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.

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.

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.

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.

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.

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.

MDR quinone oxidoreductases: the human and yeast zeta-crystallins.

The medium-chain dehydrogenase/reductase (MDR) superfamily can be divided into Zn-containing and Zn-lacking proteins. Zn-containing MDRs are generally well-known enzymes, mostly acting as dehydrogenases. The non-Zn MDR are much less studied, and classified in several families of NADP(H)-dependent reductases, including quinone oxidoreductases (QOR). zeta-Crystallins are the best studied group of QOR, have a structural function in the lens of several mammals, exhibit ortho-quinone reductase activity, and bind to specific adenine-uracil-rich elements (ARE) in RNA. In the present work, we have further characterized human zeta-crystallin and Saccharomyces cerevisiae Zta1p, the only QOR in yeast. Subcellular localization using a fluorescent protein tag indicates that zeta-crystallin is distributed in the cytoplasm but not in nucleus. The protein may also be present in mitochondria. Zta1p localizes in both cytoplasm and nucleus. NADPH, but not NADH, competitively prevents binding of zeta-crystallin to RNA, suggesting that the cofactor-binding site is involved in RNA binding. Interference of NADPH on Zta1p binding to RNA is much lower, consistent with a weaker binding of NADPH to the yeast enzyme. Disruption of the yeast ZTA1 gene does not affect cell growth under standard conditions but makes yeast more sensitive to oxidative stress agents. Sequence alignments, phylogenetic tree analysis and kinetic properties reveal a close relationship between zeta-crystallin and Zta1p. Amino acid conservation, between the substrate-binding sites of the two proteins and that of an E. coli QOR, indicates that zeta-crystallins maintained their kinetic function throughout evolution. Quinones are toxic compounds and a relevant step in their detoxification is reduction to their corresponding hydroquinones. Many enzymes of several superfamilies can reduce quinones, including NAD(P)H:quinone oxidoreductase 1 (NQO1 or DT-diaphorase), aldo-keto reductases and short-chain dehydrogenases/reductases. In this context, the physiological role of zeta-crystallins is discussed.

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.

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.

pH-responsive stabilization of glutamate dehydrogenase mRNA in LLC-PK1-F+ cells.

During chronic metabolic acidosis, the adaptive increase in rat renal ammoniagenesis is sustained, in part, by increased expression of mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH) enzymes. The increase in GA activity results from the pH-responsive stabilization of GA mRNA. The 3'-untranslated region (3'-UTR) of GA mRNA contains a direct repeat of an eight-base AU-rich element (ARE) that binds zeta-crystallin/NADPH:quinone reductase (zeta-crystallin) with high affinity and functions as a pH-response element. RNA EMSAs established that zeta-crystallin also binds to the full-length 3'-UTR of GDH mRNA. This region contains four eight-base sequences that are 88% identical to one of the two GA AREs. Direct binding assays and competition studies indicate that the two individual eight-base AREs from GA mRNA and the four individual GDH sequences bind zeta-crystallin with different affinities. Insertion of the 3'-UTR of GDH cDNA into a beta-globin expression vector (pbetaG) produced a chimeric mRNA that was stabilized when LLC-PK1-F+ cells were transferred to acidic medium. A pH-responsive stabilization was also observed using a betaG construct that contained only the single GDH4 ARE and a destabilizing element from phosphoenolpyruvate carboxykinase mRNA. Therefore, during acidosis, the pH-responsive stabilization of GDH mRNA may be accomplished by the same mechanism that affects an increase in GA mRNA.

The NADPH:quinone oxidoreductase P1-zeta-crystallin in Arabidopsis catalyzes the alpha,beta-hydrogenation of 2-alkenals: detoxication of the lipid peroxide-derived reactive aldehydes.

P1-zeta-crystallin (P1-ZCr) is an oxidative stress-induced NADPH:quinone oxidoreductase in Arabidopsis thaliana, but its physiological electron acceptors have not been identified. We found that recombinant P1-ZCr catalyzed the reduction of 2-alkenals of carbon chain C(3)-C(9) with NADPH. Among these 2-alkenals, the highest specificity was observed for 4-hydroxy-(2E)-nonenal (HNE), one of the major toxic products generated from lipid peroxides. (3Z)-Hexenal and aldehydes without alpha,beta-unsaturated bonds did not serve as electron acceptors. In the 2-alkenal molecules, P1-ZCr catalyzed the hydrogenation of alpha,beta-unsaturated bonds, but not the reduction of the aldehyde moiety, to produce saturated aldehydes, as determined by gas chromatography/mass spectrometry. We propose the enzyme name NADPH:2-alkenal alpha,beta-hydrogenase (ALH). A major portion of the NADPH-dependent HNE-reducing activity in A. thaliana leaves was inhibited by the specific antiserum against P1-ZCr, indicating that the endogenous P1-ZCr protein has ALH activity. Because expression of the P1-ZCr gene in A. thaliana is induced by oxidative stress treatments, we conclude that P1-ZCr functions as a defense against oxidative stress by scavenging the highly toxic, lipid peroxide-derived alpha,beta-unsaturated aldehydes.