Crystallin Research Articles

A New Functional Model for Prediction of Chaperone Activity of the Recombinant M. tb Acr (α-Crystallin) Using Insulin as Substrate.

Mycobacterium tuberculosis Acr is an important protein expressed in latent tuberculosis which is active as an oligomer in preventing misfolding of cellular proteins. In this study, Mycobacterium alpha crystallin (acr) gene was cloned and expressed in Escherichia coli (E. coli). The recombinant Acr protein was purified by Nickel-NTA resin. The oligomeric state of Acr was confirmed by gel filtration chromatography using Sephacryl S-200 and Native-PAGE. Studies of chaperone activity were performed with insulin as a substrate at different mole ratios of Acr with 2 types of samples, His tag elutes (H) and His tag elutes with gel filtration (G). It was observed that the ratio of different sizes of oligomers (9 to 24 mers) had a significant effect on chaperone activity. Using the mole ratio of Acr for both (H) and (G) samples to insulin B chain and ratio of oligomers, we determined the number of Acr molecules binding to insulin as a model substrate. We found that if 1.5% of the insulin B chains are covered completely by the (G) samples, aggregation is completely inhibited as compared to 6% with (H) samples. Pre-heat treatment studies were carried out at 37°C, 60°C, and 70°C. Far-ultraviolet Circular Dichroism (UV-CD) analysis provided fresh insights into the role of β-sheets and α-helices in chaperone activity, particularly in (H) samples suggesting a reversible conformational transition from helices to sheets. This enabled us to formulate a functional model for binding of Acr to insulin B chains which incorporated 4 types of secondary structure molecules. This might be a useful tool for analyzing in vitro preparations of recombinant Acr and build more consensuses on the structure-activity relationship especially in terms of oligomeric ratios.

Enhanced pH Dependent Modulation of Alpha Crystallin Chaperone Function and Subunit Exchange in an N-Terminal Phosphorylation Mimic

Enhanced pH Dependent Modulation of Alpha Crystallin Chaperone Function and Subunit Exchange in an N-Terminal Phosphorylation Mimic

Induced expression of small heat shock proteins is associated with thermotolerance in female Laodelphax striatellus planthoppers.

Insects are often exposed to high temperature stress in natural environments, but the mechanisms involved in thermotolerance in many insect groups like Hemiptera are not well known. To explore possible mechanisms of thermotolerance in the hemipteran pest Laodelphax striatellus, which damages rice through direct feeding and viral transmission, small heat shock proteins (sHsps) implicated in thermotolerance in other insect groups were identified. The seven sHsps identified have a conserved alpha crystallin domain, a variable N-terminal region, and shared relative low identities to each other. Three of the sHsp genes (LsHsp20.5, LsHsp21.5, and LsHsp21.6) exhibited higher basal expression than the other four genes but showed weak or no heat-induced expression. The other four genes (LsHsp20.1, LsHsp21.2, LsHsp21.4, and LsHsp22.0) were induced up to 3306-fold by heat. Injection of dsRNA indicated that expression of these sHsps was associated with thermotolerance, and Escherichia coli transformed with LsHsp21.2 and LsHsp20.1 showed relatively higher thermotolerance. These results point to an important functional role of these sHsps for thermotolerance in L. striatellus.

Computational genome-wide identification of heat shock protein genes in the bovine genome

Background: Heat shock proteins (HSPs) are molecular chaperones known to bind and sequester client proteins under stress. Methods: To identify and better understand some of these proteins, we carried out a computational genome-wide survey of the bovine genome. For this, HSP sequences from each subfamily (sHSP, HSP40, HSP70 and HSP90) were used to search the Pfam (Protein family) database, for identifying exact HSP domain sequences based on the hidden Markov model. ProtParam tool was used to compute potential physico-chemical parameters detectable from a protein sequence. Evolutionary trace (ET) method was used to extract evolutionarily functional residues of a homologous protein family. Results: We computationally identified 67 genes made up of 10, 43, 10 and 4 genes belonging to small HSP, HSP40, HSP70 and HSP90 families respectively. These genes were widely dispersed across the bovine genome, except in chromosomes 24, 26 and 27, which lack bovine HSP genes. We found an uncharacterized outer dense fiber ( ODF1) gene in cattle with an intact alpha crystallin domain, like other small HSPs. Physico-chemical characteristic of aliphatic index was higher in HSP70 and HSP90 gene families, compared to small HSP and HSP40. Grand average hydropathy showed that small HSP (sHSP), HSP40, HSP70 and HSP90 genes had negative values except for DNAJC22, a member of HSP40 gene family. The uniqueness of DNAJA3 and DNAJB13 among HSP40 members, based on multiple sequence alignment, evolutionary trace analysis and sequence identity dendrograms, suggests evolutionary distinct structural and functional features, with unique roles in substrate recognition and chaperone functions. The monophyletic pattern of the sequence identity dendrograms of cattle, human and mouse HSP sequences suggests functional similarities. Conclusions: Our computational results demonstrate the first-pass in-silico identification of heat shock proteins and calls for further investigation to better understand their functional roles and mechanisms in Bovidae.

Hydropathic wave ordering of alpha crystallin—Membrane interactions enhances human lens transparency and resists cataracts

Granted that neural networks are nature’s most amazing invention, what is in second place? One candidate is the human eye, and specifically its transparent lens. Here we use the evolution of the lens’ dominant proteins to quantify subtle improvements in alpha crystallins between chickens and humans. These improvements are different in αA and αB crystallins, and suggest that their functions are complementary. We relate crystallin evolution to the fiber cell structure of the eye, and its enhanced resistance to cataracts in humans, with weakened resistance in rats, which improves to elephants and finally humans. Our results show that the 2007 Brazilian hydropathicity scale based on self-organized criticality is qualitatively superior to the >100 classical scales based on protein unfolding.

Possible association of oestrogen and Cryba4 with masticatory muscle tendon-aponeurosis hyperplasia.

Masticatory muscle tendon-aponeurosis hyperplasia, which is associated with limited mouth opening, progresses very slowly from adolescence. The prevalence rates of this disease are higher among women than among men, suggesting oestrogen involvement. As parafunctional habits are frequently observed, mechanical stress is likely involved in the pathogenesis and advancement of this disease. To elucidate the pathological condition, we examined the effect of oestrogen on tenocyte function and the relationship between mechanical stress and crystallin beta A4 (Cryba4), using murine TT-D6 tenocytes. Cell proliferation assays, RT-PCR, real-time RT-PCR, Western blot analysis and mechanical loading experiments were performed. The physiological dose of oestrogen increased the levels of scleraxis and tenomodulin in TT-D6 tenocytes. In contrast, forced expression of Cryba4 inhibited scleraxis expression in these cells. Surprisingly, oestrogen significantly promoted cell differentiation in the Cryba4-overexpressing TT-D6 tenocytes. Moreover, tensile force induced Cryba4 expression in these tendon cells. Oestrogen and Cryba4 may be associated with the progression of masticatory muscle tendon-aponeurosis hyperplasia.

Computational genome-wide identification of heat shock protein genes in the bovine genome.

Background: Heat shock proteins (HSPs) are molecular chaperones known to bind and sequester client proteins under stress. Methods: To identify and better understand some of these proteins, we carried out a computational genome-wide survey of the bovine genome. For this, HSP sequences from each subfamily (sHSP, HSP40, HSP70 and HSP90) were used to search the Pfam (Protein family) database, for identifying exact HSP domain sequences based on the hidden Markov model. ProtParam tool was used to compute potential physico-chemical parameters detectable from a protein sequence. Evolutionary trace (ET) method was used to extract evolutionarily functional residues of a homologous protein family. Results: We computationally identified 67 genes made up of 10, 43, 10 and 4 genes belonging to small HSP, HSP40, HSP70 and HSP90 families respectively. These genes were widely dispersed across the bovine genome, except in chromosomes 24, 26 and 27, which lack bovine HSP genes. We found an uncharacterized outer dense fiber ( ODF1) gene in cattle with an intact alpha crystallin domain, like other small HSPs. Physico-chemical characteristic of aliphatic index was higher in HSP70 and HSP90 gene families, compared to small HSP and HSP40. Grand average hydropathy showed that small HSP (sHSP), HSP40, HSP70 and HSP90 genes had negative values except for DNAJC22, a member of HSP40 gene family. The uniqueness of DNAJA3 and DNAJB13 among HSP40 members, based on multiple sequence alignment, evolutionary trace analysis and sequence identity dendrograms, suggests evolutionary distinct structural and functional features, with unique roles in substrate recognition and chaperone functions. The monophyletic pattern of the sequence identity dendrograms of cattle, human and mouse HSP sequences suggests functional similarities. Conclusions: Our computational results demonstrate the first-pass in-silico identification of heat shock proteins and calls for further investigation to better understand their functional roles and mechanisms in Bovidae.

Sequence specific 1H, 13C and 15N resonance assignments of the C-terminal domain of human Gamma S-Crystallin

Sequence specific 1H, 13C and 15N resonance assignments of the C-terminal domain of human Gamma S-Crystallin

Accumulation of perfluorobutane sulfonate (PFBS) and impairment of visual function in the eyes of marine medaka after a life-cycle exposure

As an alternative to perfluorooctane sulfonate (PFOS), increasing usage of perfluorobutane sulfonate (PFBS) has led to ubiquitous presence in the environment. PFBS is also shown to potently disrupt the thyroid endocrine system. Considering the regulation of thyroid hormones in visual development, PFBS is likely to adversely affect the development and function of visual systems, which is a sensitive target of environmental pollutants. Therefore, the present study exposed marine medaka embryos to environmentally realistic concentrations of PFBS (0, 1.0, 2.9 and 9.5 μg/L) for an entire life-cycle. After exposure until sexual maturity, eyes of adult medaka were dissected to directly investigate the ocular accumulation and toxicity of PFBS. For the first time, substantial accumulation of an environmental pollutant (i.e., PFBS) was observed in the eye tissue. PFBS exposure was also found to impair the visual development and function in a sex-dependent manner. In female medaka, weight of eyes was significantly decreased, while content of water was increased, probably resulting in higher intraocular pressure. Multiple neural signaling processes were also disturbed by PFBS life-cycle exposure, including cholinergic, glutamatergic, GABAergic and monoaminergic systems. Increased levels of norepinephrine and epinephrine neurotransmitters may adaptively decrease the intraocular hypertension in female eyes. In addition, proteomic profiling identified the visual proteins of differential expressions (e.g., beta and gamma crystallins, arrestin and lumican), which were significantly associated with visual perception and motor activity of eyes. Overall, this study found that PFBS was able to accumulate in the eyes and induce ocular toxicities. The susceptibility and sex-specific responses of visual systems to environmental pollutants warrants more works for a comprehensive risk assessment.

P-87 - Ditryptophan cross-links are detected in bovine beta crystallin irradiated with a solar simulator and in human lenses with advanced cataract

We first showed the formation of a ditryptophan (Trp-Trp) cross-link through recombination of human superoxide dismutase-tryptophanyl radicals in 2010. This and related publications received limited attention up to very recently, when similar cross-links have been reported by other investigators in different proteins. Since Trp-Trp cross-links may play a role in protein aggregation processes, they deserve further studies. Here, we report the presence of Trp-Trp cross-links (f3B2-Trp151-Trp151-f3B2 and f3B2-Trp82-Trp85) in beta crystallin irradiated in a solar simulator (SOL-UV-2) for 2–8 hours. Moreover, Trp-Trp cross-links (f3B2-Trp151-Trp151-f3B2; f3B1-Trp217-Trp220 and f3B1-Trp124-Trp127) were detectable in human lenses with advanced cataract (LOCS II). In both cases, monomer and dimers of the proteins constituting the samples were separated by SDS-PAGE and the tryptic peptides analyzed by Orbitrap Lumos Fusion. To the best of our knowledge, this is the first direct evidence for the formation of Trp-Trp cross-links in vivo.

Hard-sphere-like dynamics in highly concentrated alpha-crystallin suspensions.

The dynamics of concentrated suspensions of the eye-lens protein alpha crystallin have been measured using x-ray photon correlation spectroscopy. Measurements were made at wave vectors corresponding to the first peak in the hard-sphere structure factor and volume fractions close to the critical volume fraction for the glass transition. Langevin dynamics simulations were also performed in parallel to the experiments. The intermediate scattering function f(q,τ) could be fit using a stretched exponential decay for both experiments and numerical simulations. The measured relaxation times show good agreement with simulations for polydisperse hard-sphere colloids.

Chromosomal Micro-aberration in a Saudi Family with Juvenile Myoclonic Epilepsy.

Epilepsy is etiologically and genetically complex neurological disorder affecting millions of people worldwide. Juvenile myoclonic epilepsy (JME) is the most common epilepsy syndrome that starts in the teen age group commonly between ages 12, 18, and lasts till adulthood. One out of fourteen people with epilepsy suffers with JME. Myoclonic seizures and muscle twitching or uncontrolled jerking are the most common type of seizures in the people suffering with JME. To observe the novel CNVs involved in JME, we investigated a Saudi family with nine siblings including one male and one female affected members. In this study we used high density whole genome Agilent sure print G3 Hmn CGH 2x 400K array-CGH chips. Our results showed CNVs including the amplifications and deletions in different chromosomal regions in the patients as compared to the normal members of the family. Amplifications were observed in the chromosome 22 cytoband 22q11.23 with LDL receptor related protein 5 like (LRP5L), Immunoglobulin Lambda-Like Polypeptide 3 (IGLL3) and crystallin beta B2 pseudogene (CRYBB2P) genes respectively whereas the deletions were observed in the chromosomal regions 4q22.2 with Glutamate receptor, ionotropic, delta 2 (GRID2) as potential gene cytoband 1p31.1 with potential Neuronal Growth Regulator 1 gene (NEGR1) gene in this region and NME/NM23 family member (NME7) gene cytoband 1q24. Moreover, the array CGH resulting in deletions and duplication were also validated by using primer for simple PCR or also by using quantitative real time PCR analysis. We found deletions and duplication in JME patients in our study for the first time in Saudi population. The findings in this study suggest that the array-CGH may be considered as a first line of genetic testing for diagnosis of epilepsy unless strong evidence is presented for a monogenic syndrome. The use of high throughput technique in this study will help to identify novel mechanisms underlying epileptic disorder in order to lower the burden of epilepsy in Saudi Arabia.

Associations between CRYBA4 gene variants and high myopia in a Japanese population.

PurposeThe crystallin beta A4 (CRYBA4) gene variant, rs2009066, was previously reported to be associated with high myopia in a southern Chinese population. In the present study, we investigated whether CRYBA4 variants were associated with high myopia in a Japanese population.MethodsWe recruited 1,063 Japanese patients with high myopia (spherical equivalent [SE] ≤−9.00 D in both eyes) and 1,009 healthy Japanese subjects (SE >−1.00 D). We genotyped rs2009066 and three tagging single-nucleotide polymorphisms (SNPs), rs16982456, rs2071861, and rs4276, in the CRYBA4 region.ResultsWe did not find any significant association between these four SNPs and high myopia in an allele analysis. However, rs2009066 and rs2071861, which were in strong linkage disequilibrium (LD; r2=0.86), showed a marginal association with high myopia in the recessive genotype model of risk alleles (rs2009066 G allele: P=0.032, odds ratio [OR] =1.31; rs2071861 A allele: P=0.037, OR =1.31). Nevertheless, this association became insignificant after correcting for multiple testing (Pc >0.05).ConclusionThis study showed no significant association between CRYBA4 variants and high myopia in a Japanese population. Our findings did not correspond with a previous study. Further genetic studies with other populations are needed to elucidate a potential contribution of the CRYBA4 region in the development of high myopia.

Ractopamine-induced changes in sarcoplasmic proteome profile of post-rigor pork <i>semimembranosus</i> muscle

Ractopamine is a beta-adrenergic agonist that increases leanness and carcass weight in finishing pigs. Our previous study observed that dietary ractopamine increased the abundance of several glycolytic enzymes in the sarcoplasmic proteome of post-rigor pork longissimus thoracis muscle. Pork semimembranosus is an economically important muscle and demonstrates differences in biochemistry compared with longissimus thoracis. Nonetheless, the effects of ractopamine on sarcoplasmic proteome of semimembranosus have not been evaluated yet. Therefore, this study examined the influence of ractopamine on sarcoplasmic proteome of post-rigor pork semimembranosus. Analyses of sarcoplasmic proteome of semimembranosus muscles from control (CON; diet without ractopamine) and ractopamine-fed (RAC; 7.4 mg/kg for 14 days followed by 10.0 mg/kg for 14 days) barrows revealed that haemoglobin subunit beta, alpha-crystallin B, and titin fragments were over-abundant in CON. In contrast, myosin light chain 1/3 and tripartite motif-containing protein 72 were over-abundant in RAC. The low abundance of haemoglobin subunit beta and alpha crystallin B in RAC could be attributed to fibre type shift (from oxidative to glycolytic) in response to ractopamine. The over-abundance of MLC 1/3 and tripartite motif-containing protein 72 in RAC could be due to the increased myofibrillar protein synthesis and muscle mass in ractopamine-fed pigs. Dietary ractopamine decreased the abundance of sarcoplasmic proteins involved in oxygen transport and chaperone activity, but increased the abundance of proteins involved in muscle contraction and plasma membrane repair in pork semimembranosus muscle. Keywords : Pork, ractopamine, sarcoplasmic proteome, semimembranosus

Association of alpha A-crystallin polymorphisms with susceptibility to nuclear age-related cataract in a Han Chinese population

BackgroundAlpha A-crystallin (CRYAA) is considered critical for the maintenance of lens transparency and is related to the pathogenesis of age-related cataracts (ARCs), especially the nuclear subtype. As the 5′ untranslated region (5′ UTR) modulates gene expression, the purpose of current study was to investigate whether single nucleotide polymorphisms (SNPs) in the 5′ UTR of CRYAA were associated with susceptibility to ARC in a Han Chinese population and to clarify the mechanism of this association.MethodsSNPs in the 5′ UTR (−1 to −1000) of CRYAA were identified in 243 nuclear ARC patients and 263 controls using polymerase chain reaction and DNA sequencing. Allele and genotype frequencies were calculated and compared between two groups. Haploview 4.2 was used to calculate the linkage disequilibrium index, and the SHEsis analysis platform was used to infer haplotype construction. A dual-luciferase reporter gene assay was used for transcription of CRYAA in the presence of a protective haplotype with individual SNP alteration, Chromatin immunoprecipitation (ChIP) was employed to determine whether SNPs regulated CRYAA expression by altering the binding affinity of transcription factors.ResultsThree polymorphisms were identified in the 5′ UTR of CRYAA: rs3761381 (P = 0.000357, odds ratio [OR] = 1.837), rs13053109 (P = 0.788, OR = 1.086), and rs7278468 (P = 0.00136, OR = 0.652). The haplotype C-G-T (P = 0.0014, OR = 1.536) increased the risk of nuclear ARC, whereas the haplotype T-G-G (P = 0.00029, OR = 0.535) decreased the risk. The haplotype C-G-T decreased CRYAA transcription through rs7278468, which is located in the binding site of specificity protein 1 (Sp1). Furthermore, the G allele of rs7278468 increased CRYAA transcription by enhancing the binding affinity of Sp1.ConclusionsThese data indicate that the CRYAA polymorphism is a genetic marker of inter-individual differences in the risk of nuclear ARC.

A silent mutation in human alpha-A crystallin gene in patients with age-related nuclear or cortical cataract.

A cataract is a complex multifactorial disease that results from alterations in the cellular architecture, i.e. lens proteins. Genes associated with the development of lens include crystallin genes. Although crystallins are highly conserved proteins among vertebrates, a significant number of polymorphisms exist in human population. In this study, we screened for polymorphisms in crystallin alpha A (CRYAA) and alpha B (CRYAB) genes in 200 patients over 40 years of age, diagnosed with age-related cataract (ARC; nuclear and cortical cataracts). Genomic DNA was extracted from the peripheral blood. The coding regions of the CRYAA and CRYAB gene were amplified using polymerase chain reaction and subjected to restriction digestion. Restriction fragment length polymorphism (RFLP) was performed using known restriction enzymes for CRYAA and CRYAB genes. Denaturing high performance liquid chromatography and direct sequencing were performed to detect sequence variation in CRYAA gene. In silico analysis of secondary CRYAA mRNA structure was performed using CLC RNA Workbench. RFLP analysis did not show any changes in the restriction sites of CRYAA and CRYAB genes. In 6 patients (4 patients with nuclear cataract and 2 with cortical cataract), sequence analysis of the exon 1 in the CRYAA gene showed a silent single nucleotide polymorphism [D2D] (CRYAA: C to T transition). One of the patients with nuclear cataract was homozygous for this allele. The in silico analysis revealed that D2D mutation results in a compact CRYAA mRNA secondary structure, while the wild type CRYAA mRNA has a weak or loose secondary structure. D2D mutation in the CRYAA gene may be an additional risk factor for progression of ARC.

A novel frameshift mutation in CX46 associated with hereditary dominant cataracts in a Chinese family.

To investigate the genetic mutations that are associated the hereditary autosomal dominant cataract in a Chinese family. A Chinese family consisting of 20 cataract patients (including 9 male and 11 female) and 2 unaffected individuals from 5 generations were diagnosed to be a typical autosomal dominant cataract pedigree. Genomic DNA samples were extracted from the peripheral blood cells of the participants in this pedigree. Exon sequence was used for genetic mutation screening. In silico analysis was used to study the structure characteristics of connexin 46 (CX46) mutant. Immunoblotting was conduceted for testing the expression of CX46. To determine the involved genetic mutations, 11 well-known cataract-associated genes (cryaa, cryab, crybb1, crybb2, crygc, crygd, Gja3, Gja8, Hsf4, Mip and Pitx3) were chosen for genetic mutation test by using exon sequencing. A novel cytosine insertion at position 1195 of CX46 cDNA (c.1194_1195ins C) was found in the samples of 5 tested cataract patients but not in the unaffected 2 individuals nor in normal controls, which resulted in 30 amino acids more extension in CX46C-terminus (cx46fs400) compared with the wild-type CX46. In silico protein structure analysis indicated that the mutant showed distinctive hydrophobicity and protein secondary structure compared with the wild-type CX46. The immunoblot results revealed that CX46 protein, which expressed in the aging cataract lens tissues, was absence in the proband lens. In contrast, CX50, alpha A-crystallin and alphaB-crystallin expressed equally in both proband and aging cataract tissues. Those results revealed that the cx46fs400 mutation could impair CX46 protein expression. The insertion of cytosine at position 1195 of CX46 cDNA is a novel mutation site that is associated with the autosomal dominant cataracts in this Chinese family. The C-terminal frameshift mutation is involved in regulating CX46 protein expression.

Controlling the Biophysical Properties and Functionality of Phase-Separated Elastin-Based Droplets through Amino Acid Sequence Mutations

Liquid-liquid phase separation underlies the spontaneous formation of protein-rich droplets in a protein-poor solution phase, concomitant with a sharp increase in solution turbidity. Phase separation is typically triggered in response to a stimulus such as change in protein concentration, ionic strength, or temperature, and may be reversible or precede gelation, fibre formation, or aggregation. Phase separation is a well-known property of synthetic polymers, and is exploited for material design, for example, capsules for drug delivery and fast-setting hydrogels, but is not well understood for proteins. Some examples of phase-separating protein-based systems include lens gamma crystallin (implicated in cataract formation), ‘membraneless organelles’ (transiently formed compartments that regulate molecular interactions), and self-assembling elastomeric proteins, including some spider silks, insect resilin and vertebrate elastin, for which phase separation is on-pathway for the formation of elastic materials. Here we studied the phase separation of model polypeptides based on the protein elastin. These polypeptides contain both regions of well-defined secondary structure and intrinsic disorder. Droplet growth and biophysical properties were monitored using a variety of microscopy and optical spectroscopy techniques. We report the effect of amino acid sequence mutations on the size, stability, reversibility and interactions of phase-separated droplets, and address implications for functional tunability, including loading of small molecules.

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.

1 - The Overlooked Ditryptophan Cross-Link Is Present in Bovine Beta Crystalline Submitted to UVC or Solar Simulator Irradiation

During aging and pathological conditions associated with oxidative stress, the levels of over-oxidized and cross-linked proteins build-up because these species are poor substrates for the intracellular proteolytic systems, leading to protein aggregation. The chemical nature of protein cross-links remains largely uncharacterized despite their potential importance in protein aggregation. Even for characterized cross-links, such as dityrosine, information about their occurrence on specific proteins and on specific residues remains limited because bioinformatic tools to analyze LC-MS/MS proteomic data are under development. Not surprisingly, the ditryptophan cross-links (Trp-Trp), which are produced by recombination of protein-tryptophanyl radicals (protein-Trp•), have been largely disregarded in the literature. Previously, we showed that Trp-Trp cross-links are formed on human SOD1 and lysozyme submitted to the carbonate radical and on lysozyme irradiated with UVC light. Here, we report that Trp-Trp cross-links are also produced upon bovine beta crystalline irradiation. Beta crystalline (40 μM) and DTPA (0.1 mM) in phosphate buffer, pH 7.0 was irradiated for different times with UVC (254 nm, 6.3 mW/cm2) (0.5-10 min) or with a solar simulator (SOL-UV-2 (Newport); 5.0 mW/cm2 at 315 nm) (1-120 min). After irradiation, the samples analyzed by SDS-PAGE showed formation of dimers (~50 kDa) that attained a maximum at 2 min and decreased to produce higher oligomers (> 100 kDa); in the case of the simulator, dimers were clearly noted after 60 min irradiation. The bands corresponding to the irradiated samples at the required time were excised and pooled from the gels, reduced, alkylated, digested with trypsin and submitted to nano-ESI-Q-TOF MS/MS analysis. In the case of the samples irradiated with UVC, the results characterized the residues oxidized in the monomer at chain B3 (Trp82, Trp151, Trp158andTyr161) and chain A3 (Trp198) and in the dimer, a Trp151-Trp151crosslink was characterized in chain B2. In the case of samples irradiated with the simulator, the results characterized the residues oxidized in the monomer at chain B2 (Trp82) and chain A3 (Trp198). In the dimer, a Trp150-Trp150cross-link was characterized in chain A2. Although different dimers were produced under the 2 irradiation conditions, the results indicate that the formation of Trp-Trp cross-links is more common than previously considered.