction in its thiol content, resulting in a concentration- and temperature-dependent aggregation of ß-cry, whereas only little aggregation of α-cry induced by 1,2-NQ was seen. Interestingly, addition of α-cry to the reaction mixture of ß-cry and 1,2-NQ markedly blocked ß-cry aggregation induced by 1,2-NQ in a concentration-dependent manner. These results suggest that ß-cry predominantly undergoes chemical modification by 1,2-NQ, causing its aggregation, which is suppressed by the chaperone-like protein, α-cry. This ß-cry aggregation may be, at least in part, involved in the induction of cataract caused by 1,2-NQ.

MALDI imaging mass spectrometry of ß- and γ-crystallins in the ocular lens.

Lens crystallin proteins make up 90% of expressed proteins in the ocular lens and are primarily responsible for maintaining lens transparency and establishing the gradient of refractive index necessary for proper focusing of images onto the retina. Age-related modifications to lens crystallins have been linked to insolubilization and cataractogenesis in human lenses. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been shown to provide spatial maps of such age-related modifications. Previous work demonstrated that, under standard protein IMS conditions, α-crystallin signals dominated the mass spectrum and age-related modifications to α-crystallins could be mapped. In the current study, a new sample preparation method was optimized to allow imaging of ß- and γ-crystallins in ocular lens tissue. Acquired images showed that γ-crystallins were localized predominately in the lens nucleus whereas ß-crystallins were primarily localized to the lens cortex. Age-related modifications such as truncation, acetylation, and carbamylation were identified and spatially mapped. Protein identifications were determined by top-down proteomics analysis of lens proteins extracted from tissue sections and analyzed by LC-MS/MS with electron transfer dissociation. This new sample preparation method combined with the standard method allows the major lens crystallins to be mapped by MALDI IMS.

Divalent Cations and the Divergence of ßγ-Crystallin Function.

The ßγ-crystallin superfamily contains both ß- and γ-crystallins of the vertebrate eye lens and the microbial calcium-binding proteins, all of which are characterized by a common double-Greek key domain structure. The vertebrate ßγ-crystallins are long-lived structural proteins that refract light onto the retina. In contrast, the microbial ßγ-crystallins bind calcium ions. The ßγ-crystallin from the tunicate Ciona intestinalis (Ci-ßγ) provides a potential link between these two functions. It binds calcium with high affinity and is found in a light-sensitive sensory organ that is highly enriched in metal ions. Thus, Ci-ßγ is valuable for investigating the evolution of the ßγ-crystallin fold away from calcium binding and toward stability in the apo form as part of the vertebrate lens. Here, we investigate the effect of Ca2+ and other divalent cations on the stability and aggregation propensity of Ci-ßγ and human γS-crystallin (HγS). Beyond Ca2+, Ci-ßγ is capable of coordinating Mg2+, Sr2+, Co2+, Mn2+, Ni2+, and Zn2+, although only Sr2+ is bound with comparable affinity to its preferred metal ion. The extent to which the tested divalent cations stabilize Ci-ßγ structure correlates strongly with ionic radius. In contrast, none of the tested divalent cations improved the stability of HγS, and some of them induced aggregation. Zn2+, Ni2+, and Co2+ induce aggregation by interacting with cysteine residues, whereas Cu2+-mediated aggregation proceeds via a different binding site.

ßγ-Crystallination Endows a Novel Bacterial Glycoside Hydrolase 64 with Ca2+-Dependent Activity Modulation.

The prokaryotic ßγ-crystallins are a large group of uncharacterized domains with Ca2+-binding motifs. We have observed that a vast number of these domains are found appended to other domains, in particular, the carbohydrate-active enzyme (CAZy) domains. To elucidate the functional significance of these prospective Ca2+ sensors in bacteria and this widespread domain association, we have studied one typical example from Clostridium beijerinckii, a bacterium known for its ability to produce acetone, butanol, and ethanol through fermentation of several carbohydrates. This novel glycoside hydrolase of family 64 (GH64), which we named glucanallin, is composed of a ßγ-crystallin domain, a GH64 domain, and a carbohydrate-binding module 56 (CBM56). The substrates of GH64, ß-1,3-glucans, are the targets for industrial biofuel production due to their plenitude. We have examined the Ca2+-binding properties of this protein, assayed its enzymatic activity, and analyzed the structural features of the ß-1,3-glucanase domain through its high-resolution crystal structure. The reaction products resulting from the enzyme reaction of glucanallin reinforce the mixed nature of GH64 enzymes, in contrast to the prevailing notion of them being an exotype. Upon disabling Ca2+ binding and comparing different domain combinations, we demonstrate that the ßγ-crystallin domain in glucanallin acts as a Ca2+ sensor and enhances the glycolytic activity of glucanallin through Ca2+ binding. We also compare the structural peculiarities of this new member of the GH64 family to two previously studied members.IMPORTANCE We have biochemically and structurally characterized a novel glucanase from the less studied GH64 family in a bacterium significant for fermentation of carbohydrates into biofuels. This enzyme displays a peculiar property of being distally modulated by Ca2+ via assistance from a neighboring ßγ-crystallin domain, likely through changes in the domain interface. In addition, this enzyme is found to be optimized for functioning in an acidic environment, which is in line with the possibility of its involvement in biofuel production. Multiple occurrences of a similar domain architecture suggest that such a "ßγ-crystallination"-mediated Ca2+ sensitivity may be widespread among bacterial proteins.

The mechanism of the interaction of α-crystallin and UV-damaged ßL-crystallin.

α-Crystallin maintains the transparency of the lens by preventing the aggregation of damaged proteins. The aim of our work was to study the chaperone-like activity of native α-crystallin in near physiological conditions (temperature, ionic power, pH) using UV-damaged ßL-crystallin as the target protein. α-Crystallin in concentration depended manner inhibits the aggregation of UV-damaged ßL-crystallin. DSC investigation has shown that refolding of denatured UV-damaged ßL-crystallin was not observed under incubation with α-crystallin. α-Crystallin and UV-damaged ßL-crystallin form dynamic complexes with masses from 75 to several thousand kDa. The content of UV-damaged ßL-crystallin in such complexes increases with the mass of the complex. Complexes containing >10% of UV-damaged ßL-crystallin are prone to precipitation whereas those containing <10% of the target protein are relatively stable. Formation of a stable 75 kDa complex is indicative of α-crystallin dissociation. We suppose that α-crystallin dissociation is the result of an interaction of comparable amounts of the chaperone-like protein and the target protein. In the lens simultaneous damage of such amounts of protein, mainly ß and gamma-crystallins, is impossible. The authors suggest that in the lens rare molecules of the damaged protein interact with undissociated oligomers of α-crystallin, and thus preventing aggregation.

Polyphenol-enriched fraction of Vaccinium uliginosum L. protects selenite-induced cataract formation in the lens of Sprague-Dawley rat pups.

Purpose: As the aging population is increasing, the incidence of age-related cataract is expected to increase globally. The surgical intervention, a treatment for cataract, still has complications and is limited to developed countries. In this study, we investigated whether the polyphenol-enriched fraction of Vaccinium uliginosum L. (FH) prevents cataract formation in Sprague-Dawley (SD) rat pups. Methods: Sixty rat pups were randomly divided into six groups: CTL, Se, FH40, FH80, FH120, and Cur80. The cataract was induced with subcutaneous injection of sodium selenite (18 µmol/kg bodyweight) on postnatal (P) day 10. All groups, except CTL, were injected with sodium selenite, and the FH40, FH80, and FH120 groups were given gastric intubation with FH40 mg/kg, 80 mg/kg, and 120 mg/kg on P9, P10, and P11. The Cur80 group was also given gastric intubation with curcumin 80 mg/kg on P9, P10, and P11. All rat pups were euthanized on P30. Results: Lens morphological analysis showed that FH dose-dependently inhibited cataract formation. In the Se group, soluble proteins were insolubilized, and the gene expression of the α-, ß-, and γ-crystallins was downregulated. However, FH treatment statistically significantly inhibited insolubilization of soluble proteins and downregulation of the gene expression of the α-, ß-, and γ-crystallins. In the Se group, the gene and protein levels of m-calpain were downregulated, which were attenuated with FH treatment. In addition, sodium selenite injection caused reduced antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GPx)), glutathione (GSH) depletion, and malondialdehyde (MDA) production in the lens. The administration of FH inhibited sodium selenite-induced oxidative stress in a dose-dependent manner. The mechanism of protection against oxidative stress by FH involves NF-E2-related factor (Nrf-2) and hemoxygenase-1 (HO-1). FH treatment inhibited decrease of Nrf-2 in the nucleus fraction and HO-1 in the cytosol fraction. Finally, the FH treatment protected poly (ADP)-ribose polymerase (PARP) from cleavage, determined with western blotting. Conclusions: FH showed a preventive effect against cataract formation by inhibiting m-calpain-mediated proteolysis and oxidative stress in the lens. These results suggest that FH could be a potential anticataract agent in age-related cataract.

TREK­TRAAK two­pore domain potassium channels protect human retinal pigment epithelium cells from oxidative stress.

The aim of the current study was to explore the potential of TREK­TRAAK two­pore domain potassium (K2P) channels in protecting human retinal pigment epithelium (hRPE) cells against oxidative stress. hRPE cells were obtained from donors, and then cell identification and detection of the expression levels of TREK­TRAAK K2P channels in hRPE cells were conducted. Subsequently, tert­butyl hydroperoxide (t­BH) was used to induce oxidative stress in hRPE cells. Docosahexaenoic acid (DHA) was used to stimulate and fluoxetine was used to inhibit the TREK­TRAAK K2P channels. The survival rates of hRPE cells under oxidative stress were examined using flow cytometry. Apoptosis­associated factors, including Bax, Bcl­2, cleaved­caspase­3, αB­crystallin and their mRNAs, were examined using immunofluorescence, western blot and reverse transcription­polymerase chain reaction analyses. Variations in the cytoarchitecture were observed by immunofluorescence and electron microscopy. The cells examined in the present study were identified as hRPE cells. All members in the TREK­TRAAK K2P channel family (including TREK­1, TREK­2 and TRAAK) were found to be expressed in hRPE cells. Stimulation of TREK­TRAAK K2P channels increased the survival rates of hRPE cells under oxidative stress and the levels of intracellular protective factors, such as Bcl­2 and αB­crystallin. By contrast, inhibition of these channels decreased the cell survival rates and increased apoptosis enhancing factors, such as Bax and cleaved­caspase­3. Further examination of the cytoarchitecture revealed that TREK­TRAAK K2P channels protected the integrity of the hRPE cell structure against oxidative stress. In conclusion, the present study suggested that the activated TREK­TRAAK K2P channels serve a role in protecting hRPE cells against the oxidative stress induced by t­BH, which indicated that these K2P channels are potential novel targets in retinal protection and provided a new direction for research and therapy in retinal degeneration diseases.

Asp 58 modulates lens αA-crystallin oligomer formation and chaperone function.

Senile cataract onset is caused by insolubilization of lens proteins. The lens crystallin protein family correctly orders the formation of homo- or hetero-oligomers in lens fiber cells. Because lens fiber cells do not divide, covalent post-translational modifications, such as isomerization of aspartate residues, accumulate with aging. Although many isomerization sites of αA-crystallin have been reported, their structural and functional contributions have never been identified. In this study, αA-crystallin was extracted from aged human lens and separated into each oligomeric state by size exclusion chromatography and electrophoresis. The novel combination methodology of in-solution/gel tryptic digestion with liquid chromatography equipped with mass spectrometry (LC-MS/MS) was used to evaluate the isomerization of Asp 58. The contributions of isomerization to assembly, solubility, and chaperone functions of αA-crystallin were estimated using a series of mutations of Asp 58 in αA-crystallin. The results indicated that the isomerization of Asp 58 depended on the oligomer size and age of the lens. The substitution of Asp 58 for hydrophobic residues increased αA-crystallin oligomer size and decreased solubility. All substitutions decreased the chaperone function of αA-crystallin for aggregates of bovine ßL-crystallin and alcohol dehydrogenase. The data indicated that Asp 58 in αA-crystallin was critical for intermolecular interactions in the lens. Our results also suggested that LC-MS/MS-based isomerization analyses of in-gel-digested products could be useful for investigating the isomerization of Asp residues in oligomeric states. This method could also be used to analyze d/l ratios of amino acid residues in soluble protein aggregates.

Crim1 is required for maintenance of the ocular lens epithelium.

The development and growth of the vertebrate ocular lens is dependent on the regulated proliferation of an anterior monolayer of epithelial cells, and their subsequent differentiation into elongate fiber cells. The growth factor rich ocular media that bathes the lens mediates these cellular processes, and their respective intracellular signaling pathways are in turn regulated to ensure that the proper lens architecture is maintained. Recent studies have proposed that Cysteine Rich Motor Neuron 1 (Crim1), a transmembrane protein involved in organogenesis of many tissues, might influence cell adhesion, polarity and proliferation in the lens by regulating integrin-signaling. Here, we characterise the lens and eyes of the Crim1KST264 mutant mice, and show that the loss of Crim1 function in the ocular tissues results in inappropriate differentiation of the lens epithelium into fiber cells. Furthermore, restoration of Crim1 levels in just the lens tissue of Crim1KST264 mice is sufficient to ameliorate most of the dysgenesis observed in the mutant animals. Based on our findings, we propose that tight regulation of Crim1 activity is required for maintenance of the lens epithelium, and its depletion leads to ectopic differentiation into fiber cells, dramatically altering lens structure and ultimately leading to microphthalmia and aphakia.

A role for Hippo/YAP-signaling in FGF-induced lens epithelial cell proliferation and fibre differentiation.

Recent studies indicate an important role for the transcriptional co-activator Yes-associated protein (YAP), and its regulatory pathway Hippo, in controlling cell growth and fate during lens development; however, the exogenous factors that promote this pathway are yet to be identified. Given that fibroblast growth factor (FGF)-signaling is an established regulator of lens cell behavior, the current study investigates the relationship between this pathway and Hippo/YAP-signaling during lens cell proliferation and fibre differentiation. Rat lens epithelial explants were cultured with FGF2 to induce epithelial cell proliferation or fibre differentiation. Immunolabeling methods were used to detect the expression of Hippo-signaling components, Total and Phosphorylated YAP, as well as fibre cell markers, Prox-1 and ß-crystallin. FGF-induced lens cell proliferation was associated with a strong nuclear localisation of Total-YAP and low-level immuno-staining for phosphorylated-YAP. FGF-induced lens fibre differentiation was associated with a significant increase in cytoplasmic phosphorylated YAP (inactive state) and enhanced expression of core Hippo-signaling components. Inhibition of YAP with Verteporfin suppressed FGF-induced lens cell proliferation and ablated cell elongation during lens fibre differentiation. Inhibition of either FGFR- or MEK/ERK-signaling suppressed FGF-promoted YAP nuclear translocation. Here we propose that FGF promotes Hippo/YAP-signaling during lens cell proliferation and differentiation, with FGF-induced nuclear-YAP expression playing an essential role in promoting the proliferation of lens epithelial cells. An FGF-induced switch from proliferation to differentiation, hence regulation of lens growth, may play a key role in mediating Hippo suppression of YAP transcriptional activity.

Transcriptome Analysis and Differential Gene Expression on the Testis of Orange Mud Crab, Scylla olivacea, during Sexual Maturation.

Adequate genetic information is essential for sustainable crustacean fisheries and aquaculture management. The commercially important orange mud crab, Scylla olivacea, is prevalent in Southeast Asia region and is highly sought after. Although it is a suitable aquaculture candidate, full domestication of this species is hampered by the lack of knowledge about the sexual maturation process and the molecular mechanisms behind it, especially in males. To date, data on its whole genome is yet to be reported for S. olivacea. The available transcriptome data published previously on this species focus primarily on females and the role of central nervous system in reproductive development. De novo transcriptome sequencing for the testes of S. olivacea from immature, maturing and mature stages were performed. A total of approximately 144 million high-quality reads were generated and de novo assembled into 160,569 transcripts with a total length of 142.2 Mb. Approximately 15-23% of the total assembled transcripts were annotated when compared to public protein sequence databases (i.e. UniProt database, Interpro database, Pfam database and Drosophila melanogaster protein database), and GO-categorised with GO Ontology terms. A total of 156,181 high-quality Single-Nucleotide Polymorphisms (SNPs) were mined from the transcriptome data of present study. Transcriptome comparison among the testes of different maturation stages revealed one gene (beta crystallin like gene) with the most significant differential expression-up-regulated in immature stage and down-regulated in maturing and mature stages. This was further validated by qRT-PCR. In conclusion, a comprehensive transcriptome of the testis of orange mud crabs from different maturation stages were obtained. This report provides an invaluable resource for enhancing our understanding of this species' genome structure and biology, as expressed and controlled by their gonads.

A Transition Metal-Binding, Trimeric ßγ-Crystallin from Methane-Producing Thermophilic Archaea, Methanosaeta thermophila.

ßγ-Crystallins are important constituents of the vertebrate eye lens, whereas in microbes, they are prevalent as Ca2+-binding proteins. In archaea, ßγ-crystallins are conspicuously confined to two methanogens, viz., Methanosaeta and Methanosarcina. One of these, i.e., M-crystallin from Methanosarcina acetivorans, has been shown to be a typical Ca2+-binding ßγ-crystallin. Here, with the aid of a high-resolution crystal structure and isothermal titration calorimetry, we report that "Methallin", a ßγ-crystallin from Methanosaeta thermophila, is a trimeric, transition metal-binding protein. It binds Fe, Ni, Co, or Zn ion with nanomolar affinity, which is consistent even at 55 °C, the optimal temperature for the methanogen's growth. At the center of the protein trimer, the metal ion is coordinated by six histidines, two from each protomer, leading to an octahedral geometry. Small-angle X-ray scattering analysis confirms that the trimer seen in the crystal lattice is a biological assembly; this assembly dissociates to monomers upon removal of the metal ion. The introduction of two histidines (S17H/S19H) into a homologous ßγ-crystallin, Clostrillin, allows it to bind nickel at the introduced site, though with micromolar affinity. However, because of the lack of a compatible interface, nickel binding could not induce trimerization, affirming that Methallin is a naturally occurring trimer for high-affinity transition metal binding. While ßγ-crystallins are known to bind Ca2+ and form homodimers and oligomers, the transition metal-binding, trimeric Methallin is a new paradigm for ßγ-crystallins. The distinct features of Methallin, such as nickel or iron binding, are also possible imprints of biogeochemical changes during the period of its origin.

Calcium Binding Dramatically Stabilizes an Ancestral Crystallin Fold in Tunicate ßγ-Crystallin.

The tunicate (Ciona intestinalis) ßγ-crystallin represents an intermediate case between the calcium-binding proteins ancestral to the vertebrate ßγ-crystallin fold and the vertebrate structural crystallins. Unlike the structural ßγ-crystallins in the vertebrate eye lens, this ßγ-crystallin strongly binds Ca2+. Furthermore, Ca2+ binding greatly stabilizes the protein, an effect that has previously been observed in microbial ßγ-crystallins but not in those of vertebrates. This relationship between binding and protein stabilization makes the tunicate ßγ-crystallin an interesting model for studying the evolution of the human ßγ-crystallin. We also compare and contrast the binding sites of tunicate ßγ-crystallin with those of other ßγ-crystallins to develop hypotheses about the functional origin of the lack of Ca2+-binding sites in human crystallins.

Selenite and ebselen supplementation attenuates D-galactose-induced oxidative stress and increases expression of SELR and SEP15 in rat lens.

Selenite and ebselen supplementation has been shown to possess anti-cataract potential in some experimental animal models of cataract, however, the underlying mechanisms remain unclear. The present study was designed to evaluate the anti-cataract effects and the underlying mechanisms of selenite and ebselen supplementation on galactose induced cataract in rats, a common animal model of sugar cataract. Transmission electron microscopy images of lens fiber cells (LFC) and lens epithelial cells (LEC) were observed in D-galactose-induced experimental cataractous rats treated with or without selenite and ebselen, also redox homeostasis and expression of proteins such as selenoprotein R (SELR), 15kD selenoprotein (SEP15), superoxide dismutase 1 (SOD1), catalase (CAT), ß-crystallin protein, aldose reductase (AR) and glucose-regulated protein 78 (GRP78) were estimated in the lenses. The results showed that D-galactose injection injured rat lens and resulted in cataract formation; however, selenite and ebselen supplementation markedly alleviated ultrastructural injury of LFC and LEC. Moreover, selenite and ebselen supplementation could mitigate the oxidative damage in rat lens and increase the protein expressions of SELR, SEP15, SOD1, CAT and ß-crystallin, as well as decrease the protein expressions of AR and GRP78. Taken together, these findings for the first time reveal the anti-cataract potential of selenite and ebselen in galactosemic cataract, and provide important new insights into the anti-cataract mechanisms of selenite and ebselen in sugar cataract.

One-shot LC-MS/MS analysis of post-translational modifications including oxidation and deamidation of rat lens α- and ß-crystallins induced by γ-irradiation.

The eye lens is a transparent organ that functions to focus light and images on the retina. The transparency and high refraction of the lens are maintained by the function of α-, ß-, and γ-crystallins. These long-lived proteins are subject to various post-translational modifications, such as oxidation, deamidation, truncation and isomerization, which occur gradually during the aging process. Such modifications, which are generated by UV light and oxidative stress, decrease crystallin solubility and lens transparency, and ultimately lead to the development of age-related cataracts. Here, we irradiated young rat lenses with γ-rays (5-500 Gy) and extracted the water-soluble (WS) and water-insoluble (WI) protein fractions. The WS and WI lens proteins were digested with trypsin, and the resulting peptides were analyzed by one-shot LC-MS/MS to determine the specific sites of oxidation of methionine and tryptophan, deamidation sites of asparagine and glutamine, and isomerization of aspartyl in rat α- and ß-crystallins in the WS and WI fractions. Oxidation and deamidation occurred in several crystallins after irradiation at more than, respectively, 50 and 5 Gy; however, isomerization did not occur in any crystallin even after exposure to 500 Gy of irradiation. The number of oxidation and deamidation sites was much higher in the WI than in the WS fraction. Furthermore, the oxidation and deamidation sites in rat crystallins resemble those reported in crystallins from human age-related cataracts. Thus, this study on post-translational modifications of crystallins induced by ionizing irradiation may provide useful information relevant to the formation of human age-related cataracts.