24 results on '"Desjobert C"'
Search Results
2. Circulating t(2;5)-positive cells can be detected in cord blood of healthy newborns
- Author
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Laurent, C, Lopez, C, Desjobert, C, Berrebi, A, Damm-Welk, C, Delsol, G, Brousset, P, and Lamant, L
- Published
- 2012
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3. Biochemical and random mutagenesis analysis of the region carrying the catalytic E152 amino acid of HIV-1 integrase
- Author
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Calmels, C., de Soultrait, V. Richard, Caumont, A., Desjobert, C., Faure, A., Fournier, M., Tarrago-Litvak, L., and Parissi, V.
- Published
- 2004
4. Leukemogenic mechanisms and targets of a NUP98/HHEX fusion in acute myeloid leukemia
- Author
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Jankovic, D., Gorello, Paolo, Liu, T., Ehret, T., LA STARZA, Roberta, Desjobert, C., Baty, F., Brutsche, M., Jayaraman, P. S., Santoro, A., Mecucci, Cristina, and Schwaller, J.
- Published
- 2008
5. Circulating t(2;5)-positive cells can be detected in cord blood of healthy newborns
- Author
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Laurent, C, primary, Lopez, C, additional, Desjobert, C, additional, Berrebi, A, additional, Damm-Welk, C, additional, Delsol, G, additional, Brousset, P, additional, and Lamant, L, additional
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- 2011
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6. R15 – Oral miR-16 participe à l’angiogenèse des tumeurs exprimant l’oncogène de fusion X-ALK
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Dejean, E., primary, Rénalier, M.H., additional, Foisseau, M., additional, Agirre, X., additional, De Paiva, G.R., additional, Al Saati, T., additional, Soulier, J., additional, Desjobert, C., additional, Lamant, L., additional, Felsher, D.W., additional, Cavaillé, J., additional, Prats, H., additional, Delsol, G., additional, Giuriato, S., additional, and Meggetto, F., additional
- Published
- 2010
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7. Peptides as New Inhibitors of HIV-1 Reverse Transcriptase and Integrase
- Author
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Soultrait, V., primary, Desjobert, C., additional, and Tarrago-Litvak, L., additional
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- 2003
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8. HIV-1 integrase and RNase H activities as therapeutic targets
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Andréola, ML, primary, De Soultrait, V Richard, additional, Fournier, M, additional, Parissi, V, additional, Desjobert, C, additional, and Litvak, S, additional
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- 2002
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9. miR-16 participates in tumor angiogenesis expressing X-ALK oncogene fusion
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Dejean, E., Renalier, M. H., Foisseau, M., Agirre, X., Paiva, G. R., Al Saati, T., Soulier, J., Desjobert, C., Lamant, L., Felsher, D. W., Cavaille, J., Prats, H., Delsol, G., Sylvie Giuriato, and Meggetto, F.
10. Epigenetically regulated PCDHB15 impairs aggressiveness of metastatic melanoma cells.
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Carrier A, Desjobert C, Lobjois V, Rigal L, Busato F, Tost J, Ensenyat-Mendez M, Marzese DM, Pradines A, Favre G, Lamant L, Lanfrancone L, Etievant C, Arimondo PB, and Riond J
- Subjects
- Humans, DNA Methylation, Signal Transduction, Exons, Melanoma genetics, Lung Neoplasms genetics
- Abstract
The protocadherin proteins are cell adhesion molecules at the crossroad of signaling pathways playing a major role in neuronal development. It is now understood that their role as signaling hubs is not only important for the normal physiology of cells but also for the regulation of hallmarks of cancerogenesis. Importantly, protocadherins form a cluster of genes that are regulated by DNA methylation. We have identified for the first time that PCDHB15 gene is DNA-hypermethylated on its unique exon in the metastatic melanoma-derived cell lines and patients' metastases compared to primary tumors. This DNA hypermethylation silences the gene, and treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine reinduces its expression. We explored the role of PCDHB15 in melanoma aggressiveness and showed that overexpression impairs invasiveness and aggregation of metastatic melanoma cells in vitro and formation of lung metastasis in vivo. These findings highlight important modifications of the methylation of the PCDHβ genes in melanoma and support a functional role of PCDHB15 silencing in melanoma aggressiveness., (© 2022. The Author(s).)
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- 2022
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11. DNA methylome combined with chromosome cluster-oriented analysis provides an early signature for cutaneous melanoma aggressiveness.
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Carrier A, Desjobert C, Ponger L, Lamant L, Bustos M, Torres-Ferreira J, Henrique R, Jeronimo C, Lanfrancone L, Delmas A, Favre G, Daunay A, Busato F, Hoon DSB, Tost J, Etievant C, Riond J, and Arimondo PB
- Subjects
- Animals, Chromosomes, CpG Islands, Cytosine, DNA Methylation, Epigenesis, Genetic, Epigenome, Gene Expression Regulation, Neoplastic, Guanine, Humans, Mice, Phosphates, Rats, Melanoma, Cutaneous Malignant, Melanoma genetics, Melanoma pathology, Skin Neoplasms genetics
- Abstract
Aberrant DNA methylation is a well-known feature of tumours and has been associated with metastatic melanoma. However, since melanoma cells are highly heterogeneous, it has been challenging to use affected genes to predict tumour aggressiveness, metastatic evolution, and patients' outcomes. We hypothesized that common aggressive hypermethylation signatures should emerge early in tumorigenesis and should be shared in aggressive cells, independent of the physiological context under which this trait arises. We compared paired melanoma cell lines with the following properties: (i) each pair comprises one aggressive counterpart and its parental cell line and (ii) the aggressive cell lines were each obtained from different host and their environment (human, rat, and mouse), though starting from the same parent cell line. Next, we developed a multi-step genomic pipeline that combines the DNA methylome profile with a chromosome cluster-oriented analysis. A total of 229 differentially hypermethylated genes was commonly found in the aggressive cell lines. Genome localization analysis revealed hypermethylation peaks and clusters, identifying eight hypermethylated gene promoters for validation in tissues from melanoma patients. Five Cytosine-phosphate-Guanine (CpGs) identified in primary melanoma tissues were transformed into a DNA methylation score that can predict survival (log-rank test, p=0.0008). This strategy is potentially universally applicable to other diseases involving DNA methylation alterations., Competing Interests: AC, CD, LP, LL, MB, JT, RH, CJ, LL, AD, GF, AD, FB, DH, JT, CE, JR, PA No competing interests declared, (© 2022, Carrier et al.)
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- 2022
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12. Demethylation by low-dose 5-aza-2'-deoxycytidine impairs 3D melanoma invasion partially through miR-199a-3p expression revealing the role of this miR in melanoma.
- Author
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Desjobert C, Carrier A, Delmas A, Marzese DM, Daunay A, Busato F, Pillon A, Tost J, Riond J, Favre G, Etievant C, and Arimondo PB
- Subjects
- Animals, Cell Line, Tumor, Cell Survival drug effects, Female, Gene Expression Profiling methods, Gene Expression Regulation, Neoplastic drug effects, Gene Regulatory Networks drug effects, Humans, Lung Neoplasms drug therapy, Lung Neoplasms genetics, Melanoma drug therapy, Mice, Neoplasm Invasiveness, Neoplasm Transplantation, Promoter Regions, Genetic, Sequence Analysis, RNA, Spheroids, Cellular drug effects, Up-Regulation, DNA Methylation drug effects, Decitabine pharmacology, Lung Neoplasms secondary, Melanoma genetics, MicroRNAs genetics, Spheroids, Cellular cytology
- Abstract
Background: Efficient treatments against metastatic melanoma dissemination are still lacking. Here, we report that low-cytotoxic concentrations of 5-aza-2'-deoxycytidine, a DNA demethylating agent, prevent in vitro 3D invasiveness of metastatic melanoma cells and reduce lung metastasis formation in vivo., Results: We unravelled that this beneficial effect is in part due to MIR-199A2 re-expression by promoter demethylation. Alone, this miR showed an anti-invasive and anti-metastatic effect. Throughout integration of micro-RNA target prediction databases with transcriptomic analysis after 5-aza-2'-deoxycytidine treatments, we found that miR-199a-3p downregulates set of genes significantly involved in invasion/migration processes. In addition, analysis of data from melanoma patients showed a stage- and tissue type-dependent modulation of MIR-199A2 expression by DNA methylation., Conclusions: Thus, our data suggest that epigenetic- and/or miR-based therapeutic strategies can be relevant to limit metastatic dissemination of melanoma.
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- 2019
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13. Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2'-deoxycytidine in human leukemic KG1 cells.
- Author
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Vispé S, Deroide A, Davoine E, Desjobert C, Lestienne F, Fournier L, Novosad N, Bréand S, Besse J, Busato F, Tost J, De Vries L, Cussac D, Riond J, and Arimondo PB
- Subjects
- Azacitidine pharmacology, Cell Line, Tumor, Cell Proliferation genetics, CpG Islands genetics, DNA (Cytosine-5-)-Methyltransferase 1, DNA Damage genetics, DNA Methylation drug effects, DNA Methyltransferase 3A, DNA-Binding Proteins genetics, Decitabine, G2 Phase Cell Cycle Checkpoints drug effects, G2 Phase Cell Cycle Checkpoints genetics, Histones metabolism, Humans, Nuclear Proteins genetics, Phosphorylation, Promoter Regions, Genetic genetics, RNA Interference, RNA, Small Interfering, Tumor Protein p73, Tumor Suppressor Proteins genetics, DNA Methyltransferase 3B, Azacitidine analogs & derivatives, DNA (Cytosine-5-)-Methyltransferases antagonists & inhibitors, DNA (Cytosine-5-)-Methyltransferases genetics, DNA Methylation genetics, Leukemia drug therapy
- Abstract
5-azacytidine and 5-aza-2'-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2'-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2'-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2'-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2'-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia.
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- 2015
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14. Combined analysis of DNA methylation and cell cycle in cancer cells.
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Desjobert C, El Maï M, Gérard-Hirne T, Guianvarc'h D, Carrier A, Pottier C, Arimondo PB, and Riond J
- Subjects
- Cell Line, Tumor, Humans, Cell Cycle, DNA Methylation, DNA, Neoplasm genetics, Flow Cytometry methods
- Abstract
DNA methylation is a chemical modification of DNA involved in the regulation of gene expression by controlling the access to the DNA sequence. It is the most stable epigenetic mark and is widely studied for its role in major biological processes. Aberrant DNA methylation is observed in various pathologies, such as cancer. Therefore, there is a great interest in analyzing subtle changes in DNA methylation induced by biological processes or upon drug treatments. Here, we developed an improved methodology based on flow cytometry to measure variations of DNA methylation level in melanoma and leukemia cells. The accuracy of DNA methylation quantification was validated with LC-ESI mass spectrometry analysis. The new protocol was used to detect small variations of cytosine methylation occurring in individual cells during their cell cycle and those induced by the demethylating agent 5-aza-2'-deoxycytidine (5AzadC). Kinetic experiments confirmed that inheritance of DNA methylation occurs efficiently in S phase and revealed a short delay between DNA replication and completion of cytosine methylation. In addition, this study suggests that the uncoupling of 5AzadC effects on DNA demethylation and cell proliferation might be related to the duration of the DNA replication phase.
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- 2015
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15. MiR-29a down-regulation in ALK-positive anaplastic large cell lymphomas contributes to apoptosis blockade through MCL-1 overexpression.
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Desjobert C, Renalier MH, Bergalet J, Dejean E, Joseph N, Kruczynski A, Soulier J, Espinos E, Meggetto F, Cavaillé J, Delsol G, and Lamant L
- Subjects
- Anaplastic Lymphoma Kinase, Animals, Cell Line, Tumor, Cells, Cultured, Down-Regulation genetics, Female, Gene Expression Regulation, Neoplastic, Humans, Lymphoma, Large-Cell, Anaplastic metabolism, Lymphoma, Large-Cell, Anaplastic pathology, Mice, Mice, Inbred NOD, Mice, SCID, Mice, Transgenic, MicroRNAs metabolism, MicroRNAs physiology, Myeloid Cell Leukemia Sequence 1 Protein, Proto-Oncogene Proteins c-bcl-2 metabolism, Receptor Protein-Tyrosine Kinases metabolism, Up-Regulation genetics, Xenograft Model Antitumor Assays, Apoptosis genetics, Lymphoma, Large-Cell, Anaplastic genetics, MicroRNAs genetics, Proto-Oncogene Proteins c-bcl-2 genetics, Receptor Protein-Tyrosine Kinases genetics
- Abstract
Although deregulated expression of specific microRNAs (miRNAs) has been described in solid cancers and leukemias, little evidence of miRNA deregulation has been reported in ALK-positive (ALK(+)) anaplastic large cell lymphomas (ALCL). These tumors overexpress the major antiapoptotic protein myeloid cell leukemia 1 (MCL-1), a situation that could compensate for the lack of BCL-2. We report that ALK(+) ALCL cell lines and biopsy specimens (n = 20) express a low level of miR-29a and that this down-modulation requires an active NPM-ALK kinase. Murine models (transgenic mice and mouse embryonic fibroblast [MEF] cells), which allow conditional NPM-ALK fusion protein expression, showed an increase of miR-29a expression in the absence of NPM-ALK. Concordant results were observed after the abolition of NPM-ALK kinase activity (siALK or PF-2341066) in NPM-ALK(+) ALCL cell lines. In addition, we showed that low expression of miR-29a, probably through methylation repression, plays an important regulatory role in MCL-1 overexpression that could promote tumor cell survival by inhibiting apoptosis. Enforced miR-29a expression was found to modulate apoptosis through inhibition of MCL-1 expression in ALCL cell lines and in a xenografted model, with a concomitant tumor growth reduction. Thus, synthetic miR-29a represents a potential new tool to affect tumorigenesis in these lymphomas.
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- 2011
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16. HuR-mediated control of C/EBPbeta mRNA stability and translation in ALK-positive anaplastic large cell lymphomas.
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Bergalet J, Fawal M, Lopez C, Desjobert C, Lamant L, Delsol G, Morello D, and Espinos E
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- 3' Untranslated Regions genetics, Anaplastic Lymphoma Kinase, Animals, Antigens, Surface genetics, CCAAT-Enhancer-Binding Proteins metabolism, Cell Line, Tumor, Cell Transformation, Neoplastic genetics, ELAV Proteins, ELAV-Like Protein 1, Humans, Lymphoma, Large-Cell, Anaplastic metabolism, Mice, NIH 3T3 Cells, Protein Biosynthesis genetics, Protein-Tyrosine Kinases genetics, Protein-Tyrosine Kinases metabolism, RNA-Binding Proteins genetics, Receptor Protein-Tyrosine Kinases genetics, Antigens, Surface metabolism, CCAAT-Enhancer-Binding Proteins genetics, Gene Expression Regulation, Neoplastic, Lymphoma, Large-Cell, Anaplastic genetics, RNA Stability, RNA-Binding Proteins metabolism, Receptor Protein-Tyrosine Kinases metabolism
- Abstract
The CCAAT/enhancer-binding protein β (C/EBPβ) plays a major role in the pathogenesis of anaplastic large cell lymphomas (ALCL) that express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) tyrosine kinase (ALK(+)). Although ALK-mediated C/EBPβ transcriptional activation has been reported, C/EBPβ mRNA possesses U- and AU-rich domains in its 3'-untranslated region (3'-UTR) that might be privileged targets for posttranscriptional control in ALK(+) ALCLs. The purpose of this study was to explore this possibility. By using human ALCL-derived cells and a murine model of ALK-transformed cells, we show that the AU-binding protein HuR binds to the 3'-UTR of C/EBPβ mRNA, as previously reported in adipocytes, and that NPM-ALK enhances this interaction. Interaction between HuR and C/EBPβ mRNA impacts on C/EBPβ gene expression at both the mRNA and protein levels. Indeed, C/EBPβ mRNA stability following HuR silencing is reduced and reaches the value observed in ALK-inactivated cells. Remarkably, HuR expression is not modified by NPM-ALK, but its association with actively translating polysomes is dramatically increased in ALK(+) cells. HuR/polysomes association diminishes when NPM-ALK activity is inhibited and is accompanied by a concomitant decrease of C/EBPβ mRNA translation. Finally, we show that HuR and NPM-ALK colocalized in cytoplasmic granules and HuR is phosphroylated on tyrosine residues in ALK(+) ALCL cells. Our study thus demonstrates that C/EBPβ is indeed regulated at the posttranscriptional level by HuR in ALK(+) cells, leading us to propose that part of NPM-ALK oncogenic properties relies on its ability to modify HuR properties in the cytoplasm and hence to alter expression of key actors of transformation., (©2011 AACR.)
- Published
- 2011
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17. The PRH/Hex repressor protein causes nuclear retention of Groucho/TLE co-repressors.
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Desjobert C, Noy P, Swingler T, Williams H, Gaston K, and Jayaraman PS
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- Blotting, Western, Cell Line, Tumor, Co-Repressor Proteins, DNA metabolism, Electrophoretic Mobility Shift Assay, Fluorescent Antibody Technique, Homeodomain Proteins genetics, Humans, Immunoprecipitation, Mutation, Protein Binding, Transcription Factors genetics, Cell Nucleus metabolism, Homeodomain Proteins metabolism, Repressor Proteins metabolism, Transcription Factors metabolism
- Abstract
The PRH (proline-rich homeodomain) [also known as Hex (haematopoietically expressed homeobox)] protein is a transcription factor that functions as an important regulator of vertebrate development and many other processes in the adult including haematopoiesis. The Groucho/TLE (transducin-like enhancer) family of co-repressor proteins also regulate development and modulate the activity of many DNA-binding transcription factors during a range of diverse cellular processes including haematopoiesis. We have shown previously that PRH is a repressor of transcription in haematopoietic cells and that an Eh-1 (Engrailed homology) motif present within the N-terminal transcription repression domain of PRH mediates binding to Groucho/TLE proteins and enables co-repression. In the present study we demonstrate that PRH regulates the nuclear retention of TLE proteins during cellular fractionation. We show that transcriptional repression and the nuclear retention of TLE proteins requires PRH to bind to both TLE and DNA. In addition, we characterize a trans-dominant-negative PRH protein that inhibits wild-type PRH activity by sequestering TLE proteins to specific subnuclear domains. These results demonstrate that transcriptional repression by PRH is dependent on TLE availability and suggest that subnuclear localization of TLE plays an important role in transcriptional repression by PRH.
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- 2009
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18. Leukemogenic mechanisms and targets of a NUP98/HHEX fusion in acute myeloid leukemia.
- Author
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Jankovic D, Gorello P, Liu T, Ehret S, La Starza R, Desjobert C, Baty F, Brutsche M, Jayaraman PS, Santoro A, Mecucci C, and Schwaller J
- Subjects
- Animals, Bone Marrow Cells pathology, Bone Marrow Cells physiology, COS Cells, Chlorocebus aethiops, Gene Expression Profiling, HeLa Cells, Humans, Leukemia, Myeloid, Acute pathology, Leukemia, Myeloid, Acute therapy, Male, Mice, Mice, Inbred BALB C, Middle Aged, NIH 3T3 Cells, Neoplasm Transplantation, Gene Expression Regulation, Leukemic, Homeodomain Proteins genetics, Leukemia, Myeloid, Acute genetics, Nuclear Pore Complex Proteins genetics, Oncogene Proteins, Fusion genetics, Transcription Factors genetics
- Abstract
We have studied a patient with acute myeloid leukemia (AML) and t(10;11)(q23;p15) as the sole cytogenetic abnormality. Molecular analysis revealed a translocation involving nucleoporin 98 (NUP98) fused to the DNA-binding domain of the hematopoietically expressed homeobox gene (HHEX). Expression of NUP98/HHEX in murine bone marrow cells leads to aberrant self-renewal and a block in normal differentiation that depends on the integrity of the NUP98 GFLG repeats and the HHEX homeodomain. Transplantation of bone marrow cells expressing NUP98/HHEX leads to transplantable acute leukemia characterized by extensive infiltration of leukemic blasts expressing myeloid markers (Gr1(+)) as well as markers of the B-cell lineage (B220(+)). A latency period of 9 months and its clonal character suggest that NUP98/HHEX is necessary but not sufficient for disease induction. Expression of EGFP-NUP98/HHEX fusions showed a highly similar nuclear localization pattern as for other NUP98/homeodomain fusions, such as NUP98/HOXA9. Comparative gene expression profiling in primary bone marrow cells provided evidence for the presence of common targets in cells expressing NUP98/HOXA9 or NUP98/HHEX. Some of these genes (Hoxa5, Hoxa9, Flt3) are deregulated in NUP98/HHEX-induced murine leukemia as well as in human blasts carrying this fusion and might represent bona fide therapeutic targets.
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- 2008
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19. HIV-1 integrase crosslinked oligomers are active in vitro.
- Author
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Faure A, Calmels C, Desjobert C, Castroviejo M, Caumont-Sarcos A, Tarrago-Litvak L, Litvak S, and Parissi V
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- Cross-Linking Reagents, DNA metabolism, HIV Integrase genetics, HIV Integrase isolation & purification, HIV Long Terminal Repeat, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Yeasts genetics, HIV Integrase metabolism, HIV-1 enzymology
- Abstract
The oligomeric state of active human immunodeficiency virus type 1 (HIV-1) integrase (IN) has not been clearly elucidated. We analyzed the activity of the different purified oligomeric forms of recombinant IN obtained after stabilization by platinum crosslinking. The crosslinked tetramer isolated by gel chromatography was able to catalyze the full-site integration of the two viral LTR ends into a target DNA in vitro, whereas the isolated dimeric form of the enzyme was involved in the processing and integration of only one viral end. Accurate concerted integration by IN tetramers was confirmed by cloning and sequencing. Kinetic studies of DNA-integrase complexes led us to propose a model explaining the formation of an active complex. Our data suggest that the tetrameric IN bound to the viral DNA ends is the minimal complex involved in the concerted integration of both LTRs and should be the oligomeric form targeted by future inhibitors.
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- 2005
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20. Identification by phage display selection of a short peptide able to inhibit only the strand transfer reaction catalyzed by human immunodeficiency virus type 1 integrase.
- Author
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Desjobert C, de Soultrait VR, Faure A, Parissi V, Litvak S, Tarrago-Litvak L, and Fournier M
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- Binding, Competitive, Capsid Proteins, Catalysis, Catalytic Domain, DNA-Binding Proteins chemical synthesis, DNA-Binding Proteins metabolism, Dimerization, HIV Integrase metabolism, HIV Integrase Inhibitors chemical synthesis, HIV Integrase Inhibitors metabolism, Oligopeptides chemical synthesis, Oligopeptides metabolism, Protein Binding, Substrate Specificity, Viral Fusion Proteins chemical synthesis, Viral Fusion Proteins metabolism, Bacteriophage M13, HIV Integrase chemistry, HIV Integrase Inhibitors chemistry, Oligopeptides chemistry, Peptide Library, Transcription, Genetic, Virus Integration
- Abstract
Human immunodeficiency virus type 1 integrase catalyzes the integration of proviral DNA into the infected cell genome, so it is an important potential target for antiviral drug design. In an attempt to search for peptides that specifically interact with integrase (IN) and inhibit its function, we used an in vitro selection procedure, the phage display technique. A phage display library of random heptapeptides was used to screen for potential peptide ligands of HIV-1 IN. Several phage clones were identified that specifically bound IN. Two of the selected peptides (FHNHGKQ and HLEHLLF) exhibited a high affinity for IN and were chemically synthesized. High affinity was confirmed by a displacement assay which showed that these two synthetic peptides were able to compete with the phages expressing the corresponding peptide. These agents were assayed on the in vitro IN activities. While none of them inhibited the 3'-processing reaction, the FHNHGKQ peptide was found to be an inhibitor of the strand transfer reaction. Despite its high affinity for IN, the HLEHLLF peptide selected and assayed under the same conditions was unable to inhibit this reaction. We showed that the FHNHGKQ peptide inhibits specifically the strand transfer activity by competing with the target DNA for binding to IN. These IN-binding agents could be used as a base for developing new anti-integrase compounds as well as for structural studies of the still unknown three-dimensional structure of the entire integrase molecule.
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- 2004
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21. The lethal phenotype observed after HIV-1 integrase expression in yeast cells is related to DNA repair and recombination events.
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Parissi V, Caumont A, de Soultrait VR, Desjobert C, Calmels C, Fournier M, Gourgue G, Bonneu M, Tarrago-Litvak L, and Litvak S
- Subjects
- Cell Division genetics, DNA, Fungal genetics, DNA, Fungal metabolism, DNA, Viral genetics, DNA, Viral metabolism, Deoxyribonuclease I metabolism, Diploidy, Gene Expression Regulation, Enzymologic, HIV Integrase genetics, HIV Long Terminal Repeat genetics, Haploidy, Mutation, Phenotype, Plasmids genetics, Saccharomyces cerevisiae growth & development, DNA Repair, HIV Integrase metabolism, Recombination, Genetic, Saccharomyces cerevisiae genetics
- Abstract
Human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalyzes the insertion of the viral genome into the host cell DNA, an essential reaction during the retroviral cycle. We described previously that expression of HIV-1 IN in some yeast strains may lead to the emergence of a lethal phenotype which was not observed when the catalytically crucial residues D, D, (35)E were mutated. The lethal effect in yeast seems to be related to the mutagenic effect of the recombinant HIV-1 IN, most probably via the non-sequence-specific endonucleolytic activity carried by this enzyme. This non-sequence-specific endonuclease activity was further characterized. Although the enzyme was active on DNA substrates devoid of viral long terminal repeat (LTR) sequences, the presence of LTR regions stimulated significantly this activity. Genetic experiments were designed to show that both the mutagenic effect and the level of recombination events were affected in cells expressing the active retroviral enzyme, while expression of the mutated inactive IN D116A has no significant effect. A close interaction was demonstrated between integrase activity and in vivo/in vitro recombination process, suggesting that retroviral integration and recombination mechanism are linked in the infected cell. Our results show that the yeast system is a powerful cellular model to study the non-sequence-specific endonucleolytic activity of IN. Its characterization is essential since this activity might represent a very important step in the retroviral infectious cycle and would provide further insights into the function of IN. Indeed, effectors of this activity should be sought as potential antiviral agents since stimulation of this enzymatic activity would induce the destruction of early synthesized proviral DNA.
- Published
- 2003
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22. Peptides as new inhibitors of HIV-1 reverse transcriptase and integrase.
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de Soultrait VR, Desjobert C, and Tarrago-Litvak L
- Subjects
- Anti-HIV Agents chemistry, Anti-HIV Agents therapeutic use, Drug Design, HIV Infections drug therapy, HIV Infections virology, HIV Integrase Inhibitors chemistry, HIV Integrase Inhibitors therapeutic use, HIV-1 enzymology, Humans, Models, Biological, Peptide Library, Peptides chemistry, Peptides therapeutic use, Reverse Transcriptase Inhibitors chemistry, Reverse Transcriptase Inhibitors therapeutic use, Anti-HIV Agents pharmacology, HIV Integrase Inhibitors pharmacology, HIV Reverse Transcriptase antagonists & inhibitors, HIV-1 drug effects, Peptides pharmacology, Reverse Transcriptase Inhibitors pharmacology
- Abstract
Current treatments of human immunodeficiency virus type 1 (HIV-1) infection consist in the combination of drugs targeting reverse transcriptase (RT) and protease (PR). Despite the multiple clinical benefits of this combination therapy, the emergence of resistance highlights the need for new anti-HIV agents. Agents able to interfere with additional steps of viral replication, such as integration of viral DNA in the host genome, would improve the antiviral potency of the treatment. In this regard, we have focused our interest on peptide-based compounds that have been shown to exhibit potential inhibition of RT and integrase (IN) activities in vitro and in vivo. Recently, the expansion of powerful technologies which allow the selection of peptides exhibiting high affinity for a target protein have provided a new approach to selecting potential anti-HIV drugs. Furthermore, efforts to characterize the protein-protein interactions involved in efficient reverse transcription and integration, as well as the determination of the enzyme structure, have generated a very useful source of data for the development of peptide inhibitors. Finally, while this class of compounds has long been considered as poor drug candidates, current knowledge on improving the stability and bioavailability of these agents would lead to the effective use of peptides in therapy.
- Published
- 2003
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23. Dynamic, thermodynamic, and kinetic basis for recognition and transformation of DNA by human immunodeficiency virus type 1 integrase.
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Bugreev DV, Baranova S, Zakharova OD, Parissi V, Desjobert C, Sottofattori E, Balbi A, Litvak S, Tarrago-Litvak L, and Nevinsky GA
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- DNA, Single-Stranded chemistry, DNA, Single-Stranded metabolism, Enzyme Activation, Humans, Kinetics, Models, Chemical, Nucleic Acid Heteroduplexes genetics, Nucleic Acid Heteroduplexes metabolism, Oligonucleotides chemistry, Oligonucleotides metabolism, Protein Binding, Substrate Specificity, DNA, Viral chemistry, DNA, Viral metabolism, HIV Integrase chemistry, HIV Integrase genetics, HIV-1 enzymology, HIV-1 genetics, Thermodynamics, Transformation, Genetic
- Abstract
Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3'-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn(2+), the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide approximately 8 orders of magnitude in the affinity (Delta G degrees approximately equal to -10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to approximately 1.5 orders of magnitude (Delta G degrees approximately equal to - 2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the k(cat) term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.
- Published
- 2003
- Full Text
- View/download PDF
24. HIV-1 integrase interacts with yeast microtubule-associated proteins.
- Author
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de Soultrait VR, Caumont A, Durrens P, Calmels C, Parissi V, Recordon P, Bon E, Desjobert C, Tarrago-Litvak L, and Fournier M
- Subjects
- Binding Sites genetics, HIV Integrase genetics, Humans, Microtubule-Associated Proteins genetics, Protein Binding, Recombinant Proteins genetics, Recombinant Proteins metabolism, HIV Integrase metabolism, HIV-1 enzymology, Microtubule-Associated Proteins metabolism, Saccharomyces cerevisiae metabolism
- Abstract
The human immunodeficiency virus type 1 (HIV-1) integrase (IN) mediates the insertion of viral DNA into the human genome. In addition to IN, cellular and viral proteins are associated to proviral DNA in the so-called preintegration complex (PIC). We previously reported that the expression of HIV-1 IN in yeast leads to the emergence of a lethal phenotype. This effect may be linked to the IN activity on infected human cells where integration requires the cleavage of genomic DNA. To isolate and characterize potential cellular partners of HIV-1 IN, we used it as a bait in a two-hybrid system with a yeast genomic library. IN interacted with proteins belonging to the microtubule network, or involved in the protein synthesis apparatus. We focused our interest on one of the selected inserts, L2, which corresponds to the C-end half of the yeast STU2p, a microtubule-associated protein (MAP). STU2p is an essential component of the yeast spindle pole body (SPB), which is able to bind microtubules in vitro. After expressing and purifying L2 as a recombinant protein, we showed its binding to IN by ELISA immunodetection. L2 was also able to inhibit IN activity in vitro. In addition, the effect of L2 was tested using the "lethal yeast phenotype". The coexpression of IN and the L2 peptide abolished the lethal phenotype, thus showing important in vivo interactions between IN and L2. The identification of components of the microtubule network associated with IN suggest a role of this complex in the transport of HIV-1 IN present in the PIC to the nucleus, as already described for other human viruses.
- Published
- 2002
- Full Text
- View/download PDF
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