Selenoproteins in Archaea and Gram-positive bacteria

Selenium is an essential trace element for many organisms by serving important catalytic roles in the form of the 21st co-translationally inserted amino acid selenocysteine. It is mostly found in redox-active proteins in members of all three domains of life and analysis of the ever-increasing number of genome sequences has facilitated identification of the encoded selenoproteins. Available data from biochemical, sequence, and structure analyses indicate that Gram-positive bacteria synthesize and incorporate selenocysteine via the same pathway as enterobacteria. However, recent in vivo studies indicate that selenocysteine-decoding is much less stringent in Gram-positive bacteria than in Escherichia coli. For years, knowledge about the pathway of selenocysteine synthesis in Archaea and Eukarya was only fragmentary, but genetic and biochemical studies guided by analysis of genome sequences of Sec-encoding archaea has not only led to the characterization of the pathways but has also shown that they are principally identical. This review summarizes current knowledge about the metabolic pathways of Archaea and Gram-positive bacteria where selenium is involved, about the known selenoproteins, and about the respective pathways employed in selenoprotein synthesis.     

Dendritic LSm1/CBP80-mRNPs mark the early steps of transport commitment and translational control

Messenger RNA (mRNA) transport to neuronal dendrites is crucial for synaptic plasticity, but little is known of assembly or translational regulation of dendritic messenger ribonucleoproteins (mRNPs). Here we characterize a novel mRNP complex that is found in neuronal dendrites throughout the central nervous system and in some axonal processes of the spinal cord. The complex is characterized by the LSm1 protein, which so far has been implicated in mRNA degradation in nonneuronal cells. In brain, it associates with intact mRNAs. Interestingly, the LSm1-mRNPs contain the cap-binding protein CBP80 that associates with (pre)mRNAs in the nucleus, suggesting that the dendritic LSm1 complex has been assembled in the nucleus. In support of this notion, neuronal LSm1 is partially nuclear and inhibition of mRNA synthesis increases its nuclear localization. Importantly, CBP80 is also present in the dendrites and both LSm1 and CBP80 shift significantly into the spines upon stimulation of glutamergic receptors, suggesting that these mRNPs are translationally activated and contribute to the regulated local protein synthesis.     

The Hepatitis C Virus Non-structural NS5A Protein Impairs Both the Innate and Adaptive Hepatic Immune Response in Vivo

The role of hepatitis C virus (HCV) protein non-structural (NS) 5A in HCV-associated pathogenesis is still enigmatic. To investigate the in vivo role of NS5A for viral persistence and virus-associated pathogenesis a transgenic (Tg) mouse model was established. Mice with liver-targeted NS5A transgene expression were generated using the albumin promoter. Alterations in the hepatic immune response were determined by Western blot, infection by lymphocytic choriomeningitis virus (LCMV), and using transient NS3/4A Tg mice generated by hydrodynamic injection. Cytotoxic T lymphocyte (CTL) activity was investigated by the Cr-release assay. The stable NS5A Tg mice did not reveal signs of spontaneous liver disease. The intrahepatic immunity was disrupted in the NS5A Tg mice as determined by clearance of LCMV infection or transiently NS3/4A Tg hepatocytes in vivo. This impaired immunity was explained by a reduced induction of interferon β, 2′,5′-OAS, and PKR after LCMV infection and an impairment of the CTL-mediated elimination of NS3-expressing hepatocytes. In conclusion, these data indicate that in the present transgenic mouse model, NS5A does not cause spontaneous liver disease. However, we discovered that NS5A could impair both the innate and the adaptive immune response to promote chronic HCV infection.Chronic hepatitis C virus (HCV)⁴ infection is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma (HCC). The HCV genome is a single-stranded positive-sense RNA molecule of ∼9600 bp (1). The viral RNA codes for one large polyprotein of ∼3100 amino acids that is post-translationally processed by cellular and viral proteases, leading to the structural proteins core, E1 and E2, the p7 protein, and the non-structural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (2). The mature NS5A protein is generated by the action of the NS3/NS4A serine protease. NS5A is a phosphoprotein that exists in a basal or in a hyperphosphorylated state (p56 and p58) (3). Through an amphipathic α-helix, NS5A is associated with the cytoplasmic face of the ER (4) and is an integral part of the replication complex (5). Mutations in NS5A affect the rate of HCV replication suggesting a role of NS5A in modulating viral expression and replication (6). Moreover, NS5A is able to interfere with a variety of cellular proteins. Some of these interaction partners, such as Grb2, PI3K, p53, or Raf-1 are important key players in host cell signal transduction, enabling NS5A to deregulate important cellular check points (7–10). Recent reports even suggest that NS5A may deregulate cell cycle progression by modulating the expression of cell cycle regulatory genes (11). In light of these observations and that it has been suggested to transform murine fibroblasts (12), it is speculated that NS5A could represent an important factor for the development of HCV-associated HCC (13).Infection of transgenic mice expressing the complete HCV polyprotein with lymphocytic choriomeningitis virus (LCMV) showed a reduced IFN response and a delayed viral elimination (14). Cell culture-based experiments have shown that NS5A interacts directly with the interferon-dependent induced protein kinase R (PKR), a key player in the cellular antiviral response and that this interaction results in an inhibition of PKR function (15). Therefore, a role of NS5A for the establishment of a chronic HCV infection by inhibiting the innate immunity is conceivable.To enable in vivo studies of NS5A-specific effects transgenic mice were generated with a liver-specific expression of NS5A. We used these mice to show that NS5A affects both the innate and the adaptive hepatic immunity.     

KCNE beta subunits determine pH sensitivity of KCNQ1 potassium channels.

BACKGROUND/AIMS: Heteromeric KCNEx/KCNQ1 (=KvLQT1, Kv7.1) K(+) channels are important for repolarization of cardiac myocytes, endolymph secretion in the inner ear, gastric acid secretion, and transport across epithelia. They are modulated by pH in a complex way: homomeric KCNQ1 is inhibited by external acidification (low pH(e)); KCNE2/KCNQ1 is activated; and for KCNE1/KCNQ1, variable effects have been reported. Methods: The role of KCNE subunits for the effect of pH(e) on KCNQ1 was analyzed in transfected COS cells and cardiac myocytes by the patch-clamp technique. RESULTS: In outside-out patches of transfected cells, hKCNE2/hKCNQ1 current was increased by acidification down to pH 4.5. Chimeras with the acid-insensitive hKCNE3 revealed that the extracellular N-terminus and at least part of the transmembrane domain of hKCNE2 are needed for activation by low pH(e). hKCNE1/hKCNQ1 heteromeric channels exhibited marked changes of biophysical properties at low pH(e): The slowly activating hKCNE1/hKCNQ1 channels were converted into constitutively open, non-deactivating channels. Experiments on guinea pig and mouse cardiac myocytes pointed to an important role of KCNQ1 during acidosis implicating a significant contribution to cardiac repolarization under acidic conditions. CONCLUSION: External pH can modify current amplitude and biophysical properties of KCNQ1. KCNE subunits work as molecular switches by modulating the pH sensitivity of human KCNQ1.     

Two protein-protein interaction sites on the spliceosome-associated human cyclophilin CypH

Cyclophilins are a family of proteins that share a common, highly conserved sequence motif. Cyclophilins bind transiently to other proteins and facilitate their folding. One member of the family, hCypH, is part of the human spliceosomal [U4/U6·U5] tri-snRNP complex; it associates specifically and stably with the U4/U6-specific protein 60K. Here, we demonstrate that recombinant hCypH exhibits peptidyl–prolyl isomerase (PPIase) activity, and describe mutagenesis studies demonstrating that it shares the catalytic pocket with other members of the cyclophilin family. However, neither the PPIase activity nor the catalytic pocket is required for binding of protein 60K. Rather, hCypH contains a small insertion in a loop of the otherwise conserved cyclophilin backbone, and this minor change creates a highly specific binding site that is responsible for the association of this cyclophilin, but not others, with protein 60K. hCypH is thus the first small cyclophilin shown to have a second protein–protein interaction site and the ability to bind stably to another protein. Since the catalytic pocket and the second binding site are located on opposite sides of the cyclophilin structure, this opens up the interesting possibility that hCypH may serve as a bridge mediating interactions between protein 60K of the U4/U6 snRNP and other as yet unknown factors.     

Induction of larval settlement in the serpulid polychaete Hydroides elegans (Haswell): role of bacterial extracellular polymers

Larval settlement in the marine polychaete Hydroides elegans is effectively mediated upon contact with the surface of marine bacterial films. Using the bacterium Roseobacter litoralis as a model strain, the effect of bacterial extracellular polymers (exopolymers) on larval settlement of H. elegans was investigated. Bioassays with exopolymer fractions dissociated from bacterial films evoked the initial stages of the larval settlement process, i.e. larvae slowed down, secreted a mucous thread and crawled over the surface. This response is typical of larvae that encounter an attractive bacterial film. In contrast, bioassays with exopolymers in association with UV-irradiated, metabolically inactive bacterial films evoked complete settlement. However, the percentage of responding larvae was negatively correlated with the magnitude of UV-dosage. Since UV energy crosslinks both intra- and extracellular proteinaceous components, it could not be distinguished whether the decrease in larval settlement was due to a modification of proteinaceous components of exopolymers or due the elimination of cellular activity. Nevertheless, the results ascribe bacterial exopolymers the role of an indicator of substratum suitability and provide evidence that the polysaccharide moiety of exopolymers does not complement this effect.     

RNAi knockdown of hPrp31 leads to an accumulation of U4/U6 di-snRNPs in Cajal bodies

Cajal bodies (CBs) are subnuclear organelles of animal and plant cells. A role of CBs in the assembly and maturation of small nuclear ribonucleoproteins (snRNP) has been proposed but is poorly understood. Here we have addressed the question where U4/U6.U5 tri-snRNP assembly occurs in the nucleus. The U4/U6.U5 tri-snRNP is a central unit of the spliceosome and must be re-formed from its components after each round of splicing. By combining RNAi and biochemical methods, we demonstrate that, after knockdown of the U4/U6-specific hPrp31 (61 K) or the U5-specific hPrp6 (102 K) protein in HeLa cells, tri-snRNP formation is inhibited and stable U5 mono-snRNPs and U4/U6 di-snRNPs containing U4/U6 proteins and the U4/U6 recycling factor p110 accumulate. Thus, hPrp31 and hPrp6 form an essential connection between the U4/U6 and U5 snRNPs in vivo. Using fluorescence microscopy, we show that, in the absence of either hPrp31 or hPrp6, U4/U6 di-snRNPs as well as p110 accumulate in Cajal bodies. In contrast, U5 snRNPs largely remain in nucleoplasmic speckles. Our data support the idea that CBs may play a role in tri-snRNP recycling.