BJ Gene via MedWorm.com

A naturally occurring nonapeptide functionally compensates the CP1 domain of leucyl-tRNA synthetase to modulate aminoacylation activity

BJ Gene — Wed, 01 Feb 2012 05:00:00 +0100

Aminoacyl-tRNA synthetases (aaRSs) establish the rules of the genetic code by catalyzing the formation of aminoacyl-tRNA. The quality control for aminoacylation reaction is achieved by editing activity, which is usually carried out by a discrete editing domain. For leucyl-tRNA synthetase (LeuRS), the connective peptide 1 (CP1) domain is the editing domain responsible for hydrolyzing mis-charged tRNA. The CP1 domain is universally present in LeuRSs except LeuRS from Mycoplasma mobile (MmLeuRS). The substitute of CP1 in MmLeuRS is a nonapeptide (MmLinker). We show here that the MmLinker, which is critical for aminoacylation activity of MmLeuRS, could confer remarkable tRNA charging activity to the inactive CP1-deleted LeuRS from Escherichia coli (EcLeuRS) and Aquifex aeolicus (AaLeuRS). Furt...

Cross-talk between TGF{beta}1 and EGFR signaling pathways induces TM4SF5 expression and Epithelial-Mesenchymal Transition

BJ Gene — Tue, 31 Jan 2012 05:00:00 +0100

The epithelial-mesenchymal transition (EMT) is involved in fibrosis and cancer, and regulated by different signaling pathways mediated through soluble factors, actin reorganization, and transcription factor actions. Because tetraspan(in) transmembrane 4 L6 family member 5 (TM4SF5) is highly expressed in hepatocellular carcinoma and induces EMT, understanding how TM4SF5 expression in hepatocytes is regulated is important. We explored the mechanisms that induce TM4SF5 expression and whether impaired signaling pathways for TM4SF5 expression inhibit acquisition of mesenchymal cell features, using human and mouse normal hepatocytes. We found that transforming growth factor beta 1 (TGFβ1)-mediated Smad activation caused TM4SF5 expression and EMT, and activation of the epidermal growth fac...

Involvement of PGE2 and cyclic AMP signaling pathway in the up-regulation of COX-2 and mPGES-1 expression in LPS -activated macrophages

BJ Gene — Mon, 23 Jan 2012 05:00:00 +0100

In this study, we describe a PGE2 positive feedback for Cyclooxygenase (COX) -2 and microsomal PGE Synthase (mPGES) -1 expression in the macrophage cell line RAW 264.7. Our results show that PGE2 induces COX-2 and mPGES-1 expression, an effect mimicked by dibutyryl-cAMP (dbcAMP) or Forskolin. Furthermore, cAMP signaling pathway cooperates with LPS in the induction of COX-2 and mPGES-1 transcriptional activation. Analysis of the involvement of EP receptors showed that incubation with EP2 agonists up-regulated both COX-2 and mPGES-1 mRNA levels. Moreover, EP2 receptor over expression enhanced the transcriptional activation of COX-2 and mPGES-1 promoters, being this induction abolished by the PKA inhibitor, H89. Activation of PGE2/EP2/PKA signaling pathway induced the phosphorylation of the c...

Regulation of Human Microsomal Prostaglandin E Synthase-1 by IL-1{beta} requires a Distal Enhancer Element with a Unique Role for C/EBP{beta}

BJ Gene — Fri, 20 Jan 2012 05:00:00 +0100

The studies of prostaglandin E2 (PGE2) biosynthesis have primarily focused on the role of cyclooxygenases. Efforts have shifted towards the specific PGE2 terminal synthases, particularly microsomal PGE synthase (mPGES-1), which has emerged as the crucial inducible synthase with roles in pain, cancer and inflammation. mPGES-1 is induced by proinflammatory cytokines with studies focusing on the proximal promoter, mediated specifically through Egr-1. Numerous studies demonstrate that the mPGES-1 promoter alone cannot account for the level of IL-1β induction. We identified two DNase I hypersensitive sites within the proximal promoter near the Egr-1 element and a novel distal site near -8.6kb. Functional analysis of the distal site revealed two elements that cooperate with basal promoter...

Activating transcription factor 4-dependent induction of FGF21 during amino acid deprivation.

BJ Gene — Wed, 11 Jan 2012 05:00:00 +0100

Nutrient deprivation or starvation frequently correlates with amino acid limitation. Amino acid starvation initiates a signal transduction cascade starting with the activation of the kinase GCN2 phosphorylation of eukaryotic initiation factor 2, global protein synthesis reduction and increased activating transcription factor (ATF) 4. ATF4 modulates a wide spectrum of genes involved in the adaptation to dietary stress. The hormone FGF21 is induced during fasting in liver, and its expression induces a metabolic state that mimics long-term fasting. Thus, FGF21 is critical for the induction of hepatic fat oxidation, ketogenesis and gluconeogenesis, metabolic processes which are essential for the adaptive metabolic response to starvation. In this article, we show that FGF21 is induced by amino ...

Serum Copper as a Novel Biomarker for Resistance to Thyroid Hormone

BJ Gene — Fri, 06 Jan 2012 05:00:00 +0100

Thyroid hormone action is mediated by the thyroid hormone receptors TRa1 and TRb. Defects in TRb lead to “Resistance to Thyroid Hormone” (RTHb), a syndrome characterized by high levels of thyroid hormone and non-suppressed thyroid-stimulating hormone (TSH). However, a correct diagnosis of RTHb patients is difficult as the clinical picture varies. A biochemical serum marker indicative of defects in TRb signaling is needed and could simplify the diagnosis of RTHb, in particular the differentiation to TSH-secreting pituitary adenomas, which present with clinically similar symptoms. Here we show that serum copper levels are regulated by thyroid hormone, which stimulates the synthesis and the export of the hepatic Cu-transport protein ceruloplasmin into the serum. This is accomp...

Transcriptional control of glyoxalase 1 by Nrf2 provides a stress responsive defence against dicarbonyl glycation

BJ Gene — Thu, 22 Dec 2011 05:00:00 +0100

Abnormal cellular accumulation of the dicarbonyl metabolite methylglyoxal occurs on exposure to high glucose concentration, inflammation, cell ageing and senescence. It is associated with increased methylglyoxal-adduct content of protein and DNA linked to increased DNA strand breaks and mutagenesis, mitochondrial dysfunction and reactive oxygen species formation and cell detachment from the extracellular matrix. Methylglyoxal–mediated damage is countered by glutathione-dependent metabolism by glyoxalase-1. It is not known, however, if glyoxalase-1 has stress responsive up-regulation to counter periods of high methylglyoxal concentration or dicarbonyl stress. We identified a functional antioxidant response element in the 5’-untranslated region of exon-1 of the mammalian glyoxa...

Crystal structure of the Sox4 HMG/DNA complex suggests a mechanism for the positional interdependence in DNA recognition

BJ Gene — Mon, 19 Dec 2011 05:00:00 +0100

It has recently been proposed that the sequence preferences of DNA-binding transcription factors can be well described by models that include the positional interdependence of the nucleotides of the target sites. Such binding models allow for multiple motifs to be invoked, such as principal and secondary motifs differing at two or more nucleotide positions. However, the structural mechanisms underlying the accommodation of such variant motifs by TFs remain elusive. Here we present the crystal structure of the high-mobility group (HMG) domain of Sox4 bound to DNA. By comparing this structure with previously solved structures of Sox17 and Sox2 we observed subtle conformational differences at the DNA binding interface. Furthermore, using quantitative electrophoretic mobility shift assays (EMS...

Staphylococcus aureus DinG, a helicase that has evolved into a nuclease

BJ Gene — Wed, 14 Dec 2011 05:00:00 +0100

DinG is a bacterial superfamily 2 helicase with 5’ to 3’ polarity. DinG is related to the XPD helicase family, and they have in common an iron-sulphur (FeS) binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. Here we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3’ to 5’ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modifi...

Functional analysis of membraneous Fo-a subunit of F1Fo-ATP synthase by in vitro protein synthesis

BJ Gene — Wed, 14 Dec 2011 05:00:00 +0100

Fo-a subunit of F1Fo-ATP synthase (F1Fo) is a highly hydrophobic protein with five putative transmembrane helices and it plays a central role in H+-translocation that is coupled with ATP synthesis/hydrolysis. Here, we show that Fo-a subunit produced by the in vitro protease-free protein synthesis system (PURE system) is integrated into a preformed Fo-a-less F1Fo complex in the Escherichia coli membrane vesicles and in liposomes. The resulting F1Fo has H+-coupled ATP synthesis/hydrolysis activity that is approximately half of that of the native F1Fo. By using this procedure, we analyzed five mutations of F1Fo, where the conserved residues in Fo-a subunit (N90, D112, R169, N173, and Q217) were individually replaced with alanine. All of the mutant Fo-a subunits were successfully...

The chromatin binding protein HMGN3 stimulates histone acetylation and transcription across the Glyt1 gene

BJ Gene — Mon, 12 Dec 2011 05:00:00 +0100

HMGNs are nucleosome-binding proteins that alter the pattern of histone modifications and modulate the binding of linker histones to chromatin. The HMGN3 family member exists as two splice forms, HMGN3a which is full length and HMGN3b which lacks the C-terminal regulatory domain. Here, we have used the Glyt1 gene as a model system to investigate where HMGN proteins are bound across the locus in vivo, and to study how the two HMGN3 splice variants affect histone modifications and gene expression. We demonstrate that HMGN1, HMGN2, HMGN3a and HMGN3b are bound across the Glyt1 gene locus and surrounding regions, and are not enriched more highly at the promoter or putative enhancer. We conclude that the peaks of H3K4me3 and H3K9ac at the active Glyt1a promoter do not play a major role in recrui...

RCD1-DREB2A interaction in leaf senescence and stress responses in Arabidopsis thaliana

BJ Gene — Mon, 12 Dec 2011 05:00:00 +0100

Transcriptional regulation of gene expression is one major determinant of developmental control and stress adaptation in virtually all living organisms. In recent years numerous transcription factors controlling various aspects of plant life have been identified. The activity of transcription factors needs to be regulated to prevent unspecific, prolonged or inappropriate responses. The transcription factor DEHYDRATION-RESPONSIVE ELEMENT BINDING 2A (DREB2A) has been identified as one of the main regulators of drought and heat responses, and it is regulated through protein stability. Here we present evidence that the interaction with RADICAL-INDUCED CELL DEATH1 (RCD1) contributes to the control of DREB2A under a range of conditions. The interaction is mediated by a novel protein motif in DRE...

Epidermal Growth Factor Induces Tumor Marker AKR1B10 Expression through Activator Protein-1 Signaling in Hepatocellular Carcinoma Cells

BJ Gene — Fri, 02 Dec 2011 05:00:00 +0100

This study showed that AKR1B10 is induced by mitogens epidermal growth factor (EGF) and insulin through the activator protein-1 (AP-1) signaling pathway. In human hepatocellular carcinoma cells (HepG2 and Hep3B), EGF (50ng/ml) and insulin (10nM) stimulated endogenous AKR1B10 expression and promoter activity. In the AKR1B10 promoter, a putative AP-1 element was found at -222 to -212bp. Deletion or mutations of this AP-1 element abrogated the basal promoter activity and response to EGF and AP-1 proteins. This AP-1 element bound to nuclear proteins extracted from HepG2 cells, and this binding was stimulated by EGF and insulin in a dose-dependent manner. Chromatin immunoprecipitation showed that AP-1 proteins c-Fos and c-Jun were predominant factors bound to the AP-1 consensus, followed by Jun...

Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains

BJ Gene — Thu, 24 Nov 2011 05:00:00 +0100

Eukaryotic elongation factor 2 kinase (eEF2K) is a calcium-calmodulin dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called ‘a-kinases’ which are distinct from the main eukaryotic protein kinase superfamily. In addition to the a-kinase catalytic domain, other domains have been identified in eEF2K: a calmodulin-binding region, N-terminal to the kinase domain, a C-terminal region containing several predicted a-helices (resembling SEL1 domains) and a probably rather unstructured ‘linker’ region connecting them. Here, we demonstrate (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/calmodulin enhance the abi...

ZraP is a periplasmic molecular chaperone and a repressor of the zinc responsive two component regulator ZraSR

BJ Gene — Tue, 15 Nov 2011 05:00:00 +0100

In this study we demonstrate that the periplasmic protein ZraP contributes to envelope homeostasis and assign both chaperone and regulatory function to ZraP purified from Salmonella Typhimurium. The ZraP chaperone mechanism is catalytic and independent of ATP; the chaperone activity is dependent on the presence of zinc, which is shown to be responsible for the stabilisation of an oligomeric ZraP complex. Furthermore, ZraP can act to repress the two component regulatory system ZraSR, which itself is responsive to zinc concentrations. Through structural homology, ZraP is a member of the bacterial CpxP family of periplasmic proteins, which also consists of CpxP and Spy. We demonstrate environmental co-expression of the CpxP family and identify an important role for these proteins in Salmo...

New Insights into the Structure of the Reaction Centre from Blastochloris viridis: Evolution in the Laboratory

BJ Gene — Fri, 04 Nov 2011 04:00:00 +0100

Newly determined crystal structures of the photosynthetic reaction centre (RC) from two substrains of the non-sulphur purple bacterium Blastochloris viridis strain DSM 133, together with analysis of their gene sequences, has revealed intra-species evolutionary changes over a time period of 14 years. Over one hundred point mutations were identified between these two substrains in the four genes encoding the protein subunits of the RC, of which about one fifth resulted in a total of 16 amino acid changes. The most interesting difference was in the M subunit where the change from leucine to glycine in the carotenoid binding pocket allowed dihydroneurosporene to adopt a more sterically favoured conformation and similar to the carotenoid conformation found in other related reaction centres. Our...

The multifunctional poly(A)-binding protein (PABP) 1 is subject to extensive dynamic post-translational modification, which molecular modelling suggests plays an important role in co-ordinating its activities

BJ Gene — Tue, 18 Oct 2011 04:00:00 +0100

Poly(A)-binding protein 1 (PABP1) is a central regulator of mRNA translation and stability and is required for miRNA-mediated regulation and nonsense-mediated decay. Numerous protein, as well as RNA, interactions underlie its multi-functional nature, however it is unclear how its different activities are coordinated since many partners interact via overlapping binding sites. Here, we show that human PABP1 is subject to elaborate post-translational modification, identifying 14 modifications located throughout the functional domains, all but one of which are conserved in mouse. Intriguingly, PABP1 contains glutamate and aspartate methylations, modifications of unknown function in eukaryotes, as well as lysine and arginine methylations and lysine acetylations. The latter dramatically alter th...

The multifunctional poly(A)-binding protein (PABP) 1 is subject to extensive dynamic post-translational modification, which molecular modelling suggests plays an important a role in coordinating its activities.

BJ Gene — Tue, 18 Oct 2011 04:00:00 +0100

ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites

BJ Gene — Tue, 11 Oct 2011 04:00:00 +0100

Poly(ADP-ribosyl)ation (PARylation) is involved in the maintenance of genomic methylation patterns through its control of DNA methyltranferase 1 (Dnmt1) activity. Our previous findings indicated that the CCCTC binding factor/Ctcf may be an important player in key events whereby PARylation controls the unmethylated status of some CpG-rich regions. Ctcf is able to activate Poly (ADP-ribose) polymerase 1 (Parp1) which ADP-ribosylates itself and, in turn, inhibits DNA methylation via non-covalent interaction between its ADP-ribose polymers and Dnmt1. By such a mechanism, Ctcf may preserve the epigenetic pattern at promoters of important housekeeping genes. Data here reported evidence Dnmt1 as a new protein partner of Ctcf. Moreover, we show that Ctcf forms a complex with Dnmt1 and PARylated Pa...

Translation of a minigene in the 5{'} leader sequence of the enterohaemorrhagic Escherichia coli LEE1 transcription unit affects expression of the neighbouring downstream gene

BJ Gene — Thu, 06 Oct 2011 04:00:00 +0100

The 5’ end of the major RNA transcript of the LEE1 operon of enterohaemorrhagic Escherichia coli contains ~170 bases before the AUG translation start codon of the first recognised gene, ler. This unusually long leader sequence carries three potential alternative AUG start codons. Using a lac fusion expression vector, we confirmed that the ler gene AUG is functional for translation initiation, and we checked for translation initiation at the three alternative AUG codons. Whilst two of the alternative AUG codons appear incompetent for translation initiation, we detected strong initiation at the third AUG, which is followed by one AAA codon and a UAG stop codon. The location of this very short two-codon open reading frame with respect to the ler translation start appears critical. Henc...

Location and dynamics of an active promoter in Escherichia coli K-12

BJ Gene — Wed, 21 Sep 2011 04:00:00 +0100

We report that transcription affects the location of a DNA target in Escherichia coli K-12. A strain whose chromosome had been engineered to encode a lac repressor-GFP fusion was used as a host for a low copy number plasmid that carries an array of five lac operator sites. Individual cells of this strain exhibited a diffuse fluorescence signal, suggesting that the plasmid is distributed throughout the cell cytoplasm. However, a derivative of this plasmid carrying a cloned promoter is targeted to a location at the edge of the nucleoid towards the pole of the host cell. We conclude that transcription from the cloned promoter is driving the location of the plasmid and that specific locations in bacterial cells may favour gene expression. (Source: BJ Gene)

BAG-1 diversely affects steroid receptor activity

BJ Gene — Thu, 15 Sep 2011 04:00:00 +0100

Part of the cellular and physiological functions of BAG-1 has been ascribed to the ability of this hsp70 cochaperone to regulate steroid receptor activity. BAG-1 has been reported to inhibit the glucocorticoid receptor (GR), to stimulate the androgen receptor but to leave the activity of the mineralocorticoid receptor (MR) unchanged. Given the high homology between MR and GR, this disparity in the actions of BAG‑1 is surprising. Here we analyzed the effect of BAG-1 on the activity of the closely related progesterone receptor (PR). Similarly to GR, the transcriptional activity of PR is inhibited by the long and middle isoforms of BAG-1, BAG-1L and BAG-1M, but not by the short isoform BAG-1S. We found this inhibition to require the hsp70 binding domain of BAG‑1. To shed light o...

Phospholipase D1 mediates bFGF-induced Bcl-2 expression leading to neurite outgrowth in H19-7 cells

BJ Gene — Wed, 14 Sep 2011 04:00:00 +0100

In conclusion, PLD1 acts as a downstream effector of bFGF/Ras/PI3K/PLCγ signaling and regulates Bcl-2 expression through JNK/STAT3, which leads to neurite outgrowth in H19-7 cells. . (Source: BJ Gene)

PTEN interacts with metal-responsive transcription factor 1 and stimulates its transcriptional activity

BJ Gene — Wed, 31 Aug 2011 23:00:00 +0100

We report here that phosphatase and tensin homologue deleted on chromosome 10 (PTEN) associates with MTF-1 in the cells. These two proteins interact via the acidic domain of MTF-1 and the phosphatase/C2 domain of PTEN. Depletion of PTEN reduced metallothionein (MT) gene expression and increased cellular sensitivity to cadmium toxicity. PTEN did not alter the nuclear translocation, protein stability or DNA-binding activity of MTF-1. Zn increased MTF-1/PTEN interaction in a dose-dependent manner. The interaction elevated within 2 h of Zn addition and declined afterward in the cells. The enhanced binding activity occurred mainly in cytoplasm and reduced after translocating the MTF-1 into nucleus. Blocking the transduction signals of phosphatidylinositol 3-kinase did not alter the Zn-induced M...

Lrpprc mutation suppresses cytochrome oxidase activity by altering mitochondrial RNA transcript stability in a mouse model

BJ Gene — Tue, 30 Aug 2011 23:00:00 +0100

LRPPRC has been shown to be essential for the maturation of COX (cytochrome oxidase) possibly by stabilizing RNA transcripts of COXI, II and III genes encoded in mtDNA.We established a mouse ‘genetrap’ model using embryonic stem cells in which the C-terminus of LRPPRC has been replaced with a b-geo construct. Mice homozygous for this modification were found to be subject to embryonic lethality, with death before 12.5 dpc. Biochemical analysis of mouse embryonic fibroblasts isolated from homozygous mutants showed a major decrease in COX activity, with slight reductions in other respiratory chain complexes with mtDNA encoded components. Constructs of LRPPRC containing different numbers of pentatricopeptide (PPR) repeats were expressed as recombinant proteins and used to assess ...

The Human Suv3 Helicase Interacts with Replication Protein A and Flap Endonuclease 1 in the Nucleus.

BJ Gene — Tue, 16 Aug 2011 23:00:00 +0100

The human Suv3 helicase (hSuv3) has been shown to be a major player in mitochondrial RNA surveillance and decay, but its physiological role might go beyond this functional niche. hSuv3 has been found to interact with BLM and WRN, members of the RecQ helicase family involved in multiple DNA metabolic processes, and in protection and stabilization of the genome. Here, we have addressed the possible role of hSuv3 in genome maintenance by examining its potential association with key interaction partners of the RecQ helicases. By analysis of hSuv3 co-immunoprecipitation complexes, we identify two new interaction partners of hSuv3: the Replication Protein A (RPA) and Flap Endonuclease 1 (FEN1). Utilizing an in vitro biochemical assay we find that low amounts of RPA inhibits helicase activity of ...

Peripheral insertion modulates editing activity of the isolated CP1 domain of leucyl-tRNA synthetase

BJ Gene — Thu, 04 Aug 2011 23:00:00 +0100

A large insertion domain called connective peptide 1 (CP1) present in class Ia aminoacyl-tRNA synthetases is responsible for post-transfer editing. Leucyl-tRNA synthetases (LeuRS) from Aquifex aeolicus and Giardia lamblia possess unique 20 and 59 amino acid insertions respectively within the CP1 that are crucial for editing activity. Crystal structures of AaLeuRS-CP1 (2.4Å), GlLeuRS-CP1 (2.6Å) and the insertion deletion mutant AaLeuRS-CP1-Δ20 (2.5Å) were solved to understand the role of these insertions in editing. Both insertions are folded as peripheral motifs located at the opposite side of the active site entrance in CP1 domain. Docking modeling and site-directed mutagenesis showed that the insertions do not interact with the substrates. Results of molecular...

The histone variant H2A.Z interconverts two stable epigenetic chromatin states

BJ Gene — Thu, 07 Jul 2011 23:00:00 +0100

The nucleosomes occupying the chromosomal start sites of transcription contain the histone H2A variant H2A.Z in place of H2A. Upon galactose induction, nucleosomes are evicted from the GAL1 locus in S. cerevisiae cells. H2A.Z (which is encoded by the HTZ1 gene in S. cerevisiae) is required for the eviction of the GAL1 promoter nucleosome and for the transcriptional activation of the GAL1 gene, however, histones are also important for transcriptional repression, and we asked here if H2A.Z plays a role in the glucose repression of the GAL1 promoter as well. With the help of a fusion of the URA3 open reading frame to the GAL1 promoter, we were able to detect two different epigenetic transcription states of the GAL1 promoter in glucose-grown cells lacking H2A.Z: a repressed state that is occup...

Helicase dissociation and annealing of RNA-DNA hybrids by Escherichia coli Cas3 protein.

BJ Gene — Wed, 22 Jun 2011 23:00:00 +0100

We report that purified E. coli Cas3 catalyses ATP-independent annealing of RNA with DNA forming R-loops, hybrids of RNA base-paired into duplex DNA. ATP abolished Cas3 R-loop formation and instead powered Cas3 helicase unwinding of the invading RNA strand of a model R-loop substrate. R-loop formation by Cas3 required magnesium as a co-factor and was inactivated by mutagenesis of a conserved amino acid motif. Cells expressing mutant Cas3 protein were more sensitive to plaque formation by phage lambda vir. A complex of CasABCDE (“Cascade”) also promoted R-loop formation and we discuss possible overlapping roles of Cas3 and Cascade in E. coli, and apparently antagonistic roles of Cas3 catalysing RNA-DNA annealing and ATP-dependent helicase unwinding. (Source: BJ Gene)

Drosophila Sal and Salr are transcriptional repressors

BJ Gene — Sun, 19 Jun 2011 23:00:00 +0100

We describe the differential subcellular localisations of Sal and Salr fragments and identify their repression domains. Surprisingly, both repressors also contain transcription activation domains. In addition, under our experimental conditions sumoylation has differential effects on Sal and Salr repressor activity. Phylogenetic comparison between nematodes, insects and vertebrates identifies conserved peptide sequences that are presumably critical for SALL protein function. (Source: BJ Gene)

Exo70, a subunit of the exocyst complex, interacts with SnevhPrp19/hPso4 and is involved in pre-mRNA splicing

BJ Gene — Thu, 02 Jun 2011 23:00:00 +0100

The CDC5L complex is a spliceosomal subcomplex that also plays a role in DNA repair. The complex contains the splicing factor hPRP19 also known as SNEV or hPso4 which is involved in cellular life span regulation and proteasomal breakdown. In a recent large scale proteomics analysis for proteins associated with this complex, proteins involved in transcription, cell cycle regulation, DNA repair, the ubiquitin/proteasome system, chromatin remodelling, cellular aging, the cytoskeleton and trafficking, including 4 members of the exocyst complex, were identified. Here we report that Exo70 interacts directly with SNEVhPrp19/hPso4 and shuttles to the nucleus, where it associates with the spliceosome. We mapped the interaction site to the N-terminal 100 amino acids of Exo70, which interfere with pr...

GATA-4 transcription factor regulates hepatic hepcidin expression.

BJ Gene — Mon, 23 May 2011 23:00:00 +0100

In conclusion, our findings: i) indicate that GATA-4 may participate to the control of hepcidin expression, and ii) suggest that alteration of its expression could contribute to the development of iron-related disorders. (Source: BJ Gene)

Gut Bitter Taste Receptor Signaling Induces ABCB1 through a Mechanism Involving CCK

BJ Gene — Wed, 18 May 2011 23:00:00 +0100

Bitter taste-sensing receptors (T2Rs) are expressed in the oral cavity to prevent ingestion of dietary toxins through taste avoidance. They are also expressed in other cell-types including gut enteroendocrine cells where their physiological role is enigmatic. Previously, we proposed T2R dependent cholecystokinin (CCK) secretion from enteroendocrine cells limits absorption of dietary toxins but an active mechanism was lacking. Here we show T2R signaling activates ATP-binding cassette B1 (ABCB1) in intestinal cells through a CCK signaling mechanism. Phenylthiocarbamide (PTC), an agonist for the T2R38 bitter receptor, increased ABCB1 expression in both intestinal cells and mouse intestine. PTC induction of ABCB1 was decreased by either T2R38 siRNA or treatment with YM022, a gastrin receptor a...

The DLK gene is a transcriptional target of PPAR{gamma}

BJ Gene — Tue, 17 May 2011 23:00:00 +0100

DLK is a key regulator of development, cell differentiation and apoptosis. Interestingly, recent studies have shown that DLK expression is up-regulated in 3T3-L1 cells induced to differentiate into adipocytes and that DLK knockdown impairs the expression of peroxisome proliferator-activated receptor-γ (PPARγ), a master regulator of adipogenesis. Because the PPARγ agonist rosiglitazone was found to increase DLK expression in 3T3-L1 cells, we hypothesized that PPARγ is required for the transcriptional activation of the DLK gene. To test this notion, we first examined the effects of pharmacological inhibition or shRNA-mediated depletion of PPARγ on DLK accumulation in 3T3-L1 cells undergoing differentiation. Besides blocking adipocyte conversion of 3T3-L1 ce...

Interleukin enhancer binding factor 3 functions as liver receptor homolog-1 coactivator in cooperation with nuclear receptor coactivators, PRMT1 and PGC-1{alpha}

BJ Gene — Mon, 09 May 2011 23:00:00 +0100

In this study, we identified an additional LRH-1-binding protein, interleukin enhancer binding factor 3 (ILF3). ILF3 formed a complex with LRH-1 and the other two nuclear receptor coactivators, protein arginine methyltransferase 1 (PRMT1) and peroxisome proliferator-activated receptor g coactivator-1 a (PGC-1a). We demonstrated that ILF3, PRMT1, and PGC-1a were recruited to the promoter region of the LRH-1-regulated small heterodimer partner (SHP) gene, encoding one of the nuclear receptors. ILF3 enhanced SHP gene expression in cooperation with PRMT1 and PGC-1a through the C-terminal region of ILF3. In addition, we found that the siRNA-mediated down-regulation of ILF3 expression led to a reduction in the occupancy of PGC-1a at the SHP promoter and the SHP expression. Taken together, our re...

Characterization of DNA-binding activity in the N-terminal domain of DNA methyltransferase Dnmt3a

BJ Gene — Wed, 20 Apr 2011 23:00:00 +0100

The Dnmt3a gene, which encodes de novo-type DNA methyltransferase, encodes two isoforms, full length Dnmt3a and Dnmt3a2, which lacks the N-terminal 219 amino acid residues. We found that Dnmt3a showed higher DNA-binding and DNA methylation activities than Dnmt3a2. The N-terminal 1-211 sequence was able to bind to DNA, but could not distinguish methylated and unmethylated CpG. Its binding to DNA was inhibited by a major groove binder. Four basic amino acid residues, K51, K53, R177, and R179, in the N-terminal region were crucial for the DNA-binding activity. The ectopically expressed N-terminal sequence (residues 1-211) was localized in nuclei, while that harbouring mutations at the four basic amino acid residues was detected even in the cytoplasm. The DNA methylation activity of Dnmt3a wit...

Repression of the RHOH gene by JunD

BJ Gene — Thu, 07 Apr 2011 23:00:00 +0100

RhoH is member of the Rho family of small GTP-binding proteins that lacks GTPase activity. Since RhoH is constantly bound by GTP, it is thought to be constitutively active and controlled predominantly by changes in quantitative expression. RhoH is produced specifically in haematopoietic cells and aberrant expression has been linked to various forms of leukaemia. Transcription of the RHOH gene is the first level at which the quantitative levels of the RhoH protein are regulated. Previous studies have demonstrated that RHOH gene transcription is initiated by three distinct promoter regions designated P1, P2 and P3 that define the 5’ end of exons 1, 2 and 4, respectively. Here we report that the P3 promoter is largely responsible for RHOH gene transcription in the B-lymphocytic...

A cryptic promoter in the LEE1 regulatory region of enterohaemorrhagic Escherichia coli: promoter specificity in AT-rich gene regulatory regions

BJ Gene — Thu, 07 Apr 2011 23:00:00 +0100

We report here that mutation of the P1 -10 element is less effective in unmasking P1A promoter activity than mutation of the P1 -35 element. This suggests that the P1 promoter -35 element, which corresponds to the consensus, can sequester RNA polymerase even when P1 is inactive and, thereby, prevent RNA polymerase from serving the P1A promoter. We propose that such promoter elements may play a role in enforcing specificity in bacterial regulatory regions that contain alternative possible promoters. (Source: BJ Gene)

Ataxia telangiectasia and Rad3 related kinase drives both the early and the late DNA damage response to the monofunctional antitumor alkylator S23906

BJ Gene — Wed, 06 Apr 2011 23:00:00 +0100

Numerous anticancer agents and environmental mutagens target DNA. Although all such compounds interfere with the progression of the replication fork and inhibit DNA synthesis, there are marked differences in the DNA damage response pathways they trigger, and the relative impact of the proximal or the distal signal transducers on cell survival is mainly lesion-specific. Accordingly, checkpoint kinase inhibitors in current clinical development show synergistic activity with some DNA-targeting agents, but not with others. Here, we characterize the DNA damage response to the antitumor acronycine derivative S23906 that forms monofunctional adducts with guanine residues in the minor groove of DNA. S23906 exposure is accompanied by specific recruitment of RPA at replication sites and rapid Chk1 a...

Effects of DNA and protein size on substrate cleavage by human tyrosyl-DNA phosphodiesterase (TDP1)

BJ Gene — Sun, 03 Apr 2011 23:00:00 +0100

Tyrosyl-DNA phosphodiesterase (TDP1) catalyzes the hydrolysis of phosphodiester linkages between a DNA 3' phosphate and a tyrosine residue as well as a variety of other DNA 3' substituents, and has been implicated in the repair of covalent complexes involving eukaryotic type IB topoisomerases. To better understand the substrate features that are recognized by TDP1, the size of either the DNA or protein component of the substrate was varied. Competition experiments and gel shift analyses comparing a series of substrates with DNA lengths increasing from 6 to 28 nucleotides indicated that, contrary to predictions based on the crystal structure of the protein, the apparent affinity for the substrate increased as the DNA length was increased over the entire range tested. It has previously been ...

Thermostable HIV-1 group O reverse transcriptase variants with the same fidelity as murine leukaemia virus reverse transcriptase

BJ Gene — Tue, 29 Mar 2011 23:00:00 +0100

Wild-type human immunodeficiency virus type 1 (HIV-1) group O reverse transcriptase (RT) shows increased thermostability in comparison with HIV-1 group M subtype B RT and murine leukemia virus (MLV) RT. However, its utility in the amplification of RNA targets is limited by the reduced accuracy of lentiviral RTs versus oncoretroviral RTs (i.e., MLV RT). The effects of mutations K65R, R78A and K65R/V75I on the fidelity of HIV-1 group O RTs were studied by using gel-based and M13mp2 lacZ forward mutation fidelity assays. Forward mutation assays demonstrated that mutant RTs K65R, R78A and K65R/V75I showed <9-fold increased accuracy in comparison with the wild-type enzyme, and were about two times more faithful than the MLV RT. Compared with MLV RT, all tested HIV-1 group O RT variants showe...

Structural basis for CARM1 inhibition by indole and pyrazole inhibitors

BJ Gene — Wed, 16 Mar 2011 00:00:00 +0100

Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine methyl transferase (PRMT) family member that catalyzes the transfer of methyl groups from S-Adenosyl-L-Methionine to the sidechain of specific arginine residues of substrate proteins. This post-translational modification of proteins regulates a variety of transcriptional events and other cellular processes. Moreover, CARM1 is a potential oncological target due to its multiple roles in transcription activation by nuclear hormone receptors and other transcription factors like p53. Here we present crystal structures of the CARM1 catalytic domain in complex with cofactors (S-adenosyl-L-homocyseine or Sinefungin) and indole or pyazole inhibitors. Analysis of the structures reveals that the inhibitors bind in the a...

The novel NRF2-interacting factor KAP1 regulates susceptibility to oxidative stress by promoting the NRF2-mediated cytoprotective response

BJ Gene — Mon, 07 Mar 2011 00:00:00 +0100

The transcription factor Nrf2 coordinately regulates antioxidant responsive element-mediated induction of cytoprotective genes in response to electrophiles and oxidative stress; however, the molecular mechanism controlling Nrf2-dependent gene expression is not fully understood. To identify factors that regulate Nrf2-dependent transcription, we searched for proteins that interact with the N-terminal Nrf2 transactivation domain (Nrf2-NT) by affinity purification from HeLa nuclear extracts. Here, we identified KAP1 (KRAB-associated protein 1) as a novel Nrf2-NT-interacting protein. Pull-down analysis confirmed the interaction between KAP1 and Nrf2 in cultured cells and demonstrated that the N-terminal region of KAP1 binds to Nrf2-NT in vitro. Reporter assays showed that KAP1 facilitates Nrf2 ...

The interplay of DNA binding, ATP hydrolysis and helicase activities of the archaeal MCM helicase

BJ Gene — Tue, 01 Mar 2011 00:00:00 +0100

The MCM proteins of Archaea are widely believed to be the replicative DNA helicase of these organisms. Most archaea possess a single MCM ortholog that forms homo-multimeric assemblies with a single hexamer believed to be the active form. Here we characterize the roles of highly conserved residues in the ATPase domain of the MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results identify a potential conduit for communicating DNA binding information to the ATPase active site. (Source: BJ Gene)

Solution structure of Taf14 YEATS domain and its roles in cell growth of Saccharomyces cerevisiae

BJ Gene — Mon, 28 Feb 2011 00:00:00 +0100

Chromatin modifications play important roles in cellular biological process. Recently, a novel conserved domain family, YEATS, was discovered in a variety of eukaryotic species ranging from yeast to human. Taf14, which is involved in a few protein complexes of chromatin remodeling and gene transcription, and essential for keeping chromosome stability, regular cell growth and transcriptional regulation, contains a YEATS domain at its N-terminus. In the present study, we determined the solution structure of Taf14 YEATS domain using NMR spectroscopy. Taf14 YEATS domain adopts a global fold of an elongated β-sandwich, similar to Yaf9 YEATS domain. Meanwhile, Taf14 YEATS domain differs significantly from Yaf9 YEATS domain in some aspects, which might indicate different structural classes...

Domain interactions of the transcription:translation coupling factor E. coli NusG are intermolecular and transient.

BJ Gene — Wed, 23 Feb 2011 00:00:00 +0100

The bacterial transcription factor NusG is suggested to act as a key coupling factor between transcription and translation (Burmann et al. 2010 Science. 328:501-504). and contributes to phage λ mediated antitermination in E. coli that enables read-through of early transcription termination sites. E. coli NusG consists of two structurally and functionally distinct domains that are connected via a flexible linker. The homologous Aquifex aeolicus NusG with a secondary structure that is highly similar to E. coli NusG shows direct interaction between its N-terminal and its C-terminal domain in a domain-swapped dimer. Here we performed NMR paramagnetic relaxation enhancement measurements and identified interdomain interactions that were concentration dependent and thus likely not only wea...

SUMO2 and SUMO3 transcription is differentially regulated by oxidative stress in an Sp1-dependent manner

BJ Gene — Thu, 03 Feb 2011 00:00:00 +0100

We report here that the expression of SUMO3, but not SUMO2, can be down regulated at the transcription level by cellular oxidative stress. In the present study, we checked SUMO2 and -3 mRNA levels in cells exposed to various doses of hydrogen peroxide and in cells bearing different levels of reactive oxygen species (ROS). We found an inverse relation between SUMO3 transcription and ROS levels. We characterized a promoter region specific for the mouse SUMO3 gene that is bound by the redox-sensitive transcriptional factor Sp1 and demonstrated oxidation of Sp1 as well as suppression of Sp1-DNA binding upon oxidative stress. This revealed for the first time that SUMO2 and SUMO3 expressions are regulated differently by ROS. These findings may enhance our understanding about the regulation of SU...

WT1 and its transcriptional cofactor BASP1 redirect the differentiation pathway of an established blood cell line

BJ Gene — Wed, 26 Jan 2011 00:00:00 +0100

The Wilms’ tumour suppressor WT1 is a transcriptional regulator that plays a central role in organogenesis, and is mutated or aberrantly expressed in several childhood and adult malignancies. We previously identified BASP1 as a WT1 cofactor that suppresses the transcriptional activation function of WT1. Here we have analysed the dynamic between WT1 and BASP1 in the regulation of gene expression in myelogenous leukemia K562 cells. Our findings reveals that BASP1 is a significant regulator of WT1 that is recruited to WT1-binding sites and suppresses WT1-mediated transcriptional activation at several WT1 target genes. We find that WT1 and BASP1 can divert the differentiation programme of K562 cells to a non-blood cell type following induction by the phorbol ester PMA. WT1 and BASP1 coo...

The fission yeast Schizosaccharomyces pombe has two distinct tRNase ZLs encoded by two different genes and differentially targeted to the nucleus and mitochondria

BJ Gene — Thu, 06 Jan 2011 00:00:00 +0100

This study provides evidence for partitioning of tRNase Z function between two different proteins in S. pombe, although we cannot rule out specialized functions for each protein. (Source: BJ Gene)

A Functional Family-Wide Screening of SP/KLF Proteins Identifies a Subset of Suppressors of KRAS-Mediated Cell Growth

BJ Gene — Mon, 20 Dec 2010 00:00:00 +0100

SP/KLF transcription factors comprise an emerging group of proteins that may behave as tumor suppressors. Incidentally, many cancers displaying alterations in certain KLF proteins are also associated with a high incidence of KRAS mutations. Therefore, we here investigate whether SP/KLF proteins suppress KRAS-mediated cell growth, and more importantly, the potential mechanisms underlying these effects. Using a comprehensive, family-wide screening of the 24 SP/KLF members, we discover that SP5, SP8, KLF2, KLF3, KLF4, KLF11, KLF13, KLF14, KLF15 and KLF16 inhibit cellular growth and suppress transformation mediated by oncogenic KRAS. Each protein in this subset of SP/KLF members individually inhibits BrdU incorporation in KRAS oncogenic mutant cancer cells. SP5, KLF3, KLF11, KLF13, KLF14 and K...

Functional characterization of hPCL3 (human Polycomb-like 3) isoforms identifies them as components of distinct EZH2 protein complexes

BJ Gene — Fri, 10 Dec 2010 00:00:00 +0100

Polycomb group (PcG) proteins are conserved transcriptional repressors essential to regulate cell fate and to maintain epigenetic cellular memory. They work in concert through two main families of chromatin-modifying complexes, PRC1 and PRC2-4 (Polycomb repressive complexes). In Drosophila, PRC2 contains the H3K27 histone methyltransferase E(Z) whose trimethylation activity towards PcG target-genes is stimulated by Polycomb-like (PCL). Here, we have examined hPCL3, one of its three human paralogues. Through alternative splicing, hPCL3 encodes a long isoform, hPCL3L containing an N-terminal TUDOR and two PHD domains and a smaller isoform, hPCL3S, lacking the second PHD finger (PHD2). By quantitative RT-PCR analyses, we showed that both isoforms are widely co-expressed at high levels in medu...

P14ARF inhibits the functions of adenovirus E1A oncoprotein

BJ Gene — Mon, 06 Dec 2010 00:00:00 +0100

The ARF tumor suppressor is one of the most important oncogenic stress sensors. ARF exerts “oncogenic checkpoint” function through both p53-dependent and p53-independent mechanisms. Here we demonstrate a novel p53-independent interaction between p14ARF and adenovirus oncoprotein E1A. P14ARF inhibits E1A transcriptional function and promotes ubiquitination-dependent degradation of E1A. P14ARF over-expression relocalizes E1A into nucleolus and inhibits E1A-induced cellular DNA replication independent of p53. Knockdown of endogenous p14ARF increase E1A transactivation. In addition, E1A can competitively inhibit ARF-mdm2 complex formation. These results identify a novel binding partner of p14ARF and reveal a mutually inhibitory interaction between p14ARF and E1A. We speculate tha...

A nucleotide insertion between two adjacent methyltransferases in H. pylori results in a bifunctional DNA methyltransferase

BJ Gene — Mon, 29 Nov 2010 00:00:00 +0100

Helicobacter pylori has a dynamic restriction-modification (R-M) system. It is capable of acquiring new R-M systems from the environment in the form of DNA released from other bacteria or other pylori strains. Random mutations in R-M genes can result in non functional R-M systems or R-M systems with new properties. hpyAVIAM and hpyAVIBM are two solitary DNA methyltransferase genes adjacent to each other and lacking a cognate restriction enzyme gene in H. pylori strain 26695. Interestingly, in an Indian strain D27, hpyAVIAM -hpyAVIBM encodes a single bifunctional polypeptide due to insertion of a nucleotide just before the stop codon of hpyAVIBM and when a similar mutation was made in hpyAVIAM -hpyAVIBM from strain 26695, a functional MTase with an N-terminal C5 cytosine MTase domain and a ...

Erk/p90RSK/14-3-3 signalling impacts on expression of PEA3 Ets transcription factors via the transcriptional repressor capic{u}a

BJ Gene — Thu, 18 Nov 2010 00:00:00 +0100

Compounds that inhibit signalling upstream of Erk are promising anti-cancer therapies, motivating research to define how this pathway promotes cancers. Here, we show that human capicúa represses mRNA expression for PEA3 Ets transcription factors ETV1, ETV4 and ETV5, and this repression is relieved by multisite controls of capicúa by Erk, p90RSK and 14-3-3 proteins. Specifically, 14-3-3 binds to p90RSK-phosphorylated Ser173 of capicúa thereby inhibiting DNA binding to its HMG box, while Erk phosphorylations prevent binding of a C-terminal NLS to importin alpha 4 (KPNA3). ETV1, 4 and 5 mRNA levels in melanoma cells are elevated by siRNA knockdown of capicúa, and decreased by inhibiting Erk and/or expressing a form of capicúa that cannot bind to 14-3-3s. Cap...

Crystal structure of the catalytic core of Saccharomyces cerevesiae histone demethylase Rph1: insights into the substrate specificity and catalytic mechanism

BJ Gene — Wed, 10 Nov 2010 00:00:00 +0100

We report here the crystal structures of c-Rph1 in apo form and in complex with Ni2+ and α-ketoglutarate (α-KG). The structure of c-Rph1 is composed of a JmjN domain, a long β-hairpin, a mixed structural motif, and a JmjC domain. The α-KG co-factor forms hydrogen-bonding interactions with the side chains of conserved residues, and the Ni2+ ion at the active site is chelated by conserved residues and the cofactor. Structural comparison of Rph1 with JMJD2A indicates that the substrate-binding cleft of Rph1 is formed with several structural elements of the JmjC domain, the long β-hairpin, and the mixed structural motif; and the methylated Lys36 of H3 is recognized by several conserved residues of the JmjC domain. In vitro biochemical data show ...

Escherichia coli glycogen genes are organized in a single glgBXCAP transcriptional unit possessing an alternative suboperonic promoter within glgC that directs glgAP expression

BJ Gene — Thu, 28 Oct 2010 23:00:00 +0100

Although it is generally accepted that E. coli glycogen genes are organized in two tandemly arranged, differentially regulated glgBX and glgCAP operons, RT-PCR analyses carried out in this work showed that E. coli cells possess transcripts comprising the five glgBXCAP genes. glg::lacZY expression analyses in cells lacking the region immediately upstream of the glgB gene revealed an almost total abolishment of glgB, glgX and glgC expression, but only a 50-60% reduction of the wild type glgA and glgP expression levels. Furthermore, similar type of analyses showed that glgA and glgP expression was almost totally abolished in cells lacking glgA upstream sequences including glgC, glgB and the asd-glgB intergenic region upstream of glgB. The overall data thus indicated that E. coli glgBXCAP gene...

The PKC-{zeta} interacting protein ZIP3 is generated by intronic polyadenylation and is expressed in brain and retina of the rat

BJ Gene — Wed, 27 Oct 2010 23:00:00 +0100

Scaffold proteins contain multiple protein-protein interaction modules that physically assemble functional related proteins into larger complexes. Protein kinase C-ζ interacting proteins (ZIP) link the enzymatic activity of the atypical protein kinase C isoforms PKC-λ/ι or PKC-ζ to target proteins and are associated with neurodegenerative disorders. In the rat, alternative splicing generates 3 ZIP variants. Previously, we identified the ZIP3 transcript containing 13 C-terminal amino acids encoded by intron 4 in the rat central nervous system (CNS). Here, we identified intronic polyadenylation signals in rat and human ZIP genes and detected the corresponding ZIP3-like transcripts. In addition, we generated ZIP3 specific immunesera and observed expression of the p...

SUMO modification selectively regulates transcriptional activity of peroxisome proliferator-activated receptor gamma in C2C12 myotubes

BJ Gene — Sun, 17 Oct 2010 23:00:00 +0100

In conclusion, these results indicate that SENP2 desumoylates PPARgamma in myotubes and desumoylation of PPARgamma selectively increases the expression of some PPARgamma target genes. (Source: BJ Gene)

The pancreatic islet beta cell-enriched transcription factor Pdx-1 regulates Slc30a8 gene transcription through an intronic enhancer

BJ Gene — Wed, 13 Oct 2010 23:00:00 +0100

The SLC30A8 gene encodes the zinc transporter ZnT-8, which provides zinc for insulin-hexamer formation. Genome-wide association studies have shown that a polymorphic variant in SLC30A8 is associated with altered susceptibility to type 2 diabetes and we recently reported that glucose-stimulated insulin secretion is decreased in islets isolated from Slc30a8 knockout mice. The present study examines the molecular basis for the islet-specific expression of Slc30a8. VISTA analyses identified two conserved regions in Slc30a8 introns 2 and 3, designated enhancers A and B, respectively. Transfection experiments demonstrated that enhancer B confers elevated fusion gene expression in both βTC-3 cells and αTC-6 cells. In contrast, enhancer A confers elevated fusion gene expression selec...

Inactivation of eukaryotic initiation factor 5A (eIF5A) by specific acetylation of its hypusine residue by spermidine/spermine acetyltransferase 1 (SSAT1)

BJ Gene — Tue, 12 Oct 2010 23:00:00 +0100

In this study, we report selective acetylation of the hypusine and/or deoxyhypusine residue of eIF5A by a key polyamine catabolic enzyme, spermidine/spermine- N1-acetyltransferase 1 (SSAT1). This enzyme normally catalyzes the N1-acetylation of spermine and spermidine to form acetyl-derivatives, which in turn are degraded to lower polyamines. Although SSAT1 has been reported to exert other effects in cells by its interaction with other cellular proteins, eIF5A is the first target protein specifically acetylated by SSAT1. Hypusine or deoxyhypusine, as the free amino acid, does not act as a substrate for SSAT1, suggesting a macromolecular interaction between eIF5A and SSAT1. Indeed, the binding of eIF5A and SSAT1 was confirmed by pull-down assays. The effect of the acetylation of hypusine on ...

Mycobacterium tuberculosis WhiB1 is an essential DNA-binding protein with a nitric oxide sensitive iron-sulphur cluster

BJ Gene — Thu, 07 Oct 2010 23:00:00 +0100

Mycobacterium tuberculosis is a major pathogen that has the ability to establish, and emerge from, a persistent state. Wbl family proteins are associated with developmental processes in actinomycetes, and M. tuberculosis has seven such proteins. Here it is shown that the M. tuberculosis H37Rv whiB1 gene is essential. The WhiB1 protein possesses a [4Fe-4S]2+ cluster that is stable in air but reacts rapidly with eight equivalents of nitric oxide to yield two dinuclear dinitrosyl-iron thiol complexes. The [4Fe-4S] form of WhiB1 did not bind whiB1 promoter DNA, but the reduced and oxidized apo-WhiB1, and nitric oxide-treated holo-WhiB1 did bind to DNA. Mycobacterium smegmatis RNA polymerase induced transcription of whiB1 in vitro; however in the presence of apo-WhiB1 transcription was s...

The carbohydrate response element binding protein (ChREBP) and not the liver X receptor {alpha} (LXR{alpha}) mediates elevated hepatic lipogenic gene expression in a mouse model of glycogen storage disease type 1.

BJ Gene — Sun, 19 Sep 2010 23:00:00 +0100

Glycogen storage disease type 1 (GSD-1) is caused by an inherited defect in glucose-6-phosphatase activity, resulting in massive accumulation of hepatic glycogen content and an induction of de novo lipogenesis. The chlorogenic acid derivative S4048 is a pharmacological inhibitor of the glucose-6-phosphate transporter, which is part of glucose-6-phosphatase, and allows for mechanistic studies concerning metabolic defects in GSD-1. Treatment of mice with S4048 resulted in ~60% reduction of blood glucose, increased hepatic glycogen and triglyceride content, and a markedly enhanced hepatic lipogenic gene expression. In mammals, hepatic expression of lipogenic genes is regulated by the coordinated action of the transcription factors sterol-regulatory element-binding protein-1c (SREBP-1c), liver...

Recombinant mammalian DNA methyltransferase activity on model transcriptional gene silencing short RNA:DNA heteroduplex substrates

BJ Gene — Thu, 16 Sep 2010 23:00:00 +0100

The biochemical mechanism of short RNA induced transcriptional gene silencing (TGS) in mammals is unknown. Two competing models exist; one suggesting the short RNA interacts with a nascent transcribed RNA strand (RNA:RNA model) and the other implying that short RNA forms a heteroduplex with DNA from the unwound double helix – an R-loop structure (RNA:DNA model). Likewise, the requirement for DNA methylation to enact TGS is still controversial. In vitro assays using purified recombinant murine DNA methyltransferase 1-dN, 3a and 3b enzymes and annealed oligonucleotides were designed to question whether DNA methyltransferases methylate DNA in a DNA:RNA heteroduplex context and if a DNA:RNA heteroduplex R-loop is a good substrate for DNA methyltransferases. Specifically, model synthetic...

MicroRNA-7 targets insulin-like growth factor 1 receptor (IGF1R) in tongue squamous cell carcinoma cells

BJ Gene — Mon, 06 Sep 2010 23:00:00 +0100

MicroRNA-7 (miR-7) has been characterized as a tumor suppresser in several human cancers. It targets a number of proto-oncogenes that contribute to cell proliferation and survival. However, the mechanism(s) by which miR-7 suppresses tumorigenesis in tongue squamous cell carcinoma (TSCC) is unknown. Our bioinformatics analysis revealed that the insulin-like growth factor 1 receptor (IGF1R) mRNA is a potential target for miR-7. Ectopic transfection of miR-7 led to significant reduction of IGF1R at both the mRNA and protein levels in the TSCC cells. Knockdown of miR-7 in the TSCC cells enhanced the IGF1R expression. Direct targeting of miR-7 to three candidate binding sequences located in the 3’-untranslated region of IGF1R mRNA was confirmed using luciferase reporter gene assays. The ...

Antagonistic Roles of the ERK and p38 MAPK Signaling Pathways in Globin Expression, Heme Biosynthesis and Iron Uptake

BJ Gene — Wed, 25 Aug 2010 23:00:00 +0100

Late stage erythroid cells synthesize large quantities of hemoglobin, a process requiring the coordinated regulation of globin and heme synthesis as well as iron uptake. Here, we investigated the role of the Erk and p38 mitogen-activated protein kinase (MAPK) signaling pathways in murine erythroleukemia (MEL) cell differentiation. We found that treatment of hexamethylene bisacetamide (HMBA) induced MEL cells with the Erk pathway inhibitor UO126 results in the increase of intracellular heme and hemoglobin levels. The transcript levels of the genes coding for betamajor-globin, the heme biosynthesis enzyme 5-aminolevulinate synthase2 and the mitochondrial iron transporter mitoferrin 1 are upregulated. We also showed enhanced expression of globin and transferrin receptor 1 proteins upon UO126 ...

The HDHD1 gene, often deleted in X-linked ichthyosis, encodes a pseudouridine-5{'}-phosphatase

BJ Gene — Thu, 19 Aug 2010 23:00:00 +0100

In conclusion, HDHD1 is a phosphatase specifically involved in dephosphorylation of a modified nucleotide present in RNA. (Source: BJ Gene)

Epidermal growth factor down-regulates the expression of human hepatic stimulator substance via CCAAT/enhancer binding protein {beta} in HepG2 cells

BJ Gene — Thu, 05 Aug 2010 23:00:00 +0100

In this study, we investigated transcription of hHSS triggered by epidermal growth factor (EGF) and the role of CCAAT/enhancer-binding protein β (C/EBP β) as a potential core factor responsible for hHSS transcription in HepG2 cells. The results show that EGF suppresses hHSS mRNA expression at early time points. Using a promoter deletion assay, we identified a proximal region (-358/-212) that is required for EGF suppression. Overexpression of C/EBPβ enhances EGF suppression of hHSS, and mutation of the C/EBPβ binding site at -292/-279 or siRNA interference abolishes EGF suppression. Furthermore, using an electrophoresis mobility shift assay and chromatin immunoprecipitation analysis, we found that C/EBPβ specifically binds to the -292/-279 site that is res...

Cadmium regulates copper homeostasis by inhibiting the activity of Mac1, a transcriptional activator of the copper regulon, in Saccharomyces cerevisiae

BJ Gene — Thu, 29 Jul 2010 23:00:00 +0100

Cadmium is a toxic metal, and the mechanism of its toxicity has been studied in various model systems from bacteria to mammals. We employed S. cerevisiae as a model system to study cadmium toxicity at the molecular level because it has been used to identify the molecular mechanisms of toxicity in higher organisms. cDNA microarray and northern blot analyses revealed that cadmium salts inhibited the expression of genes related to copper metabolism. Western blots, northern blots, and chromatin immunoprecipitation experiments indicated that CTR1 expression was inhibited at the transcriptional level through direct inhibition of the Mac1 transcriptional activator. The decreased expression of CTR1 results in cellular copper deficiency and inhibition of Fet3 activity and eventually impairs iron up...

The PedR transcriptional regulator interacts with thioredoxin to connect photosynthesis with gene expression in cyanobacteria

BJ Gene — Mon, 26 Jul 2010 23:00:00 +0100

In this study, we found that the conformational change of PedR and the change in transcript level of its target gene were minimal when mutants of Synechocystis that lack ferredoxin-Trx reductase or NADPH-Trx reductase were exposed to high light. These results indicate that the reduction of PedR by Trx causes transient inactivation of PedR upon the shift of cyanobacterial cells to high-light conditions. (Source: BJ Gene)

SREBP-1c Mediates the Effect of Insulin on the Expression of Cidea in Mouse Hepatocytes

BJ Gene — Wed, 23 Jun 2010 23:00:00 +0100

Members of the cell death-inducing DFF45-like effector (Cide) gene family have been reported to be associated with lipid metabolism. In this article, we show that Cidea mRNA levels are markedly reduced by fasting and are restored upon refeeding in mouse livers. To elucidate the molecular mechanism, the promoter region of the mouse Cidea gene was analyzed and a putative sterol regulatory element (SRE) was identified. Studies using luciferase reporter constructs together with electrophoretic mobility shift assays and chromatin immunoprecipitation confirmed the binding of SREBP-1c to the putative SRE. Furthermore, adenovirus-mediated overexpression of SREBP-1c led to a dramatic increase in Cidea mRNA. In contrast to the induction of Cidea expression by insulin and TO901317 in wild type mouse ...

Post-transfer editing by a eukaryotic leucyl-tRNA synthetase resistant to the broad-spectrum drug AN2690

BJ Gene — Thu, 17 Jun 2010 23:00:00 +0100

Some aminoacyl-tRNA synthetases (aaRSs) develop editing mechanism to correct mis-charged tRNA. The connective peptide 1 (CP1) domain of leucyl-tRNA synthetase (LeuRS) contains the editing active site, which is the proven target for the broad-spectrum drug AN2690. The eukarya-specific insertion 1 (ESI) in the CP1 domain of Giardia lamblia LeuRS (GlLeuRS) has been identified. Similar substitution with ESI from Homo sapiens LeuRS (HsLeuRS) impeded the leucine activation, aminoacylation, and post-transfer editing of the enzyme, but had no effect on the editing specificity toward nonspecific amino acid. The Thr341 in GlLeuRS served as a specificity discriminator as in other LeuRS systems although its Ala substitution did not destroy Leu-tRNALeu synthesis in vitro and in vivo. The Arg338 was cru...

Unusual organisation, complexity and redundancy at the Escherichia coli hcp-hcr operon promoter

BJ Gene — Wed, 09 Jun 2010 23:00:00 +0100

Expression from the Escherichia coli hcp-hcr operon promoter is optimally induced during anaerobic conditions in the presence of nitrite. This expression depends on transcription activation by FNR, which binds to a target centred at position ‑72.5 upstream of the transcript start site. Mutational analysis was exploited to identify the corresponding -10 and -35 hexamer elements. A DNA site for NarL and NarP, located at position ‑104.5, plays only a minor role, whilst NsrR binding to a DNA target centred at position +6 plays a major role in induction of the hcp‑hcr operon promoter. Electromobility shift assays show that NsrR binds to this target. The consequences of this for the kinetics of induction of the hcp‑hcr operon are discussed. (Source: BJ Gene)

Effect of 8-oxo-guanine and abasic site DNA lesions on in vitro elongation by human DNA polymerase {epsilon} in the presence of replication protein A and proliferating cell nuclear antigen

BJ Gene — Mon, 07 Jun 2010 23:00:00 +0100

DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes. Here we show that human DNA pol ε can efficiently bypass an 8-oxo-G lesion on the template strand by inserting either dCMP or dAMP opposite to it, but it cannot bypass an abasic site. During replication DNA polymerases associate with accessory proteins that may alter their bypass ability. We investigated the role of the human DNA sliding clamp PCNA and of the human single-stranded DNA binding protein RPA in the modulation of the DNA synthesis and translesion capacity of DNA pol ε. RPA inhibited the elongation by human DNA pol ε on templates annealed to short primers. PCNA did not influence the elongation by DNA pol e and had no effect on inhibition of elongation caused by RPA. RP...

Human Pif1 helicase is a G-quadruplex DNA binding protein with G-quadruplex DNA unwinding activity

BJ Gene — Thu, 03 Jun 2010 23:00:00 +0100

Pif1 proteins are helicases that in yeast are implicated in the maintenance of genome stability. One activity of Saccharomyces cerevisiae Pif1 is to stabilize DNA sequences that could otherwise form deleterious G-quadruplex (G4) structures by acting as a G4 resolvase. Evidence is presented here that human Pif1 (hPif1, nuclear form) is a G4 DNA binding and resolvase protein and that these activities are properties of the conserved helicase domain (amino acids 206-620 of 641, hPifHD). hPif1 preferentially bound synthetic G4-DNA relative to ssDNA, dsDNA and a partially single-stranded duplex DNA helicase substrate. G4 DNA unwinding but not binding required an extended (<10 nucleotide) 5’ ssDNA tail and in competition assays G4 DNA was an ineffective suppressor of helicase activity c...

Amino acid residues in the {beta}3 strand and subsequent loop of the conserved ETS domain that mediate bZIP recruitment and potentially distinguish functional attributes of Ets proteins

BJ Gene — Wed, 02 Jun 2010 23:00:00 +0100

Ets family members share a conserved DNA-binding ETS domain, and serve a variety of roles in development, differentiation, and oncogenesis. Besides DNA binding, the ETS domain also participates in protein-protein interactions with other structurally unrelated transcription factors. Although such mechanism appears to confer tissue- or development stage-specific functions on individual Ets proteins, the biological significance of many of these interactions remains to be evaluated, because their molecular basis has been elusive. We previously demonstrated a direct interaction between the ETS domain of widely expressed GABPα (GA-binding protein α) and the granulocyte inducer C/EBPα (CCAAT/enhancer-binding protein α), and suggested its involvement in cooperative tran...

Regulation of plasma membrane-associated sialidase NEU3 gene by SP1/SP3 transcription factors

BJ Gene — Tue, 01 Jun 2010 23:00:00 +0100

Gene expression of the human plasma membrane-associated sialidase (NEU3), a key enzyme for ganglioside degradation, is relatively high in brain and is modulated in response to many cellular processes including neuronal cell differentiation and tumorigenesis. We previously demonstrated NEU3 to be markedly up-regulated in various human cancers and showed that NEU3 transgenic mice developed a diabetic phenotype and were susceptible to azoxymethane-induced aberrant crypt foci in their colon tissues. These results suggest that appropriate control of NEU3 gene expression is required for homeostasis of cellular functions. To gain insights into regulation mechanisms, we determined the gene structure and assessed transcription factor involvement. Oligo-capping analysis indicated the existence of al...

Translational Control of the sterol regulatory transcription factor SREBP-1 mRNA in response to serum starvation or ER stress is mediated by an internal ribosome entry site

BJ Gene — Mon, 31 May 2010 23:00:00 +0100

In this study evidences for SREBP-1 regulation at translational level have been reported. By several experimental approaches, we demonstrated that 5' UTR of the SREBP-1a mRNA contains an internal ribosome entry site (IRES). Transfection experiments with SREBP-1a UTR inserted in a dicistronic reporter vector showed a remarkable increase of the downstream cistron translation, through a cap-independent mechanism. Insertion of the SREBP-1c 5’ UTR in the same vector also stimulated the translation of the downstream cistron, but the observed effect can be ascribed, at least in part, to a cryptic promoter activity. Cellular stress conditions, such as serum starvation, caused in both Hep G2 and HeLa cells an increase in the level of SREBP-1 precursor and mature form, despite the overall red...

Regulation of renal sodium-dependent phosphate co-transporter genes (Npt2a and Npt2c) by all-trans retinoic acid and its receptors

BJ Gene — Wed, 26 May 2010 23:00:00 +0100

In this study, we observed the effects of a vitamin A-deficient (VAD) diet on Pi homeostasis and the expression of Npt2a and Npt2c genes in rat kidney. There was no change in the plasma levels of Pi, but VAD rats significantly increased renal Pi excretion. Renal brush border membrane Pi uptake activity and renal Npt2a and Npt2c expressions were significantly decreased in VAD rats. The transcriptional activity of a luciferase reporter plasmid containing the promoter region of human Npt2a and Npt2c genes was increased markedly by ATRA and a retinoic acid receptor (RAR)-specific analog (TTNPB) in renal proximal tubular cells over-expressing RAR and RXR. Furthermore, we identified retinoic acid response elements (RAREs) in both gene promoters. Interestingly, the half-site sequences (5’-...

Functional characterization of leucine-specific domain 1 from eukaryal and archaeal leucyl-tRNA synthetases

BJ Gene — Tue, 18 May 2010 23:00:00 +0100

Leucyl-tRNA synthetase (LeuRS) catalyzes the esterification of tRNAsLeu with leucine. This family of enzymes is divided into prokaryotic and eukaryal/archaeal groups according to the presence and position of specific insertions and extensions. Here, we investigate the function of leucine-specific domain 1 (LSD1) which is naturally present in eukaryal/archaeal LeuRSs but absent in prokaryotic LeuRSs. When mutated in their common domain, the eukaryal and archaeal LeuRSs exhibited defects in the first reaction step of amino acid activation with variations of leucine or ATP binding strength, whereas the tRNA aminoacylation was moderately affected. When the eukaryal extension was mutated, severe tRNA charging defects were observed, suggesting that eukaryotes evolved this LSD1 extension in order...

Sterol regulatory element-binding protein 2 and nuclear factor Y controls human farnesyl diphosphate synthase expression and affects cell proliferation in hepatoblastoma cells

BJ Gene — Thu, 06 May 2010 23:00:00 +0100

In this study, we characterized the sterol-response element and nuclear factor Y-binding site in the human FDPS promoter. Using a luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay, we demonstrated that these elements are responsible for the transcription of the FDPS gene, and that its transcriptional activation is mediated by sterol regulatory element-binding protein 2 (SREBP-2) and nuclear factor Y (NF-Y). We also investigated whether sterol-mediated FDPS expression is involved in the cell proliferation induced by zoledronic acid, an FDPS inhibitor. We show that the SREBP-2 and NF-Y-mediated regulation of FDPS gene transcription modulates cell proliferation. These results suggest that SREBP-2 and NF-Y are required to trigger cell proliferation...

SUMO Modification Regulates Transcriptional Activity of XBP1

BJ Gene — Wed, 21 Apr 2010 23:00:00 +0100

The unfolded protein response (UPR), a cellular defense mechanism against misfolded protein accumulation in the endoplasmic reticulum (ER), is associated with many human diseases such as aging, cancer and diabetes. X-box binding protein 1 (XBP1), a key transcription factor of the UPR, is critical in maintaining ER homeostasis. Nonetheless, the mechanism by which XBP1 transcriptional activity is regulated remains unexplored. Here we show that the active form of XBP1 protein, XBP1s, is SUMOylated mainly by the protein inhibitors of activated STAT 2 (PIAS2) at two lysine residues located at the C-terminal transactivation domain. Ablation of these SUMOylation events significantly enhances the transcriptional activity of XBP1s towards UPR target genes. Thus, our results reveal a previously unex...

Temporal regulation of Cat-1 gene transcription during endoplasmic reticulum stress

BJ Gene — Tue, 20 Apr 2010 23:00:00 +0100

Expression of the cationic amino acid transporter-1 gene (Cat-1) is induced in proliferating cells and in response to a variety of stress conditions. The expression of the gene is mediated via a TATA-less promoter. It is shown here that an Sp1-binding site within a GC-rich region of the Cat-1 gene controls its basal expression and is important for induction of the gene during the unfolded protein response (UPR). We have previously shown that induction of Cat-1 gene expression during the UPR requires phosphorylation of the translation initiation factor eIF2α by PERK kinase, one of the signaling pathways activated during the UPR. This leads to increased translation of the transcription factor ATF4. We also show that a second signaling pathway is required for sustained transcriptional ...

Cellular nucleic acid binding protein, a transcriptional enhancer of c-Myc, promotes the formation of parallel G-quadruplexes

BJ Gene — Wed, 14 Apr 2010 23:00:00 +0100

G-rich sequences that contain stretches of tandem guanines can form four-stranded, intramolecular, stable DNA structures called G-quadruplexes (G4s). Regulation of the equilibrium between single-stranded and G4 DNA in promoter regions is essential for control of gene expression in the cell. G4s are highly stable structures, however, their folding kinetics are slow under physiological conditions. Cellular Nucleic Acid Binding Protein (CNBP) is a nucleic acid chaperone that binds the G4-forming G-rich sequence located within the nuclease hypersensitivity element (NHE) III of the c-Myc proto-oncogene promoter. Several reports have demonstrated that CNBP enhances the transcription of c-Myc in vitro and in vivo. However, none of these reports have assessed the molecular mechanisms responsible f...

SENP1 participates in the dynamic regulation of Elk-1 sumoylation.

BJ Gene — Thu, 25 Mar 2010 00:00:00 +0100

The modification of proteins with SUMO plays an important role in determining their functional properties. Importantly though, sumoylation is a highly dynamic process enabling transient responses to be elicited. This dynamism is controlled by two competing conjugating and deconjugating activities. The latter activity is mediated by the SENP family of SUMO-specific proteases. The transcription factor Elk-1 undergoes rapid desumoylation following cellular stimulation with growth factors, and this contributes to its conversion from a SUMO-dependent repressor to a potent transcriptional activator. Here we demonstrate an important role for SENP1 in the desumoylation of Elk-1 and therefore an integral role in determining the Elk-1-dependent transcriptional programme. Amongst the SENPs, Elk-1 pre...

Myogenic regulatory factors transactivate the Tceal7 gene and modulate muscle differentiation

BJ Gene — Tue, 23 Mar 2010 00:00:00 +0100

Recurrent injuries eventually exhaust the capacity of skeletal muscle to fully restore or regenerate its cellular architecture. Therefore, a comprehensive understanding of the muscle regeneration program is needed to provide a platform for new therapies for devastating diseases such as Duchenne Muscle Dystrophy. To begin to decipher the molecular program that directs muscle regeneration, we undertook an unbiased strategy using microarray analysis of cardiotoxin injured skeletal muscle at defined time periods in the adult mouse. Using this strategy, we identified Tceal7, which was dynamically regulated during muscle regeneration. Our studies revealed that Tceal7 was restricted to the skeletal muscle lineage during embryogenesis. Using transgenic technologies and transcriptional assays, we d...

Regulation of the miR-212/132 locus by MSK and CREB in response to neurotrophins.

BJ Gene — Tue, 23 Mar 2010 00:00:00 +0100

Neurotrophins are growth factors that are important in neuronal development and survival as well as synapse formation and plasticity. Many of the effects of neurotrophins are mediated by changes in protein expression as a result of altered transcription or translation. To determine if neurotrophins regulate the production of microRNAs, small RNA species that modulate protein translation or mRNA stability, we used deep sequencing to identify brain derived neurotrophic factor (BDNF) induced microRNAs in cultured primary cortical mouse neurons. This revealed that the miR-212/132 cluster contained the microRNAs most responsive to BDNF treatment. This cluster was found to produce four microRNAs; miR-132, miR-132*, miR-212, miR-212*. Using specific inhibitors, mouse models and promoter analysis ...

The DNA-binding activity of mouse DNA methyltransferase 1 is regulated by phosphorylation with casein kinase 1{delta}/{epsilon}

BJ Gene — Mon, 01 Mar 2010 00:00:00 +0100

DNA methyltansferase 1 (Dnmt1) is an enzyme that recognizes and methylates hemimethylated DNA during DNA replication to maintain methylation patterns. The N-terminal region of Dnmt1 is known to form an independent domain structure that interacts with various regulatory proteins and DNA. In the present study, we investigated protein kinases in the mouse brain that could bind and phosphorylate the N-terminal regulatory domain of Dnmt1. A protein fraction containing protein kinase activity for phosphorylation of Dnmt1(1-290) was prepared using Dnmt1(1-290)-affinity, DNA-cellulose and gel-filtration columns. When the proteins in this fraction were analyzed by LC-MS/MS, casein kinase 1δ/ε (CK1δ/ε) was the only protein kinase identified. Recombinant CK1δ/;...

The small heterodimer partner suppresses the transcriptional activity and nuclear localization of hedgehog signaling protein Gli1

BJ Gene — Tue, 23 Feb 2010 00:00:00 +0100

Glioma-associated oncogene homologue (Gli) acts as a terminal effector of hedgehog signaling pathway which is implicated in the development of many human malignancies. Gli activation is important for cell proliferation and anti-apoptosis in various cancers. Several studies have suggested that nuclear receptors have anti-cancer effects by inhibiting the activation of various oncoproteins. However, the involvement of nuclear receptors on Hedgehog-Gli signal pathway is poorly known. Here, we identified small heterodimer partner (SHP) as a nuclear receptor that decreased the expression of Gli target genes by repressing the transcriptional activity of Gli1. The inhibitory effect of SHP was associated with the inhibition of Gli1 nuclear localization by protein-protein interaction. Finally, SHP o...

ZEB1 and CtBP form a repressive complex at a distal promoter element of the BCL6 locus

BJ Gene — Tue, 23 Feb 2010 00:00:00 +0100

BCL6 is essential for normal antibody responses and is highly expressed in germinal centre B-cells. Constitutive expression due to chromosomal translocations or mutations of cis-acting regulatory elements contributes to diffuse large B-cell lymphoma. BCL6 expression is, therefore, tightly regulated in a lineage and developmental stage specific manner and disruption of normal controls can contribute to lymphomagenesis. In order to discover potential cis-acting control regions we carried out DNase I hypersensitive site mapping. Gel shift assays and chromatin immuno-precipitation of the core region of a hypersensitive site 4.4kb upstream of BCL6 transcription initiation (HSS-4.4) showed an E-box element binding ZEB1 and the co-repressor CtBP. As compared to peripheral blood B-cells, ZEB1, a t...

Structural and functional analysis of amino-terminal enhancer of split in androgen receptor-driven transcription

BJ Gene — Wed, 17 Feb 2010 00:00:00 +0100

We previously demonstrated that the Groucho protein amino-terminal enhancer of split (AES) functions as a co-repressor of the androgen receptor (AR). It physically interacts with the N-terminal domain of AR and inhibits AR-driven transcription, but the molecular mechanism of its action remains unclear. Herein we report that the AES protein contains one inhibitory domain and one positive and one negative regulatory domain. The negative regulatory domain inhibits AES dimerization and AES-mediated inhibition of AR-driven transcription through the interaction with the inhibitory domain. Also, the positive regulatory domain blocked this interaction and relieved the inhibitory effect. In addition, we discovered mechanisms by which AES regulates AR transcriptional activity, which included disrupt...

Genomic organization and regulation of the human orexin (hypocretin) receptor 2 gene: identification of alternative promoters

BJ Gene — Mon, 15 Feb 2010 00:00:00 +0100

In conclusion, we demonstrate expression of the hOX2R is regulated by a complexity involving a proximal PKA/PKC regulated promoter and a distal promoter regulating tissue specific expression of alternate transcripts which in turn post transcriptionally regulate receptor levels. (Source: BJ Gene)

MMP28 gene expression is regulated by Sp1 transcription factor acetylation

BJ Gene — Tue, 09 Feb 2010 00:00:00 +0100

This study demonstrates that MMP28 expression is induced by histone deacetylase (HDAC) inhibitors and that this effect is mediated through the GT-box. Transient transfection assays have shown that the induction of MMP28 expression by the HDAC inhibitior trichostatin A (TSA) is mediated via Sp1 at the GT-box. Immunoprecipitation experiments have shown that the acetylation of Sp1 and Sp3 is increased by TSA treatment, however no effect on DNA binding was observed. Histone acetyltransferases such as p300 and P/CAF increased induction of the MMP28 promoter by Sp1. Knockdown of HDAC1 using siRNA also induces the MMP28 promoter. Oligonucleotide pulldown identified serine threonine receptor-associated protein (STRAP) as a further protein recruited to the MMP28 promoter and acting functionally wit...

Crz1 destabilizes Msn2 and Msn4 in the nucleus in response to Ca2{+} in Saccharomyces cerevisiae: FK506 has an additive effect on the Ca2{+}-induced expression of GLO1 via Msn2, Hog1, and the stress response element

BJ Gene — Tue, 02 Feb 2010 00:00:00 +0100

Although methylglyoxal is derived from glycolysis, it has adverse effects on cellular function. Hence, the intrinsic role of methylglyoxal in vivo remains to be determined. Glyoxalase I is a pivotal enzyme in the metabolism of methylglyoxal in all types of organisms. To learn about the physiological roles of methylglyoxal, we have screened conditions that alter the expression of the gene encoding glyoxalase I (GLO1) in Saccharomyces cerevisiae. Here we show that the expression of GLO1 is induced following treatment with CaCl2 dependent on Hog1-MAP kinase and the Msn2/Msn4 transcription factors. Intriguingly, the Ca2+-induced expression of GLO1 was enhanced in the presence of FK506, a potent inhibitor of calcineurin. Consequently, the Ca2+-induced expression of GLO1 in a mutan...

Transcriptional regulation of the human acetoacetyl-CoA synthetase gene by PPAR{gamma}

BJ Gene — Tue, 26 Jan 2010 00:00:00 +0100

In the cytosol of lipogenic tissue, ketone bodies are activated by acetoacetyl-CoA synthetase (AACS) and incorporated into cholesterol and fatty acids. AACS gene expression is particularly abundant in white adipose tissue, as it is induced during adipocyte differentiation. In order to elucidate the mechanism controlling the gene expression of human AACS and to clarify its physiological role, we isolated the human promoter, characterized the elements required to initiate transcription and analyzed the expression of the gene in response to PPARγ, an inducer of adipogenesis. We show that the human AACS promoter is a PPARγ target gene and that this nuclear receptor is recruited to the AACS promoter by direct interaction with Sp1. (Source: BJ Gene)

The cathepsin L gene is a direct target of FOXO1 in the skeletal muscle

BJ Gene — Wed, 20 Jan 2010 00:00:00 +0100

This study provides in vivo and in vitro evidence that cathepsin L is a direct target of FOXO1 in the skeletal muscle, thereby suggesting a role of the FOXO1/cathepsin L pathway in fasting-induced skeletal muscle metabolic change and atrophy. (Source: BJ Gene)

Dimer:dimer stacking Interactions are important for nucleic acid binding by the archaeal chromatin protein Alba

BJ Gene — Mon, 18 Jan 2010 00:00:00 +0100

Archaea use a variety of small basic proteins to package their DNA. One of the most widespread and highly conserved is the Alba (Sso10b) protein. Alba interacts with both DNA and RNA in vitro, and we show here that it binds more tightly to dsDNA than to either ssDNA or RNA. The Alba protein is dimeric in solution, and forms distinct ordered complexes with DNA that have been visualised by electron microscopy studies, these studies suggest that on binding dsDNA the protein forms extended helical protein fibres. An end-to-end association of consecutive Alba dimers is suggested by the presence of a dimer:dimer interface in crystal structures of Alba from several species, and by the strong conservation of the interface residues, centred on Arg-59 and Phe-60. Here we map perturbation of the poly...

Thyroid hormones regulate phosphate homeostasis through transcriptional control of the renal type IIa sodium-dependent phosphate co-transporter (Npt2a) gene

BJ Gene — Fri, 15 Jan 2010 00:00:00 +0100

The renal type IIa sodium-dependent phosphate (NaPi) co-transporter Npt2a, is implicated in the control of serum phosphate levels. Previously, it has been demonstrated that renal Npt2a protein and mRNA expression are upregulated by 3, 5, 3’-tri-iodothyronine (T3) in rats. However, it has never been established whether the induction was mediated by a direct effect of thyroid hormones on the Npt2a promoter. To address the role of Npt2a in T3-dependent regulation of phosphate homeostasis and to identify the molecular mechanisms by which thyroid hormones modulate Npt2a gene expression, mice were rendered pharmacologically hypo- and hyperthyroid. Hypothyroid mice showed low levels of serum phosphate and a marked decrease in renal Npt2a protein abundance. Importantly, Npt2a-deficient mice...

Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase(pCMAH) for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig-to-human xenotransplantation

BJ Gene — Tue, 05 Jan 2010 00:00:00 +0100

In this study, pig CMP-N-acetylneuraminic acid hydroxylase gene (pcmah), a key enzyme for the synthesis of N-glycolylneuraminic acid (NeuGc), was cloned from pig small intestine and characterized. The open reading frame (ORF) of pcmah was 1,734 bp encoded 577 amino acids and consists of fourteen exons. Organ expression pattern analysis reveals that pig CMAH mRNA is mainly expressed in pig rectum, tongue, spleen, and colon tissues, being the most highly expressed in small intestine. In the ectopic expression of pcmah, when pig kidney PK15 cells and human vascular endothelial ECV304 cells were transfected with the cloned pcmah, NeuGc contents of these transfectants were greater in comparison to vector transfectants used as control. In addition, in the functional analysis of NeuGc, human seru...

Characterization of RNase HII substrate recognition using RNase HII-Argonaute chimeric enzymes from Pyrococcus furiosus

BJ Gene — Tue, 05 Jan 2010 00:00:00 +0100

Ribonuclease H (RNase H) is an endonuclease that cleaves the RNA strand of RNA/DNA duplexes. It has been reported that the three-dimensional (3D) structure of RNase H is similar to that of the PIWI domain of the Pyrococcus furiosus Argonaute (Pf-Ago) protein, although the two enzymes show almost no similarity in their amino-acid sequences. Eukaryotic Ago proteins are key components of the RNA-induced silencing complex and are involved in microRNA or small interfering RNA recognition. In contrast, prokaryotic Ago proteins show greater affinity to RNA/DNA hybrids than for RNA/RNA hybrids. Interestingly, we found that wild-type Pf-RNase HII digests an RNA/RNA duplex in the presence of Mn2+ ion. To characterize the substrate specificities of Pf-RNase HII, we aligned the amino-acid seque...

Euglena ascorbate peroxidase forms an intra-molecular dimeric structure: its unique molecular characterization

BJ Gene — Thu, 17 Dec 2009 00:00:00 +0100

Euglena gracilis lacks a catalase and contains a single ascorbate peroxidase (APX) and enzymes related to the redox cycle of ascorbate in the cytosol. In the present study, a full-length cDNA clone encoding the Euglena APX was isolated and found to contain an open reading frame encoding a protein of 649 amino acids with a calculated molecular weight of 70.5 k. Interestingly, the enzyme consisted of two entirely homologous catalytic domains, designated APX-N and APX-C, and a 102-amino acid extension in the N-terminal region, which had a typical class II signal for plastid targeting proposed in Euglena. A computer-assisted analysis indicated a novel protein structure with an intra-molecular dimeric structure. The analysis of cell fractionation showed that the APX protein is distributed in th...

Thr-435 phosphorylation regulates RelA (p65) NF-{kappa}B subunit transactivation

BJ Gene — Tue, 15 Dec 2009 00:00:00 +0100

Phosphorylation of the RelA (p65) NF-κB subunit has been previously shown to modulate its ability to induce or repress transcription. Here we have investigated the consequences of Thr-435 phosphorylation within the C-terminal transactivation domain of RelA. We confirm that Thr-435 is phosphorylated in cells and is induced by TNFα treatment. Mutational analysis of this site revealed gene specific effects on transcription, with a T435D phosphomimetic mutant significantly enhancing Cxcl2 mRNA levels in reconstituted Rela-/- mouse embryonic fibroblasts. Chromatin immunoprecipitation analysis revealed that this mutation results in enhanced levels of histone acetylation, associated with decreased recruitment of the histone deacetylase HDAC1. Moreover, mutation of this site disrupte...

Caspase cleavage of Ets-1 p51 generates fragments with transcriptional dominant-negative function

BJ Gene — Thu, 10 Dec 2009 00:00:00 +0100

In this study, we demonstrate that caspase cleavage of Ets-1 within the exon VII-encoded region leads to specific down-regulation of the Ets-1 p51 isoform during apoptosis. Further, our results establish that caspase cleavage generates a stable C-terminal fragment that acts as a natural dominant-negative of the full-length Ets-1 p51 protein. (Source: BJ Gene)

Androgen regulation of the prostatic tumour suppressor NKX3.1 is mediated by its 3{'}untranslated region

BJ Gene — Tue, 03 Nov 2009 00:00:00 +0100

The homeodomain transcription factor NKX3.1 is a prostate-specific tumour suppressor, expression of which is reduced or undetectable in the majority of metastatic prostate tumours. In the normal prostate and in prostate cancer cells, NKX3.1 expression is under tight androgenic control that we have shown to be mediated by its ~2.5kb 3’ untranslated region (3’UTR). Reporter deletion analysis of the NKX3.1 3’UTR identified three regions that were transactivated by 5α-dihydrotestosterone (DHT) in the androgen receptor (AR)-expressing prostate cancer cell line, LNCaP. Reversal of DHT effects by the antiandrogen, bicalutamide supported an AR-mediated mechanism and bioinformatic analysis of the NKX3.1 3’UTR identified canonical androgen response elements (AREs) ...

Chloroplast HCF101 is a scaffold protein for [4Fe-4S] cluster assembly

BJ Gene — Thu, 08 Oct 2009 23:00:00 +0100

Oxygen evolving chloroplasts possess their own iron-sulfur cluster assembly proteins including members of the SUF and the NFU family. Recently, the chloroplast protein HCF101 (high chlorophyll fluorescence) has been shown to be essential for the accumulation of the membrane complex photosystem I and the soluble ferredoxin-thioredoxin reductases, both containing [4Fe-4S] clusters. The protein belongs to the FSC ([4Fe-4S]-cluster) superfamily of P-loop NTPases, several members of which play a crucial role in Fe/S cluster biosynthesis. Although the C-terminal Fe/S cluster binding site conserved in other members of the FSC family is not present in chloroplast HCF101 homologues, using Mössbauer and EPR spectroscopy, we provide evidence that HCF101 binds a [4Fe-4S] cluster. 55Fe incorpora...

Regulatory factors controlling transcription of Saccharomyces cerevisiae IXR1 (ORD1) by oxygen levels. A model of transcriptional adaptation from aerobiosis to hypoxia implicating ROX1 and IXR1 cross-regulation

BJ Gene — Mon, 05 Oct 2009 23:00:00 +0100

In this study it is shown that IXR1 mRNA levels are controlled by oxygen availability, increasing during hypoxia. In aerobiosis, low levels of IXR1 expression are maintained by Rox1p repression through the general co-repressor complex Tup1-Ssn6. Ixr1p is necessary for full IXR1 expression under hypoxic conditions. Deletion analyses have identified the region in the IXR1 promoter responsible for this positive auto-control (-557 to -376). EMSA and ChIP assays show that Ixr1p binds to the IXR1 promoter both in vitro and in vivo. Ixr1p is required for hypoxic repression of ROX1 and binds to its promoter. UPC2 deletion has opposite effects on IXR1 and ROX1 transcription during hypoxia. Ixr1p is also necessary for resistance to oxidative stress generated by H2O2. IXR1 expression is moderately ac...

PBX1 and MEIS1 up-regulate SOX3 gene expression by direct interaction with a consensus binding site within the basal promoter region

BJ Gene — Thu, 01 Oct 2009 23:00:00 +0100

Sox3/SOX3 is considered to be one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. We previously reported characterization of the SOX3 promoter and demonstrated that the general transcription factors NF-Y, Sp1 and USF are involved in transcriptional regulation of SOX3 promoter activity. Here we present the first evidence that the TALE transcription factors PBX1 and MEIS1 participate in regulating human SOX3 gene expression in NT2/D1 cells by direct interaction with the consensus Pbx/Meis binding site, which is conserved in all analysed mammalian orthologue promoters. PBX1 is present in the protein complex formed at this site with nuclear proteins from uninduced cells, while both PBX1 and MEIS1 proteins were detected in the complex created with extr...

Micro-RNA mir346 targets the 5{prime}UTR of RIP140 mRNA and up-regulates its protein expression

BJ Gene — Thu, 24 Sep 2009 23:00:00 +0100

Receptor-interacting protein 140 (RIP140) is a transcriptional corepressor that regulates diverse genes such as those responsive to hormones and involved in metabolic processes. The expression of RIP140 is regulated by multiple hormonal activities in adipose tissue and cancer cell lines. However, it is unclear if and how RIP140 is regulated posttranscriptionally. Using rapid amplification of 5′ complementary DNA ends (5′RACE), we have identified a novel 5′ splice variant of RIP140 mRNA in mouse brain and P19 cells. A target sequence for microRNA (miRNA) mir-346 was found in the 5′-untranslated region (5′UTR) of RIP140 mRNA; this miRNA is also expressed endogenously in mouse brain and P19 cells. Gain- and loss-of-function studies demonstrated that mir-346 ...

Mutational analysis of human heat shock transcription factor 1 reveals a regulatory role for oligomerization in DNA-binding specificity

BJ Gene — Tue, 15 Sep 2009 23:00:00 +0100

In this study, we isolated suppressors of the temperature-sensitive growth defect of hHSF1-expressing yeast cells. Intragenic suppressors contained amino acid substitutions in the DNA-binding domain of hHSF1 that enabled hHSF1 to regulate the transcription of genes containing discontinuous HSEs. The substitutions facilitated hHSF1 oligomerization, suggesting that the DNA-binding domain is important for this conformational change. Furthermore, other oligomerization-prone derivatives of hHSF1 were capable of recognizing discontinuous HSEs. These results suggest that modulation of oligomerization is important for the HSE specificity of hHSF1 and imply that hHSF1 possesses the ability to bind to and regulate gene expression via various types of HSEs in diverse cellular processes. (Source: BJ G...

Activating transcription factor 2 increases transactivation and protein stability of hypoxia-inducible factor 1 alpha in hepatocytes

BJ Gene — Wed, 26 Aug 2009 23:00:00 +0100

Hypoxia inducible factor-1(HIF-1) performs a crucial role in mediating hypoxia response. However, other transcription factors are also capable of regulating hypoxia-induced target gene transcription. In a previous report, we demonstrated that the transcription factor ATF-2 regulates hypoxia-induced gene transcription, along with HIF-1α. In the current study, we show that the protein stability of ATF-2 is induced by hypoxia and the hypoxia-mimic cobalt chloride (CoCl2), and that ATF-2 induction enhances HIF-1α protein stability via direct protein interaction. The knockdown of ATF-2 using siRNA and translation inhibition experiments demonstrated that ATF-2 plays a key role in the maintenance of the expression level and transcriptional activity of HIF-1α. Furthermore, we ...

The human urocortin 2 gene is regulated by hypoxia: identification of a hypoxia-responsive element in the 3{'}-flanking region

BJ Gene — Tue, 25 Aug 2009 23:00:00 +0100

In conclusion, our study demonstrates that hypoxia induces hUcn2 expression via a specific HRE in the 3’FLR of the hUcn2 gene, which interacts with the transcription factor HIF1α. Hypoxia-mediated stimulation of cardioprotective Ucn2 may help to preserve cardiac function and prevent apoptosis in ischemic conditions in the heart. (Source: BJ Gene)

Effect of pyruvate, lactate and insulin on ATP supply and demand in unpaced perfused rat heart

BJ Gene — Mon, 17 Aug 2009 23:00:00 +0100

Mitochondrial respiration/oxidative phosphorylation is the main source of energy in the form of ATP in heart under physiological conditions. Different respiratory substrates were used in various experiments during heart perfusion: glucose, pyruvate, lactate, glucose + pyruvate, glucose + lactate, glucose + insulin etc. Also under physiological conditions the concentration of respiratory substrates/hormones in blood can vary significantly. In the present study we tested the effect of pyruvate, lactate and insulin (all in the presence of glucose) and glucose (in the presence of pyruvate) on the ATP-producing and consuming blocks in perfused rat heart, in a system where heart rate (HR) was allowed to vary (no pacing). Changes in rate-pressure product (RPP) and phosphocrea...

Functional characterization of the interactions between endosomal adaptor protein APPL1 and the NuRD co-repressor complex

BJ Gene — Sun, 16 Aug 2009 23:00:00 +0100

Multifunctional adaptor APPL1 protein belongs to a growing group of endocytic proteins which actively participate in various stages of signaling pathways. Due to its interaction with the small GTPase Rab5, APPL1 localizes predominantly to a subpopulation of early endosomes but is also capable of nucleocytoplasmic shuttling. Among its various binding partners, APPL1 was reported to associate with the nuclear co-repressor complex NuRD, containing nucleosome remodeling and histone deacetylase activities, but the biochemical basis or functional relevance of this interaction remained unknown. Here we characterized the binding between APPL1 and NuRD in more detail, identifying histone deacetylase 2 (HDAC2) as the key NuRD subunit responsible for this association. APPL1 interacts with the NuRD co...

Evolution of vertebrate glucokinase regulatory protein from a bacterial N-acetylmuramate 6-phosphate etherase

BJ Gene — Tue, 11 Aug 2009 23:00:00 +0100

Mammalian glucokinase regulatory protein (GKRP), a fructose 6-phosphate and fructose 1-phosphate sensitive inhibitor of glucokinase (GK), appears to have resulted from the duplication of a gene similar to bacterial N-acetylmuramate 6-phosphate etherase (MurQ). In the present work, we show that several genomes of primitive eukaryotes encode a GKRP-like protein with two MurQ repeats. Recombinant Haemophilus influenzae MurQ and the GKRP homologue of the amoebaflagellate Naegleria gruberi both behaved as excellent N-acetylmuramate 6-phosphate etherases, with Kcat values (83 and 20 sec-1) at least as high as that reported for Escherichia coli MurQ. By contrast, rat and xenopus GKRP displayed much lower etherase activities (Kcat = 0.08 and 0.05 sec-1, respectively). The etherase activity of rat ...

RNase R mutants elucidate the catalysis of structured RNA: RNA-binding domains select the RNAs targeted for degradation

BJ Gene — Sun, 26 Jul 2009 23:00:00 +0100

We report that Asp-280 is crucial for RNase R activity without affecting RNA binding. When Tyr-324 was changed to alanine the final product changed from 2 to 5nts showing that this residue is responsible for setting the end-product. We have shown that the RNB domain of RNase II has catalytic activity. The most striking result is that the RNase R RNB domain per se degrades double stranded substrates even in the absence of a 3’-overhang. Moreover, we demonstrate for the first time that the substrate recognition of RNase R depends on the RNA binding domains that target the degradation of RNAs that are “tagged” by a 3’-tail. These results can have important implications for the study of poly(A)-dependent RNA degradation mechanisms. (Source: BJ Gene)

Fibulin-4 regulates the expression of the tropoelastin gene and consequent elastic fiber formation by human fibroblasts

BJ Gene — Wed, 22 Jul 2009 23:00:00 +0100

Elastic fibers are essential for normal physiology in numerous tissues including arteries, lungs, and skin. Fibulin-4 is an elastic fiber-associated glycoprotein that is indispensable for elastic fiber formation in mice. However, the mechanism by which fibulin-4 executes this function remains to be determined. Here, we established an in vitro functional assay system in which fibulin-4 was knocked down in human foreskin fibroblasts using siRNA technology. With two different siRNAs, substantial knock down of fibulin-4 was achieved, and this suppression was associated with impaired elastic fiber formation by the fibroblasts. Real-time RT-PCR analysis showed that knock-down of fibulin-4 expression was accompanied by reduced expression of tropoelastin mRNA. Further analysis showed that this dec...

Human Ccr4-Not complexes contain variable deadenylase subunits

BJ Gene — Thu, 25 Jun 2009 23:00:00 +0100

In this study, we examine the composition of the human Ccr4-Not complex in an in-depth proteomic approach using stable cell lines expressing tagged CNOT proteins. We find at least four different variants of the human complex, consisting of seven stable core proteins and mutually-exclusive associated mRNA deadenylase subunits. Interestingly, human CNOT4 is in a separate ~200 kDa complex. Furthermore, analyses of associated proteins indicate involvement of Ccr4-Not complexes in splicing, transport and localization of RNA molecules. Taken together, human Ccr4-Not complexes are heterogeneous in composition due to differences in their deadenylase subunits, which may reflect the multi-functionality of these complexes in cellular processes. (Source: BJ Gene)

Functional conservation of tRNase ZL among Saccharomyces cerevisiae, Schizosaccharomyces pombe and humans

BJ Gene — Wed, 24 Jun 2009 23:00:00 +0100

We report that both sptrz1+ and sptrz2+ are essential for growth. Moreover, sptrz1+ is required for cell viability in absence of Sla1p which is thought to be required for endonuclease-mediated maturation of pre-tRNA 3' ends in yeast. Both scTRZ1 and ELAC2 can complement a temperature sensitive allele of sptrz1+, sptrz1-1, but not the sptrz1 null mutant, indicating that despite exhibiting species specificity, tRNase ZLs are functionally conserved among S. cerevisiae, S. pombe and humans. Overexpression of sptrz1+, scTRZ1 and ELAC2 can increase suppression of the UGA nonsense mutation ade6-704 through facilitating 3' end processing of the defective suppressor tRNA that mediates suppression. Our findings reveal that 3' end processing is a limiting step for d...

A novel p53 target gene, S100A9, induces p53-dependent cellular apoptosis and mediates p53 apoptosis pathway

BJ Gene — Tue, 16 Jun 2009 23:00:00 +0100

S100A9 is a calcium-binding protein of the S100 family, and its differential expression has been associated with acute and chronic inflammation and several human cancers. Our previous work showed that S100A9 was severely down-regulated in human esophageal squamous cell carcinoma. To further investigate the transcriptional regulation of S100A9, we analyzed S100A9 promoter region and found several putative p53 binding sites (p53 BS). Luciferase reporter assay showed that the constructs carrying p53 BS exhibited enhanced luciferase activity in response to wild type p53 activation. Further study demonstrated that S100A9 mRNA and protein expression could be positively regulated in a p53-dependent manner and p53 could bind to p53 BS on S100A9 promoter. Overexpression of S100A9 could induce cellu...

P/CAF rescues Stra13 mediated repression of MyoD transactivation

BJ Gene — Thu, 11 Jun 2009 23:00:00 +0100

Recently, we found that myogenic regulatory factors (MRFs) regulated the expression of Peroxisome Proliferation Activation Receptor γ Coactivator 1α (PGC-1α) by targeting a short region (-49~+2) adjacent to the transcription initiation site that contained two E-boxes. However, only the E2-box shown significant affinity to MRFs and the E1-box was predicted to be the target of Stra13, a transcriptional repressor implicated in the regulation of several physiological processes. Using electrophoresis mobility shift assay (EMSA), we confirmed that Stra13 targeted the E1-box and formed a complex with MyoD on PGC-1α core promoter. Binding of Stra13 to PGC-1α and Myogenin promoters in vivo was also demonstrated. Apart from the promoter of PGC-1α, Str...

NCU-G1 is a highly glycosylated integral membrane protein of the lysosome

BJ Gene — Tue, 02 Jun 2009 04:00:00 +0100

Until recently, a modest number of approximately 40 lysosomal membrane proteins had been identified and even fewer were characterized in their function. In a proteomic study, using lysosomal membranes from human placenta we identified several candidate lysosomal membrane proteins and proved the lysosomal localization of two of them. Here, we demonstrate the lysosomal localization of the mouse ortholog of the human C1orf85 protein that had been termed kidney predominant protein NCU-G1 (Acc.No: AB027141). NCU-G1 encodes a 404 amino acid protein with a calculated molecular mass of 39 kDa. The bioinformatics analysis of its amino acid sequence suggests it is a type I transmembrane protein containing a single tyrosine-based consensus lysosomal sorting motif at position 400 within the 12-residue...

Spermine analogs regulated expression of spermidine/spermine N1-acetyltransferase and its effects on depletion of intracellular polyamine pools in mouse fetal fibroblasts

BJ Gene — Wed, 27 May 2009 04:00:00 +0100

In this study, the regulation of SSAT by spermine analogs, the inducers of the enzyme, was studied in the wild-type mouse fetal fibroblasts expressing endogenous SSAT and in the SSAT-deficient mouse fetal fibroblasts transiently expressing the SSAT- EGFP (enhanced green fluorescent protein) fusion gene. In both cell lines treatments with DENSpm (N1, N11 –bis(ethyl)norspermine), CPENSpm (N1-ethyl-N11-((cyclopropyl)methyl)-4,8-diazaundecane) and CHENSpm (N1-ethyl-N11-((cycloheptyl)methyl)-4,8-diazaundecane) led to high, moderate or low induction of SSAT activity, respectively. The detected levels of activities correlated with the presence of SSAT and SSAT-EGFP proteins, the last one localizing both in the cytoplasm and nucleus. Reverse transcriptase -PCR results suggested that the ana...

Elevated levels of IL-1{beta} in Fanconi Anemia group A patients due to a constitutively active PI3K-AKT pathway are capable of promoting tumor cell proliferation

BJ Gene — Wed, 27 May 2009 04:00:00 +0100

Fanconi Anemia is a hereditary disease characterized by congenital malformations, progressive bone marrow failure and an extraordinary elevated predisposition to develop cancer. In the present article we describe an anomalous high level of the proinflammatory cytokine IL-1β present in the serum of Fanconi Anemia patients. The elevated levels of IL-1β were completely reverted by transduction of a wild type copy of the FancA cDNA into FA-A lymphocytes. Although the transcription factor NF-κB is a well established regulator of IL-1β expression, our experiments did not show any proof of elevated NF-κB activity in FA-A cells. However, we found that the overexpression of IL-1β in FA-A cells is related to a constitutively activated PI3K-AKT pathway in the...

A novel function of Aft1 in regulating ferrioxamine B (FOB) uptake; Aft1 modulates Arn3 ubiquitination in Saccharomyces cerevisiae

BJ Gene — Tue, 26 May 2009 04:00:00 +0100

Aft1 is a transcriptional activator in Saccharomyces cerevisiae that responds to iron availability and regulates the expression of genes in the iron regulon, such as FET3, FTR1, and the ARN family. Using a two-hybrid screen, we found that Aft1 physically interacts with the ferrioxamine B (FOB) transporter Arn3. This interaction modulates the ability of Arn3 to take up FOB. The interaction between Arn3 and Aft1 was confirmed by β-galactosidase, co-immunoprecipitation, and surface plasmon resonance (SPR) assays. Truncated Aft1 interacted more strongly with Arn3 and caused a higher FOB-uptake activity than full-length Aft1. Interestingly, only full-length Aft1 induced the correct localization of Arn3 in response to FOB. Furthermore, we found Aft1 affected Arn3 ubiquitination. These res...

Overexpression of Drosophila mitoferrin in l(2)mbn cells results in dysregulation of Fer1HCH expression

BJ Gene — Tue, 19 May 2009 04:00:00 +0100

Mrs3p and Mrs4p (Mrs3/4p) are mitochondrial iron carrier proteins that play important roles in iron-sulphur cluster (ISC) and haem biosynthesis. At low iron conditions mitochondrial and cytoplasmic ISC protein maturation is correlated with MRS3/4 expression. Zebrafish mitoferrin1 (mfrn1), one of two MRS3/4 orthologues, is essential for erythropoiesis. Little is known about the ubiquitously expressed paralogue mfrn2. We identified only one mitoferrin gene (dmfrn) in the genome of Drosophila melanogaster; moste likely an orthologue of mfrn2. Overexpression of dmfrn in the Drosophila l(2)mbn cell line (mbn-dmfrn) resulted in decreased IRP-1A/IRE binding activity, increased cytoplasmic aconitase activity and slightly decreased iron content when compared to control cell lines, whereas iron load...

Sumoylation enhances DNA methyltransferase 1 activity

BJ Gene — Mon, 18 May 2009 04:00:00 +0100

DNA methylation regulates gene expression through a complex network of protein/protein and protein/DNA interactions in chromatin. The maintenance methylase, DNA methyltransferase 1 (DNMT1), is a prominent enzyme in the process that is linked to DNA replication and drives the heritable nature of epigenetic modifications. The mechanistic details that explain how DNMT1 catalytic action is directed and regulated in chromatin are important in our overall understanding of gene control. In this work, we show that DNMT1 is modified by sumoylation and we have mapped these sumoylation sites by defined mutations. Sumoylated DNMT1 is catalytically active on genomic DNA in vivo and we find that sumoylation significantly enhances the methylase activity of DNMT1 both in vitro and in chromatin. These data...

Hsp70 is required for optimal cell proliferation in mouse A6 mesoangioblast stem cells

BJ Gene — Fri, 01 May 2009 04:00:00 +0100

Mouse Hsp70 is preferentially induced by heat or stress stimuli. We previously found that Hsp70 is constitutively expressed in A6 mouse mesoangioblast stem cells, but its possible role in these cells and the control of its basal transcription remained unexplored. Here we report that in the absence of stress, Ku factor is able to bind the heat shock element (HSE) consensus sequence in vitro, and in vivo it is bound to the proximal hsp70 promoter. In addition, we show that constitutive hsp70 transcription depends on the cooperative interaction of different factors such as Sp1 and GAGA binding protein with Ku factor, which binds the HSE consensus sequence. We used mRNA interference assays to select knockdown cell clones. These cells were able to respond to heat stress by producing a large amo...

Characterization of protein arginine methyltransferase I from Plasmodium falciparum

BJ Gene — Fri, 03 Apr 2009 04:00:00 +0100

Arginine methylation is a posttranslational modification that affects many cellular processes in eukaryotes. The malaria parasite Plasmodium falciparum encodes three conserved protein arginine methyltransferases (PfPRMTs). We have determined that PfPRMT1 had authentic type I PRMT activity to form monomethylarginines and asymmetric dimethylarginines. Compared to mammalian PRMT1s, PfPRMT1 possesses a distinctive, ~50 amino acids longer N-terminal sequence, which is essential for enzyme activity. Recombinant PfPRMT1 methylated histones H4 and H2A, and several conserved substrates involved in RNA metabolism, including fibrillarin, poly(A) binding protein II, ribosomal protein S2, and a putative splicing factor. Using synthetic peptides and mass spectrometry, we determined target arginines in s...

Drosophila expresses a CD98 transporter with evolutionarily conserved structure and amino acid transport properties

BJ Gene — Wed, 01 Apr 2009 04:00:00 +0100

Mammalian CD98 heterodimeric amino acid transporters consist of a promiscuous single-pass transmembrane glycoprotein, CD98hc, the ‘heavy chain’, and one of six multipass transmembrane proteins, or ‘light chains’. The heterodimeric complexes of CD98hc and the light chains LAT1 or LAT2 specifically promote sodium-independent System L exchange of neutral amino acids, including leucine. CD98hc is also implicated in other processes, including cell fusion, cell adhesion and activation of TOR signalling. Recent reports surprisingly suggested that insects lack a membrane-bound CD98hc, but here we show that Drosophila CG2791 encodes a functional CD98hc orthologue with conservation in intracellular, transmembrane and extracellular domains. We demonstrate by RNAi knockdown...

Kr{u}ppel-like factor 4 (KLF4) positively regulates human ghrelin expression

BJ Gene — Fri, 27 Mar 2009 04:00:00 +0100

Ghrelin, an endogenous ligand of the GH secretagogue receptor, influences many metabolic processes including GH secretion, food intake, energy balance, insulin secretion, and adipogenesis. Although ghrelin exhibits a variety of biological functions, the mechanism by which ghrelin expression is regulated is unknown. Ghrelin is expressed in the gastrointestinal tract, predominantly the stomach, as is Krüppel-like factor 4 (KLF4). Therefore, we investigated whether ghrelin expression is associated with KLF4. We found that the tissue distribution of ghrelin was in accordance with that of KLF4. Furthermore, treatment with butyrate, an inducer of KLF4 expression, stimulated ghrelin expression, and fasting, which induces ghrelin expression, also increased KLF4 expression, suggesting that g...

Desmosterol can replace cholesterol in sustaining cell proliferation and regulating the SREBP pathway in a sterol-{Delta}24-reductase deficient cell line

BJ Gene — Thu, 05 Mar 2009 05:00:00 +0100

Cholesterol homeostasis is critical for cell viability and proliferation. The sterol regulatory element-binding protein (SREBP) pathway is crucial for the maintenance of cholesterol homeostasis. This pathway is controlled by cholesterol and cholesterol-derived oxysterols. J774 cells cannot convert desmosterol into cholesterol, a defect resulting from the absence of mRNA for sterol-Δ24-reductase. Using J774 cells, we addressed the capacity of desmosterol to replace cholesterol in sustaining cell proliferation and regulating the SREBP pathway. J774 cells were able to grow indefinitely after the virtually total replacement of cholesterol by desmosterol (J774-D cells). Inhibition of sterol biosynthesis with lovastatin suppressed J774-D cell proliferation. Desmosterol prevented this effe...

Competition between NarL-dependent activation and Fis-dependent repression controls expression from the Escherichia coli yeaR and ogt promoters

BJ Gene — Thu, 26 Feb 2009 05:00:00 +0100

The Escherichia coli NarL protein is a global gene regulatory factor that activates transcription at many target promoters in response to nitrate and nitrite ions. Although most NarL-dependent promoters are also co-dependent on a second transcription factor, FNR, two targets, the yeaR and ogt promoters are activated by NarL alone with no involvement of FNR. Biochemical and genetic studies presented here show that activation of the yeaR promoter is dependent on the binding of NarL to a single target centred at position 43.5, whilst activation at the ogt promoter requires NarL binding to tandem DNA targets centred at position -45.5 and -78.5. NarL dependent activation at both the yeaR and ogt promoters is decreased in rich medium and this depends on Fis, a nucleoid-associated protein. DNAse ...

Proper expression of helix-loop-helix protein Id2 is important to chondrogenic differentiation of ATDC5 cells

BJ Gene — Wed, 28 Jan 2009 05:00:00 +0100

The process of chondrogenesis can be mimicked in vitro by insulin treatment of mouse ATDC5 chondroprogenitor cells. To identify novel factors that are involved in the control of chondrogenesis, we carried out a large-scale screening through retroviral insertion mutagenesis and isolated a fast-growing ATDC5 clone incapable of chondrogenic differentiation. Inverse-PCR analysis of this clone revealed that the retroviral DNA was inserted into the promoter region of mouse Id2 gene. This retroviral insertion increased Id2 protein level to twice as much as that in normal ATDC5 cells. To investigate whether an elevated level of Id2 protein was responsible for inhibition of chondrogenic differentiation, ATDC5 cells were infected with a retrovirus to stably express Id2. ATDC5 cells expressing ectopi...

Resistance of Helicoverpa armigera to CRY1Ac toxin from Bacillus thuringiensis is due to improper processing of the protoxin

BJ Gene — Fri, 16 Jan 2009 05:00:00 +0100

The bacterium Bacillus thuringiensis produces insecticidal crystal proteins (ICP) that are deposited in their spore mother cells (SMC). When susceptible lepidopteran larvae ingest these SMC, the ICP get solubilised in the alkaline gut environment. Of about 140 insecticidal proteins described thus far, insecticidal protein Cry1Ac has been applied extensively as the main ingredient of spray formulation as well as the principal ICP introduced into crops as transgene for agricultural crop protection. The 135 kDa Cry1Ac protein upon ingestion by the insect is processed successively at the N and C- termini by the insect midgut proteases to generate a 65 kDa bioactive core protein. The activated core protein interacts with specific receptors located at the midgut epithilium resulting in the lysis...

hCASK regulates the expression of p21 via E2A transcription factor

BJ Gene — Tue, 06 Jan 2009 05:00:00 +0100

This study revealed that hCASK mediates the protein and mRNA level of p21wafi/cip1 (p21), and activates the expression of p21 in a time-dependent manner. Two E-boxes in the proximal region at the transcription start site (TSS) play key roles in regulating hCASK-mediated p21 expression. We suggest that E2A (E12 and E47), a representative of the E proteins that binds the E-box elements, is an accomplice in the mediation of p21 expression by hCASK. The data suggest that hCASK regulation of cell growth might be involved in p21expression, while the bHLH transcription factor E2A probably participates in hCASK mediation of p21expression. From these findings, we propose a novel proliferation signal pathway mediated by hCASK. (Source: BJ Gene)

Characterization of key residues and membrane association domains in RDH10

BJ Gene — Mon, 22 Dec 2008 05:00:00 +0100

RDH10 was originally identified from the retinal pigment epithelium (RPE) and retinal Müller cells. It has retinoid oxidoreductase activity and is thought to play a role in the retinoid visual cycle. A recent study showed that RDH10 is essential for generating retinoic acid at early embryonic stages. The present study demonstrated that wild-type (wt) RDH10 catalyzed both oxidation of all-trans retinol and reduction of all-trans retinal in a cofactor-dependent manner in vitro. In cultured cells, however, oxidation is the favored reaction catalyzed by RDH10. Substitution of any of the predicted key residues in the catalytic center conserved in the retinol dehydrogenase (RDH) family abolished the enzymatic activity of RDH10 without affecting its protein level. Unlike other RDH members,...

Physarum nitric oxide synthases: Genomic structures and enzymology of recombinant proteins

BJ Gene — Mon, 01 Dec 2008 05:00:00 +0100

Physarum polycephalum expresses two closely related, calcium-independent nitric oxide synthases. In previous work, we showed that both nitric oxide synthases are induced during starvation and apparently play a functional role for sporulation. Here, we characterized the genomic structures of both Physarum nitric oxide synthases, expressed both enzymes recombinantly in bacteria, and characterized their biochemical properties. Whereas the overall genomic organization of Physarum nitric oxide synthase genes is comparable to various animal nitric oxide synthases, none of the exon-intron boundaries are conserved. Recombinant expression of clones with various N-termini identified N-terminal amino acids essential for enzyme activity, but not required for heme binding or dimerization, and suggests ...

The Drosophila nuclear factor DREF positively regulates the expression of the mitochondrial transcription termination factor DmTTF

BJ Gene — Tue, 25 Nov 2008 05:00:00 +0100

In this study, by in silico analysis, we have identified DNA Replication-related Elements (DRE) in the promoter region of a gene participating in mtDNA transcription, the Drosophila mitochondrial Transcription Termination Factor (DmTTF). Transient transfection assays in Drosophila S2 cells, with mutated versions of DmTTF promoter region, showed that DRE elements control DmTTF transcription; moreover, gel-shift and ChIP assays demonstrated that the analysed DRE sites interact with DREF either in vitro and in vivo. Accordingly, DREF knockdown in S2 cells by RNA interference induced a considerable decrease in DmTTF mRNA level. These results clearly demonstrate that DREF positively controls DmTTF expression. On the contrary, mitochondrial RNA polymerase (mtRNApol) lacks DRE elements in its pro...

Active site substitutions delineate distinct classes of eubacterial flap endonuclease

BJ Gene — Tue, 11 Nov 2008 05:00:00 +0100

We present evidence of paralogous FEN-encoding genes present in many eubacteria. Two distinct classes of these independent FEN-encoding genes exist with four groups of eubacteria being identified based on the number and type of FEN gene encoded. The respective proteins possess distinct motifs hallmarking their differentiation. Crucially, based on primary sequence and predicted secondary structural motifs, we reveal key differences at their active site. These data are supported by biochemical characterization of two family members - exonuclease IX (ExoIX) from Escherichia coli and Staphylococcus aureus flap endonuclease (SaFEN). These proteins displayed marked differences in their ability to process a range of branched and linear DNA structures. On bifurcated substrates, SaFEN exhibited sim...

The selenocysteine tRNA STAF-binding region is essential for adequate selenocysteine tRNA status, selenoprotein expression and early age survival of mice

BJ Gene — Thu, 30 Oct 2008 04:00:00 +0100

STAF is a transcription activating factor for a number of RNA Pol III- and RNA Pol II-dependent genes including the selenocysteine (Sec) tRNA gene, which in turn controls the expression of all selenoproteins. Here, the role of STAF in regulating expression of Sec tRNA and selenoproteins was examined. We generated transgenic mice expressing the Trsp transgene lacking the STAF binding site and made these mice dependent on the transgene for survival by removing the wild type Sec tRNA gene. The level of Sec tRNA was unaffected or slightly elevated in heart and testis, but reduced ~60% in liver and kidney, ~70% in lung and spleen, and ~80% in brain and muscle compared to the corresponding organs in control mice. Moreover, the ratio of the two isoforms of Sec tRNA that differ by methylation at p...

Peptide mimics of a conserved H5N1 avian influenza virus neutralization site

BJ Gene — Thu, 30 Oct 2008 04:00:00 +0100

Conclusion. Findings inferred that 8H5 recognizes discontinuous sites presented by secondary and possibly higher structural orders of the peptides in spatially favorable positions for binding with the antibody, and that the peptides partially mimic the native 8H5 epitopes on the H5N1 virus. (Source: BJ Gene)

Insulin and epidermal growth factor suppress basal glucose-6-phosphatase catalytic subunit gene transcription through overlapping but distinct mechanisms

BJ Gene — Fri, 10 Oct 2008 04:00:00 +0100

The glucose-6-phosphatase catalytic subunit (G6Pase) catalyses the final step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate to glucose. We show here that, in HepG2 hepatoma cells, epidermal growth factor (EGF) inhibits basal mouse G6Pase fusion gene transcription. Several studies have shown that insulin represses basal mouse G6Pase fusion gene transcription through FOXO1 but Stoffel and colleagues (Proc. Natl. Acad. Sci. 100:11624, 2003) have recently suggested that insulin can also regulate gene transcription through FOXA2. A combined glucocorticoid receptor (GR)-FOXA2 binding site is located between -185 and -174 in the mouse G6Pase promoter overlapping two FOXO1 binding sites located between (-188 and -182) and (-174 and -168). Selective mutatio...

Transcriptional induction of the human asparagine synthetase gene during the unfolded protein response does not require the ATF6 and IRE1/XBP1 arms of the pathway

BJ Gene — Wed, 08 Oct 2008 04:00:00 +0100

The Unfolded Protein Response (UPR) pathway is comprised of three signaling cascades mediated by the ER stress sensor proteins PERK, IRE1, and ATF6. The present study shows that asparagine synthetase (ASNS) transcription activity was up-regulated in HepG2 cells treated with the UPR activators thapsigargin and tunicamycin. Chromatin immunoprecipitation (ChIP) analysis demonstrated that during ER stress ATF4, ATF3, and C/EBPβ bind to the ASNS proximal promoter region that includes the genomic sequences Nutrient Sensing Response Elements-1,2 (NSRE-1, NSRE-2), previously implicated by mutagenesis in UPR activation. Consistent with increased ASNS transcription, ChIP analysis also demonstrated that UPR signaling resulted in enhanced recruitment of general transcription factors, including ...

The transporter CefM involved in translocation of biosynthetic intermediates is essential for cephalosporin production

BJ Gene — Wed, 08 Oct 2008 04:00:00 +0100

The cluster of early cephalosporin biosynthesis genes (pcbAB, pcbC, cefD1, cefD2 and cefT of Acremonium chrysogenum) contains all genes required for the biosynthesis of the cephalosporin biosynthetic pathway intermediate penicillin N. Downstream of the cefD1 gene there is an unassigned open reading frame named cefM encoding a protein of the MFS family with 12 transmembrane spanning domains, different from the previously reported cefT. Targeted inactivation of cefM by gene replacement showed that it is essential for cephalosporin biosynthesis. The disrupted mutant accumulates a significant amount of penicillin N, is unable to synthesize deacetoxy-, deacetylcephalosporin C and cephalosporin C and shows impaired differentiation into arthrospores. Complementation of the disrupted mutant with t...

High incidence of DNA mutations and gene amplifications of the ALK gene in advanced sporadic neuroblastoma tumours

BJ Gene — Tue, 07 Oct 2008 04:00:00 +0100

Anaplastic lymphoma kinase (ALK) is oncogenic in several tumours and recently identified as a predisposition gene for familial neuroblastoma (NB) harbouring mutations in the tyrosine kinase domain (TKD). We have analysed a large set of sporadic human NB primary tumours of all clinical stages for chromosomal re-arrangements using array-CGH (n=108) and mutations of the ALK gene (n=90), and expression of ALK and related genes (n=19). ALK amplification or in-gene re-arrangements were found in 5% of NB tumours and mutations in 11% including two novel not previously published mutations in the TKD; c.3733T>A and c.3735C>A. DNA mutations in the TKD and gene amplifications were only found in advanced large primaries or metastatic tumours and correlated with the expression levels of ALK and downstre...

P/CAF modulates the activity of the transcription factor p48/PTF1a involved in pancreatic acinar differentiation

BJ Gene — Mon, 06 Oct 2008 04:00:00 +0100

p48, also called Ptf1a, is a tissue-restricted basic helix-loop-helix transcription factor critical for pancreatic commitment during development and for the activation/maintenance of the acinar differentiation program in exocrine pancreas. High-level expression of exocrine digestive enzymes, a hallmark of mature acinar cells, depends largely on the trimeric complex PTF1 (pancreas transcription factor 1), formed by p48, RBP-L and a class A bHLH protein. In addition, p48 induces cell cycle exit by controlling G1-S progression. However, the mechanisms that mediate the PTF1-dependent gene activation are poorly understood. Here, we report that p48 increases transcription through two activation domains located in its N-terminal region by recruiting transcriptional coactivators. The histone acety...

Two biochemically distinct and tissue-specific twinfilin isoforms are generated from mouse twinfilin-2 gene by alternative promoter usage

BJ Gene — Mon, 06 Oct 2008 04:00:00 +0100

Twinfilin is an evolutionarily conserved regulator of actin dynamics composed of two actin-depolymerizing factor homology (ADF-H) domains. Twinfilin binds actin monomers and heterodimeric capping protein with high affinity. Previous studies demonstrated that mammals express two twinfilin isoforms, twinfilin-1 and twinfilin-2, of which at least twinfilin-1 also regulates cytoskeletal dynamics by capping actin filament barbed ends. Here, we show that alternative promoter usage of the mouse twinfilin-2 gene generates two isoforms, which differ from each other only at their very N-terminal region. Of these isoforms, twinfilin-2a is predominantly expressed in non-muscle tissues, whereas expression of twinfilin-2b is restricted to heart and skeletal muscle. Both proteins bind actin monomers and ...

BCL11B enhances TCR/CD28-triggered NF-kB activation through upregulation of Cot kinase gene expression in T lymphocytes

BJ Gene — Fri, 03 Oct 2008 04:00:00 +0100

BCL11B is a transcriptional regulator with important role in T cell development and leukemogenesis. Recently we demonstrated that BCL11B controls expression from IL-2 promoter through direct binding to the US1 site. Here we provide evidence that BCL11B also participates in the activation of IL-2 gene expression by enhancing NF-kB activity in the context of TCR/CD28–triggered T cell activation. Enhanced NF-kB activation is not a consequence of BCL11B binding to the NF-kB response elements or association with the NF-kB-DNA complexes, but rather the result of higher translocation of NF-kB to the nucleus caused by enhanced degradation of the IkB inhibitors. The enhanced IkB degradation in cells with increased levels of BCL11B was specific for T cells activated through TCR, but not throu...

A chimeric glutamyl: glutaminyl-tRNA synthetase: implications for evolution

BJ Gene — Fri, 26 Sep 2008 04:00:00 +0100

Aminoacyl-tRNA synthetases (aaRS) are multi-domain proteins that have evolved by domain acquisition. The anti-codon binding domain was added to the more ancient catalytic domain during aaRS evolution. Unlike in eukaryotes, the anti-codon binding domains of GluRS and GlnRS in bacteria are structurally distinct. This originates from the unique evolutionary history of GlnRSs. Starting from the catalytic domain, eukaryotic GluRS evolved by acquiring the archaea/eukaryote-specific anti-codon binding domain after branching away from the eubacteria family. Subsequently, eukaryotic GlnRS evolved from GluRS by gene duplication and horizontally transferred to bacteria. In order to study the properties of the putative ancestral GluRS in eukaryotes, formed immediately after acquiring the anti-codon bi...

A cysteine-rich metal-binding domain from rubella virus nonstructural protein is essential for viral protease activity and virus replication

BJ Gene — Wed, 17 Sep 2008 04:00:00 +0100

In this study, we carried out metal-binding and conformational studies on a purified cysteine-rich minidomain of the RUBV NS protease containing the putative Zn binding ligands. This minidomain bound to zinc ions with a stoichiometry of ~0.7 and an apparent dissociation constant of < 500 nM. Fluorescence quenching and ANS fluorescence methods revealed that zinc binding resulted in conformational changes characterized by shielding of hydrophobic regions from the solvent. Mutational analyses using the minidomain identified residues C1175, C1178, C1225 and C1227 were required for the binding of zinc ions. Corresponding mutational analyses using a RUBV replicon confirmed that these residues were necessary for both proteolytic activity of the NS protease and viability. The present study demonst...

Hepatitis B virus X protein induces lipogenic transcription factor SREBP1 and fatty acid synthase through the activation of nuclear receptor LXR alpha

BJ Gene — Wed, 10 Sep 2008 04:00:00 +0100

In this study, we demonstrate that the HBx protein interacts with LXRα and enhances the binding of LXRα to the LXR-response element (LXRE), thereby resulting in the upregulation of SREBP1 and fatty acid synthase (FAS) in the presence or absence of the LXR agonist T0901317 in the hepatic cells and HBx-transgenic mice. Furthermore, HBx also augments the ability to recruit the activating signal cointegrator 2 (ASC2), a transcriptional coactivator that controls liver lipid metabolic pathways, to the LXRE with LXRα. These studies place LXRα in a key position within the HBx-induced lipogenic pathways, and suggest a molecular mechanism via which HBV infection can stimulate the SREBP1-mediated control of hepatic lipid accumulation. (Source: BJ Gene)