Monique Benaïssa

Université des Sciences et Technologies de Lille 1, Lille, Nord-Pas-de-Calais, France

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Publications (16)55.24 Total impact

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    ABSTRACT: Delta-lactoferrin (DeltaLf) is a transcription factor that up-regulates DcpS, Skp1, and Bax genes, provoking cell cycle arrest and apoptosis. It is post-translationally modified either by O-GlcNAc or phosphate, but the effects of the O-GlcNAc/phosphorylation interplay on DeltaLf function are not yet understood. Here, using a series of glycosylation mutants, we showed that Ser(10) is O-GlcNAcylated and that this modification is associated with increased DeltaLf stability, achieved by blocking ubiquitin-dependent proteolysis, demonstrating that O-GlcNAcylation protects against polyubiquitination. We highlighted the (391)KSQQSSDPDPNCVD(404) sequence as a functional PEST motif responsible for DeltaLf degradation and defined Lys(379) as the main polyubiquitin acceptor site. We next investigated the control of DeltaLf transcriptional activity by the O-GlcNAc/phosphorylation interplay. Reporter gene analyses using the Skp1 promoter fragment containing a DeltaLf response element showed that O-GlcNAcylation at Ser(10) negatively regulates DeltaLf transcriptional activity, whereas phosphorylation activates it. Using a chromatin immunoprecipitation assay, we showed that O-GlcNAcylation inhibits DNA binding. Deglycosylation leads to DNA binding and transactivation of the Skp1 promoter at a basal level. Basal transactivation was markedly enhanced by 2-3-fold when phosphorylation was mimicked at Ser(10) by aspartate. Moreover, using double chromatin immunoprecipitation assays, we showed that the DeltaLf transcriptional complex binds to the DeltaLf response element and is phosphorylated and/or ubiquitinated, suggesting that DeltaLf transcriptional activity and degradation are concomitant events. Collectively, our results indicate that reciprocal occupancy of Ser(10) by either O-phosphate or O-GlcNAc coordinately regulates DeltaLf stability and transcriptional activity.
    Journal of Biological Chemistry 06/2010; 285(25):19205-18. · 4.65 Impact Factor
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    ABSTRACT: The expression of the transcription factor DeltaLf is deregulated in cancer cells. Its overexpression provokes cell cycle arrest along with antiproliferative effects and we recently showed that the Skp1 gene promoter was a target of DeltaLf. Skp1 belongs to the Skp1/Cullin-1/F-box ubiquitin ligase complex responsible for the ubiquitination and the proteosomal degradation of numerous cellular regulators. The transcriptional activity of DeltaLf is highly controlled and negatively regulated by O-GlcNAc, a dynamic post-translational modification known to regulate the functions of many intracellular proteins. We, therefore, constructed a DeltaLf-M4 mutant corresponding to a constitutively active DeltaLf isoform in which all the glycosylation sites were mutated. In order to discover novel targets of DeltaLf transcriptional activity and to investigate the impact of the O-GlcNAc regulation on this activity in situ we compared the proteome profiles of DeltaLf- and DeltaLf-M4-expressing HEK293 cells versus null plasmid transfected cells. A total of 14 differentially expressed proteins were visualized by 2D electrophoresis and silver staining and eight proteins were identified by mass spectrometry analyses (MALDI-TOF; LC-MS/MS), all of which were upregulated. The identified proteins are involved in several processes such as mRNA maturation and stability, cell viability, proteasomal degradation, protein and mRNA quality control. Among these proteins, only DcpS and TCPB were also upregulated at the mRNA level. Analysis of their respective promoters led to the detection of a cis-regulating element in the DcpS promoter. The S1(DcpS) is 80% identical to the S1 sequence previously described by He and Furmanski [Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA, Nature 373 (1995) 721-724]. Reporter gene analyses and ChIP assays demonstrated that DeltaLf interacts specifically with the DcpS promoter in vivo. These data established that DcpS, a key enzyme in mRNA decay, is a new target of DeltaLf transcriptional activity.
    Biochimie 09/2008; 91(1):109-22. · 3.14 Impact Factor
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    ABSTRACT: Delta-lactoferrin is a cytoplasmic lactoferrin isoform that can locate to the nucleus, provoking antiproliferative effects and cell cycle arrest in S phase. Using macroarrays, the expression of genes involved in the G(1)/S transition was examined. Among these, Skp1 showed 2-3-fold increased expression at both the mRNA and protein levels. Skp1 (S-phase kinase-associated protein) belongs to the Skp1/Cullin-1/F-box ubiquitin ligase complex responsible for the ubiquitination of cellular regulators leading to their proteolysis. Skp1 overexpression was also found after delta-lactoferrin transient transfection in other cell lines (HeLa, MDA-MB-231, HEK 293) at comparable levels. Analysis of the Skp1 promoter detected two sequences that were 90% identical to those previously known to interact with lactoferrin, the secretory isoform of delta-lactoferrin (GGCACTGTAC-S1(Skp1), located at - 1067 bp, and TAGAAGTCAA-S2(Skp1), at - 646 bp). Both gel shift and chromatin immunoprecipitation assays demonstrated that delta-lactoferrin interacts in vitro and in vivo specifically with these sequences. Reporter gene analysis confirmed that delta-lactoferrin recognizes both sequences within the Skp1 promoter, with a higher activity on S1(Skp1). Deletion of both sequences totally abolished delta-lactoferrin transcriptional activity, identifying them as delta-lactoferrin-responsive elements. Delta-lactoferrin enters the nucleus via a short bipartite RRSDTSLTWNSVKGKK(417-432) nuclear localization signal sequence, which was demonstrated to be functional using mutants. Our results show that delta-lactoferrin binds to the Skp1 promoter at two different sites, and that these interactions lead to its transcriptional activation. By increasing Skp1 gene expression, delta-lactoferrin may regulate cell cycle progression via control of the proteasomal degradation of S-phase actors.
    FEBS Journal 05/2007; 274(8):2038-53. · 4.25 Impact Factor
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    ABSTRACT: We investigated the expression levels of human lactoferrin (Lf), a steroid hormone-inducible gene product the expression of which is often altered during oncogenesis, and of Delta-lactoferrin (DeltaLf), its alternative isoform, which has been shown to be absent from tumor cell lines in commonly used human breast epithelial cell lines, using semiquantitative RT-PCR. Both mRNAs were detected but with levels of expression lower than those found in normal breast epithelial cells. This downregulation was much more visible for DeltaLf since its expression was either significantly diminished (BT-20, MCF-7 cell lines) or practically absent (MDA-MB-231, T-47D, HBL 100 cell lines). In order to determine whether Lf gene products are useful prognosic tools, we further analyzed their expression levels in 99 primary breast cancer biopsies. DeltaLf transcripts were found in all of the samples, whereas Lf transcripts were found in 88% of them. Lf and DeltaLf expression levels were positively correlated (p = 0.003). Lf expression was related to tumor type with a higher recovery in lobular-type tumors (p = 0.04). DeltaLf expression was related to the histoprognostic grading (p = 0.02). In univariate analyses, DeltaLf and Lf expressions were prognosis parameters, high concentrations being associated with a longer overall survival.
    International Journal of Cancer 04/2005; 114(2):299-306. · 6.20 Impact Factor
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    ABSTRACT: The screening of a bovine submaxillary gland cDNA library yielded 25 clones coding for bovine lactotransferrin. The nucleotide sequence of the longest insert contained a protein-coding region of 2115 nucleotides and a 3′ non-coding region of 194 nucleotides followed by a poly(A) tract of about 55 nucleotides. The predicted peptide sequence included a 16-amino-acid signal sequence upstream of the first amino acid of the native protein. The identity of the clone was confirmed by matching the amino acid sequence predicted from the cDNA with the N-terminal and tryptic peptide sequences derived from purified bovine milk lactotransferrin, and also by similarity with human and murine lactotransferrins. The cDNA described corresponds to a 705-amino-acid-long preprotein that lacks the start methionine. The sequence of the secreted protein is 689 amino acids long and contains five potential glycosylation sites. Bovine lactotransferrin is 69% and 64% identical to human and murine lactotransferrins, respectively.
    European Journal of Biochemistry. 03/2005; 196(1):177 - 184.
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    ABSTRACT: Delta-lactoferrin (deltaLf) mRNA is the product of alternative splicing of the Lf gene. It has been found in normal tissues and was reported to be absent from their malignant counterparts. Our recent investigations have shown that deltaLf expression is a good prognostic indicator in human breast cancer. However, deltaLf has up till now only been identified as a transcript, and in order to characterize the deltaLf protein and determine its function we have used a deltaLf cDNA construct to produce the protein in vitro and in vivo. A 73 kDa protein was immunoprecipitated from in vitro translation products and this molecular weight is in accordance with the use of the first in frame AUG start codon located in exon 2. We also produced a cell line expressing deltaLf under doxycycline induction. Using this model we have been able to show that deltaLf is mainly distributed in the cytoplasm. Its expression induces cell cycle arrest and inhibits cell proliferation. Our results suggest that deltaLf may play an important role in the regulation of normal cell growth.
    BioMetals 07/2004; 17(3):325-9. · 3.28 Impact Factor
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    ABSTRACT: Abstract : Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation, which accumulates in the brain during neurodegenerative disorders. Prior to determining Lf function in pathological brain tissues, we investigated its transport through the blood-brain barrier (BBB) in inflammatory conditions. For this purpose, we used a reconstituted BBB model consisting of the coculture of bovine brain capillary endothelial cells (BBCECs) and astrocytes in the presence of tumor necrosis factor-α (TNF-α). As TNF-α can be either synthesized by brain glial cells or present in circulating blood, BBCECs were exposed to this cytokine at their luminal or abluminal side. We have been able to demonstrate that in the presence of TNF-α, whatever the type of exposure, BBCECs were activated and Lf transport through the activated BBCECs was markedly increased. Lf was recovered intact at the abluminal side of the cells, suggesting that increased Lf accumulation may occur in immune-mediated pathophysiology. This process was transient as 20 h later, cells were in a resting state and Lf transendothelial traffic was back to normal. The enhancement of Lf transcytosis seems not to involve the up-regulation of the Lf receptor but rather an increase in the rate of transendothelial transport.
    Journal of Neurochemistry 01/2002; 73(6):2491 - 2500. · 3.97 Impact Factor
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    ABSTRACT: The presence of the iron-binding protein lactoferrin (Lf) in some specific areas of the central nervous system and particularly in the normal human substantia nigra, where it is found in dopaminergic (DA) neurons and some glial cells, led us to investigate Lf synthesis in this area. Lf mRNA were identified using in situ hybridization and found in small ameboid cells. These cells were identified using immunocytochemistry as activated microglia since they exhibited macrophage markers such as the CD68 and the CR1 antigens. Double immunofluorescent labeling confirmed that the two Lf immunostained cell populations were activated microglia and DA neurons. Since activated microglia contained both Lf and its messenger, these cells are the Lf producing cells. The presence of Lf in DA neurons in which no Lf messengers were visible, might be due to an endocytosis mechanism, DA neurons probably internalizing Lf produced in microglial cells located in their neighborhood. In neuropathological disorders, such as Alzheimer's and Parkinson's diseases, inflammatory process and oxidative stress are events that contribute to neuronal death. Since Lf concentration increases during these pathologies, we studied the level of Lf expression under these different stresses and showed, using RT-PCR, that the immortalized human embryonic microglial CHME cell line produced Lf transcripts under tumor necrosis factor alpha or 1-methyl-4-phenylpyridinium treatment whereas untreated cells did not. These data confirm that Lf is produced only when microglia are activated.
    Molecular Brain Research 12/2001; 96(1-2):103-13. · 2.00 Impact Factor
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    ABSTRACT: Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation, which accumulates in the brain during neurodegenerative disorders. Prior to determining Lf function in pathological brain tissues, we investigated its transport through the blood-brain barrier (BBB) in inflammatory conditions. For this purpose, we used a reconstituted BBB model consisting of the coculture of bovine brain capillary endothelial cells (BBCECs) and astrocytes in the presence of tumor necrosis factor-alpha (TNF-alpha). As TNF-alpha can be either synthesized by brain glial cells or present in circulating blood, BBCECs were exposed to this cytokine at their luminal or abluminal side. We have been able to demonstrate that in the presence of TNF-alpha, whatever the type of exposure, BBCECs were activated and Lf transport through the activated BBCECs was markedly increased. Lf was recovered intact at the abluminal side of the cells, suggesting that increased Lf accumulation may occur in immune-mediated pathophysiology. This process was transient as 20 h later, cells were in a resting state and Lf transendothelial traffic was back to normal. The enhancement of Lf transcytosis seems not to involve the up-regulation of the Lf receptor but rather an increase in the rate of transendothelial transport.
    Journal of Neurochemistry 01/2000; 73(6):2491-500. · 3.97 Impact Factor
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    ABSTRACT: The presence of iron in brain tissue in increased concentrations in Parkinson's disease cases, where it might be responsible for oxidative stress, and the parallel observation that the iron transporter lactoferrin (Lf) was present in increased amounts in surviving neurons, led us to study the synthesis of Lf in a mouse model of Parkinson's disease. In this context, the origin and expression of brain Lf in normal, aged and MPTP (1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine)-treated mice were investigated. Lf immunostaining was observed mainly on microvessels in the cerebral cortex of the adult mice and to a greater extent in older mice. Lf immunoreactivity was also present in the hippocampus only in the aged mouse brains, associated with structures which seemed to be pyramidal neurons and fibers. After RT-PCR (polymerase chain reaction), Lf transcripts were found in mouse brain tissue whatever the age of the animals studied but the level of their expression was very low. No up-regulation of Lf was detectable during aging. Lf distribution and expression in the MPTP-induced Parkinsonian mouse model were also investigated. A marked depletion of dopamine (DA) occurred in the high dose MPTP-treated mice. The level of Lf expression was found to be markedly increased in the same animals and this up-regulation occurred on the first day after MPTP administration. When the brain was stressed by the neurotoxin MPTP, Lf expression increased in line with antioxidant enzymes such as catalase and gamma-glutamylcysteine synthetase, which may permit the protection of brain tissue from oxidative damage induced by the drug.
    Molecular Brain Research 11/1999; 72(2):183-94. · 2.00 Impact Factor
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    ABSTRACT: Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation; it accumulates in the brain during neurodegenerative disorders. Before determining Lf function in brain tissue, we investigated its origin and demonstrate here that it crosses the blood-brain barrier. An in vitro model of the blood-brain barrier was used to examine the mechanism of Lf transport to the brain. We report that differentiated bovine brain capillary endothelial cells exhibited specific high (Kd = 37.5 nM; n = 90,000/cell) and low (Kd = 2 microM; n = 900,000 sites/cell) affinity binding sites. Only the latter were present on nondifferentiated cells. The surface-bound Lf was internalized only by the differentiated cell population leading to the conclusion that Lf receptors were acquired during cell differentiation. A specific unidirectional transport then occurred via a receptor-mediated process with no apparent intraendothelial degradation. We further report that iron may cross the bovine brain capillary endothelial cells as a complex with Lf. Finally, we show that the low density lipoprotein receptor-related protein might be involved in this process because its specific antagonist, the receptor-associated protein, inhibits 70% of Lf transport.
    Journal of Biological Chemistry 04/1999; 274(11):7011-7. · 4.65 Impact Factor
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    ABSTRACT: The biological role and origin of human lactoferrin (Lf) within the brain in normal and disease processes are as yet uncharted. In this context the origin and expression of brain Lf in normal and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated mice were investigated using immunohisto chemistry, PCR amplification and in situ hybridization. Lf immunostaining was observed both on sections of mouse lactating mammary gland, which was used as a positive control, and brains from young, adult and aged mice. Lf immunoreactivity was present in the pituitary gland, the hippocampus and the cortex of mouse brains and to a greater extent in older mice. After reverse transcription, Lf transcripts were also found in these brain sections. Lf distribution and expression in the MPTP-induced parkinsonian mouse model were next investigated. A marked depletion of dopamine and its metabolites: dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxy indole acetic acid (5-HIAA) occurs in the high dose MPTP-treated mice. The level of Lf expression was found to be greatly increased in the same animals but Lf immunoreactivity detected in the same brain region was not found increased in the affected areas.
    Advances in experimental medicine and biology 02/1998; 443:293-300. · 1.83 Impact Factor
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    ABSTRACT: A full-length cDNA coding for human lactoferrin was isolated from a mammary gland library and the recombinant protein was expressed in BHK cells as described by Stowell K. M. et al. [1991, Biochem. J. 276, 349-355]. Two N-linked glycans from purified recombinant lactoferrin were released by hydrazinolysis and analyzed by 400-MHz 1H-NMR spectroscopy. The identified structures corresponded to N-acetyllactosaminic biantennary glycans and were alpha-2,3-disialylated forms (80%) or alpha-2,3-monosialylated (20%) forms. Moreover, 70% of total glycans were alpha-1,6-fucosylated at the GlcNAc residue linked to asparagine. In regard to its glycan moiety, the recombinant glycoprotein is close to native lactoferrins from milk or leucocytes but shows specific structural features which should be taken into account prior to in vivo and in vitro biological studies.
    FEBS Letters 06/1995; 365(1):57-60. · 3.58 Impact Factor
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    ABSTRACT: The complete cDNA for rat mammary-gland transferrin (Tf) has been sequenced and also the native protein isolated from milk in order to analyse the structure of the main glycan variants present. A lactating-rat mammary-gland cDNA library in lambda gt10 was screened with a partial cDNA copy of rat liver Tf and subsequently rescreened with 5' fragments of the longest clones. This produced a 2275 bp insert coding for an open reading frame of 695 amino acid residues. This includes a 19-amino acid signal sequence and the mature protein containing 676 amino acids and one N-glycosylation site in the C-terminal domain at residue 490. Phylogenetic analysis was carried out using 14 translated Tf nucleotide sequences, and the derived evolutionary tree shows that at least three gene duplication events have occurred during Tf evolution, one of which generated the N- and C-terminal domains and occurred before separation of arthropods and chordates. The two halves of human melanotransferrin are more similar to each other than to any other sequence, which contrasts with the pattern shown by the remaining sequences. Native rat milk Tf is separated into four bands on native PAGE that differ only in their sialic acid content: one biantennary glycan is present containing either no sialic acid residues or up to three. The complete structures of the two major variants were determined by methylation, m.s. and 400 MHz 1H-n.m.r. spectroscopy. They contain either one or two neuraminic acid residues (alpha 2-->6)-linked to galactose in conventional biantennary N-acetyl-lactosamine-type glycans. Most contain fucose (alpha 1-->6)-linked to the terminal non-reducing N-acetylglucosamine.
    Biochemical Journal 04/1995; 307 ( Pt 1):47-55. · 4.65 Impact Factor
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    ABSTRACT: In order to determine whether the human lactotransferrin receptor recently described on platelets was also present on hematopoietic precursors, we investigated its presence and characteristics on the megakaryocytic Dami cell line. The reversible binding of human 5-(([2-(carbo(hydrazino)methyl]thio)acetyl)aminofluorescein-labele d lactotransferrin showed that such a receptor was only present on the subpopulation of the largest cells. The increase in numbers of large cells during culture was paralleled by a concurrent increase in lactotransferrin receptor positive cells. Scatchard analysis of the binding of [125I]-labeled lactotransferrin showed that a single affinity class of binding site was present (Kd = 446 +/- 40 nM) and that there were 52 +/- 3 x 10(5) sites per cell. The mouse monoclonal antibody DP5B3G10, specific for the human lactotransferrin receptor, allowed its characterization as a 105 kDa protein on Western blots. The same monoclonal antibody was used to separate the small and large cell subpopulations of Dami cells by panning. Separate culture of the small cells showed that the receptor appeared prior to and independent from endomitosis. In contrast, GPIb was expressed only by large megakaryocytes. The use of conditioned medium from cultures of whole Dami cell populations indicated that a soluble factor is involved in differentiation, but not in the appearance of the lactotransferrin receptor.
    Biology of the Cell 02/1994; 82(2-3):149-59. · 3.49 Impact Factor
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    ABSTRACT: The screening of a bovine submaxillary gland cDNA library yielded 25 clones coding for bovine lactotransferrin. The nucleotide sequence of the longest insert contained a protein-coding region of 2115 nucleotides and a 3' non-coding region of 194 nucleotides followed by a poly(A) tract of about 55 nucleotides. The predicted peptide sequence included a 16-amino-acid signal sequence upstream of the first amino acid of the native protein. The identity of the clone was confirmed by matching the amino acid sequence predicted from the cDNA with the N-terminal and tryptic peptide sequences derived from purified bovine milk lactotransferrin, and also by similarity with human and murine lactotransferrins. The cDNA described corresponds to a 705-amino-acid-long preprotein that lacks the start methionine. The sequence of the secreted protein is 689 amino acids long and contains five potential glycosylation sites. Bovine lactotransferrin is 69% and 64% identical to human and murine lactotransferrins, respectively.
    European Journal of Biochemistry 03/1991; 196(1):177-84. · 3.58 Impact Factor

Publication Stats

446 Citations
55.24 Total Impact Points

Institutions

  • 1994–2010
    • Université des Sciences et Technologies de Lille 1
      • Unité de Glycobiologie Structurale et Fonctionnelle (UGSF)
      Lille, Nord-Pas-de-Calais, France
  • 1991–2005
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France