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See detailProteomic discovery of substrates of the cardiovascular protease ADAMTS7.
Colige, Alain ULiege; Monseur, Christine; Crawley, JTB et al

in Journal of Biological Chemistry (2019)

The protease ADAMTS7 functions in the extracellular matrix (ECM) of the cardiovascular system. However, its physiological substrate specificity and mechanism of regulation remain to be explored. To ... [more ▼]

The protease ADAMTS7 functions in the extracellular matrix (ECM) of the cardiovascular system. However, its physiological substrate specificity and mechanism of regulation remain to be explored. To address this, we conducted an unbiased substrate analysis using terminal amine isotopic labeling of substrates (TAILS). The analysis identified candidate substrates of ADAMTS7 in the human fibroblast secretome, including proteins with a wide range of functions, such as collagenous and non-collagenous ECM proteins, growth factors, proteases, and cell-surface receptors. It also suggested that autolysis occurs at Glu729-Val730 and Glu732-Ala733 in the ADAMTS7 spacer domain, which was corroborated by N-terminal sequencing and western blotting. Importantly, TAILS also identified proteolysis of the latent TGF-β-binding proteins 3 and 4 (LTBP3/4) at a Glu-Val and Glu-Ala site, respectively. Using purified enzyme and substrate, we confirmed ADAMTS7-catalyzed proteolysis of recombinant LTBP4. Moreover, we identified multiple additional scissile bonds in an N-terminal linker region of LTBP4 that connects fibulin-5/tropoelastin and fibrillin-1-binding regions, which have an important role in elastogenesis. ADAMTS7-mediated cleavage of LTBP4 was efficiently inhibited by the metalloprotease inhibitor TIMP-4, but not by TIMP-1 and less efficiently by TIMP-2 and TIMP-3. As TIMP-4 expression is prevalent in cardiovascular tissues, we propose that TIMP-4 represents the primary endogenous ADAMTS7 inhibitor. In summary, our findings reveal LTBP4 as an ADAMTS7 substrate, whose cleavage may potentially impact elastogenesis in the cardiovascular system. We also identify TIMP-4 as a likely physiological ADAMTS7 inhibitor. [less ▲]

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See detailA dynamic and screening-compatible nanoluciferase-based complementation assay enables profiling of individual GPCR-G protein interactions
Laschet, Céline ULiege; Dupuis, Nadine; Hanson, Julien ULiege

in Journal of Biological Chemistry (2019), 294(11), 4079-4090

G protein-coupled receptors (GPCRs) are currently the target of more than 30% of the marketed medicines. However, there is an important medical need for ligands with improved pharmacological activities on ... [more ▼]

G protein-coupled receptors (GPCRs) are currently the target of more than 30% of the marketed medicines. However, there is an important medical need for ligands with improved pharmacological activities on validated drug targets. Moreover, most of these ligands remain poorly characterized, notably because of a lack of pharmacological tools. Thus, there is an important demand for innovative assays that can detect and drive the design of compounds with novel or improved pharmacological properties. In particular, a functional and screening-compatible GPCR-G protein interaction assay is still unavailable. Here, we report on a nanoluciferase-based complementation technique to detect ligands that promote a GPCR-G protein interaction. We demonstrate that our system can be used to profile compounds with regard to the G proteins they activate through a given GPCR. Furthermore, we established a proof of applicability of screening for distinct G proteins on dopamine receptor D2 whose differential coupling to Gαi/o family members has been extensively studied. In a D2-Gαi1 versus D2- Gαo screening, we retrieved five agonists that are currently being used in antiparkinsonian medications. We determined that in this assay, piribedil and pergolide are full agonists for the recruitment of Gαi1 but are partial agonists for Gαo, that the agonist activity of ropinirole is biased in favor of Gαi1 recruitment, and that the agonist activity of apomorphine is biased for Gαo. We proposed that this newly developed assay could be used to develop molecules that selectively modulate a particular G protein pathway. [less ▲]

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See detailDirect interactions between the secreted effector and the T2SS components GspL and GspM reveal a new effector-sensing step during type 2 secretion
Michel-Souzy, S.; Douzi, B.; Cadoret, F. et al

in Journal of Biological Chemistry (2018), 293(50), 19441-19450

In many Gram-negative bacteria, the type 2 secretion system (T2SS) plays an important role in virulence because of its capacity to deliver a large amount of fully folded protein effectors to the ... [more ▼]

In many Gram-negative bacteria, the type 2 secretion system (T2SS) plays an important role in virulence because of its capacity to deliver a large amount of fully folded protein effectors to the extracellular milieu. Despite our knowledge of most T2SS components, the mechanisms underlying effector recruitment and secretion by the T2SS remain enigmatic. Using complementary biophysical and biochemical approaches, we identified here two direct interactions between the secreted effector CbpD and two components, XcpYL and XcpZM, of the T2SS assembly platform (AP) in the opportunistic pathogen Pseudomonas aeruginosa. Competition experiments indicated that CbpD binding to XcpYL is XcpZM-dependent, suggesting sequential recruitment of the effector by the periplasmic domains of these AP components. Using a bacterial two-hybrid system, we then tested the influence of the effector on the AP protein–protein interaction network. Our findings revealed that the presence of the effector modifies the AP interactome and, in particular, induces XcpZM homodimerization and increases the affinity between XcpYL and XcpZM. The observed direct relationship between effector binding and T2SS dynamics suggests an additional synchronizing step during the type 2 secretion process, where the activation of the AP of the T2SS nanomachine is triggered by effector binding. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc. [less ▲]

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See detailCover Letter of The journal of Biological Chemistry
Sautrey, Guillaume; El Khoury, M; Dos Giros, A et al

in Journal of Biological Chemistry (2016), 291(26),

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See detailNegatively Charged Lipids as a Potential Target for New Amphiphilic Aminoglycoside Antibiotics: A BIOPHYSICAL STUDY*
Sautrey, Guillaume; El Khoury, Micheline; Dos Giros, Andrea et al

in Journal of Biological Chemistry (2016), 291(26), 13864-13874

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See detailThe role of active site flexible loops in catalysis and of zinc in conformational stability of Bacillus cereus 569/H/9 beta-lactamase.
Montagner, Caroline ULiege; Nigen, Michaël; Jacquin, Olivier et al

in Journal of Biological Chemistry (2016), 291(31), 16124-16137

Metallo-beta-lactamases catalyse the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the ... [more ▼]

Metallo-beta-lactamases catalyse the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus beta-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (DeltaG degrees ) of 32 +/- 2 kJ.mol11. For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site and the protein displays a well-organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. 2D NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with DeltaG degrees value of 65 +/- 1.4 kJ.mol11. These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well-defined conformation for both active site loops in order to maintain enzymatic activity. [less ▲]

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See detailThe N-terminal region of CHD4 is essential for activity and contains a HMG-box-like-domain that can bind poly(ADP-ribose).
Silva, Ana; Ryan, Daniel; Galanty, Y et al

in Journal of Biological Chemistry (2015)

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See detailTransforming Growth Factor-beta and Interleukin-1beta Signaling Pathways Converge on the Chemokine CCL20 Promoter.
Brand, Oliver J.; Somanath, Sangeeta; Moermans, Catherine ULiege et al

in Journal of Biological Chemistry (2015), 290(23), 14717-28

CCL20 is the only chemokine ligand for the chemokine receptor CCR6, which is expressed by the critical antigen presenting cells, dendritic cells. Increased expression of CCL20 is likely involved in the ... [more ▼]

CCL20 is the only chemokine ligand for the chemokine receptor CCR6, which is expressed by the critical antigen presenting cells, dendritic cells. Increased expression of CCL20 is likely involved in the increased recruitment of dendritic cells observed in fibroinflammatory diseases such as chronic obstructive pulmonary disease (COPD). CCL20 expression is increased by the proinflammatory cytokine IL-1beta. We have determined that IL-1beta-dependent CCL20 expression is also dependent on the multifunctional cytokine TGF-beta. TGF-beta is expressed in a latent form that must be activated to function, and activation is achieved through binding to the integrin alphavbeta8 (itgb8). Here we confirm correlative increases in alphavbeta8 and IL-1beta with CCL20 protein in lung parenchymal lysates of a large cohort of COPD patients. How IL-1beta- and alphavbeta8-mediated TGF-beta activation conspire to increase fibroblast CCL20 expression remains unknown, because these pathways have not been shown to directly interact. We evaluate the 5'-flanking region of CCL20 to determine that IL-1beta-driven CCL20 expression is dependent on alphavbeta8-mediated activation of TGF-beta. We identify a TGF-beta-responsive element (i.e. SMAD) located on an upstream enhancer of the human CCL20 promoter required for efficient IL-1beta-dependent CCL20 expression. By chromatin immunoprecipitation, this upstream enhancer complexes with the p50 subunit of NF-kappaB on a NF-kappaB-binding element close to the transcriptional start site of CCL20. These interactions are confirmed by electromobility shift assays in nuclear extracts from human lung fibroblasts. These data define a mechanism by which alphavbeta8-dependent activation of TGF-beta regulates IL-1beta-dependent CCL20 expression in COPD. [less ▲]

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See detailTumor suppressive p53 signaling empowers metastatic inhibitor KLF17-dependent transcription to overcome tumorigenesis in non-small cell lung cancer.
Ali, Amjad ULiege; Zeeshan Bhatti M; Saboor Shah A et al

in Journal of Biological Chemistry (2015)

Metastasis, which is controlled by concerted action of multiple genes, is a complex process, and is important cause of cancer death. KLF17 is a negative regulator of metastasis and epithelial-mesenchymal ... [more ▼]

Metastasis, which is controlled by concerted action of multiple genes, is a complex process, and is important cause of cancer death. KLF17 is a negative regulator of metastasis and epithelial-mesenchymal-transition (EMT) during cancer progression. However, the underlying molecular mechanism and biological relevance of KLF17 in cancer cells are poorly understood. Here, we show that tumor suppressor protein p53 plays an integral role to induce KLF17 expression in NSCLC. p53 is recruited to KLF17 promoter and results in the formation of p53-DNA complex. p53 enhances binding of p300, and favors histone acetylation on KLF17 promoter. Mechanistically, p53 physically interacts with KLF17 and thereby enhances anti-metastatic function of KLF17. p53 empowers KLF17 mediated EMT genes transcription via enhancing physical association of KLF17 to target gene promoters. Nutlin-3 recruits KLF17 to EMT target gene promoters and results in the formation of KLF17-DNA complex via p53-dependent pathway. p53 depletion abrogates DNA binding affinity of KLF17 to EMT target gene promoters. KLF17 is critical for p53 cellular activities in NSCLC. Importantly, KLF17 enhances p53 transcription to generate a novel positive feedback loop. KLF17 depletion accelerates lungs cancer cells growth in response to chemotherapy. Mechanistically, we found that KLF17 increases tumor suppressor genes p53, p21 and pRB expressions in NSCLC. Functionally, KLF17 required p53 to suppress cancer cell invasion and migration in NSCLC. In sum, our study highlights novel insight into anti-EMT affect of KLF17 via p53-dependent pathway in NSCLC, and KLF17 may be a new therapeutic target in NSCLC with p53 status. [less ▲]

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See detailMutant p53 promotes tumor cell malignancy by both positive and negative regulation of TGF-β pathway
Lei Ji; Jinjin Xu; Jian Liu et al

in Journal of Biological Chemistry (2015)

Specific p53 mutations abrogate the tumor suppressive functions by gaining new abilities to promote tumorigenesis. Inactivation of p53 is known to distort the TGF-β signaling, which paradoxically displays ... [more ▼]

Specific p53 mutations abrogate the tumor suppressive functions by gaining new abilities to promote tumorigenesis. Inactivation of p53 is known to distort the TGF-β signaling, which paradoxically displays both tumor-suppressive and pro-oncogenic function. The molecular mechanisms how mutant p53 simultaneously antagonizes tumor-suppressive and synergizes tumor-promoting function of TGF-β pathway remain elusive. Here we demonstrate that mutant p53 differentially regulates subsets of TGF-β target genes, by an enhanced binding to the MH2 domain in Smad3 upon the integration of ERK signaling, therefore disrupting the Smad3/Smad4 complex formation. Silencing Smad2, inhibition of ERK or introducing a phosphorylation defective mutation at Ser392 in p53 abrogates the R175H mutant p53 dependent regulation of these TGF- β target genes. Our study provide a mechanism to reconcile the seemingly contradictory observations that mutant p53 can both attenuate and cooperate with the TGF-β pathway to promote cancer cell malignancy in a same cell type. [less ▲]

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See detailThe involvement of hydrogen-producing and ATP-dependent NADPH-consuming pathways in setting the redox poise in the chloroplast of Chlamydomonas reinhardtii in anoxia.
Clowez, Sophie; Godaux, Damien ULiege; Cardol, Pierre ULiege et al

in Journal of Biological Chemistry (2015), 290(13), 8666-76

Photosynthetic microalgae are exposed to changing environmental conditions. In particular, microbes found in ponds or soils often face hypoxia or even anoxia, and this severely impacts their physiology ... [more ▼]

Photosynthetic microalgae are exposed to changing environmental conditions. In particular, microbes found in ponds or soils often face hypoxia or even anoxia, and this severely impacts their physiology. Chlamydomonas reinhardtii is one among such photosynthetic microorganisms recognized for its unusual wealth of fermentative pathways and the extensive remodeling of its metabolism upon the switch to anaerobic conditions. As regards the photosynthetic electron transfer, this remodeling encompasses a strong limitation of the electron flow downstream of photosystem I. Here, we further characterize the origin of this limitation. We show that it stems from the strong reducing pressure that builds up upon the onset of anoxia, and this pressure can be relieved either by the light-induced synthesis of ATP, which promotes the consumption of reducing equivalents, or by the progressive activation of the hydrogenase pathway, which provides an electron transfer pathway alternative to the CO2 fixation cycle. [less ▲]

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See detailThe Structure of the Cyprinid herpesvirus 3 ORF112-Zalpha.Z-DNA Complex Reveals a Mechanism of Nucleic Acids Recognition Conserved with E3L, a Poxvirus Inhibitor of Interferon Response.
Kus, Krzysztof; Rakus, Krzysztof; Boutier, Maxime ULiege et al

in Journal of Biological Chemistry (2015), 290(52), 30713-25

In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In ... [more ▼]

In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In some teleost species another protein kinase, Z-DNA-dependent protein kinase (PKZ), plays a similar role but instead of dsRNA binding domains, PKZ has Zalpha domains. These domains recognize the left-handed conformer of dsDNA and dsRNA known as Z-DNA/Z-RNA. Cyprinid herpesvirus 3 infects common and koi carp, which have PKZ, and encodes the ORF112 protein that itself bears a Zalpha domain, a putative competitive inhibitor of PKZ. Here we present the crystal structure of ORF112-Zalpha in complex with an 18-bp CpG DNA repeat, at 1.5 A. We demonstrate that the bound DNA is in the left-handed conformation and identify key interactions for the specificity of ORF112. Localization of ORF112 protein in stress granules induced in Cyprinid herpesvirus 3-infected fish cells suggests a functional behavior similar to that of Zalpha domains of the interferon-regulated, nucleic acid surveillance proteins ADAR1 and DAI. [less ▲]

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See detailDifferential role of snail1 and snail2 zinc fingers in E-cadherin repression and epithelial to mesenchymal transition
Villarejo; Cortés-Cabrera, Alvaro; Molina Ortiz, Patricia ULiege et al

in Journal of Biological Chemistry (2014), 289(2), 930-941

Snail1 (Snail) and Snail2 (Slug) are transcription factors that share a similar DNA binding structure of four and five C2H2 zinc finger motifs (ZF), respectively. Both factors bind specifically to a ... [more ▼]

Snail1 (Snail) and Snail2 (Slug) are transcription factors that share a similar DNA binding structure of four and five C2H2 zinc finger motifs (ZF), respectively. Both factors bind specifically to a subset of E-box motifs (E2-box: CAGGTG/CACCTG) in target promoters like the E-cadherin promoter and are key mediators of epithelial-to-mesenchymal transition (EMT). However, there are differences in the biological actions, in binding affinities to E-cadherin promoter, and in the target genes of Snail1 and Snail2, although the molecular bases are presently unknown. In particular, the role of each Snail1 and Snail2 ZF in the binding to E-boxes and in EMT induction has not been previously explored. We have approached this question by modeling Snail1 and Snail2 protein-DNA interactions and through mutational and functional assays of different ZFs. Results show that Snail1 efficient repression and binding to human and mouse E-cadherin promoter as well as EMT-inducing ability require intact ZF1 and ZF2, while for Snail2, either ZF3 or ZF4 is essential for those functions. Furthermore, the differential distribution of E2-boxes in mouse and human E-cadherin promoters also contributes to the differential Snail factor activity. These data indicate a non-equivalent role of Snail1 and Snail2 ZFs in gene repression, contributing to the elucidation of the molecular differences between these important EMT regulators. [less ▲]

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See detailA structural analysis of DNA binding by myelin transcription factor 1 double zinc fingers.
Gamsjaeger, R.; O'Connell, M.R.; Cubeddu, L. et al

in Journal of Biological Chemistry (2013)

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See detailNovel association between vasoactive intestinal peptide and CRTH2 receptor in recruiting eosinophils: a possible biochemical mechanism for allergic eosinophilic inflammation of the airways.
EL SHAZLY, Amr ULiege; Begon, Dominique ULiege; KUSTERMANS, Gaëlle ULiege et al

in Journal of Biological Chemistry (2013), 288(2), 1374-84

We explored the relation between vasoactive intestinal peptide (VIP), CRTH2, and eosinophil recruitment. It is shown that CRTH2 expression by eosinophils from allergic rhinitis (AR) patients and ... [more ▼]

We explored the relation between vasoactive intestinal peptide (VIP), CRTH2, and eosinophil recruitment. It is shown that CRTH2 expression by eosinophils from allergic rhinitis (AR) patients and eosinophils cell line (Eol-1 cells) was up-regulated by VIP treatment. This was functional and resulted into exaggerated migratory response of cells against PGD2. Nasal challenge of AR patients resulted into significant increase of VIP contents in nasal secretion (ELISA), and the immunohistochemical studies of allergic nasal tissues, showed significant expression of VIP in association with intense eosinophil recruitment. Biochemical assays showed that VIP-induced eosinophils chemotaxis from AR patients and Eol-1 cells, was mediated through CRTH2 receptor. Cells migration against VIP was sensitive to protein kinase C (PKC) and protein kinase A (PKA) inhibition, but not to tyrosine kinase or P38 MAP-kinase inhibition, or calcium chelation. Western blot demonstrated a novel CRTH2 mediated cytosol to membrane translocation of PKC-epsilon, PKC-delta and PKA-alpha, gamma and IIalpha reg in Eol-1 cells upon stimulation with VIP. Confocal images and FACS demonstrated a strong association and co-localization between VIP peptide and CRTH2 molecules. Further, VIP induced PGD2 secretion from eosinophils. Our results demonstrate the first evidence of association between VIP and CRTH2 in recruiting eosinophils. [less ▲]

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See detailNew insights into DNA recognition by zinc fingers revealed by structural analysis of the oncoprotein ZNF217
Vandevenne, Marylène ULiege; Jacques, D.A; Artuz, C.D. et al

in Journal of Biological Chemistry (2013)

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See detailSynuclein Membrane Association Is Regulated by the Rab3a Recycling Machinery and Presynaptic Activity.
Wislet, Sabine ULiege; Chen, R; Samuel, F et al

in Journal of Biological Chemistry (2013)

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See detailTargeting a single function of the multifunctional matrix metalloprotease MT1-MMP. Impact on lymphangiogenesis.
Ingvarsen, Signe; Porse, Astrid; Erpicum, Charlotte ULiege et al

in Journal of Biological Chemistry (2013), sous presse

The group of matrix metalloproteases (MMPs) is responsible for multiple processes of extracellular matrix remodeling in the healthy body but also for matrix and tissue destruction during cancer invasion ... [more ▼]

The group of matrix metalloproteases (MMPs) is responsible for multiple processes of extracellular matrix remodeling in the healthy body but also for matrix and tissue destruction during cancer invasion and metastasis. The understanding of the contributions from each individual MMP, both in healthy and pathological events, has been complicated by the lack of specific inhibitors and the fact that some of the potent MMPs are multifunctional enzymes. These factors have also hampered the setup of therapeutic strategies targeting MMP activity. A tempting target is the membrane-associated MT1-MMP which has well-documented importance in matrix degradation but which takes part in more than one pathway in this regard. In this report, we describe the selective targeting of a single function of this enzyme by means of a specific monoclonal antibody against MT1-MMP, raised in an MT1-MMP knock-out mouse. The antibody blocks the enzyme ability to activate proMMP-2 without interfering with the collagenolytic function or the general proteolytic activity of MT1-MMP. Using this antibody, we have shown that the MT1-MMP-catalyzed activation of proMMP- 2 is involved in the outgrowth of cultured lymphatic endothelial cells in a collagen matrix in vitro, as well as in lymphatic vessel sprouting assayed ex vivo. This is the first example of the complete inactivation of a single function of a multifunctional MMP and the use of this strategy to pursue its role. [less ▲]

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See detailDERP6 (ELP5) and C3ORF75 (ELP6) regulate tumorigenicity and migration of melanoma cells as subunits of Elongator
Close, Pierre ULiege; Gillard, Magali; Ladang, Aurélie ULiege et al

in Journal of Biological Chemistry (2012)

The Elongator complex is composed of 6 subunits (Elp1-Elp6) and promotes RNAPII transcript elongation through histone acetylation in the nucleus as well as tRNA modification in the cytoplasm. This ... [more ▼]

The Elongator complex is composed of 6 subunits (Elp1-Elp6) and promotes RNAPII transcript elongation through histone acetylation in the nucleus as well as tRNA modification in the cytoplasm. This acetyltransferase complex directly or indirectly regulates numerous biological processes ranging from exocytosis and resistance to heat shock in yeast to cell migration and neuronal differentiation in higher eukaryotes. The identity of human ELP1 through ELP4 has been reported but human ELP5 and ELP6 have remained uncharacterized. Here, we report that DERP6 (ELP5) and C3ORF75 (ELP6) encode these subunits of human Elongator. We further investigated the importance and function of these two subunits by a combination of biochemical analysis and cellular assays. Our results show that DERP6/ELP5 is required for the integrity of Elongator and directly connects ELP3 to ELP4. Importantly, the migration and tumorigenicity of melanomaderived cells are significantly decreased upon Elongator depletion through ELP1 or ELP3. Strikingly, DERP6/ELP5 and C3ORF75/ELP6-depleted melanoma cells have similar defects, further supporting the idea that DERP6/ELP5 and C3ORF75/ELP6 are essential for Elongator function. Together, our data identify DERP6/ELP5 and C3ORF75/ELP6 as key players for migration, invasion and tumorigenicity of melanoma cells, as integral subunits of Elongator. [less ▲]

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See detailRetinoic acid receptors recognise the mouse genome through binding elements with diverse spacing and topology
Moutier, Emmanuel; Ye, Tao; Choukrallah, Mohamed-Amin et al

in Journal of Biological Chemistry (2012)

Retinoic Acid Receptors (RARs) heterodimerise with Retinoid X Receptors (RXRs) and bind to RA-response elements (RAREs) in the regulatory regions of their target genes. While previous studies on limited ... [more ▼]

Retinoic Acid Receptors (RARs) heterodimerise with Retinoid X Receptors (RXRs) and bind to RA-response elements (RAREs) in the regulatory regions of their target genes. While previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separated by 1, 2 or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodies or F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8 and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half sites with DR2 and DR0 spacings. This specific half site organisation constitutes a previously unrecognised, but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, while DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5 and DR8 to mediate RA-dependent transcriptional activation indicates that half site spacing allosterically regulates RAR function. [less ▲]

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