The cellular protein P58IPK regulates influenza virus mRNA translation and replication through a PKR-mediated mechanism

We previously hypothesized that efficient translation of influenza virus mRNA requires the recruitment of P58(IPK), the cellular inhibitor of PKR, an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2alpha. P58(IPK) also inhibits PERK, an eIF2alpha kinase that is localized in the endoplasmic reticulum (ER) and induced during ER stress. The ability of P58(IPK) to interact with and inhibit multiple eIF2alpha kinases suggests it is a critical regulator of both cellular and viral mRNA translation. In this study, we sought to definitively define the role of P58(IPK) during viral infection of mammalian cells. Using mouse embryo fibroblasts from P58(IPK-/-) mice, we demonstrated that the absence of P58(IPK) led to an increase in eIF2alpha phosphorylation and decreased influenza virus mRNA translation. The absence of P58(IPK) also resulted in decreased vesicular stomatitis virus replication but enhanced reovirus yields. In cells lacking the P58(IPK) target, PKR, the trends were reversed-eIF2alpha phosphorylation was decreased, and influenza virus mRNA translation was increased. Although P58(IPK) also inhibits PERK, the presence or absence of this kinase had little effect on influenza virus mRNA translation, despite reduced levels of eIF2alpha phosphorylation in cells lacking PERK. Finally, we showed that influenza virus protein synthesis and viral mRNA levels decrease in cells that express a constitutively active, nonphosphorylatable eIF2alpha. Taken together, our results support a model in which P58(IPK) regulates influenza virus mRNA translation and infection through a PKR-mediated mechanism which is independent of PERK.     

Salinity regulates claudin mRNA and protein expression in the teleost gill

The teleost gill carries out NaCl uptake in freshwater (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight-junctional claudins during salinity acclimation in fish. We identified claudin 3- and claudin 4-like immunoreactive proteins and examined their expression and that of select ion transporters by performing Western blot in tilapia (Oreochromis mossambicus) gill during FW and SW acclimation. Transfer of FW tilapia to SW increased plasma osmolality, which was corrected after 4 days, coinciding with increased gill Na+-K+-ATPase and Na+-K+-2Cl(-) cotransporter expression. Gill claudin 3- and claudin 4-like proteins were reduced with exposure to SW. Transfer to FW increased both claudin-like proteins. Immunohistochemistry shows that claudin 3-like protein was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer, and staining appears more intense in the gill of FW versus SW fish. In addition, tilapia claudin 28a and 30 genes were characterized, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated with salinity acclimation and possibly the formation of deeper tight junctions in FW gill. This may reduce ion permeability, which is a critical facet of FW osmoregulation.     

Sig1R protein regulates hERG channel expression through a post-translational mechanism in leukemic cells

Sig1R (Sigma-1receptor) is a 25-kDa protein structurally unrelated to other mammalian proteins. Sig1R is present in brain, liver, and heart and is overexpressed in cancer cells. Studies using exogenous sigma ligands have shown that Sig1R interacts with a variety of ion channels, but its intrinsic function and mechanism of action remain unclear. The human ether-à-gogo related gene (hERG) encodes a cardiac channel that is also abnormally expressed in many primary human cancers, potentiating tumor progression through the modulation of extracellular matrix adhesive interactions. We show herein that sigma ligands inhibit hERG current density and cell adhesion to fibronectin in K562 myeloid leukemia cells. Heterologous expression in Xenopus oocytes demonstrates that Sig1R potentiates hERG current by stimulating channel subunit biosynthesis. Silencing Sig1R in leukemic K562 cells depresses hERG current density and cell adhesion to fibronectin by reducing hERG membrane expression. In K562 cells, Sig1R silencing does not modify hERG mRNA contents but reduces hERG mature form densities. In HEK cells expressing hERG and Sig1R, both proteins co-immunoprecipitate, demonstrating a physical association. Finally, Sig1R expression enhances both channel protein maturation and stability. Altogether, these results demonstrate for the first time that Sig1R controls ion channel expression through the regulation of subunit trafficking activity.     

Up-regulation of per mRNA expression by parathyroid hormone through a protein kinase A-CREB-dependent mechanism in chondrocytes

In bone, clock genes are involved in the circadian oscillation of bone formation and extracellular matrix expression. However, to date little attention has been paid to circadian rhythm in association with expression of clock genes during chondrogenesis in cartilage. In this study, we investigated the functional expression of different clock genes by chondrocytes in the course of cartilage development. The mRNA expression of types I, II, and X collagens exhibited a 24-h rhythm with a peak at zeitgeber time 6, in addition to a 24-h rhythmicity of all the clock genes examined in mouse femurs in vivo. Marked expression of different clock genes was seen in both osteoblastic MC3T3-E1 and chondrogenic ATDC5 cells in vitro, whereas parathyroid hormone (PTH) transiently increased period 1 (per1) mRNA expression at 1 h in both cell lines. Similar increases were seen in the mRNA levels for both per1 and per2 in prehypertrophic chondrocytes in metatarsal organotypic cultures within 2 h of exposure to PTH. PTH significantly activated the mouse per1 (mper1) and mper2 promoters but not the mper3 promoter in a manner sensitive to both a protein kinase A inhibitor and deletion of the cAMP-responsive element sequence (CRE) in ATDC5 cells. In HEK293 cells, introduction of brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (bmal1)/clock enhanced mouse type II collagen first intron reporter activity without affecting promoter activity, with reduction effected by either per1 or per2. These results suggest that PTH directly stimulates mper expression through a protein kinase A-CRE-binding protein signaling pathway for subsequent regulation of bmal1/clock-dependent extracellular matrix expression in cartilage.     

Enhancement of soluble protein expression through the use of fusion tags

The soluble expression of heterologous proteins in Escherichia coli remains a serious bottleneck in protein production. Although alteration of expression conditions can sometimes solve the problem, the best available tools to date have been fusion tags that enhance the solubility of expressed proteins. However, a systematic analysis of the utility of these solubility fusions has been difficult, and it appears that many proteins react differently to the presence of different solubility tags. The advent of high-throughput structural genomics programs and advances in cloning and expression technology afford us a new way to compare the effectiveness of solubility tags. This data should allow us to better predict the effectiveness of tags currently in use, and might also provide the information needed to identify new fusion tags.     

Control of protein expression through mRNA stability in calcium signalling

Specific sequences (cis-acting elements) in the 3'-untranslated region (UTR) of RNA, together with stabilizing and destabilizing proteins (trans-acting factors), determine the mRNA stability, and consequently, the level of expression of several proteins. Such interactions were discovered initially for short-lived mRNAs encoding cytokines and early genes like c-jun and c-myc. However, they may also determine the fate of more stable mRNAs in a tissue and disease-dependent manner. The interactions between the cis-acting elements and the trans-acting factors may also be modulated by Ca(2+) either directly or via a control of the phosphorylation status of the trans-acting factors. We focus initially on the basic concepts in mRNA stability with the trans-acting factors AUF1 (destabilizing) and HuR (stabilizing). Sarco/endoplasmic reticulum Ca(2+) pumps, SERCA2a (cardiac and slow twitch muscles) and SERCA2b (most cells including smooth muscle cells), are pivotal in Ca(2+) mobilization during signal transduction. SERCA2a and SERCA2b proteins are encoded by relatively stable mRNAs that contain cis-acting stability determinants in their 3'-regions. We present several pathways where 3'-UTR mediated mRNA decay is key to Ca(2+) signalling: SERCA2a and beta-adrenergic receptors in heart failure, renin-angiotensin system, and parathyroid hormones. Other examples discussed include cytokines vascular endothelial growth factor, endothelin and endothelial nitric oxide synthase. Roles of Ca(2+) and Ca(2+)-binding proteins in mRNA stability are also discussed. We anticipate that these novel modes of control of protein expression will form an emerging area of research that may explore the central role of Ca(2+) in cell function during development and in disease.     

Hypoxia regulates glutamate receptor trafficking through an HIF-independent mechanism

Oxygen influences behaviour in many organisms, with low levels (hypoxia) having devastating consequences for neuron survival. How neurons respond physiologically to counter the effects of hypoxia is not fully understood. Here, we show that hypoxia regulates the trafficking of the glutamate receptor GLR-1 in C. elegans neurons. Either hypoxia or mutations in egl-9, a prolyl hydroxylase cellular oxygen sensor, result in the internalization of GLR-1, the reduction of glutamate-activated currents, and the depression of GLR-1-mediated behaviours. Surprisingly, hypoxia-inducible factor (HIF)-1, the canonical substrate of EGL-9, is not required for this effect. Instead, EGL-9 interacts with the Mint orthologue LIN-10, a mediator of GLR-1 membrane recycling, to promote LIN-10 subcellular localization in an oxygen-dependent manner. The observed effects of hypoxia and egl-9 mutations require the activity of the proline-directed CDK-5 kinase and the CDK-5 phosphorylation sites on LIN-10, suggesting that EGL-9 and CDK-5 compete in an oxygen-dependent manner to regulate LIN-10 activity and thus GLR-1 trafficking. Our findings demonstrate a novel mechanism by which neurons sense and respond to hypoxia.     

Three soluble form messages of murine CD46 are produced through alternative mRNA splicing.

Murine CD46 (mCD46) is a type 1 membrane protein expressed predominantly in testicular germ cells, the distribution profile of which is in contrast to that of human CD46 showing a ubiquitous tissue distribution. We have identified an additional message of mCD46 that encodes a putative secretory form [Nomura et al. (1999) Immunogenetics 50, 245-254]. Here, we cloned three cDNAs encoding putative soluble CD46 from murine testis. These soluble form messages were yielded on insertion of unidentified nucleotide sequences, 77, 179, and 73 ntds, into the junctions between the SCR3 and SCR4 (variant 2), ST(c) and UK (variant 3), and SCR4 and ST(c) (variant 1) domains, respectively, the last one corresponding to the reported soluble form. The exons corresponding to these three inserts were identified in the murine CD46 genome, indicating that the alternative splicing of mRNA participates in the generation of these various CD46 messages. In normal mouse sera and cell lines, however, virtually no soluble CD46 was detected on immunoblotting. On Northern blotting analysis with specific probes, on the other hand, variant 1 was found to be predominantly expressed in the liver and heart. In addition, all variant messages were detected on PCR in all organs examined. When a rabbit cell line, RK13 cells, was transfected with cDNA of variant 1, protein synthesis was detected on immunoblotting. Although the mCD46 protein production was inefficient, this variant 1 exhibited factor I-cofactor activity as to inhibition of the complement cascade. Since the mCD46 protein was reported to be markedly up-regulated on infection of murine cells with mCMV, the soluble mCD46 proteins may act as a complement regulator that controls the systemic complement system under the conditions of a viral infection.     

Isolation of an mRNA encoding a soluble form of the human interleukin-6 receptor.

Soluble forms of the interleukin (IL)-2, IL-4 and IL-7 receptors which lack the transmembrane domain have been described. IL-6 is a growth factor important in the final differentiation of B-cells into plasma cells and in the pathogenesis of multiple myeloma. To determine whether the receptor for IL-6 may exist as a soluble molecule, RNA was analysed from the transformed B-cell lines U266, CESS and Daudi, from bone marrow from two myeloma patients, and from normal leukocytes. Using polymerase chain reaction, oligonucleotide primers which flank the transmembrane domain were selected to generate a 339 bp fragment. All samples produced equivalent amounts of the expected 339 bp fragment plus a smaller 245 bp fragment except Daudi which exhibited virtual absence of both. Sequence analysis of the smaller fragments from each of the five samples demonstrated the deletion of the entire transmembrane region from codons 356 (G-TG) to 387 (AG-G). The boundaries of this deletion were identical in all cases. Partial sequence analysis of the ligand-binding domain for U266 demonstrated identical sequences for the membrane-bound and soluble forms of the IL-6 receptor cDNAs. In summary, an mRNA which encodes a soluble form of the IL-6 receptor is expressed in both normal and myeloma cells.     

The hereditary hemochromatosis protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa cells

HFE is the protein product of the gene mutated in the autosomal recessive disease hereditary hemochromatosis (Feder, J. N., Gnirke, A., Thomas, W., Tsuchihashi, Z., Ruddy, D. A., Basava, A., Dormishian, F., Domingo, R. J., Ellis, M. C., Fullan, A., Hinton, L. M., Jones, N. L., Kimmel, B. E., Kronmal, G. S., Lauer, P., Lee, V. K., Loeb, D. B., Mapa, F. A., McClelland, E., Meyer, N. C., Mintier, G. A., Moeller, N., Moore, T., Morikang, E., Prasss, C. E., Quintana, L., Starnes, S. M., Schatzman, R. C., Brunke, K. J., Drayna, D. T., Risch, N. J., Bacon, B. R., and Wolff, R. R. (1996) Nat. Genet. 13, 399-408). At the cell surface, HFE complexes with transferrin receptor (TfR), increasing the dissociation constant of transferrin (Tf) for its receptor 10-fold (Gross, C. N., Irrinki, A., Feder, J. N., and Enns, C. A. (1998) J. Biol. Chem. 273, 22068-22074; Feder, J. N., Penny, D. M., Irrinki, A., Lee, V. K., Lebron, J. A., Watson, N. , Tsuchihashi, Z., Sigal, E., Bjorkman, P. J., and Schatzman, R. C. (1998) Proc. Natl. Acad. Sci. U S A 95, 1472-1477). HFE does not remain at the cell surface, but traffics with TfR to Tf-positive internal compartments (Gross et al., 1998). Using a HeLa cell line in which the expression of HFE is controlled by tetracycline, we show that the expression of HFE reduces 55Fe uptake from Tf by 33% but does not affect the endocytic or exocytic rates of TfR cycling. Therefore, HFE appears to reduce cellular acquisition of iron from Tf within endocytic compartments. HFE specifically reduces iron uptake from Tf, as non-Tf-mediated iron uptake from Fe-nitrilotriacetic acid is not altered. These results explain the decreased ferritin levels seen in our HeLa cell system and demonstrate the specific control of HFE over the Tf-mediated pathway of iron uptake. These results also have implications for the understanding of cellular iron homeostasis in organs such as the liver, pancreas, heart, and spleen that are iron loaded in hereditary hemochromatotic individuals lacking functional HFE.     

The soluble form of Bax regulates mitochondrial fusion via MFN2 homotypic complexes

In mammals, fusion of the mitochondrial outer membrane is controlled by two DRPs, MFN1 and MFN2, that function in place of a single outer membrane DRP, Fzo1 in yeast. We addressed the significance of two mammalian outer membrane fusion DRPs using an in?vitro mammalian mitochondrial fusion assay. We demonstrate that heterotypic MFN1-MFN2 trans complexes possess greater efficacy in fusion as compared to homotypic MFN1 or MFN2 complexes. In addition, we show that the soluble form of the proapoptotic Bcl2 protein, Bax,?positively regulates mitochondrial fusion exclusively through homotypic MFN2 trans complexes. Together, these data demonstrate functional and regulatory distinctions between MFN1 and MFN2 and provide insight into their unique physiological roles.     

Insulin regulates the expression of the insulin-like growth factor binding protein 2 mRNA in rat hepatocytes.

The goal of this study was to find out whether GH or insulin regulate the mRNA expression of the fetal binding protein of insulin-like growth factor (IGFBP-2). Primary hepatocytes from adult rats were used as a test system. IGFBP-2 mRNA was abundant in cells cultured in the absence of hormones and markedly reduced in cultures containing insulin. Addition of GH had no effect on IGFBP-2 mRNA levels although the cells are responsive to GH as demonstrated by a GH mediated elevation of IGF l mRNA levels. Half-maximal down-regulation of IGFBP-2 mRNA levels occurred at an insulin concentration of 1 to 2 x 10(-10) M. The finding that insulin is a potent negative regulator of hepatic IGFBP-2 mRNA levels suggests a physiologically important regulatory link between the two hormones insulin and IGF l.     

Age-dependent expression of hephaestin in the brain of ceruloplasmin-deficient mice

Aceruloplasminemia is an autosomal recessive disorder caused by mutations in the ceruloplasmin (CP) gene. It is characterized by iron accumulation in the brain and in visceral organs. However, little is known about the mechanism of iron transport in these regions. Adult CP null (CP^−/−) mice show increased iron deposition in several regions of brain, such as the cerebellum and brainstem. In this study, we investigated the expression of the ceruloplasmin homolog hephaestin (Heph) in the brain of CP^−/− mice as a function of age. In the cerebral cortex and caudate putamen of 80-week-old CP^−/− mice, the expression of Heph increased significantly whilst iron levels remain normal [Patel BN, Dunn RJ, Jeong SY, Zhu Q, Julien JP, David S. Ceruloplasmin regulates iron levels in the CNS and prevents free radical injury. J Neurosci 2002;22(15):6578–6], indicating that Heph might compensate for the loss of CP. In contrast, the substantia nigra and cerebellum of 80-week-old CP^−/− mice accumulate iron but do not express high levels or significant decrease of Heph, suggesting that Heph does not replace CP in these regions. These data suggest that Heph may compensate for the loss of CP in a region-specific manner.     

The URE2 protein regulates nitrogen catabolic gene expression through the GATAA-containing UASNTR element in Saccharomyces cerevisiae.

Many of the gene products that participate in nitrogen metabolism are sensitive to nitrogen catabolite repression (NCR), i.e., their expression is decreased to low levels when readily used nitrogen sources such as asparagine are provided. Previous work has shown this NCR sensitivity requires the cis-acting UASNTR element and trans-acting GLN3. Here, we extend the analysis to include the response of their expression to deletion of the URE2 locus. The expression of these nitrogen catabolic genes becomes, to various degrees, NCR insensitive in the ure2 deletion. This response is shown to be mediated through the GATAA-containing UASNTR element and supports the current idea that the NCR regulatory circuit involves the following steps: environmental signal-->URE2-->GLN3-->UASNTR operation-->NCR-sensitive gene expression. The various responses of the nitrogen catabolic genes' expression to deletion of the URE2 locus also indicate that not all NCR is mediated through URE2.