Zeta-crystallin: a moonlighting player in cancer.

Crystallins were firstly found as structural proteins of the eye lens. To this family belong proteins, such as ζ-crystallin, expressed ubiquitously, and endowed with enzyme activity. ζ-crystallin is a moonlighting protein endowed with two main different functions: (1) mRNA binding with stabilizing activity; (2) NADPH:quinone oxidoreductase. ζ-crystallin has been clearly demonstrated to stabilize mRNAs encoding proteins involved in renal glutamine catabolism during metabolic acidosis resulting in ammoniagenesis and bicarbonate ion production that concur to compensate such condition. ζ-crystallin binds also mRNAs encoding for antiapoptotic proteins, such as Bcl-2 in leukemia cells. On the other hand, the physiological role of its enzymatic activity is still elusive. Gathering research evidences and data mined from public databases, we provide a framework where all the known ζ-crystallin properties are called into question, making it a hypothetical pivotal player in cancer, allowing cells to hijack or subjugate the acidity response mechanism to increase their ability to resist oxidative stress and apoptosis, while fueling their glutamine addicted metabolism.

Activation of farnesoid X receptor (FXR) induces crystallin zeta expression in mouse medullary collecting duct cells.

Crystallin zeta (CRYZ) is a phylogenetically restricted water-soluble protein and provides cytoprotection against oxidative stress via multiple mechanisms. Increasing evidence suggests that CRYZ is high abundantly expressed in the kidney where it acts as a transacting factor in increasing glutaminolysis and the Na+/K+/2Cl- cotransporter (BSC1/NKCC2) expression to help maintain acid-base balance and medullary hyperosmotic gradient. However, the mechanism by which CRYZ is regulated in the kidney remains largely uncharacterized. Here, we show that CRYZ is a direct target of farnesoid X receptor (FXR), a nuclear receptor important for renal physiology. We found that CRYZ was ubiquitously expressed in mouse kidney and constitutively expressed in the cytoplasm of medullary collecting duct cells (MCDs). In primary cultured mouse MCDs, CRYZ expression was significantly upregulated by the activation and overexpression of FXR. FXR-induced CRYZ expression was almost completely abolished in the MCD cells with siRNA-mediated FXR knockdown. Consistently, treatment with FXR agonists failed to induce CRYZ expression in the MCDs isolated from mice with global and collecting duct-specific FXR deficiency. We identified a putative FXR response element (FXRE) on the CRYZ gene promoter. The luciferase reporter and ChIP assays revealed that FXR can bind directly to the FXRE site, which was further markedly enhanced by FXR activation. Furthermore, we found CRYZ overexpression in MCDs significantly attenuated hypertonicity-induced cell death possibly via increasing Bcl-2 expression. Collectively, our findings demonstrate that CRYZ is constitutively expressed in renal medullary collecting duct cells, where it is transcriptionally controlled by FXR. Given a critical role of FXR in MCDs, CRYZ may be responsible for protective effect of FXR on the survival of MCDs under hypertonic condition during dehydration.

Genome-wide association analysis identifies TYW3/CRYZ and NDST4 loci associated with circulating resistin levels.

Resistin is a polypeptide hormone that was reported to be associated with insulin resistance, inflammation and risk of type 2 diabetes and cardiovascular disease. We conducted a genome-wide association (GWA) study on circulating resistin levels in individuals of European ancestry drawn from the two independent studies: the Nurses' Health Study (n = 1590) and the Health, Aging and Body Composition Study (n = 1658). Single-nucleotide polymorphisms (SNPs) identified in the GWA analysis were replicated in an independent cohort of Europeans: the Gargano Family Study (n = 659). We confirmed the association with a previously known locus, the RETN gene (19p13.2), and identified two novel loci near the TYW3/CRYZ gene (1p31) and the NDST4 gene (4q25), associated with resistin levels at a genome-wide significant level, best represented by SNP rs3931020 (P = 6.37 × 10(-12)) and SNP rs13144478 (P = 6.19 × 10(-18)), respectively. Gene expression quantitative trait loci analyses showed a significant cis association between the SNP rs3931020 and CRYZ gene expression levels (P = 3.68 × 10(-7)). We also found that both of these two SNPs were significantly associated with resistin gene (RETN) mRNA levels in white blood cells from 68 subjects with type 2 diabetes (both P = 0.02). In addition, the resistin-rising allele of the TYW3/CRYZ SNP rs3931020, but not the NDST4 SNP rs13144478, showed a consistent association with increased coronary heart disease risk [odds ratio = 1.18 (95% CI, 1.03-1.34); P = 0.01]. Our results suggest that genetic variants in TYW3/CRYZ and NDST4 loci may be involved in the regulation of circulating resistin levels. More studies are needed to verify the associations of the SNP rs13144478 with NDST4 gene expression and resistin-related disease.

The Candida albicans ζ-crystallin homolog Zta1 promotes resistance to oxidative stress.

IMPORTANCE: Candida albicans is an important human pathogen that can cause lethal systemic infections. The ability of C. albicans to colonize and establish infections is closely tied to its highly adaptable nature and capacity to resist various types of stress, including oxidative stress. Previous studies showed that four C. albicans proteins belonging to the flavodoxin-like protein family of quinone reductases are needed for resistance to quinones and virulence. Therefore, in this study, we examined the role of a distinct type of quinone reductase, Zta1, and found that it acts in conjunction with the flavodoxin-like proteins to protect against oxidative stress.

Elucidation of kinetic and structural properties of eye lens ζ-crystallin: an in vitro and in silico approach.

The Arabian Camelus dromedarius contains significant concentration of eye lens ζ-crystallin. This enzyme is also present in other life forms including humans, however in lower catalytic amounts. The recombinant camel ζ-crystallin was expressed in the E. coli BL21 (DE3) pLysS strain and purified using HisTrap column. The Km of the enzyme for 9,10-phenanthrenequinone (9,10-PQ) substrate and NADPH cofactor was determined to be 11.66 and 50.93 µM, respectively. The Vmax for 9,10-PQ and NADPH was obtained as 23.19 and 19.98 µM min-1, respectively. The optimum activity of the purified enzyme was found to be at pH 6.0 and at 55 °C. Different physico-chemical parameters were analysed including instability index (II), aliphatic index (AI) and the GRAVY index to establish proper characterization. The sequence of the recombinant ζ-crystallin was subjected to homology modelling using SWISS-MODEL webserver followed by validation of the modelled target structure. The evaluation of the modelled ζ-crystallin was performed by several parameters including Ramachandran plot, Z-score values followed by molecular dynamics (MD) simulation. The cumulative analysis of the physico-chemical, quantitative, qualitative and the essential dynamics of simulation of ζ-crystallin and its complexes with 9,10-PQ and NADPH helped in verifying the acceptable quality and stability of the ζ-crystallin structure.Communicated by Ramaswamy H. Sarma.

Crystallin distribution patterns in Litoria infrafrenata and Phyllomedusa sauvagei lenses.

The eye lens remains transparent because of soluble lens proteins known as crystallins. For years γ-crystallins have been known as the main lens proteins in lower vertebrates such as fish and amphibians. The unique growth features of the lens render it an ideal structure to study ageing; few studies have examined such changes in anuran lenses. This study aimed to investigate protein distribution patterns in Litoria infrafrenata and Phyllomedusa sauvagei species. Lenses were fractionated into concentric layers by controlled dissolution. Water-soluble proteins were separated into high (HMW), middle (MMW) and low molecular weight (LMW) fractions by size-exclusion HPLC and constituents of each protein class revealed by 1DE and 2DE. Spots were selected from 2DE gels on the basis of known ranges of subunit molecular weights and pH ranges and were identified by MALDI-TOF/TOF MS following trypsin digestion. Comparable lens distribution patterns were found for each species studied. Common crystallins were detected in both species; the most prominent of these was γ-crystallin. Towards the lens centre, there was a decrease in α- and ß-crystallin proportions and an increase in γ-crystallins. Subunits representing taxon-specific crystallins demonstrating strong sequence homology with ζ-crystallin/quinone oxidoreductase were found in both L. infrafrenata and P. sauvagei lenses. Further work is needed to determine which amphibians have taxon-specific crystallins, their evolutionary origins, and their function.

Kinetic and structural evidence of the alkenal/one reductase specificity of human ζ-crystallin.

Human ζ-crystallin is a Zn(2+)-lacking medium-chain dehydrogenase/reductase (MDR) included in the quinone oxidoreductase (QOR) family because of its activity with quinones. In the present work a novel enzymatic activity was characterized: the double bond α,ß-hydrogenation of medium-chain 2-alkenals and 3-alkenones. The enzyme is especially active with lipid peroxidation products such as 4-hydroxyhexenal, and a role in their detoxification is discussed. This specificity is novel in the QOR family, and it is similar to that described in the distantly related alkenal/one reductase family. Moreover, we report the X-ray structure of ζ-crystallin, which represents the first structure solved for a tetrameric Zn(2+)-lacking MDR, and which allowed the identification of the active-site lining residues. Docking simulations suggest a role for Tyr53 and Tyr59 in catalysis. The kinetics of Tyr53Phe and Tyr59Phe mutants support the implication of Tyr53 in binding/catalysis of alkenal/one substrates, while Tyr59 is involved in the recognition of 4-OH-alkenals.

zeta-Crystallin is a bcl-2 mRNA binding protein involved in bcl-2 overexpression in T-cell acute lymphocytic leukemia.

The human antiapoptotic bcl-2 gene has been discovered in t(14;18) B-cell leukemias/lymphomas because of its overexpression caused at a transcriptional control level by the bcl-2/IgH fusion gene. We were the first to disclose the post-transcriptional control of bcl-2 expression mediated by interactions of an adenine + uracil (AU)-rich element (ARE) in the 3'-UTR of bcl-2 mRNA with AU-binding proteins (AUBPs). Here, we identify and characterize zeta-crystallin as a new bcl-2 AUBP, whose silencing or overexpression has impact on bcl-2 mRNA stability. An increased Bcl-2 level observed in normal phytohemagglutinin (PHA)-activated T lymphocytes, acute lymphatic leukemia (ALL) T-cell lines, and T cells of patients with leukemia in comparison with normal non-PHA-activated T lymphocytes was concomitant with an increase in zeta-crystallin level. The specific association of zeta-crystallin with the bcl-2 ARE was significantly enhanced in T cells of patients with ALL, which accounts for the higher stability of bcl-2 mRNA and suggests a possible contribution of zeta-crystallin to bcl-2 overexpression occurring in this leukemia.

Protection of ζ-crystallin by α-crystallin under thermal stress.

Cataract is one of the major causes of blindness worldwide. Several factors including post-translational modification, thermal and solar radiations promote cataractogenesis. The camel lens proteins survive very harsh desert conditions and resist cataractogenesis. The folding and aggregation mechanism of camel lens proteins are poorly characterized. The camel lens contains three ubiquitous crystallins (α-, ß-, and γ-crystallin) and a novel protein (ζ-crystallin) in large amounts. In this study, a sequence similarity search of camel α-crystallin with that of other organisms showed that the camel αB-crystallin consists of an extended N-terminal domain. Our results indicate that camel α-crystallin efficiently prevented aggregation of ζ-crystallin, with or without an obligate cofactor up to 89 °C. It performed a quick and efficient holdase function irrespective of the unfolding stage or aggregation. Camel α-crystallin exhibits approximately 20% chaperone activity between 30 and 40 °C and is completely activated above 40 °C. Camel α-crystallin underwent a single reversible thermal transition without loss of ß-sheet secondary structure. Intrinsic tryptophan fluorescence and ANS binding experiments revealed two transitions which corresponded to activation of its chaperone function. In contrast to earlier studies, camel α-crystallin completely protected lens proteins during thermal stress.

Expression and purification of his-tagged recombinant mouse zeta-crystallin.

Zeta-crystallin is an NADPH-binding protein consisting of four identical 35kD subunits. The protein possesses quinone oxidoreductase activity, and is present in large amounts in the lenses of camelids, certain hystricomorphic rodents, and the Japanese tree frog, and in lower catalytic amounts in certain tissues of various species. In this study, recombinant methods were used to produce substantial quantities of his-tagged recombinant mouse zeta-crystallin, which was then purified to homogeneity. The yield of pure recombinant mouse zeta-crystallin was five times that obtained previously for purification of recombinant guinea pig zeta-crystallin. The quinone oxidoreductase activity of purified his-tagged recombinant mouse zeta-crystallin was comparable to that of purified native guinea pig lens zeta-crystallin, and to that previously reported for recombinant guinea pig zeta-crystallin. The method permits production of substantial amounts of recombinant zeta-crystallin for conducting studies on the biological role of this interesting protein, which exists in such high concentration in the lenses of certain species.

Zeta-crystallin mediates the acid pH-induced increase of BSC1 cotransporter mRNA stability.

The Na+/K+/2Cl- cotransporter (BSC1/NKCC2) is the major transporter mediating sodium chloride and ammonium absorption in the medullary thick ascending limb. A loss-of-function mutation of BSC1 is responsible for Bartter's syndrome. We previously showed both in vivo and in vitro that acidosis increases the expression and activity of BSC1 and that acid pH enhances the stability of BSC1 mRNA by mechanisms involving its 3'-untranslated region (UTR). zeta-Crystallin is a pH response factor that protects the mitochondrial glutaminase mRNA by a specific interaction with AU-rich motifs. Here we identified the molecular determinant(s) within the 3'-UTR that are responsible for BSC1-mRNA expression and assessed the involvement of zeta-crystallin in this regulation. Deleting three out of six conserved AU-rich motifs drastically reduced the expression of BSC1-mRNA with maximal effect for motif 3 at position 870 of the 3'UTR. This motif was responsible for pH and zeta-crystallin-induced stability of BSC1 mRNA. The abundance of zeta-crystallin was increased by acid pH and its overexpression increased the stability of BSC1 mRNA, but its RNA silencing inhibited acid pH-induced BSC1 expression. Therefore the 3'UTR of BSC1-mRNA is a target for zeta-crystallin. The induction of zeta-crystallin by an acid pH plays an important role in preventing BSC1 mRNA decay, thus increasing its expression and activity.

Zeta-crystallin: a tale of two cells.

Szutkowska et al. demonstrate that zeta-crystallin plays an essential role in the stabilization of the Na(+)/K(+)/2Cl(-) cotransporter mRNA in the medullary thick ascending limb. However, differential effects of experiments using small interfering RNA to knock down zeta-crystallin in proximal tubule and thick ascending limb cells suggest that additional proteins must contribute to the rapid turnover and selective stabilization of the various mRNAs during metabolic acidosis.

Isolation of a single-stranded DNA-binding protein from the methylotrophic yeast, Pichia pastoris and its identification as zeta crystallin.

A single-stranded DNA (ssDNA)-binding protein (SSB) that binds to specific upstream sequences of alcohol oxidase (AOX1) promoter of the methylotrophic yeast Pichia pastoris has been isolated and identified as zeta crystallin (ZTA1). The cDNA encoding P.pastoris ZTA1 (PpZTA1) was cloned into an Escherichia coli expression vector, the recombinant PpZTA1 was expressed and purified from E.coli cell lysates. The DNA-binding properties of recombinant PpZTA1 are identical to those of the SSB present in P.pastoris cell lysates. PpZTA1 binds to ssDNA sequences >24 nt and its DNA-binding activity is abolished by NADPH. This is the first report on the characterization of DNA-binding properties of a yeast ZTA1.

Spectral and thermal properties of novel eye lens ζ-crystallin.

Eye lenses are exposed to thermal, solar radiations, dryness that enhances cataractogenesis. Some animal lenses contain novel proteins in bulk quantities. ζ-crystallin occurred in three ecologically divergent species, but it's physiological role not known. The truncated variant of ζ-crystallin causes hereditary cataract. Guinea pig ζ-crystallin is temperature-sensitive and rapidly aggregates at 41°C. Camels adopted to survive above 50°C, which raises an interesting question about how it retains lens proteins in the soluble state? Here, we have optimized expression and purification of recombinant camel ζ-crystallin. We have studied thermodynamic and spectroscopic properties using orthogonal techniques. Dynamic multimode spectroscopy results showed that camel ζ-crystallin unfolds via single transition with Tm value of 60.8±0.1°C and van't Hoff enthalpy of 714.7±7.1kJ/mol. Thermal-shift assay calculates Tm value of 62°C at pH 7. Additionally, the conformational stability of ζ-crystallin increases with ionic-strength. The influence of pH on ζ-crystallin was evaluated where the protein was found to be stable in the pH range of 6-9, but its stability drastically decreases below pH 6. Our results also showed that quaternary structure of ζ-crystallin drastically changed as a result of lowering pH. This study provides significant understandings onto the conformational, thermodynamic and unfolding pathway of camel ζ-crystallin.

Expression, Purification and Properties of Redox-Sensitive Eye Lens Zeta-Crystallin of Arabian Camel.

The high protein concentration, unique composition and complex geometry of the lens makes it transparent. α-, β-, and γ-crystallins are present in all the lenses. In addition, taxon-specific crystallins are present in lenses in bulk quantity. Zeta (ζ)-crystallin is an NADPH-dependent quinone oxidoreductase, which constitutes nearly 10 % of the total eye lens protein in the evolutionary divergent animals (Camel, guinea pig and Japanese frog eye lenses) living in different ecological conditions. ζ -Crystallin is also present in human and other animal lenses but at catalytic amount. The physiological role of γ-crystallin in the eye lens is not well understood, however, truncated ζ-crystallin causes congenital cataract in guinea pig. In earlier study, redox regulated reversible activity of ζ-crystallin was reported. In this study, recombinant camel ζ-crystallin was overexpressed in E.coli and purified to homogeneity. Effect of different concentrations of reducing agent, dithiothretol (DTT) on the quinone oxidoreductase activity of recombinant ζ-crystallin was studied by enzymatic assay. To evaluate the effect of the reducing agent on the ζ-crystallin conformation, we have used far-UV and near-UV CD, intrinsic fluorescence, ANS binding assay and size exclusion chromatography. Our results showed that nearly 50% of the of ζ-crystallin activity was lost at 50 µM DTT. However, no detectable changes in secondary structure were observed. No changes in the tertiary structure and surface hydrophobicity of ζ-crystallin were detected; however, marginal changes were seen at saturating concentration of DTT (1 mM).

Inhibition of camel lens zeta-crystallin/NADPH:quinone oxidoreductase by pyridoxal-5'-phosphate.

Camel lens zeta-crystallin was inhibited by pyridoxal-5'-phosphate (PAL-P) and o-phthalaldehyde. PAL-P inactivated zeta-crystallin in a time- and concentration-dependent manner. The initial rate of inactivation followed pseudo-first-order kinetics with the second-order rate constant of 91 M-1 s-1. The modified enzyme showed the characteristic absorption peak at 325 nm indicative of the formation of phosphopyridoxallysine. Quantitative analysis suggested the incorporation of 1 mole of PAL-P/subunit of enzyme. NADPH was able to substantially protect zeta-crystallin against PAL-P inactivation, whereas the substrate 9,10-phenanthrenequinone (PQ) did not provide any protection. Inhibition of zeta-crystallin by PAL-P was uncompetitive with NADPH (Ki=37 microM) and non-competitive with respect to the substrate (Ki=57 microM). Inhibition of zeta-crystallin by o-phthalaldehyde was used to establish the location of an essential lysine residue. Incubation of zeta-crystallin with o-phthalaldehyde resulted in the formation of an isoindole derivative that had a characteristic fluorescence spectrum. This suggested that a lysine residue is located within 3 A of a cysteine residue at the NADPH binding region. SDS-PAGE showed the o-phthalaldehyde-modified enzyme remained largely monomer (approx. 80%), although bands corresponding to dimer and tetramer forms were also present. These results suggested that an essential lysine residue is located in the vicinity of the NADPH binding site. This residue may simply ensure the proper binding of NADPH to the active site of zeta-crystallin.

Hydrophobicity of the NADPH binding domain of camel lens zeta-crystallin.

Interaction of camel lens zeta-crystallin with the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) enhanced the ANS fluorescence and quenched the protein fluorescence. Both of these events were concentration-dependent and showed typical saturation curves suggesting specific ANS-zeta-crystallin binding. Quantitative analysis indicated that 1 mole zeta-crystallin bound at most 1 mole ANS. NADPH but not 9,10-phenanthrenequinone (PQ) was able to displace zeta-crystallin-bound ANS. These results suggested the presence of a hydrophobic domain in zeta-crystallin, possibly at the NADPH binding site. alpha-Crystallin as well as NADPH protected zeta-crystallin against thermal inactivation suggesting the importance of this site for enzyme stability. The NADPH:quinone oxidoreductase activity of zeta-crystallin was inhibited by ANS with NADPH as electron donor and PQ as electron acceptor. Lineweaver-Burk plots indicated mixed-type inhibition with respect to NADPH, with a K(i) of 2.3 microM. Secondary plots of inhibition with respect to NADPH indicated a dissociation constant (K'I) of 12 microM for the zeta-crystallin-NADPH-ANS complex. The K(i) being smaller than K'I suggested that competitive inhibition at the NADPH binding site was predominant over non-competitive inhibition. Like ANS-zeta-crystallin binding, inhibition was dependent on ANS concentration but independent of incubation time.

The yeast ζ-crystallin/NADPH:quinone oxidoreductase (Zta1p) is under nutritional control by the target of rapamycin pathway and is involved in the regulation of argininosuccinate lyase mRNA half-life.

The yeast ζ-crystallin (Zta1p) is a quinone oxidoreductase belonging to the ζ-crystallin family, with activity in the reduction of alkenal/alkenone compounds. Various biological functions have been ascribed to the members of this protein family, such as their ability to interact specifically with AU-rich sequences in mRNA, and thus they have been proposed to act as AU-rich element-binding proteins (AREBPs). In this study, we evaluated the specificity of Zta1p for RNA versus DNA by means of a novel nonisotopic method for the in vitro quantitative detection of protein · RNA complexes. Through comparative transcriptomic analysis, we found that the lack of Zta1p negatively affects the expression of a group of genes involved in amino acid biosynthesis, the argininosuccinate lyase (ARG4) gene being one of them. Here, we propose that Zta1p participates in the post-transcriptional regulation of ARG4 expression by increasing the ARG4 mRNA half-life. In addition, expression of the ζ-crystallin gene (ZTA1) is itself regulated by nutrient availability through the general amino acid control and target of rapamycin pathways. Our results shed new light on the ζ-crystallin family members from yeast to humans as stress response proteins with a bifunctional role in the detoxification of alkenal and alkenone compounds, and the regulation of gene expression.

Stage-independent splicing of transcripts two heterogeneous neighboring genes in Leishmania amazonensis.

Gene expression in trypanosomatid protozoa is largely regulated posttranscriptionally, e.g., 5' splice leader addition and 3' polyadenylation of mRNAs. We examined these events in Leishmania by mapping the splice sites of the transcripts from two different, but closely linked single-copy genes 2.3 kb apart. The coding regions of the approx. 1 kb upstream gene (P36) and the approx. 1.4 kb downstream gene (NAGT) produce approx. 2 and 3 kb mRNAs, respectively. Both genes were overexpressed in cells that were transfected with this bicistronic unit (> or = 7.5 kb), taking advantage of the NAGT as a selectable marker for tunicamycin-resistance. The transcripts from both genes were spliced constitutively at both ends, irrespective of their episomal or chromosomal expression in both leishmanial stages. Primer extension of the 5' UTRs and S1 nuclease protection of the 3' UTRs initially identified the major splice sites, corresponding to the genomic sequence at -205 bp and + approx. 900 bp of P36, and -1012 bp and + approx. 600 bp of NAGT. These splice sites, consistent with the size of the major transcripts, are among those mapped precisely by sequencing RT-PCR amplified 5' and 3' UTRs. The additional sites mapped by the latter are minor alternatives, especially abundant for transcripts of the downstream NAGT. All these minor splice sites are closer than the major splice sites to the coding region, indicating that the most distant splice sites are preferentially used. This preference creates a 387 bp 'gap' with polypyrimidine tracts in the intergenic region consistent with the model coupling splice leader addition with polyadenylation in pre-mRNA processing. The stage-independence of these events suggests that the 7.5 kb dicistronic unit is suitable for constructing Leishmania-specific constitutive expression vectors.