Cardiac expression of the cystic fibrosis transmembrane conductance regulator involves novel Exon 1 usage to produce a unique amino-terminal protein

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel present in many cells. In cardiomyocytes, we report that multiple exon 1 usage and alternative splicing produces four CFTR transcripts, with different 5'-untranslated regions, CFTRTRAD-139, CFTR-1C/-1A, CFTR-1C, and CFTR-1B. CFTR transcripts containing the novel upstream exons (exons -1C, -1B, and -1A) represent more than 90% of cardiac expressed CFTR mRNA. Regulation of cardiac CFTR expression, in response to developmental and pathological stimuli, is exclusively due to the modulation of CFTR-1C and CFTR-1C/-1A expression. Upstream open reading frames have been identified in the 5'-untranslated regions of all CFTR transcripts that, in conjunction with adjacent stem-loop structures, modulate the efficiency of translation initiation at the AUG codon of the main CFTR coding region in CFTRTRAD-139 and CFTR-1C/-1A transcripts. Exon(-1A), only present in CFTR-1C/-1A transcripts, encodes an AUG codon that is in-frame with the main CFTR open reading frame, the efficient translation of which produces a novel CFTR protein isoform with a curtailed amino terminus. As the expression of this CFTR transcript parallels the spatial and temporal distribution of the cAMP-activated whole-cell current density in normal and diseased hearts, we suggest that CFTR-1C/-1A provides the molecular basis for the cardiac cAMP-activated chloride channel. Our findings provide further insight into the complex nature of in vivo CFTR expression, to which multiple mRNA transcripts, protein isoforms, and post-transcriptional regulatory mechanisms are now added.

Novel characteristics of the biological properties of the yeast Saccharomyces cerevisiae eukaryotic initiation factor 2A

Eukaryotic initiation factor 2A (eIF2A) has been shown to direct binding of the initiator methionyl-tRNA (Met-tRNA(i)) to 40 S ribosomal subunits in a codon-dependent manner, in contrast to eIF2, which requires GTP but not the AUG codon to bind initiator tRNA to 40 S subunits. We show here that yeast eIF2A genetically interacts with initiation factor eIF4E, suggesting that both proteins function in the same pathway. The double eIF2A/eIF4E-ts mutant strain displays a severe slow growth phenotype, which correlated with the accumulation of 85% of the double mutant cells arrested at the G(2)/M border. These cells also exhibited a disorganized actin cytoskeleton and elevated actin levels, suggesting that eIF2A might be involved in controlling the expression of genes involved in morphogenic processes. Further insights into eIF2A function were gained from the studies of eIF2A distribution in ribosomal fractions obtained from either an eIF5BDelta (fun12Delta) strain or a eIF3b-ts (prt1-1) strain. It was found that the binding of eIF2A to 40 and 80 S ribosomes was not impaired in either strain. We also found that eIF2A functions as a suppressor of Ure2p internal ribosome entry site-mediated translation in yeast cells. The regulation of expression from the URE2 internal ribosome entry site appears to be through the levels of eIF2A protein, which has been found to be inherently unstable with a half-life of approximately 17 min. It was hypothesized that this instability allows for translational control through the level of eIF2A protein in yeast cells.

Saccharomyces Cerevisiae as a biotechnological tool for ageing research:studies on translation and metabolism

The yeast Saccharomyces cerevisiae is an important model organism for the study of cell biology. The similarity between yeast and human genes and the conservation of fundamental pathways means it can be used to investigate characteristics of healthy and diseased cells throughout the lifespan. Yeast is an equally important biotechnological tool that has long been the organism of choice for the production of alcoholic beverages, bread and a large variety of industrial products. For example, yeast is used to manufacture biofuels, lubricants, detergents, industrial enzymes, food additives and pharmaceuticals such as anti-parasitics, anti-cancer compounds, hormones (including insulin), vaccines and nutraceuticals. Its function as a cell factory is possible because of the speed with which it can be grown to high cell yields, the knowledge that it is generally recognized as safe (GRAS) and the ease with which metabolism and cellular pathways, such as translation can be manipulated. In this thesis, these two pathways are explored in the context of their biotechnological application to ageing research: (i) understanding translational processes during the high-yielding production of membrane protein drug targets and (ii) the manipulation of yeast metabolism to study the molecule, L-carnosine, which has been proposed to have anti-ageing properties. In the first of these themes, the yeast strains, spt3?, srb5?, gcn5? and yTHCBMS1, were examined since they have been previously demonstrated to dramatically increase the yields of a target membrane protein (the aquaporin, Fps1) compared to wild-type cells. The mechanisms underlying this discovery were therefore investigated. All high yielding strains were shown to have an altered translational state (mostly characterised by an initiation block) and constitutive phosphorylation of the translational initiation factor, eIF2a. The relevance of the initiation block was further supported by the finding that other strains, with known initiation blocks, are also high yielding for Fps1. A correlation in all strains between increased Fps1 yields and increased production of the transcriptional activator protein, Gcn4, suggested that yields are subject to translational control. Analysis of the 5´ untranslated region (UTR) of FPS1 revealed two upstream open reading frames (uORFs). Mutagenesis data suggest that high yielding strains may circumvent these control elements through either a leaky scanning or a re-initiation mechanism. In the second theme, the dipeptide L-carnosine (ß-alanyl-L-histidine) was investigated: it has previously been shown to inhibit the growth of cancer cells but delay senescence in cultured human fibroblasts and extend the lifespan of male fruit flies. To understand these apparently contradictory properties, the effects of L-carnosine on yeast were studied. S. cerevisiae can respire aerobically when grown on a non-fermentable carbon source as a substrate but has a respiro-fermentative metabolism when grown on a fermentable carbon source; these metabolisms mimic normal cell and cancerous cell metabolisms, respectively. When yeast were grown on fermentable carbon sources, in the presence of L-carnosine, a reduction in cell growth and viability was observed, which was not apparent for cells grown on a non-fermentable carbon source. The metabolism-dependent mechanism was confirmed in the respiratory yeast species Pichia pastoris. Further analysis of S. cerevisiae yeast strains with deletions in their nutrient-sensing pathway, which result in an increase in respiratory metabolism, confirmed the metabolism-dependent effects of L-carnosine.

Epigenetic and oncogenic regulation of SLC16A7 (MCT2) results in protein over-expression, impacting on signalling and cellular phenotypes in prostate cancer

Monocarboxylate Transporter 2 (MCT2) is a major pyruvate transporter encoded by the SLC16A7 gene. Recent studies pointed to a consistent overexpression of MCT2 in prostate cancer (PCa) suggesting MCT2 as a putative biomarker and molecular target. Despite the importance of this observation the mechanisms involved in MCT2 regulation are unknown. Through an integrative analysis we have discovered that selective demethylation of an internal SLC16A7/MCT2 promoter is a recurrent event in independent PCa cohorts. This demethylation is associated with expression of isoforms differing only in 5'-UTR translational control motifs, providing one contributing mechanism for MCT2 protein overexpression in PCa. Genes co-expressed with SLC16A7/MCT2 also clustered in oncogenic-related pathways and effectors of these signalling pathways were found to bind at the SLC16A7/MCT2 gene locus. Finally, MCT2 knock-down attenuated the growth of PCa cells. The present study unveils an unexpected epigenetic regulation of SLC16A7/MCT2 isoforms and identifies a link between SLC16A7/MCT2, Androgen Receptor (AR), ETS-related gene (ERG) and other oncogenic pathways in PCa. These results underscore the importance of combining data from epigenetic, transcriptomic and protein level changes to allow more comprehensive insights into the mechanisms underlying protein expression, that in our case provide additional weight to MCT2 as a candidate biomarker and molecular target in PCa.

Insights into the mechanisms of eukaryotic translation gained with ribosome profiling

The development of Ribosome Profiling (RiboSeq) has revolutionized functional genomics. RiboSeq is based on capturing and sequencing of the mRNA fragments enclosed within the translating ribosome and it thereby provides a â snapshotâ of ribosome positions at the transcriptome wide level. Although the method is predominantly used for analysis of differential gene expression and discovery of novel translated ORFs, the RiboSeq data can also be a rich source of information about molecular mechanisms of polypeptide synthesis and translational control. This review will focus on how recent findings made with RiboSeq have revealed important details of the molecular mechanisms of translation in eukaryotes. These include mRNA translation sensitivity to drugs affecting translation initiation and elongation, the roles of upstream ORFs in response to stress, the dynamics of elongation and termination as well as details of intrinsic ribosome behavior on the mRNA after translation termination. As the RiboSeq method is still at a relatively early stage we will also discuss the implications of RiboSeq artifacts on data interpretation.

Multiple recurrent genetic events converge on control of histone lysine methylation in medulloblastoma

We used high-resolution SNP genotyping to identify regions of genomic gain and loss in the genomes of 212 medulloblastomas, malignant pediatric brain tumors. We found focal amplifications of 15 known oncogenes and focal deletions of 20 known tumor suppressor genes (TSG), most not previously implicated in medulloblastoma. Notably, we identified previously unknown amplifications and homozygous deletions, including recurrent, mutually exclusive, highly focal genetic events in genes targeting histone lysine methylation, particularly that of histone 3, lysine 9 (H3K9). Post-translational modification of histone proteins is critical for regulation of gene expression, can participate in determination of stem cell fates and has been implicated in carcinogenesis. Consistent with our genetic data, restoration of expression of genes controlling H3K9 methylation greatly diminishes proliferation of medulloblastoma in vitro. Copy number aberrations of genes with critical roles in writing, reading, removing and blocking the state of histone lysine methylation, particularly at H3K9, suggest that defective control of the histone code contributes to the pathogenesis of medulloblastoma.

Post-translational modifications regulate distinct functions of CARMA1 and BCL10.

Activation of the transcription factor nuclear factor (NF)-kappaB is essential for the normal functioning of the immune system. Deregulated NF-kappaB signalling in lymphocytes can lead to immunodeficiency, but also to autoimmunity or lymphomas. Many of the signalling components controlling NF-kappaB activation in lymphocytes are now known, but it is less clear how distinct molecular components of this pathway are regulated. Here, we summarize recent findings on post-translational modifications of intracellular components of this pathway. Phosphorylation of the CARMA1 and BCL10 proteins and ubiquitylation of BCL10 affect the formation and stability of the CARMA1-BCL10-MALT1 (CBM) complex, and also control negative feedback regulation of the NF-kappaB signalling pathway. Moreover, the study of BCL10 phosphorylation isoforms has revealed a new mechanism controlling BCL10 nuclear translocation and an unexpected role for BCL10 in the regulation of the actin cytoskeleton.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA'-'lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Bridging basic science and clinical research - The EASL Monothematic Conference on Translational Research in Viral Hepatitis.

The EASL Monothematic Conference on Translational Research in Viral Hepatitis brought together a group of leading scientists and clinicians working on both, basic and clinical aspects of viral hepatitis, thereby building bridges from bench to bedside. This report recapitulates the presentations and discussions at the conference held in Lyon, France on November 29-30, 2013. In recent years, great advances have been made in the field of viral hepatitis, particularly in hepatitis C virus (HCV) infection. The identification of IL28B genetic polymorphisms as a major determinant for spontaneous and treatment-induced HCV clearance was a seminal discovery. Currently, hepatologists are at the doorstep of even greater advances, with the advent of a wealth of directly acting antivirals (DAAs) against HCV. Indeed, promising results have accumulated over the last months and few years, showing sustained virological response (SVR) rates of up to 100% with interferon-free DAA combination therapies. Thus, less than 25years after its identification, HCV infection may soon be curable in the vast majority of patients, highlighting the great success of HCV research over the last decades. However, viral hepatitis and its clinical complications such as liver cirrhosis and hepatocellular carcinoma (HCC) remain major global challenges. New therapeutic strategies to tackle hepatitis B virus (HBV) and hepatitis D virus (HDV) infection are needed, as current therapies have undeniable limitations. Nucleoside/nucleotide analogues (NUC) can efficiently control HBV replication and reduce or even reverse liver damage. However, these drugs have to be given for indefinite periods in most patients to maintain virological and biochemical responses. Although sustained responses off treatment can be achieved by treatment with (pegylated) interferon-α, only about 10-30% of patients effectively resolve chronic hepatitis B. It was the goal of this conference to review the progress made over the last years in chronic viral hepatitis research and to identify key questions that need to be addressed in order to close the gap between basic and clinical research and to develop novel preventive and treatment approaches for this most common cause of liver cirrhosis and HCC.

Pom1 regulates the assembly of Cdr2-Mid1 cortical nodes for robust spatial control of cytokinesis.

Proper division plane positioning is essential to achieve faithful DNA segregation and to control daughter cell size, positioning, or fate within tissues. In Schizosaccharomyces pombe, division plane positioning is controlled positively by export of the division plane positioning factor Mid1/anillin from the nucleus and negatively by the Pom1/DYRK (dual-specificity tyrosine-regulated kinase) gradients emanating from cell tips. Pom1 restricts to the cell middle cortical cytokinetic ring precursor nodes organized by the SAD-like kinase Cdr2 and Mid1/anillin through an unknown mechanism. In this study, we show that Pom1 modulates Cdr2 association with membranes by phosphorylation of a basic region cooperating with the lipid-binding KA-1 domain. Pom1 also inhibits Cdr2 interaction with Mid1, reducing its clustering ability, possibly by down-regulation of Cdr2 kinase activity. We propose that the dual regulation exerted by Pom1 on Cdr2 prevents Cdr2 assembly into stable nodes in the cell tip region where Pom1 concentration is high, which ensures proper positioning of cytokinetic ring precursors at the cell geometrical center and robust and accurate division plane positioning.

Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa.

The opportunistic human pathogen Pseudomonas aeruginosa is able to utilize a wide range of carbon and nitrogen compounds, allowing it to grow in vastly different environments. The uptake and catabolism of growth substrates are organized hierarchically by a mechanism termed catabolite repression control (Crc) whereby the Crc protein establishes translational repression of target mRNAs at CA (catabolite activity) motifs present in target mRNAs near ribosome binding sites. Poor carbon sources lead to activation of the CbrAB two-component system, which induces transcription of the small RNA (sRNA) CrcZ. This sRNA relieves Crc-mediated repression of target mRNAs. In this study, we have identified novel targets of the CbrAB/Crc system in P. aeruginosa using transcriptome analysis in combination with a search for CA motifs. We characterized four target genes involved in the uptake and utilization of less preferred carbon sources: estA (secreted esterase), acsA (acetyl-CoA synthetase), bkdR (regulator of branched-chain amino acid catabolism) and aroP2 (aromatic amino acid uptake protein). Evidence for regulation by CbrAB, CrcZ and Crc was obtained in vivo using appropriate reporter fusions, in which mutation of the CA motif resulted in loss of catabolite repression. CbrB and CrcZ were important for growth of P. aeruginosa in cystic fibrosis (CF) sputum medium, suggesting that the CbrAB/Crc system may act as an important regulator during chronic infection of the CF lung.

Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes.

In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs) is very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential (AP). Navs are composed of nine different isoforms with distinct biophysical properties. Studying the mutations associated with the increase or absence of pain sensitivity in humans, as well as other expression studies, have highlighted Nav1.7, Nav1.8, and Nav1.9 as being the most important contributors to the control of nociceptive neuronal electrogenesis. Modulating their expression and/or function can impact the shape of the AP and consequently modify nociceptive transmission, a process that is observed in persistent pain conditions. Post-translational modification (PTM) of Navs is a well-known process that modifies their expression and function. In chronic pain syndromes, the release of inflammatory molecules into the direct environment of dorsal root ganglia (DRG) sensory neurons leads to an abnormal activation of enzymes that induce Navs PTM. The addition of small molecules, i.e., peptides, phosphoryl groups, ubiquitin moieties and/or carbohydrates, can modify the function of Navs in two different ways: via direct physical interference with Nav gating, or via the control of Nav trafficking. Both mechanisms have a profound impact on neuronal excitability. In this review we will discuss the role of Protein Kinase A, B, and C, Mitogen Activated Protein Kinases and Ca++/Calmodulin-dependent Kinase II in peripheral chronic pain syndromes. We will also discuss more recent findings that the ubiquitination of Nav1.7 by Nedd4-2 and the effect of methylglyoxal on Nav1.8 are also implicated in the development of experimental neuropathic pain. We will address the potential roles of other PTMs in chronic pain and highlight the need for further investigation of PTMs of Navs in order to develop new pharmacological tools to alleviate pain.

Analysis of translational errors in frame-based and frameless cranial radiosurgery using an anthropomorphic phantom

Abstract Objective: To evaluate three-dimensional translational setup errors and residual errors in image-guided radiosurgery, comparing frameless and frame-based techniques, using an anthropomorphic phantom. Materials and Methods: We initially used specific phantoms for the calibration and quality control of the image-guided system. For the hidden target test, we used an Alderson Radiation Therapy (ART)-210 anthropomorphic head phantom, into which we inserted four 5mm metal balls to simulate target treatment volumes. Computed tomography images were the taken with the head phantom properly positioned for frameless and frame-based radiosurgery. Results: For the frameless technique, the mean error magnitude was 0.22 ± 0.04 mm for setup errors and 0.14 ± 0.02 mm for residual errors, the combined uncertainty being 0.28 mm and 0.16 mm, respectively. For the frame-based technique, the mean error magnitude was 0.73 ± 0.14 mm for setup errors and 0.31 ± 0.04 mm for residual errors, the combined uncertainty being 1.15 mm and 0.63 mm, respectively. Conclusion: The mean values, standard deviations, and combined uncertainties showed no evidence of a significant differences between the two techniques when the head phantom ART-210 was used.

Differential Regulation of c-FLIP Isoforms Through Post-translational Modifications

Cells are constantly responding to signals from the surrounding tissues and the environment. To dispose of infected and potentially dangerous cells, to ensure the optimal execution of developmental processes and to maintain tissue homeostasis, a multicellular organism needs to tightly control both the number and the quality of its cells. Apoptosis is a form of active cellular self-destruction that enables an organism to regulate its cell number by deleting damaged or potentially dangerous cells. Apoptosis can be induced by death ligands, which bind to death receptors on the cell surface. Ligation of the receptors leads to the formation of an intracellular death inducing signaling complex (DISC). One of the DISC components is caspase-8, a protease that triggers the caspase cascade and is thereby a key initiator of programmed cell death. The activation of caspase-8 is controlled by the cellular FLICE-inhibitory proteins (c-FLIPs). Consequently, sensitivity towards receptor-mediated apoptosis is determined by the amount of c-FLIP, and the c-FLIP levels are actively regulated for example during erythroid differentiation of K562 erythroleukemia cells and by hyperthermia in Jurkat leukemia cells. The aim of my thesis was to investigate how c-FLIP is regulated during these processes. We found that c-FLIP isoforms are short-lived proteins, although c-FLIPS had an even shorter half-life than c-FLIPL. In both experimental models, increased death receptor sensitivity correlated with induced ubiquitylation and consequent proteasomal degradation of c-FLIP. Furthermore, we elucidated how phosphorylation regulates the biological functions and the turnover of c-FLIP, thereby contributing to death receptor sensitivity. We mapped the first phosphorylation sites on c-FLIP and dissected how their phosphorylation affects c-FLIP. Moreover, we demonstrated that phosphorylation of serine 193, a phosphorylated residue common to all c-FLIPs, is primarily mediated by the classical PKC. Furthermore, we discovered a novel connection between the phosphorylation and ubiquitylation of c-FLIP: phosphorylation of S193 protects c-FLIP from ubiquitylation. Surprisingly, although all c-FLIP isoforms are phosphorylated on this conserved residue, the biological outcome is different for the long and short isoforms, since S193 specifically prolongs the half-lives of the short c-FLIP isoforms, but not c-FLIPL. To summarize, we show that c-FLIP proteins are modified by ubiquitylation and phosphorylation, and that the biological outcomes of these modifications are isoform-specifically determined.

Dengue virus-induced regulation of the host cell translational machinery

Dengue virus (DV)-induced changes in the host cell protein synthesis machinery are not well understood. We investigated the transcriptional changes related to initiation of protein synthesis. The human hepatoma cell line, HepG2, was infected with DV serotype 2 for 1 h at a multiplicity of infection of one. RNA was extracted after 6, 24 and 48 h. Microarray results showed that 36.5% of the translation factors related to initiation of protein synthesis had significant differential expression (Z-score ≥ ±2.0). Confirmation was obtained by quantitative real-time reverse transcription-PCR. Of the genes involved in the activation of mRNA for cap-dependent translation (eIF4 factors), eIF4A, eIF4G1 and eIF4B were up-regulated while the negative regulator of translation eIF4E-BP3 was down-regulated. This activation was transient since at 24 h post-infection levels were not significantly different from control cells. However, at 48 h post-infection, eIF4A, eIF4E, eIF4G1, eIF4G3, eIF4B, and eIF4E-BP3 were down-regulated, suggesting that cap-dependent translation could be inhibited during the progression of infection. To test this hypothesis, phosphorylation of p70S6K and 4E-BP1, which induce cap-dependent protein synthesis, was assayed. Both proteins remained phosphorylated when assayed at 6 h after infection, while infection induced dephosphorylation of p70S6K and 4E-BP1 at 24 and 48 h of infection, respectively. Taken together, these results provide biological evidence suggesting that in HepG2 cells DV sustains activation of the cap-dependent machinery at early stages of infection, but progression of infection switches protein synthesis to a cap-independent process.