Phosphatidylethanolamine is the precursor of the ethanolamine phosphoglycerol moiety bound to eukaryotic elongation factor 1A

In addition to its conventional role during protein synthesis, eukaryotic elongation factor 1A is involved in other cellular processes. Several regions of interaction between eukaryotic elongation factor 1A and the translational apparatus or the cytoskeleton have been identified, yet the roles of the different post-translational modifications of eukaryotic elongation factor 1A are completely unknown. One amino acid modification, which so far has only been found in eukaryotic elongation factor 1A, consists of ethanolamine-phosphoglycerol attached to two glutamate residues that are conserved between mammals and plants. We now report that ethanolamine-phosphoglycerol is also present in eukaryotic elongation factor 1A of the protozoan parasite Trypanosoma brucei, indicating that this unique protein modification is of ancient origin. In addition, using RNA-mediated gene silencing against enzymes of the Kennedy pathway, we demonstrate that phosphatidylethanolamine is a direct precursor of the ethanolamine-phosphoglycerol moiety. Down-regulation of the expression of ethanolamine kinase and ethanolamine-phosphate cytidylyltransferase results in inhibition of phosphatidylethanolamine synthesis in T. brucei procyclic forms and, concomitantly, in a block in glycosylphosphatidylinositol attachment to procyclins and ethanolamine-phosphoglycerol modification of eukaryotic elongation factor 1A.

MicroRNAs: a new class of regulatory genes affecting metabolism

MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression by binding to target mRNAs, which leads to reduced protein synthesis and sometimes decreased steady-state mRNA levels. Although hundreds of miRNAs have been identified, much less is known about their biological function. Several studies have provided evidence that miRNAs affect pathways that are fundamental for metabolic control in higher organisms such as adipocyte and skeletal muscle differentiation. Furthermore, some miRNAs have been implicated in lipid, amino acid, and glucose homeostasis. These studies open the possibility that miRNAs may contribute to common metabolic diseases and point to novel therapeutic opportunities based on targeting of miRNAs.

The consequences of growth hormone deficiency in adulthood, and the effects of growth hormone replacement

The availability of recombinant human growth hormone (GH) has resulted in investigation of the role of GH in adulthood and the effects of GH replacement in the GH-deficient adult. These studies have led to the recognition of a specific syndrome of GH-deficiency, characterized by symptoms, signs and investigative findings. Adults with long-standing growth hormone deficiency are often overweight, have altered body composition, with reduced lean body mass (LBM), increased fat mass (FM), reduced total body water and reduced bone mass. In addition, there is reduced physical and cardiac performance, altered substrate metabolism and an abnormal lipid profile predisposing to the development of cardiovascular disease. Adults with GH deficiency report reduced psychological well-being and quality of life. These changes may contribute to the morbidity and premature mortality observed in hypopituitary adults on conventional replacement therapy. GH treatment restores LBM, reduces FM, increases total body water and increases bone mass. Following GH therapy, increases are recorded in exercise capacity and protein synthesis, and "favourable" alterations occur in plasma lipids. In addition, psychological well-being and quality of life improve with replacement therapy. GH is well tolerated; adverse effects are largely related to fluid retention and respond to dose adjustment. It is likely that GH replacement will become standard therapy for the hypopituitary adult in the near future. The benefits of GH replacement in the GH-deficient adult have been unequivocally demonstrated in studies lasting up to 3 years. The results of longer term studies are awaited to determine whether these benefits are sustained over a lifetime.

Stimulation of MMP-1 and CCL2 by NAMPT in PDL cells

Periodontitis is an inflammatory disease caused by pathogenic microorganisms and characterized by the destruction of the periodontium. Obese individuals have an increased risk of periodontitis, and elevated circulating levels of adipokines, such as nicotinamide phosphoribosyltransferase (NAMPT), may be a pathomechanistic link between both diseases. The aim of this in vitro study was to examine the regulation of periodontal ligament (PDL) cells by NAMPT and its production under inflammatory and infectious conditions. NAMPT caused a significant upregulation of 9 genes and downregulation of 3 genes, as analyzed by microarray analysis. Eight of these genes could be confirmed by real-time PCR: NAMPT induced a significant upregulation of EGR1, MMP-1, SYT7, ITPKA, CCL2, NTM, IGF2BP3, and NRP1. NAMPT also increased significantly the MMP-1 and CCL2 protein synthesis. NAMPT was significantly induced by interleukin-1 β and the periodontal microorganism P. gingivalis. NAMPT may contribute to periodontitis through upregulation of MMP-1 and CCL2 in PDL cells. Increased NAMPT levels, as found in obesity, may therefore represent a mechanism whereby obesity could confer an increased risk of periodontitis. Furthermore, microbial and inflammatory signals may enhance the NAMPT synthesis in PDL cells and thereby contribute to the increased gingival and serum levels of this adipokine, as found in periodontitis.

Regulation of visfatin by microbial and biomechanical signals in PDL cells

OBJECTIVES This in vitro study was established to examine whether visfatin thought to be a link between periodontitis and obesity is produced by periodontal ligament (PDL) cells and, if so, whether its synthesis is modulated by microbial and/or biomechanical signals. MATERIALS AND METHODS PDL cells seeded on BioFlex® plates were exposed to the oral pathogen Fusobacterium nucleatum ATCC 25586 and/or subjected to biomechanical strain for up to 3 days. Gene expression of visfatin and toll-like receptors (TLR) 2 and 4 was analyzed by RT-PCR, visfatin protein synthesis by ELISA and immunocytochemistry, and NFκB nuclear translocation by immunofluorescence. RESULTS F. nucleatum upregulated the visfatin expression in a dose- and time-dependent fashion. Preincubation with neutralizing antibodies against TLR2 and TLR4 caused a significant inhibition of the F. nucleatum-upregulated visfatin expression at 1 day. F. nucleatum stimulated the NFκB nuclear translocation. Biomechanical loading reduced the stimulatory effects of F. nucleatum on visfatin expression at 1 and 3 days and also abrogated the F. nucleatum-induced NFκB nuclear translocation at 60 min. Biomechanical loading inhibited significantly the expression of TLR2 and TLR4 at 3 days. The regulatory effects of F. nucleatum and/or biomechanical loading on visfatin expression were also observed at protein level. CONCLUSIONS PDL cells produce visfatin, and this production is enhanced by F. nucleatum. Biomechanical loading seems to be protective against the effects of F. nucleatum on visfatin expression. CLINICAL RELEVANCE Visfatin produced by periodontal tissues could play a major role in the pathogenesis of periodontitis and the interactions with obesity and other systemic diseases.

Peptide transporter DtpA has two alternate conformations, one of which is promoted by inhibitor binding

Peptide transporters (PTRs) of the large PTR family facilitate the uptake of di- and tripeptides to provide cells with amino acids for protein synthesis and for metabolic intermediates. Although several PTRs have been structurally and functionally characterized, how drugs modulate peptide transport remains unclear. To obtain insight into this mechanism, we characterize inhibitor binding to the Escherichia coli PTR dipeptide and tripeptide permease A (DtpA), which shows substrate specificities similar to its human homolog hPEPT1. After demonstrating that Lys[Z-NO2]-Val, the strongest inhibitor of hPEPT1, also acts as a high-affinity inhibitor for DtpA, we used single-molecule force spectroscopy to localize the structural segments stabilizing the peptide transporter and investigated which of these structural segments change stability upon inhibitor binding. This characterization was done with DtpA embedded in the lipid membrane and exposed to physiologically relevant conditions. In the unbound state, DtpA adopts two main alternate conformations in which transmembrane α-helix (TMH) 2 is either stabilized (in ∼43% of DtpA molecules) or not (in ∼57% of DtpA molecules). The two conformations are understood to represent the inward- and outward-facing conformational states of the transporter. With increasing inhibitor concentration, the conformation characterized by a stabilized TMH 2 becomes increasingly prevalent, reaching ∼92% at saturation. Our measurements further suggest that Lys[Z-NO2]-Val interacts with discrete residues in TMH 2 that are important for ligand binding and substrate affinity. These interactions in turn stabilize TMH 2, thereby promoting the inhibited conformation of DtpA.

The ribosome as novel target for small regulatory RNAs

Small non-protein-coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily, not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. This is unexpected, given the central position the ribosome plays during gene expression. Furthermore it is strongly assumed that the primordial translation system in the ‘RNA world’ most likely received direct regulatory input from ncRNA-like cofactors. The fundamental question that we would like to ask is: Does the ‘RNA world still communicate’ with the ribosome? To address this question, we have analyzed the small ncRNA interactomes of ribosomes of prokaryotic (H. volcanii, S. aureus) and unicellular eukaryotic model organisms. Deep-sequencing and subsequent bioinformatic analyses revealed thousands of putative ribosome-associated ncRNAs. For a subset of these ncRNA candidates we have gathered experimental evidence that they are expressed in a stress-dependent manner and indeed directly target the ribosome. In the archaeon H. volcanii a tRNA-derived fragment was identified to target the small ribosomal subunit upon alkaline stress in vitro and in vivo. As a consequence of ribosome binding, this tRNA-fragment reduces protein synthesis by interfering with the peptidyl transferase activity. Our data reveal the ribosome as a novel target for small regulatory ncRNAs in all domains of life. Ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory sRNAs.

The ribosome as novel target for small regulatory RNAs

Small non-protein-coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily, not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. This is unexpected, given the central position the ribosome plays during gene expression. Furthermore it is strongly assumed that the primordial translation system in the ‘RNA world’ most likely received direct regulatory input from ncRNA-like cofactors. The fundamental question that we would like to ask is: Does the ‘RNA world still communicate’ with the ribosome? To address this question, we have analyzed the small ncRNA interactomes of ribosomes of organisms from all three domains of life. Deep-sequencing and subsequent bioinformatic analyses revealed thousands of putative ribosome-associated ncRNAs.1,2 For a subset of these ncRNA candidates we have gathered experimental evidence that they are expressed in a stress-dependent manner and indeed directly target the ribosome. We show that some of these ribosome-bound small ncRNAs are capable of fine tuning protein synthesis in vitro and in vivo. Our data therefore reveal the ribosome as a novel target for small regulatory ncRNAs in all domains of life and suggest the existence of a so far largely unexplored mechanism of translation regulation.

When small non-coding RNAs meet the ribosome: Tuning the translational machinery

The functions of ribosomes in translation are complex and involve different types of activities critical for decoding the genetic code, linkage of amino acids via amide bonds to form polypeptide chains, as well as the release and proper targeting of the synthesized protein. Non-protein-coding RNAs (ncRNAs) have been recognized to be crucial in establishing regulatory networks.1 However all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. The main goal of this project is to identify potential novel ncRNAs that directly bind and possibly regulate the ribosome during protein biosynthesis. To address this question we applied various stress conditions to the archaeal model organism Haloferax volcanii and deep-sequenced the ribosome-associated small ncRNA interactome. In total we identified 6.250 ncRNA candidates. Significantly, we observed the emersed presence of tRNA-derived fragments (tRFs). These tRFs have been identified in all domains of life and represent a growing, yet functionally poorly understood, class of ncRNAs. Here we present evidence that tRFs from H. volcanii directly bind to ribosomes. In the presented genomic screen of the ribosome-associated RNome a 26 residue long fragment originating from the 5’ part of valine tRNA was by far the most abundant tRF. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. As a consequence of ribosome binding, Val-tRF reduces protein synthesis by interfering with peptidyl transferase activity. Therefore this tRF functions as ribosome-bound small ncRNA capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production.2 Currently we are investigating the binding site of this tRF on the 30S subunit in more detail.

Mechanistic aspects of NMD in human cells

Eukaryotic mRNAs with premature translation termination codons (PTCs) are recognized and degraded through a process termed nonsense-mediated mRNA decay (NMD). To get more insight into the recruitment of the central NMD factor UPF1 to target mRNAs, we mapped transcriptome-wide UPF1-binding sites by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) in human cells and found that UPF1 preferentially associated with 3′ UTRs in translationally active cells but underwent significant redistribution toward coding regions (CDS) upon translation inhibition. This indicates that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. Corroborated by RNA immunoprecipitation and by UPF1 cross-linking to long noncoding RNAs, our evidence for translation-independent UPF1-RNA interaction suggests that the triggering of NMD occurs after UPF1 binding to mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination. Unlike in yeast, in mammalian cells NMD has been reported to be restricted to cap-binding complex (CBC)–bound mRNAs during the pioneer round of translation. However, we compared decay kinetics of two NMD reporter genes in mRNA fractions bound to either CBC or the eukaryotic initiation factor 4E (eIF4E) in human cells and show that NMD destabilizes eIF4E-bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event.

CTIF is involved in mRNA export in human cells

In eukaryotic cells, translation of messenger RNA (mRNA) can be initiated either on transcripts associated with the cap-binding complex (CBC; consisting of CBP80 and CBP20) or on transcripts with the eukaryotic translation initiation factor (eIF) 4E bound to the cap. Together with eIF4G and eIF4A, eIF4E forms the eIF4F-complex, which mediates translation initiation during the bulk of cellular protein synthesis. Functionally substituting for eIF4G, the CBP80/20-dependent translation initiation factor (CTIF) has been reported to be part of the CBC-dependent translation initiation complex 1,2. CTIF consists of a N-terminal CBP80-binding domain and a conserved C-terminal MIF4G domain 1. This MIF4G domain has been shown to mediate the interaction between CTIF and different factors such as eIF3g and the stem-loop binding protein (SLBP) 2,3. Here we provide evidence that CTIF, besides its function in translation initiation, is also involved in mRNA translocation from the nucleus to the cytoplasm, possibly through a direct interaction with the nuclear export factor NFX1/TAP. Taken together our results suggest that CTIF can function as a platform that interacts with proteins involved in different steps of the mRNA metabolism.

CTIF is involved in mRNA export in human cells

In eukaryotic cells translation initiation of messenger RNA (mRNA) transcripts can be initiated either by the cap-binding complex (CBC) consisting of CBP80 and CBP20, or by the eukaryotic translation initiation factor (eIF) 4E. Together with eIF4G and eIF4A, eIF4E forms the eIF4F-complex, which mediates translation initiation during the bulk of cellular protein synthesis [1,2]. Functionally analogous to eIF4G, the CBP80/20-dependent translation initiation factor (CTIF) has been reported to be part of the CBC-dependent translation initiation complex [3,4]. CTIF consists of a N-terminal CBP80-binding domain and a conserved C-terminal MIF4G domain [3]. This MIF4G domain has been shown to mediate the interaction between CTIF and different factors such as eIF3g and the stem-loop binding protein (SLBP) [4,5]. Here we show data indicating that CTIF, besides its function in translation initiation, is involved in mRNA translocation from the nucleus to the cytoplasm, possibly through a direct interaction with the nuclear export factor NFX1/TAP. Taken together our results suggest that CTIF can function as a platform that interacts with proteins involved in different steps of the mRNA metabolism. [1] Haghighat A. and Sonenberg N. (1997) JBC 272:21677-21680 [2] Gross J.D. et al. (2003) Cell 115:739-750 [3] Kim K.M. et al. (2009) Genes Dev 23:2033-2045 [4] Choe J. et al. (2012) JBC 287:18500-18509 [5] Choe J. et al. (2013) NAR 41:1307-1318

Development of the First Fluorescence Screening Assay for the SLC39A2 Zinc Transporter.

Zinc is an essential micronutrient that is crucial for many vital cellular functions such as DNA and protein synthesis, metabolism, and intracellular signaling. Therefore, the intracellular zinc concentration is tightly regulated by zinc transporters and zinc-binding proteins. The members of the SCL39 transporter family transport zinc into the cytosol. The SLC39A2 (hZIP2) protein is highly expressed in prostate epithelial cells and was found to be involved in prostate cancer development. Thus far, there is no specific modulator available for the SLC39 transporters. The aim of this study was to develop a screening assay for compound screening targeting hZIP2. Employing the pIRES2-DsRed Express 2 bicistronic vector, we detected human ZIP2 expression at the plasma membrane in transiently transfected HEK293 cells. Using the FLIPR Tetra fluorescence plate reader, we demonstrated that ZIP2 transports Cd(2+) with an apparent Km value of 53.96 nM at an extracellular pH of 6.5. The cadmium influx via hZIP2 was inhibited by zinc in a competitive manner. We found that hZIP2 activity can be measured using cadmium in the range of 0.1 to 10 µM with our assay. In summary, for the first time we developed an assay for human ZIP2 that can be adapted to other zinc transporters.

Insulin, CCAAT/enhancer-binding proteins and lactate regulate the human 11β-hydroxysteroid dehydrogenase type 2 gene expression in colon cancer cell lines.

11β-Hydroxysteroid dehydrogenases (11beta-HSD) modulate mineralocorticoid receptor transactivation by glucocorticoids and regulate access to the glucocorticoid receptor. The isozyme 11beta-HSD2 is selectively expressed in mineralocorticoid target tissues and its activity is reduced in various disease states with abnormal sodium retention and hypertension, including the apparent mineralocorticoid excess. As 50% of patients with essential hypertension are insulin resistant and hyperinsulinemic, we hypothesized that insulin downregulates the 11beta-HSD2 activity. In the present study we show that insulin reduced the 11beta-HSD2 activity in cancer colon cell lines (HCT116, SW620 and HT-29) at the transcriptional level, in a time and dose dependent manner. The downregulation was reversible and required new protein synthesis. Pathway analysis using mRNA profiling revealed that insulin treatment modified the expression of the transcription factor family C/EBPs (CCAAT/enhancer-binding proteins) but also of glycolysis related enzymes. Western blot and real time PCR confirmed an upregulation of C/EBP beta isoforms (LAP and LIP) with a more pronounced increase in the inhibitory isoform LIP. EMSA and reporter gene assays demonstrated the role of C/EBP beta isoforms in HSD11B2 gene expression regulation. In addition, secretion of lactate, a byproduct of glycolysis, was shown to mediate insulin-dependent HSD11B2 downregulation. In summary, we demonstrate that insulin downregulates HSD11B2 through increased LIP expression and augmented lactate secretion. Such mechanisms are of interest and potential significance for sodium reabsorption in the colon.