Functional and structural characterization of a eurytolerant calsequestrin from the intertidal teleost Fundulus heteroclitus.
ABSTRACT: Calsequestrins (CSQ) are high capacity, medium affinity, calcium-binding proteins present in the sarcoplasmic reticulum (SR) of cardiac and skeletal muscles. CSQ sequesters Ca²? during muscle relaxation and increases the Ca²?-storage capacity of the SR. Mammalian CSQ has been well studied as a model of human disease, but little is known about the environmental adaptation of CSQ isoforms from poikilothermic organisms. The mummichog, Fundulus heteroclitus, is an intertidal fish that experiences significant daily and seasonal environmental fluctuations and is an interesting study system for investigations of adaptation at the protein level. We determined the full-length coding sequence of a CSQ isoform from skeletal muscle of F. heteroclitus (FCSQ) and characterized the function and structure of this CSQ. The dissociation constant (K(d)) of FCSQ is relatively insensitive to changes in temperature and pH, thus indicating that FCSQ is a eurytolerant protein. We identified and characterized a highly conserved salt bridge network in FCSQ that stabilizes the formation of front-to-front dimers, a process critical to CSQ function. The functional profile of FCSQ correlates with the natural history of F. heteroclitus suggesting that the eurytolerant function of FCSQ may be adaptive.
Project description:The nuclear receptors (NRs) are ligand-dependent transcription factors that respond to various internal as well as external cues such as nutrients, pheromones, and steroid hormones that play crucial roles in regulation and maintenance of homeostasis and orchestrating the physiological and stress responses of an organism. We annotated the Fundulus heteroclitus (mummichog; Atlantic killifish) nuclear receptors. Mummichog are a non-migratory, estuarine fish with a limited home range often used in environmental research as a field model for studying ecological and evolutionary responses to variable environmental conditions such as salinity, oxygen, temperature, pH, and toxic compounds because of their hardiness. F. heteroclitus have at least 74 NRs spanning all seven gene subfamilies. F. heteroclitus is unique in that no RXR? member was found within the genome. Interestingly, some of the NRs are highly conserved between species, while others show a higher degree of divergence such as PXR, SF1, and AR?. Fundulus like other fish species show expansion of the RAR (NR1B), Rev-erb (NR1D), ROR (NR1F), COUPTF (NR2F), ERR (NR3B), RXR (NR2B), and to a lesser extent the NGF (NR4A), and NR3C steroid receptors (GR/AR). Of particular interest is the co-expansion of opposing NRs, Reverb-ROR, and RAR/RXR-COUPTF.
Project description:Most studies that examine the ontogeny of lymphoid organ development in teleostean fishes use species of interest to aquaculture or genetic research and, to date, have focused strictly on marine or freshwater species. The mummichog, Fundulus heteroclitus, also known as the estuarine killifish, is a unique model for studies on developmental immunobiology, because it is euryhaline, has a high degree of thermal tolerance, and has a unique reproductive strategy. Embryonic and larval mummichogs were examined for the ontogeny of lymphoid tissue development. The first lymphoid organ to appear was the head kidney at 1 dph, followed by the spleen at 1 wph, and then the thymus at 3 wph. Rag-1 was partially cloned and sequenced and shown to be highly conserved among other vertebrate Rag-1 genes. Using QT-PCR to monitor the temporal expression of Rag-1, it was shown to reach a maximum intensity at 3 and 4 wph and then to drop to pre-2-wph levels. Overall, this study suggests that juvenile mummichogs do not possess the ability to mount T- or B-cell responses until some time after 5 wph. Even though the estuarine killifish tolerates a wide range of salinities, the developmental patterns of lymphoid tissues are similar to what has been reported for strictly marine (stenohaline) teleosts. Thus, the mummichog should be a convenient model for understanding the developmental immunobiology of most marine teleosts.
Project description:Sarcoplasmic reticulum (SR) Ca(2+) release in striated muscle is mediated by a multiprotein complex that includes the ryanodine receptor (RyR) Ca(2+) channel and the intra-SR Ca(2+) buffering protein calsequestrin (CSQ). Besides its buffering role, CSQ is thought to regulate RyR channel function. Here, CSQ-dependent luminal Ca(2+) regulation of skeletal (RyR1) and cardiac (RyR2) channels is explored. Skeletal (CSQ1) or cardiac (CSQ2) calsequestrin were systematically added to the luminal side of single RyR1 or RyR2 channels. The luminal Ca(2+) dependence of open probability (Po) over the physiologically relevant range (0.05-1 mM Ca(2+)) was defined for each of the four RyR/CSQ isoform pairings. We found that the luminal Ca(2+) sensitivity of single RyR2 channels was substantial when either CSQ isoform was present. In contrast, no significant luminal Ca(2+) sensitivity of single RyR1 channels was detected in the presence of either CSQ isoform. We conclude that CSQ-dependent luminal Ca(2+) regulation of single RyR2 channels lacks CSQ isoform specificity, and that CSQ-dependent luminal Ca(2+) regulation in skeletal muscle likely plays a relatively minor (if any) role in regulating the RyR1 channel activity, indicating that the chief role of CSQ1 in this tissue is as an intra-SR Ca(2+) buffer.
Project description:Cr(III) is the dominant toxicant at some Superfund sites within the United States and therefore we are interested in its effects. Cr(III)s mechanisms are not well studied or understood because of its low bioavailability. We have attempted to characterize the effects of Cr(III) on gene expression in Fundulus heteroclitus (mummichog) liver. The NOEC and LOEC were determined at 32 and 64 mg/L, respectively, by measuring growth and mortality after exposing juveniles for 30 days. Secondary adult male exposures were performed at 32 mg/L, livers excised, and RNA extracted. Arrays were probed with cDNA from untreated or Cr(III)-exposed adult fish and gene expression was quantified. Cr(III) at 32 mg/L altered the expression of five genes, including GSTtau, GSTalpha, and ALDH4. Ultimately, we anticipate using this gene expression information to ascertain whether Cr(III) is bioavailable at potentially adverse concentrations in contaminated sites.
Project description:Increasing evidence suggests that polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are localized to the mitochondria. Because the toxic effects of many PAHs are the result of metabolism by cytochrome P4501A (CYP1A), it is important to investigate whether active forms of these enzymes can be identified in the mitochondria. In this study, we identified mitochondrial P450s with a monoclonal antibody against scup (Stenotomus chrysops) CYP1A in the isolated mitochondrial fraction of the liver from adult male mummichog (Fundulus heteroclitus) livers. The size of the protein in the mitochondria was similar to that of microsomal CYP1A. Fish dosed with 10mg/kg BaP had increased EROD activity in the mitochondrial fraction compared to controls. In mummichog larvae dosed with 100 microg/L BaP and 100 microg/L benzo[k]fluoranthene, CYP1A protein levels as well as enzyme activity were elevated. However, fish from a PAH-polluted Superfund site (Elizabeth River, Portsmouth VA) showed recalcitrant mitochondrial CYP1A protein levels and enzyme activity in a similar manner to microsomal CYP1A.
Project description:In a previous study [Yamaguchi, Kawasaki and Kasai (1995) Biochem. Biophys. Res. Commun. 210, 648-653], we showed that the stilbene derivative 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid activates the Ca2+ channel in the sarcoplasmic reticulum (SR) in rabbit skeletal muscle, and it does not bind to the channel protein itself but to the SR 30 kDa protein. Furthermore, the 30 kDa protein was shown to bind to calsequestrin (CSQ), which is one of the regulators of the Ca2+ release channel in the SR. In the present study, we determined the partial amino acid sequence of the CSQ-binding 30 kDa protein and, consequently, this protein was proved to be highly similar to ADP/ATP translocase (AAT) expressed in the mitochondria in a variety of cells. By Western-blotting analysis, the CSQ-binding 30 kDa protein was recognized by the antibody raised against bovine cardiac AAT and, furthermore, depolarization-induced Ca2+ release monitored in the rabbit skeletal muscle triads was significantly activated by the antibody. As a result of cloning and sequencing of the cDNA encoding AAT of the rabbit skeletal muscle, the amino acid sequence was found to be the same as that of the CSQ-binding 30 kDa protein determined above. Furthermore, the expressed product of the cDNA encoding AAT in Escherichia coli was proved to bind to CSQ. These results suggest that AAT itself is expressed in the rabbit skeletal muscle SR and regulates the Ca2+ release from the SR; that is, excitation-contraction coupling of the skeletal muscle cell.
Project description:The goals of these studies are to explore the mechanisms that enable extreme physiological plasticity and that may account for evolutionary divergence of adaptive osmotic physiologies among taxa that occupy different osmotic niches. In a common-garden environment, we track physiological and genome expression responses to hypo-osmotic (freshwater) challenge during a time-course of acclimation, and contrast these responses within and between species. We seek to identify mechanisms that facilitate osmotic acclimation that are evolutionarily conserved between basal and derived physiologies, and identify mechanisms that are uniquely derived to enable the extreme osmotic plasticity exhibited by F. heteroclitus. Importantly, previous studies using a comparable experimental design have identified physiological changes and genome expression responses that are adaptive for populations of F. heteroclitus that live in fresh water. As such, this enables us to test whether mechanisms of adaptive micro-evolutionary divergence across osmotic gradients within F. heteroclitus are shared with the mechanisms that account for patterns of macro-evolutionary divergence between F. heteroclitus and F. majalis that we identify in this study. That is, are the targets of micro-evolutionary fine-tuning the same or different as the targets of macro-evolutionary divergence across osmotic boundaries? Population comparisons include between populations from Chesapeake Bay (CB), coastal Virginia (VA), and coastal Georgia (GA).
Project description:Cr(VI) is a common bioavailable toxic metal that can cause oxidative stress, DNA adducts, and perturb normal gene expression. Changes in gene expression are useful biomarkers of toxicant exposure that provide information about the health of an organism, its ability to adapt to its environment, and indicate potential toxicant-specific effects. Therefore, we developed a toxicology array to the estuarine sentinel species Fundulus heteroclitus, or mummichog. Juvenile mummichog were exposed to potassium dichromate for thirty days at concentrations from 0 to 24 mg/L of Cr(VI), and growth was measured to determine the NOEC (1.5 mg/L or 0.0288 mM) and LOEC (3 mg/L or 0.0577 mM). Body burdens from Cr(VI) exposed fish demonstrated a dose dependent increase and were inversely correlated to body weight. Cr(VI)-exposed juvenile mummichog differentially expressed greater than 20 genes in a dose-dependent manner, including hepatic glucose transporter 2, liver fatty acid binding protein, ATPase synthase 8, type II keratin, TBT binding protein, and complement component C3-2. Many of these genes are involved in energy metabolism or growth, which is consistent with the reduced growth caused by Cr(VI). Keywords: dose response Overall design: Juvenile mummichogs (Fundulus heteroclitus) were exposed to potassium chromate at either 0, 1.5 (0.0288 mM), or 3 mg/L (0.0577 mM) Cr(VI). Thirty day exposures began within 48 hours of mummichog hatching. Each treatment group had four 500mL plastic tanks with 6 fish per tank with static media renewals every 48 hours. Mummichogs were housed at 25C in a 14/10 light/dark cycle at 18‰ salinity. At the end of the exposure, half of the fish from each tank were euthanized and whole bodies were placed in TRI-Reagent® for RNA isolation (Sigma Chemical Co, St. Louis, MO), then stored at -80C. The other three fish in each tank were used for chromium body burden determination.
Project description:The Atlantic Wood Industries Superfund site (AWI) on the Elizabeth River in Portsmouth, VA is heavily contaminated with polycyclic aromatic hydrocarbons (PAHs) from a wood treatment facility. Atlantic killifish, or mummichog (Fundulus heteroclitus), at this Superfund site are exposed to very high concentrations of several carcinogens. In this study, we measured PAH concentrations in both fish tissues and sediments. Concurrently, we assessed different aspects of genotoxicity in the killifish exposed in situ. Both sediment and tissue PAH levels were significantly higher in AWI samples, relative to a reference site, but the chemistry profile was different between sediments and tissues. Killifish at AWI exhibited higher levels of DNA damage compared to reference fish, as measured via the flow cytometric method (FCM), and the damage was consistent with sediment PAH concentrations. Covalent binding of benzo[a]pyrene (BaP) metabolites to DNA, as measured via LC-MS/MS adduct detection methods, were also elevated and could be partially responsible for the DNA damage. Using similar LC-MS/MS methods, we found no evidence that oxidative DNA adducts had a role in observed genotoxicity.