Rainbow Trout (Oncorhynchus mykiss) Na+/H+ Exchangers tNhe3a and tNhe3b Display Unique Inhibitory Profiles Dissimilar from Mammalian NHE Isoforms.

Freshwater fishes maintain an internal osmolality of ~300 mOsm, while living in dilute environments ranging from 0 to 50 mOsm. This osmotic challenge is met at least partially, by Na+/H+ exchangers (NHE) of fish gill and kidney. In this study, we cloned, expressed, and pharmacologically characterized fish-specific Nhes of the commercially important species Oncorhynchus mykiss. Trout (t) Nhe3a and Nhe3b isoforms from gill and kidney were expressed and characterized in an NHE-deficient cell line. Western blotting and immunocytochemistry confirmed stable expression of the tagged trout tNhe proteins. To measure NHE activity, a transient acid load was induced in trout tNhe expressing cells and intracellular pH was measured. Both isoforms demonstrated significant activity and recovered from an acute acid load. The effect of the NHE transport inhibitors amiloride, EIPA (5-(N-ethyl-N-isopropyl)-amiloride), phenamil, and DAPI was examined. tNhe3a was inhibited in a dose-dependent manner by amiloride and EIPA and tNhe3a was more sensitive to amiloride than EIPA, unlike mammalian NHE1. tNhe3b was inhibited by high concentrations of amiloride, while even in the presence of high concentrations of EIPA (500 µM), some activity of tNhe3b remained. Phenamil and DAPI were ineffective at inhibiting tNhe activity of either isoform. The current study aids in understanding the pharmacology of fish ion transporters. Both isoforms display inhibitory profiles uniquely different from mammalian NHEs and show resistance to inhibition. Our study allows for more direct interpretation of past, present, and future fish-specific sodium transport studies, with less reliance on mammalian NHE data for interpretation.

Evidence for convergent sensing of multiple abiotic stresses in cyanobacteria.

BACKGROUND: Bacteria routinely utilize two-component signal transduction pathways to sense and alter gene expression in response to environmental cues. While cyanobacteria express numerous two-component systems, these pathways do not regulate all of the genes within many of the identified abiotic stress-induced regulons. METHODS: Electron transport inhibitors combined with western analysis and measurement of chlorophyll a fluorescent yield, using pulse amplitude modulation fluorometry, were used to detect the effect of a diverse range of abiotic stresses on the redox status of the photosynthetic electron transport chain and the accumulation and degradation of the Synechocystis sp. PCC 6803 DEAD box RNA helicase, CrhR. RESULTS: Alterations in CrhR abundance were tightly correlated with the redox poise of the electron transport chain between QA and cytochrome b6f, with reduction favoring CrhR accumulation. CONCLUSIONS: The results provide evidence for an alternative, convergent sensing mechanism mediated through the redox poise of QB/PQH2 that senses multiple, divergent forms of abiotic stress and regulates accumulation of CrhR. The RNA helicase activity of CrhR could then function as a post-translational effector to regulate downstream gene expression. GENERAL SIGNIFICANCE: The potential for a related system in Staphylococcus aureus and higher plant chloroplasts suggest convergent sensing mechanisms may be evolutionarily conserved and occur more widely than anticipated.

Inactivation of the RNA helicase CrhR impacts a specific subset of the transcriptome in the cyanobacterium Synechocystis sp. PCC 6803.

DEAD-box RNA-helicases catalyze the reorganization of structured RNAs and the formation of RNP complexes. The cyanobacterium Synechocystis sp. PCC 6803 encodes a single DEAD-box RNA helicase, CrhR (Slr0083), whose expression is regulated by abiotic stresses that alter the redox potential of the photosynthetic electron transport chain, including temperature downshift. Despite its proposed effect on RNA metabolism and its known relevance in cold-stress adaptation, the reported impact of a CrhR knockout on the cold adaption of the transcriptome only identified eight affected genes. Here, we utilized a custom designed microarray to assess the impact of the absence of CrhR RNA helicase activity on the transcriptome, independent of cold stress. CrhR truncation impacts an RNA subset comprising ~10% of the ncRNA and also ~10% of the mRNA transcripts. While equal numbers of mRNAs showed increased as well as decreased abundance, more than 90% of the ncRNAs showed enhanced expression in the absence of CrhR, indicative of a negative effect on ncRNA transcription or stability. We further tested the effect of CrhR on the stability of strongly responding RNAs that identify examples of post-transcriptional and transcriptional regulation. The data suggest that CrhR impacts multiple aspects of RNA metabolism in Synechocystis.

The role of the Human Metabolome Database in inborn errors of metabolism.

Metabolomics holds considerable promise to advance our understanding of human disease, including our understanding of inborn errors of metabolism (IEM). The application of metabolomics in IEM research has already led to the discovery of several novel IEMs and the identification of novel IEM biomarkers. However, with hundreds of known IEMs and more than 700 associated IEM metabolites, it is becoming increasingly challenging for clinical researchers to keep track of IEMs, their associated metabolites, and their corresponding metabolic mechanisms. Furthermore, when using metabolomics to assist in IEM biomarker discovery or even in IEM diagnosis, it is becoming much more difficult to properly identify metabolites from the complex NMR and MS spectra collected from IEM patients. To that end, comprehensive, open access metabolite databases that provide up-to-date referential information about metabolites, metabolic pathways, normal/abnormal metabolite concentrations, and reference NMR or MS spectra for compound identification are essential. Over the last few years, a number of compound databases, including the Human Metabolome Database (HMDB), have been developed to address these challenges. First described in 2007, the HMDB is now the world's largest and most comprehensive metabolomic resource for human metabolic studies. The latest release of the HMDB contains 114,100 metabolite entries (with 247 being relevant to IEMs), thousands of metabolite concentrations (with 600 being relevant to IEMs), and ~33,000 metabolic and disease-associated pathways (with 202 being relevant to IEMs). Here we provide a summary of the HMDB and offer some guidance on how it can be used in metabolomic studies of IEMs.

Characterization of developmental Na(+) uptake in rainbow trout larvae supports a significant role for Nhe3b.

Developing freshwater fish must compensate for the loss of ions, including sodium (Na(+)), to the environment. In this study, we used a radiotracer flux approach and pharmacological inhibitors to investigate the role of sodium/hydrogen exchange proteins (Nhe) in Na(+) uptake in rainbow trout (Oncorhynchus mykiss) reared from fertilization in soft water (0.1mM Na(+)). For comparison, a second group of embryos/larvae reared in hard water (2.2mM Na(+), higher pH and [Ca(2+)]) were also included in the experiment but were fluxed in soft water, only. Unidirectional rates of Na(+) uptake increased throughout development and were significantly higher in embryos/larvae reared in soft water. However, the mechanisms of Na(+) uptake in both groups of larvae were not significantly different, either in larvae immediately post-hatch or later in development: the broad spectrum Na(+) channel blocker amiloride inhibited 85-90% of uptake and the Nhe-inhibitor EIPA also caused near maximal inhibitions of Na(+) uptake. These data indicated Na(+) uptake was Nhe-mediated in soft water. A role of Nhe3b (but not Nhe2 or Nhe3a) in Na(+) uptake in soft water was also supported through gene expression analyses: expression of nhe3b increased throughout development in whole embryos/larvae in both groups and was significantly higher in those reared in soft water. This pattern of expression correlated well with measurements of Na(+) uptake. Together these data indicate that in part, rainbow trout embryos/larvae reared in low Na(+) soft water maintained Na(+) homeostasis by an EIPA sensitive component of Na(+) uptake, and support a primary role for Nhe3b.

Mechanisms of Cl(-) uptake in rainbow trout: cloning and expression of slc26a6, a prospective Cl(-)/HCO3(-) exchanger.

In fresh waters, fishes continuously acquire ions to offset diffusive losses to a more dilute ambient environment and to maintain acid-base status. The objectives of the present study were to clone slc26a6, a prospective Cl(-)/HCO3(-) exchanger from rainbow trout, investigate its expression patterns in various tissues, at different developmental stages and after differential salinity exposure, and probe the mechanisms of Cl(-) uptake in rainbow trout embryos during development using a pharmacological inhibitor approach combined with (36)Cl(-) unidirectional fluxes. Results showed that the cloned gene encoded a 783 amino acid protein with conserved domains characteristic of the SLC26a family of anion exchange proteins. Phylogenetic analysis of this sequence against all subfamilies of the SLC26a family demonstrated that this translated protein shared a common ancestor with other actinopterygii and mammalian SLC26a6 isoforms and thus confirmed the identity of the cloned gene. Expression of slc26a6 was detected in all tissues and developmental stages assayed but was highest in the gill of juvenile trout. In trout embryos, Cl(-) uptake increased significantly post-hatch and was demonstrated to be mediated via an anion exchanger specific (DIDS sensitive) pathway that was also sensitive to hypercapnia. This parallels well with the predicted function of slc26a6, and the detection of the transcript in embryos and tissues of trout. In conclusion, this study is the first report of slc26a6 in rainbow trout and functional and expression analyses indicate its likely involvement in Cl(-)/HCO3(-) exchange in two life stages of rainbow trout.

Acid-sensing ion channels are involved in epithelial Na+ uptake in the rainbow trout Oncorhynchus mykiss.

A role for acid-sensing ion channels (ASICs) to serve as epithelial channels for Na(+) uptake by the gill of freshwater rainbow trout was investigated. We found that the ASIC inhibitors 4',6-diamidino-2-phenylindole and diminazene decreased Na(+) uptake in adult rainbow trout in a dose-dependent manner, with IC50 values of 0.12 and 0.96 µM, respectively. Furthermore, we cloned the trout ASIC1 and ASIC4 homologs and demonstrated that they are expressed differentially in the tissues of the rainbow trout, including gills and isolated mitochondrion-rich cells. Immunohistochemical analysis using custom-made anti-zASIC4.2 antibody and the Na(+)-K(+)-ATPase (α5-subunit) antibody demonstrated that the trout ASIC localizes to Na(+)/K(+)-ATPase-rich cells in the gill. Moreover, three-dimensional rendering of confocal micrographs demonstrated that ASIC is found in the apical region of mitochondrion-rich cells. We present a revised model whereby ASIC4 is proposed as one mechanism for Na(+) uptake from dilute freshwater in the gill of rainbow trout.

Conditional, temperature-induced proteolytic regulation of cyanobacterial RNA helicase expression.

Conditional proteolysis is a crucial process regulating the abundance of key regulatory proteins associated with the cell cycle, differentiation pathways, or cellular response to abiotic stress in eukaryotic and prokaryotic organisms. We provide evidence that conditional proteolysis is involved in the rapid and dramatic reduction in abundance of the cyanobacterial RNA helicase, CrhR, in response to a temperature upshift from 20 to 30°C. The proteolytic activity is not a general protein degradation response, since proteolysis is only present and/or functional in cells grown at 30°C and is only transiently active at 30°C. Degradation is also autoregulatory, since the CrhR proteolytic target is required for activation of the degradation machinery. This suggests that an autoregulatory feedback loop exists in which the target of the proteolytic machinery, CrhR, is required for activation of the system. Inhibition of translation revealed that only elongation is required for induction of the temperature-regulated proteolysis, suggesting that translation of an activating factor was already initiated at 20°C. The results indicate that Synechocystis responds to a temperature shift via two independent pathways: a CrhR-independent sensing and signal transduction pathway that regulates induction of crhR expression at low temperature and a CrhR-dependent conditional proteolytic pathway at elevated temperature. The data link the potential for CrhR RNA helicase alteration of RNA secondary structure with the autoregulatory induction of conditional proteolysis in the response of Synechocystis to temperature upshift.

Autoregulation of RNA helicase expression in response to temperature stress in Synechocystis sp. PCC 6803.

RNA helicases are ubiquitous enzymes whose modification of RNA secondary structure is known to regulate RNA function. The pathways controlling RNA helicase expression, however, have not been well characterized. Expression of the cyanobacterial RNA helicase, crhR, is regulated in response to environmental signals that alter the redox poise of the electron transport chain, including light and temperature. Here we analyze crhR expression in response to alteration of abiotic conditions in wild type and a crhR mutant, providing evidence that CrhR autoregulates its own expression through a combination of transcriptional and post-transcriptional mechanisms. Temperature regulates crhR expression through alteration of both transcript and protein half-life which are significantly extended at low temperature (20°C). CrhR-dependent mechanisms regulate both the transient accumulation of crhR transcript at 20°C and stability of the CrhR protein at all temperatures. CrhR-independent mechanisms regulate temperature sensing and induction of crhR transcript accumulation at 20°C and the temperature regulation of crhR transcript stability, suggesting CrhR is not directly associated with crhR mRNA turnover. Many of the processes are CrhR- and temperature-dependent and occur in the absence of a correlation between crhR transcript and protein abundance. The data provide important insights into not only how RNA helicase gene expression is regulated but also the role that rearrangement of RNA secondary structure performs in the molecular response to temperature stress. We propose that the crhR-regulatory pathway exhibits characteristics similar to the heat shock response rather than a cold stress-specific mechanism.

Inactivation of a low temperature-induced RNA helicase in Synechocystis sp. PCC 6803: physiological and morphological consequences.

Inactivation of the DEAD box RNA helicase, crhR, has dramatic effects on the physiology and morphology of the photosynthetic cyanobacterium, Synechocystis sp. PCC 6803. These effects are observed at both normal growth temperature (30°C) and under cold stress (20°C), indicating that CrhR performs crucial function(s) at all temperatures. A major physiological effect is the rapid cessation of photosynthesis upon temperature downshift from 30 to 20°C. This defect does not originate from an inability to transport or accumulate inorganic carbon or a deficiency in photosynthetic capacity as the mutant has sufficient electron transport and enzymatic capacity to sustain photosynthesis at 30°C and inorganic carbon (Ci) accumulation at 20°C. Oxygen consumption in the presence of methyl viologen indicated that while electron transport capacity is sufficient to accumulate Ci, the mutant does not possess sufficient activity to sustain carbon fixation at maximal rates. These defects are correlated with severely impaired cell growth and decreased viability, cell size and DNA content at low temperature. The ΔcrhR mutant also progressively accumulates structural abnormalities at low temperature that cannot be attributed solely to reactive oxygen species (ROS)-induced photooxidative damage, suggesting that they are manifestations of pre-existing defects that are amplified over time. The data indicate that the observed physiological and morphological effects are intimately related to crhR mutation, implying that the lack of CrhR RNA unwinding/annealing activity results in the inability to execute one or more vital steps in photosynthesis that are required at all temperatures but are crucial at low temperature.

RNA structural rearrangement via unwinding and annealing by the cyanobacterial RNA helicase, CrhR.

Rearrangement of RNA secondary structure is crucial for numerous biological processes. RNA helicases participate in these rearrangements through the unwinding of duplex RNA. We report here that the redox-regulated cyanobacterial RNA helicase, CrhR, is a bona fide RNA helicase possessing both RNA-stimulated ATPase and bidirectional ATP-stimulated RNA helicase activity. The processivity of the unwinding reaction appears to be low, because RNA substrates containing duplex regions of 41 bp are not unwound. CrhR also catalyzes the annealing of complementary RNA into intermolecular duplexes. Uniquely and in contrast to other proteins that perform annealing, the CrhR-catalyzed reactions require ATP hydrolysis. Through a combination of the unwinding and annealing activities, CrhR also catalyzes RNA strand exchange resulting in the formation of RNA secondary structures that are too stable to be resolved by helicase activity. RNA strand exchange most probably occurs through the CrhR-dependent formation and resolution of an RNA branch migration structure. Demonstration that another cyanobacterial RNA helicase, CrhC, does not catalyze annealing indicates that this activity is not a general biochemical characteristic of RNA helicases. Biochemically, CrhR resembles RecA and related proteins that catalyze strand exchange and branch migration on DNA substrates, a characteristic that is reflected in the recently reported structural similarities between these proteins. The data indicate the potential for CrhR to catalyze dynamic RNA secondary structure rearrangements through a combination of RNA helicase and annealing activities.