O-GlcNAc transferase associates with the MCM2-7 complex and its silencing destabilizes MCM-MCM interactions

International audienceO-GlcNAcylation of proteins is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The homeostasis of O-GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O-GlcNAcylation of the Mini-Chromosome Maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2-7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2-7 are O-GlcNAcylated and that O-GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilize MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2-7 complex and O-GlcNAcylation homeostasis might regulate MCM2-7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions

Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite Toxoplasma gondii

The protozoan parasite Toxoplasma gondii differentially expresses two distinct enolase isoenzymes known as ENO1 and ENO2, respectively. To understand differential gene expression during tachyzoite to bradyzoite conversion, we have characterized the two T.gondii enolase promoters. No homology could be found between these sequences and no TATA or CCAAT boxes were evident. The differential activation of the ENO1 and ENO2 promoters during tachyzoite to bradyzoite differentiation was investigated by deletion analysis of 5′-flanking regions fused to the chloramphenicol acetyltransferase reporter followed by transient transfection. Our data indicate that in proliferating tachyzoites, the repression of ENO1 involves a negative distal regulatory region (nucleotides −1245 to −625) in the promoter whereas a proximal regulatory region in the ENO2 promoter directs expression at a low level. In contrast, the promoter activity of ENO1 is highly induced following the conversion of tachyzoites into resting bradyzoites. The ENO2 promoter analysis in bradyzoites showed that there are two upstream repression sites (nucleotides −1929 to −1067 and −456 to −222). Furthermore, electrophoresis mobility shift assays demonstrated the presence of DNA-binding proteins in tachyzoite and bradyzoite nuclear lysates that bound to stress response elements (STRE), heat shock-like elements (HSE) and other cis-regulatory elements in the upstream regulatory regions of ENO1 and ENO2. Mutation of the consensus AGGGG sequence, completely abolished protein binding to an oligonucleotide containing this element. This study defines the first characterization of cis-regulatory elements and putative transcription factors involved in gene regulation of the important pathogen T.gondii

Cyclin D1 Stability Is Partly Controlled by O-GlcNAcylation

Cyclin D1 is the regulatory partner of the cyclin-dependent kinases (CDKs) CDK4 or CDK6. Once associated and activated, the cyclin D1/CDK complexes drive the cell cycle entry and G1 phase progression in response to extracellular signals. To ensure their timely and accurate activation during cell cycle progression, cyclin D1 turnover is finely controlled by phosphorylation and ubiquitination. Here we show that the dynamic and reversible O-linked β-N-Acetyl-glucosaminylation (O-GlcNAcylation) regulates also cyclin D1 half-life. High O-GlcNAc levels increase the stability of cyclin D1, while reduction of O-GlcNAcylation strongly decreases it. Moreover, elevation of O-GlcNAc levels through O-GlcNAcase (OGA) inhibition significantly slows down the ubiquitination of cyclin D1. Finally, biochemical and cell imaging experiments in human cancer cells reveal that the O-GlcNAc transferase (OGT) binds to and glycosylates cyclin D1. We conclude that O-GlcNAcylation promotes the stability of cyclin D1 through modulating its ubiquitination

Mycobacterial lipomannan induces MAP kinase phosphatase-1 expression in macrophages.

Mycobacterial lipomannan (LM) and lipoarabinomannan (LAM) regulate macrophage activation by interacting with Toll-like receptors (TLRs). The intracellular signalling pathways elicited by these complex molecules are poorly defined. We have demonstrated that LM purified from various mycobacterial species, but not LAM from Mycobacterium kansasii or Mycobacterium bovis BCG, induced expression of the MAP kinase phosphatase 1 (MKP-1) in macrophages. Anti-TLR2 antibodies, as well as specific ERK and p38 MAPK inhibitors, decreased MKP-1 transcription in LM-stimulated cells. These findings suggest that the binding of LM to TLR2 triggers MAPK activation, followed by an up-regulation of MKP-1 expression, which in turn may act as a negative regulator of MAPK activation

Synthetic ferrocenic mefloquine and quinine analogues as potential antimalarial agents

A few years ago we proposed a strategy for the synthesis of new ferrocene-chloroquine analogues replacing the carbon chain of chloroquine by hydrophobic ferrocenyl moieties. Now, this strategy has been applied to the antimalarial amino-alcohols class to afford new potentially active analogues of mefloquine and quinine bearing a substituted ferrocenic group. The pathway used for the synthesis of the mefloquine analogues includes the coupling of an aminomethyl substituted ferrocene carboxaldehyde with a lithio quinoline compound. On the other hand, the synthesis of quinine analogues was ensured by the 'inverse' reaction of a lithio aminomethyl ferrocene with a quinoline carboxaldehyde. The configurations of each diastereoisomer were unambiguously determined by spectroscopic data. The mechanistic interpretations were fully discussed. Ferrocenyl analogues of mefloquine and quinine exhibited a lower antimalarial activity than mefloquine and quinine themselves. Comparing optical isomers, those isomers dissimilar to ferrocenyl derivatives presented better antimalarial activities than those similar to ferrocenyl. (C) 2000 Editions scientifiques et medicales Elsevier SAS.info:eu-repo/semantics/publishe

TNF differentially regulates ganglioside biosynthesis and expression in breast cancer cell lines.

Gangliosides are glycosphingolipids concentrated in glycolipid-enriched membrane microdomains. Mainly restricted to the nervous system in healthy adult, complex gangliosides such as GD3 and GD2 have been shown to be involved in aggressiveness and metastasis of neuro-ectoderm derived tumors such as melanoma and neuroblastoma. GD3 synthase (GD3S), the key enzyme that controls the biosynthesis of complex gangliosides, was shown to be over-expressed in Estrogen Receptor (ER)-negative breast cancer tumors, and associated with a decreased overall survival of patients. We previously demonstrated that GD3S expression in ER-negative breast cancer cells induced a proliferative phenotype and an increased tumor growth. In addition, our results clearly indicate that Tumor Necrosis Factor (TNF) induced GD3S over-expression in breast cancer cells via NFκB pathway. In this study, we analyzed the effect of TNF on ganglioside biosynthesis and expression in breast cancer cells from different molecular subtypes. We showed that TNF up-regulated the expression of GD3S in MCF-7 and Hs578T cells, whereas no change was observed for MDA-MB-231. We also showed that TNF induced an increased expression of complex gangliosides at the cell surface of a small proportion of MCF-7 cells. These results demonstrate that TNF differentially regulates gangliosides expression in breast cancer cell lines and establish a possible link between inflammation at the tumor site environment, expression of complex gangliosides and tumor development

Novel metallocenic compounds as antimalarial agents: study of the position of ferrocene in chloroquine

The synthesis, characterization and antimalarial activity of two new ferrocene-chloroquine compounds are reported. One of them, 7-chloro-4-N-[(4-N′-ethyl-N′-ferrocenylmethyl)ammonio-1- methylbutylamino]quinolin-1-ium bi-tartrate (2) showed very promising antimalarial activity in vivo on mice infected with Plasmodium berghei N. and Plasmodium yoelii NS. and in vitro against chloroquine resistant-strains of Plasmodium falciparum. © 1999 Elsevier Science S.A.info:eu-repo/semantics/publishe

( and ) Overview of the results obtained by promoter analysis and by EMSAs

<p><b>Copyright information:</b></p><p>Taken from "Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite "</p><p>Nucleic Acids Research 2005;33(5):1722-1736.</p><p>Published online 22 Mar 2005</p><p>PMCID:PMC1903550.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The promoter regions of and are shown. The forward primers used for CAT expressing constructs are indicated in red while the common reverse primers are shown in blue. The name of these constructs are displayed in red on top of the forward primers. The DNA fragments used in EMSAs are indicated by bold bars and underlined. The transcription start sites are shown by black arrows. The -regulatory elements identified are displayed as boldface, black, underlined sequences on top and at the beginning of the EMSA fragments. () The nature of -regulatory elements and the corresponding putative -acting or transcription factors are indicated in the table. The oligonucleotide sequences were checked for homology to sequences in the TRANSFAC and TFSEARCH databases

Localization of protein binding within the 35 and 42 bp DNA of oligonucleotides 3b and 2p2

<p><b>Copyright information:</b></p><p>Taken from "Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite "</p><p>Nucleic Acids Research 2005;33(5):1722-1736.</p><p>Published online 22 Mar 2005</p><p>PMCID:PMC1903550.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () The sequence of oligonucleotide 3b and the extent of the oligonucleotides 3b1 and 3b2 used in the competition experiments using the tachyzoite nuclear extracts are shown. () Right panel: lane 1, free probe; lane 2, no competitor; lane 3, specific competitor (100-fold excess); and lanes 5 and 6 show competition with oligonucleotides 3b1 and 3b2, respectively (100-fold excess each). Arrowheads show the oligonucleotide–protein complexes. Band shift assay with oligonucleotides 2p1 and 2p2. The sequence of oligonucleotide 2p2 and the extent of the oligonucleotides 2p2a, 2p2b and 2p2c used in the competition experiments are shown in (A). Lane 1: free probe; lane 2, no competitor; and lane 3, specific competitor (100-fold excess). Lanes 4–6 show competition of labelled oligonucleotide 2p2 with the short overlapping self oligonucleotides shown in (A). Note the disappearance of the oligonucleotide protein complexes in lanes 5 and 6 (arrowed)