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.

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.

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.

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.

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).

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.

Novel alkenal/one reductase activity of yeast NADPH:quinone reductase Zta1p. Prospect of the functional role for the ζ-crystallin family.

ζ-Crystallins are a Zn(2+)-lacking enzyme group with quinone reductase activity, which belongs to the medium-chain dehydrogenase/reductase superfamily. It has been recently observed that human ζ-crystallin is capable of reducing the α,ß-double bond of alkenals and alkenones. Here we report that this activity is also shared by the homologous Zta1p enzyme from Saccharomyces cerevisiae. While the two enzymes show similar substrate specificity, human ζ-crystallin exhibits higher activity with lipid peroxidation products and Zta1p is more active with cinnamaldehyde. The presence of Zta1p has an in vivo protective effect on yeast strains exposed to the toxic substrate 3-penten-2-one. Analysis of ZTA1 gene expression indicates an induction under different types of cellular stress, including ethanol and dimethylsulfoxide exposure and by reaching the stationary growth phase. The role of Zta1p in the yeast adaptation to some stress types and the general functional significance of ζ-crystallins are discussed.

Tumor suppressor protein p53 recruits human Sin3B/HDAC1 complex for down-regulation of its target promoters in response to genotoxic stress.

Master regulator protein p53, popularly known as the "guardian of genome" is the hub for regulation of diverse cellular pathways. Depending on the cell type and severity of DNA damage, p53 protein mediates cell cycle arrest or apoptosis, besides activating DNA repair, which is apparently achieved by regulation of its target genes, as well as direct interaction with other proteins. p53 is known to repress target genes via multiple mechanisms one of which is via recruitment of chromatin remodelling Sin3/HDAC1/2 complex. Sin3 proteins (Sin3A and Sin3B) regulate gene expression at the chromatin-level by serving as an anchor onto which the core Sin3/HDAC complex is assembled. The Sin3/HDAC co-repressor complex can be recruited by a large number of DNA-binding transcription factors. Sin3A has been closely linked to p53 while Sin3B is considered to be a close associate of E2Fs. The theme of this study was to establish the role of Sin3B in p53-mediated gene repression. We demonstrate a direct protein-protein interaction between human p53 and Sin3B (hSin3B). Amino acids 1-399 of hSin3B protein are involved in its interaction with N-terminal region (amino acids 1-108) of p53. Genotoxic stress induced by Adriamycin treatment increases the levels of hSin3B that is recruited to the promoters of p53-target genes (HSPA8, MAD1 and CRYZ). More importantly recruitment of hSin3B and repression of the three p53-target promoters upon Adriamycin treatment were observed only in p53(+/+) cell lines. Additionally an increased tri-methylation of the H3K9 residue at the promoters of HSPA8 and CRYZ was also observed following Adriamycin treatment. The present study highlights for the first time the essential role of Sin3B as an important associate of p53 in mediating the cellular responses to stress and in the transcriptional repression of genes encoding for heat shock proteins or proteins involved in regulation of cell cycle and apoptosis.

Human and yeast zeta-crystallins bind AU-rich elements in RNA.

Zeta-crystallins constitute a family of proteins with NADPH:quinone reductase activity found initially in mammalian lenses but now known to be present in many other organisms and tissues. Few proteins from this family have been characterized, and their function remains unclear. In the present work, zeta-crystallins from human and yeast (Zta1p) were expressed, purified and characterized. Both enzymes are able to reduce ortho-quinones in the presence of NADPH but are not active with 2-alkenals. Deletion of the ZTA1 gene makes yeast more sensitive to menadione and hydrogen peroxide, suggesting a role in the oxidative stress response. The human and yeast enzymes specifically bind to adenine-uracil rich elements (ARE) in RNA, indicating that both enzymes are ARE-binding proteins and that this property has been conserved in zeta-crystallins throughout evolution. This supports a role for zeta-crystallins as trans-acting factors that could regulate the turnover of certain mRNAs.