Biochemical and biophysical characterization of the nucleic acid binding properties of the RNA/DNA binding protein EWS.

EWS is a member of the FET family of RNA/DNA binding proteins that regulate crucial phases of nucleic acid metabolism. EWS comprises an N-terminal low-complexity domain (LCD) and a C-terminal RNA-binding domain (RBD). The RBD is further divided into three RG-rich regions, which flank an RNA-recognition motif (RRM) and a zinc finger (ZnF) domain. Recently, EWS was shown to regulate R-loops in Ewing sarcoma, a pediatric bone and soft-tissue cancer in which a chromosomal translocation fuses the N-terminal LCD of EWS to the C-terminal DNA binding domain of the transcription factor FLI1. Though EWS was shown to directly bind R-loops, the binding mechanism was not elucidated. In the current study, the RBD of EWS was divided into several constructs, which were subsequently assayed for binding to various nucleic acid structures expected to form at R-loops, including RNA stem-loops, DNA G-quadruplexes, and RNA:DNA hybrids. EWS interacted with all three nucleic acid structures with varying affinities and multiple domains contributed to binding each substrate. The RRM and RG2 region appear to bind nucleic acids promiscuously while the ZnF displayed more selectivity for single-stranded structures. With these results, the structural underpinnings of EWS recognition and binding of R-loops and other nucleic acid structures is better understood.

Evaluation of the binding of polyhedral borane anions to representative proteins.

The ability of three polyhedral borane anions, [B20H18](2-), [B20H17SH](4-), and [B20H19](3-), to bind to proteins was evaluated by measuring the total boron content using inductively coupled plasma-atomic emission spectroscopy after purification by gel electrophoresis. Results were correlated to the known chemical reactivity of the compounds as well as the reported murine biodistributions of the liposomally encapsulated sodium salts of each of the polyhedral borane anions. Qualitative reactions were performed with the [B20H18](2-) anion to determine the potential reactivity with simple molecular building blocks.

Characterization of telomeres and telomerase expression in Xiphophorus.

Research investigating telomere lengths and telomerase expression in vertebrates has progressively become important due to the association of these two biological endpoints with cellular aging and cancer in humans. Studies that rely upon the traditional use of laboratory mice have been faced with limitations largely due to inbred mice possessing large telomeres and ubiquitous expression of telomerase. Recently, a number of small fish species have been shown to provide potentially informative models for examining the role of telomeres and telomerase within intact vertebrate animals. Xiphophorus fishes represent a new world live-bearing genus that has not previously been assessed for telomere length or telomerase expression. To add to the knowledge base of telomere and telomerase biology in vertebrates we assessed telomere length and telomerase expression among several species of Xiphophorus. The telomere lengths in several organs (gill, brain, eyes, testis, ovary and liver) in three species (Xiphophorus hellerii, Xiphophorus maculatus, Xiphophorus couchianus) and also in F(1) interspecies hybrids were approximately 2-6 kb. This size was consistent within the same organs of the same species, as well as between species and F(1) hybrids. Despite possessing relatively short telomere lengths compared to humans, the consistency of size among Xiphophorus species and organs may allow experimental detection of telomere shortening. The relative expression of telomerase reverse transcriptase (TERT) was determined by quantitative real-time PCR. Expression levels of TERT was measured in seven organs (ovary, testis, liver, gill, brain, heart, skin) from X. maculatus, X. hellerii and in control and ultraviolet light (UVB) exposed skin samples from X. maculatus, X. hellerii, and F(1) interspecies hybrids. TERT gene expression was significantly higher in ovary and testis, while all other organs showed low relative TERT expression. Detectable increases in TERT expression were found in skin samples upon UVB exposure. Our findings suggest that Xiphophorus may serve as a suitable model for future studies investigating the association of telomere length and telomerase expression in regard to aging and disease.

Proteomic analyses of the Xiphophorus Gordon-Kosswig melanoma model.

Interspecies hybridization between the platyfish X. maculatus Jp 163 A, and the swordtail X. helleri (Sarabia), generates F(1) hybrids with pronounced melanin pigmentation. Backcrossing of F(1) hybrids with the X. helleri parent results in 25% of progeny that will spontaneously develop melanoma. We have applied proteomic methods to this Gordon-Kosswig (G-K) melanoma model to identify candidate proteins that exhibit modulated expression in fin tissue due to interspecies hybridization and progression of hybrid tissues to spontaneous melanoma. Difference Gel Electrophoresis (DIGE) was used to minimize the variability commonly observed in quantitative analyses of comparative protein samples. Following identification of up- or down-regulated protein expression by DIGE, candidate protein spots were identified by mass spectrometric sequencing. Several protein expression differences displayed in interspecies hybrids were identified and compared to distinct differences that occur upon backcrossing and progression to melanoma. These studies are important for the identification of distinct biochemical pathways involved in the variety of Xiphophorus interspecies hybrid tumor models.

Identification of robust hypoxia biomarker candidates from fin of medaka (Oryzias latipes).

Aquatic hypoxia caused by organic pollution and eutrophication is a pressing worldwide water pollution problem. Better methods for monitoring oxygen levels are needed to assist efforts to maintain and protect the health of natural aquatic environments. In this project, we used a Japanese ricefish (medaka, Oryzias latipes) 8K oligonucleotide array as a platform to identify potential hypoxic biomarkers in different organs (fin, gill, liver and brain) upon exposure to hypoxia. The microarray results were validated by qRT-PCR employing a subset of candidate biomarkers. Interestingly, the largest number and most significant of hypoxia responding array features were detected in hypoxia exposed fin tissues. We identified 173 array features that exhibited a significant response (over 2 fold change in expression) upon exposure to hypoxic conditions and validated a subset of these by quantitative RT-PCR. These gene targets were subjected to annotation and gene ontology mining. Positively identifiable gene targets that may be useful for development of a rapid and accurate biomarker test using fin clips are discussed in relation to previous reports on hypoxia responsive genes.

Comparison of gene expression responses to hypoxia in viviparous (Xiphophorus) and oviparous (Oryzias) fishes using a medaka microarray.

Gene expression profiling using DNA microarray technology is a useful tool for assessing gene transcript level responses after an organism is exposed to environmental stress. Herein, we detail results from studies using an 8 k medaka (Oryzias latipes) microarray to assess modulated gene expression patterns upon hypoxia exposure of the live-bearing aquaria fish, Xiphophorus maculatus. To assess the reproducibility and reliability of using the medaka array in cross-genus hybridization, a two-factor ANOVA analysis of gene expression was employed. The data show the tissue source of the RNA used for array hybridization contributed more to the observed response of modulated gene targets than did the species source of the RNA. In addition, hierarchical clustering via heat map analyses of groupings of tissues and species (Xiphophorus and medaka) suggests that hypoxia induced similar responses in the same tissues from these two diverse aquatic model organisms. Our Xiphophorus results indicate 206 brain, 37 liver, and 925 gill gene targets exhibit hypoxia induced expression changes. Analysis of the Xiphophorus data to determine those features exhibiting a significant (p<0.05)+/-3 fold change produced only two gene targets within brain tissue and 80 features within gill tissue. Of these 82 characterized features, 39 were identified via homology searching (cut-off E-value of 1 x 10(-5)) and placed into one or more biological process gene ontology groups. Among these 39 genes, metabolic energy changes and manipulation was the most affected biological pathway (13 genes).

Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure.

The epithelial Na(+) channel/degenerin (ENaC/DEG) protein family includes a diverse group of ion channels, including nonvoltage-gated Na(+) channels of epithelia and neurons, and the acid-sensing ion channel 1 (ASIC1). In mammalian epithelia, ENaC helps regulate Na(+) and associated water transport, making it a critical determinant of systemic blood pressure and pulmonary mucosal fluidity. In the nervous system, ENaC/DEG proteins are related to sensory transduction. While the importance and physiological function of these ion channels are established, less is known about their structure. One hallmark of the ENaC/DEG channel family is that each channel subunit has only two transmembrane domains connected by an exceedingly large extracellular loop. This subunit structure was recently confirmed when Jasti and colleagues determined the crystal structure of chicken ASIC1, a neuronal acid-sensing ENaC/DEG channel. By mapping ENaC to the structural coordinates of cASIC1, as we do here, we hope to provide insight toward ENaC structure. ENaC, like ASIC1, appears to be a trimeric channel containing 1alpha, 1beta, and 1gamma subunit. Heterotrimeric ENaC and monomeric ENaC subunits within the trimer possibly contain many of the major secondary, tertiary, and quaternary features identified in cASIC1 with a few subtle but critical differences. These differences are expected to have profound effects on channel behavior. In particular, they may contribute to ENaC insensitivity to acid and to its constitutive activity in the absence of time- and ligand-dependent inactivation. Experiments resulting from this comparison of cASIC1 and ENaC may help clarify unresolved issues related to ENaC architecture, and may help identify secondary structures and residues critical to ENaC function.

Investigation of the interactions of polyhedral borane anions with serum albumins.

The retention of polyhedral borane anions within tumor cells has been attributed to the possible formation of covalent bonds with nucleophilic protein substituents. In an effort to identify the nature of possible interactions between polyhedral borane anions and proteins, three polyhedral borane anions, [B(20)H(18)](2-), [B(20)H(17)OH](4-), and [B(20)H(17)SH](4-), were allowed to react with either bovine or human serum albumin. Reaction products were analyzed with matrix assisted laser desorption ionization (MALDI) mass spectrometry and gel electrophoresis. Evidence of disulfide bond formation was observed with the [B(20)H(17)SH](4-) anion, whereas no evidence of covalent binding was observed with the [B(20)H(18)](2-) and [B(20)H(17)OH](4-) ions. The potential for disulfide bond formation was confirmed by examining the reactions of the [B(20)H(17)SH](4-) ion with both DTNB and reduced glutathione. An understanding of the nature of the binding will provide a basis for the design and synthesis of boron-containing compounds for application in boron neutron capture therapy.

Functional reconstitution of the human epithelial Na+ channel in a mammalian expression system.

Probing ion channel structure-function and regulation in native tissue can, in some instances, be experimentally challenging or impractical. To facilitate discovery and increase experimental flexibility, our laboratory routinely reconstitutes recombinant ion channels in a mammalian expression system quantifying channel activity with patch clamp electrophysiology. Here, we describe investigation of the human epithelial Na+ channel heterologously expressed in Chinese hamster ovary cells.

Epithelial Na+ channel subunit stoichiometry.

Ion channels, including the epithelial Na(+) channel (ENaC), are intrinsic membrane proteins comprised of component subunits. Proper subunit assembly and stoichiometry are essential for normal physiological function of the channel protein. ENaC comprises three subunits, alpha, beta, and gamma, that have common tertiary structures and much amino acid sequence identity. For maximal ENaC activity, each subunit is required. The subunit stoichiometry of functional ENaC within the membrane remains uncertain. We combined a biophysical approach, fluorescence intensity ratio analysis, used to assess relative subunit stoichiometry with total internal reflection fluorescence microscopy, which enables isolation of plasma membrane fluorescence signals, to determine the limiting subunit stoichiometry of ENaC within the plasma membrane. Our results demonstrate that membrane ENaC contains equal numbers of each type of subunit and that at steady state, subunit stoichiometry is fixed. Moreover, we find that when all three ENaC subunits are coexpressed, heteromeric channel formation is favored over homomeric channels. Electrophysiological results testing effects of ENaC subunit dose on channel activity were consistent with total internal reflection fluorescence/fluorescence intensity ratio findings and confirmed preferential formation of heteromeric channels containing equal numbers of each subunit.

Fluorescence resonance energy transfer analysis of subunit stoichiometry of the epithelial Na+ channel.

Activity of the epithelial Na(+) channel (ENaC) is rate-limiting for Na(+) (re)absorption across electrically tight epithelia. ENaC is a heteromeric channel comprised of three subunits, alpha, beta, and gamma, with each subunit contributing to the functional channel pore. The subunit stoichiometry of ENaC remains uncertain with electrophysiology and biochemical experiments supporting both a tetramer with a 2alpha:1beta:1gamma stoichiometry and a higher ordered channel with a 3alpha:3beta:3gamma stoichiometry. Here we used an independent biophysical approach based upon fluorescence resonance energy transfer (FRET) between differentially fluorophore-tagged ENaC subunits to determine the subunit composition of mouse ENaC functionally reconstituted in Chinese hamster ovary and COS-7 cells. We found that when all three subunits were co-expressed, ENaC contained at least two of each type of subunit. Findings showing that ENaC subunits interact with similar subunits in immunoprecipitation studies are consistent with these FRET results. Upon native polyacrylamide gel electrophoresis, moreover, oligomerized ENaC runs predominantly as a single species with a molecular mass of >600 kDa. Because single ENaC subunits have a molecular mass of approximately 90 kDa, these results also agree with the FRET results. The current results as a whole, thus, are most consistent with a higher ordered channel possibly with a 3alpha:3beta:3gamma stoichiometry.

Regulation of Na+ transport by aldosterone: signaling convergence and cross talk between the PI3-K and MAPK1/2 cascades.

Cross talk between the phosphatidylinositol 3-kinase (PI3-K) and mitogen-activating protein kinase (MAPK)1/2 signaling cascades in response to aldosterone-induced K-RasA was investigated in renal A6 epithelial cells. In addition, the contribution of these signaling pathways to aldosterone-stimulated Na(+) transport was investigated. Aldosterone increased active K-RasA levels in A6 cells resulting in activation of downstream effectors in both the MAPK1/2 and PI3-K cascades with K-RasA directly interacting with the catalytic p110 subunit of PI3-K in a steroid-dependent manner. Aldosterone-stimulated PI3-K signaling impinged on the MAPK1/2 cascade at the level of Akt-mediated phosphorylation of c-Raf at an established negative regulatory site. Aldosterone also increased Sgk levels as well as stimulated phosphorylation of this kinase in a PI3-K- and K-RasA-dependent manner. Blockade of MAPK1/2 signaling had little effect on Na(+) transport. Conversely, inhibition of PI3-K markedly suppressed transport. Likewise, suppression of K-RasA induction decreased transport. However, Na(+) transport was subsequently stimulated under these conditions with the PLA(2) inhibitor aristolochic acid, an established positive modulator of Na(+) transport, suggesting that K-RasA signaling through PI3-K does not directly affect epithelial sodium channel (ENaC) levels but the activity of this channel. Consistent with this possibility, activity of ENaC reconstituted in Chinese hamster ovary cells was increased by coexpression of constitutively active PI3-K. The current study demonstrates that aldosterone increases Na(+) transport, in part, by stimulating PI3-K signaling and that during aldosterone actions, there is both signaling convergence between the two aldosterone-induced proteins, K-RasA and Sgk, as well as cross talk between the PI3-K and MAPK1/2 cascades with the prior but not latter cascade enhancing ENaC activity.

Human glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site.

BACKGROUND: Glutaminyl cyclase (QC) forms the pyroglutamyl residue at the amino terminus of numerous secretory peptides and proteins. We previously proposed the mammalian QC has some features in common with zinc aminopeptidases. We now have generated a structural model for human QC based on the aminopeptidase fold (pdb code 1AMP) and mutated the apparent active site residues to assess their role in QC catalysis. RESULTS: The structural model proposed here for human QC, deposited in the protein databank as 1MOI, is supported by a variety of fold prediction programs, by the circular dichroism spectrum, and by the presence of the disulfide. Mutagenesis of the six active site residues present in both 1AMP and QC reveal essential roles for the two histidines (140 and 330, QC numbering) and the two glutamates (201 and 202), while the two aspartates (159 and 248) appear to play no catalytic role. ICP-MS analysis shows less than stoichiometric zinc (0.3:1) in the purified enzyme. CONCLUSIONS: We conclude that human pituitary glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site residues. In contrast to the aminopeptidase, however, QC does not appear to require zinc for enzymatic activity.

Human pituitary glutaminyl cyclase: expression in insect cells and dye affinity purification.

Human pituitary glutaminyl cyclase (hQC) was expressed in Drosophila S2 cells under the control of an inducible metallothionene promoter and fused to the Drosophila immunoglobulin-binding protein signal sequence to enable secretion into the culture media. Expression levels reached 50 microg/mL culture media after 7 days of induction. The enzyme was purified to homogeneity directly from culture media by affinity chromatography on Reactive Blue 4-agarose using a step pH elution. The identity of the expressed protein was confirmed by peptide mass mapping and Western blotting. Glutaminyl cyclase was expressed as a fully active 37 kDa enzyme with kcat/Km values of 14.3, 9.3, and 2.4 mM(-1)s(-1) for the substrates Gln-Gln, Gln-NH(2), and Gln-t-butyl ester, respectively. The two cysteines were disulfide bonded, and the lone predicted glycosylation site, asparagine 49, was shown by both enzymatic deglycosylation of the expressed enzyme and site-directed mutagenesis to be glycosylated.

A region directly following the second transmembrane domain in gamma ENaC is required for normal channel gating.

We used a yeast one-hybrid complementation screen to identify regions within the cytosolic tails of the mouse alpha, beta, and gamma epithelial Na+ channel (ENaC) important to protein-protein and/or protein-lipid interactions at the plasma membrane. The cytosolic COOH terminus of alphaENaC contained a strongly interactive domain just distal to the second transmembrane region (TM2) between Met610 and Val632. Likewise, gammaENaC contained such a domain just distal to TM2 spanning Gln573-Pro600. Interactive domains were also localized within Met1-Gln54 and the last 17 residues of alpha- and betaENaC, respectively. Confocal images of Chinese hamster ovary cells transfected with enhanced green fluorescent fusion proteins of the cytosolic tails of mENaC subunits were consistent with results in yeast. Fusion proteins of the NH2 terminus of alphaENaC and the COOH termini of all three subunits co-localized with a plasma membrane marker. The functional importance of the membrane interactive domain in the COOH terminus of gammaENaC was established with whole-cell patch clamp experiments of wild type (alpha, beta, and gamma) and mutant (alpha, beta, and gammadeltaQ573-P600) mENaC reconstituted in Chinese hamster ovary cells. Mutant channels had about 13% of the activity of wild type channels with 0.33 +/- 0.14 versus 2.5 +/- 0.80 nA of amiloridesensitive inward current at -80 mV. Single channel analysis of recombinant channels demonstrated that mutant channels had a decrease in Po with 0.16 +/- 0.03 versus 0.67 +/- 0.07 for wild type. Mutant gammaENaC associated normally with the other two subunits in co-immunoprecipitation studies and localized to the plasma membrane in membrane labeling experiments and when visualized with evanescent-field fluorescence microscopy. Similar to deletion of Gln573-Pro600, deletion of Gln573-Arg583 but not Thr584-Pro600 decreased ENaC activity. The current results demonstrate that residues within Gln573-Arg583 of gammaENaC are necessary for normal channel gating.

Targeted degradation of ENaC in response to PKC activation of the ERK1/2 cascade.

Renal A6 epithelial cells were used to determine the mechanism by which protein kinase C (PKC) decreases epithelial Na(+) channel (ENaC) activity. Activation of PKC reduced relative Na(+) reabsorption to <20% within 60 min. This decrease was sustained over the next 24-48 h. In response to PKC signaling, alpha-, beta-, and gamma-ENaC levels were 0.97, 0.36, and 0.39, respectively, after 24 h, with the levels of the latter two subunits being significantly decreased. The PKC-mediated decreases in beta- and gamma-ENaC were significantly reversed by simultaneous addition of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase-1/2 inhibitors U-0126 and PD-98059. These inhibitors, in addition, protected Na(+) reabsorption from PKC, demonstrating that the MAPK1/2 cascade, in some instances, plays a central role in downregulation of ENaC activity. The effects of PKC on beta- and gamma-ENaC levels were additive with those of inhibitors of transcription (actinomycin D) and translation (emetine and cycloheximide), suggesting that PKC promotes subunit degradation and does not affect subunit synthesis. The bulk of whole cell gamma-ENaC was degraded within 1 h after treatment with inhibitors of synthesis; however, a significant pool was "protected" from inhibitors for up to 12 h. PKC affected this protected pool of gamma-ENaC. Moreover, proteosome inhibitors (MG-132 and lactacystin) reversed PKC effects on this protected pool of gamma-ENaC. Thus PKC signaling via MAPK1/2 cascade activation in A6 cells promotes degradation of gamma-ENaC.

Identification of cytoplasmic domains within the epithelial Na+ channel reactive at the plasma membrane.

The activity of membrane proteins is controlled, in part, by protein-protein interactions localized to the plasma membrane. In the current study, domains within the epithelial Na(+) channel (ENaC) reactive at the plasma membrane were identified using a novel yeast one-hybrid screen. The cytosolic N terminus of alphaENaC and the cytosolic C termini of alpha-, beta-, and gammaENaC contained domains reactive at the plasma membrane. Fluorescent micrographs of epithelial cells overexpressing fusion proteins of enhanced green fluorescent protein and mENaC cytosolic domains were consistent with those in yeast. A novel membrane reactive domain within the cytosolic C terminus of gamma-mENaC was localized to the 17 amino acids between residues Thr(584)-Pro(600). Two overlapping internalization signals within the C terminus of gamma-mENaC, a WW-binding domain (PY motif) and a tyrosine-based endocytic signal, were additive with respect to decreasing complementation and expression levels of hybrid proteins. Decreases in expression levels of hybrid proteins containing the PY and endocytic motif were reversed with latrunculin A, an inhibitor of endosomal movement. Decreases in complementation and expression levels of hybrid proteins mediated by the combined PY and overlapping endocytic motif proceeded in the absence of established ubiquitination sites within ENaC. In addition, the endocytic motif was active in the absence of the PY motif, demonstrating that these two domains, while possibly interacting, also have discrete functions. The novel domains within the cytosolic N terminus of alphaENaC and the C termini of alpha-, beta-, and gammaENaC identified here are likely to be involved in protein-protein and/or protein-lipid interactions localized to the plasma membrane. We hypothesize that these newly identified domains play a role in modulating ENaC activity.

Aldosterone.

Aldosterone plays a pivotal role in electrolyte and fluid homeostasis and thus control of blood pressure. The "classical" view of aldosterone action is that it targets epithelia of the distal colon and renal nephron to stimulate Na(+) (re)absorption and K(+) secretion. In these cells, aldosterone binds steroid receptors, promoting translocation to the nucleus, where they modulate gene expression with the induced proteins stimulating transport. This "genomic" action is dependent on transcription and translation and has a latency of 0.5-1.0 h. Recently, more rapid actions of aldosterone that are independent of transcription and translation have been described. These "nongenomic" actions are mediated by a distinct receptor that is insensitive to inhibitors of the classical mineralocorticoid receptor, such as spironolactone. The present review describes advances in our understanding of the classical model of aldosterone action as well as those that broaden this model to encompass nongenomic actions, nonepithelial targets, production of aldosterone outside of the adrenal gland, novel mechanisms of specificity, and novel mechanisms for mediating genomic actions.