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2013
Gahoual Rabah, Burr Alicia, Busnel Jean-Marc, Kuhn Lauriane, Hammann Phillipe, Beck Alain, François Yannis-Nicolas, Leize-Wagner Emmanuelle
Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry. Article de journal
Dans: mAbs, vol. 5, no. 3, p. 479–490, 2013, ISSN: 1942-0870 1942-0862 1942-0862.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{gahoual_rapid_2013,
title = {Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry.},
author = {Rabah Gahoual and Alicia Burr and Jean-Marc Busnel and Lauriane Kuhn and Phillipe Hammann and Alain Beck and Yannis-Nicolas François and Emmanuelle Leize-Wagner},
doi = {10.4161/mabs.23995},
issn = {1942-0870 1942-0862 1942-0862},
year = {2013},
date = {2013-06-01},
journal = {mAbs},
volume = {5},
number = {3},
pages = {479--490},
abstract = {Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product - and time - consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) - tandem mass spectrometry (CESI-MS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product - and time - consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) - tandem mass spectrometry (CESI-MS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms.
Kabbage Maria, Trimeche Mounir, Bergaoui Sarra, Hammann Philippe, Kuhn Lauriane, Hamrita Bechr, ben Nasr Hela, Chaieb Anouar, Chouchane Lotfi, Chahed Karim
Calreticulin expression in infiltrating ductal breast carcinomas: relationships with disease progression and humoral immune responses. Article de journal
Dans: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, vol. 34, no. 2, p. 1177–1188, 2013, ISSN: 1423-0380 1010-4283.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{kabbage_calreticulin_2013,
title = {Calreticulin expression in infiltrating ductal breast carcinomas: relationships with disease progression and humoral immune responses.},
author = {Maria Kabbage and Mounir Trimeche and Sarra Bergaoui and Philippe Hammann and Lauriane Kuhn and Bechr Hamrita and Hela ben Nasr and Anouar Chaieb and Lotfi Chouchane and Karim Chahed},
doi = {10.1007/s13277-013-0661-y},
issn = {1423-0380 1010-4283},
year = {2013},
date = {2013-04-01},
journal = {Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine},
volume = {34},
number = {2},
pages = {1177--1188},
abstract = {The aim of this study was to evaluate calreticulin expression in infiltrating ductal breast carcinomas (IDCAs), as well as its relationships with clinicopathological parameters of the disease. Using a two-dimensional gel electrophoresis/matrix-assisted laser desorption ionization time of flight mass spectrometry investigation coupled to an immunohistochemical approach, we have assessed the expression of calreticulin in IDCAs, as well as in other types of breast tumors. The humoral immune response against calreticulin was estimated using a serological proteomics-based strategy. Proteomic analyses revealed an increased expression of calreticulin in IDCA tumors. Using immunohistochemistry, overexpression of calreticulin was confirmed in 51 additional tumor specimens. Statistical analyses revealed, however, no significant correlations between calreticulin expression and clinicopathological parameters of the disease including tumor stage, patient age, SBR grade, and lymph node metastasis occurrence. A significant association was found, however, with estrogen receptor status. This study demonstrates the upregulation of calreticulin in IDCA tissues which may highlight its involvement in breast cancer development. Our findings also support a link between calreticulin expression and estrogen transduction pathways. Our results do not, however, support the involvement of calreticulin in the development of a humoral immune response in IDCAs.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The aim of this study was to evaluate calreticulin expression in infiltrating ductal breast carcinomas (IDCAs), as well as its relationships with clinicopathological parameters of the disease. Using a two-dimensional gel electrophoresis/matrix-assisted laser desorption ionization time of flight mass spectrometry investigation coupled to an immunohistochemical approach, we have assessed the expression of calreticulin in IDCAs, as well as in other types of breast tumors. The humoral immune response against calreticulin was estimated using a serological proteomics-based strategy. Proteomic analyses revealed an increased expression of calreticulin in IDCA tumors. Using immunohistochemistry, overexpression of calreticulin was confirmed in 51 additional tumor specimens. Statistical analyses revealed, however, no significant correlations between calreticulin expression and clinicopathological parameters of the disease including tumor stage, patient age, SBR grade, and lymph node metastasis occurrence. A significant association was found, however, with estrogen receptor status. This study demonstrates the upregulation of calreticulin in IDCA tissues which may highlight its involvement in breast cancer development. Our findings also support a link between calreticulin expression and estrogen transduction pathways. Our results do not, however, support the involvement of calreticulin in the development of a humoral immune response in IDCAs.
Poirier Isabelle, Hammann Philippe, Kuhn Lauriane, Bertrand Martine
Strategies developed by the marine bacterium Pseudomonas fluorescens BA3SM1 to resist metals: A proteome analysis. Article de journal
Dans: Aquatic toxicology (Amsterdam, Netherlands), vol. 128-129, p. 215–232, 2013, ISSN: 1879-1514 0166-445X.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{poirier_strategies_2013,
title = {Strategies developed by the marine bacterium Pseudomonas fluorescens BA3SM1 to resist metals: A proteome analysis.},
author = {Isabelle Poirier and Philippe Hammann and Lauriane Kuhn and Martine Bertrand},
doi = {10.1016/j.aquatox.2012.12.006},
issn = {1879-1514 0166-445X},
year = {2013},
date = {2013-03-01},
journal = {Aquatic toxicology (Amsterdam, Netherlands)},
volume = {128-129},
pages = {215--232},
abstract = {A global proteomic evaluation of the response of the marine bacterium Pseudomonas fluorescens BA3SM1 to Cd, Zn and Cu was performed by two dimensional gel electrophoresis followed by mass spectrometry. When stressed with Cd, the most toxic metal for P. fluorescens BA3SM1, cell growth is rapidly affected and the number of proteins up-regulated (sixteen for 0.4 mM Cd) remains low in comparison with results obtained for Zn and Cu (twenty eight for 1.5mM Zn and forty four for 1.5 mM Cu). The changes in protein expression indicate that the cell adapts to metals by inducing essentially seven defense mechanisms: cell aggregation/biofilm formation (Zn=CutextbackslashtextgreaterCd); modification of envelope properties to increase the extracellular metal biosorption and/or control the uptake of metal (CutextbackslashtextgreaterZn); metal export (Cd=Zn and probably Cu); responses to oxidative stress (CutextbackslashtextgreaterZntextbackslashtextgreaterCd); intracellular metal sequestration (Zn=Cu and probably Cd); hydrolysis of abnormally folded proteins (Cd=Cu), and the over-synthesis of proteins inhibited by metal (CdtextbackslashtextgreaterCutextbackslashtextgreaterZn). To the best of our knowledge, this is the first report showing that a marine P. fluorescens is able to acquire a metal-resistant phenotype, making the strain BA3SM1 a promising agent for bioremediation processes.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
A global proteomic evaluation of the response of the marine bacterium Pseudomonas fluorescens BA3SM1 to Cd, Zn and Cu was performed by two dimensional gel electrophoresis followed by mass spectrometry. When stressed with Cd, the most toxic metal for P. fluorescens BA3SM1, cell growth is rapidly affected and the number of proteins up-regulated (sixteen for 0.4 mM Cd) remains low in comparison with results obtained for Zn and Cu (twenty eight for 1.5mM Zn and forty four for 1.5 mM Cu). The changes in protein expression indicate that the cell adapts to metals by inducing essentially seven defense mechanisms: cell aggregation/biofilm formation (Zn=CutextbackslashtextgreaterCd); modification of envelope properties to increase the extracellular metal biosorption and/or control the uptake of metal (CutextbackslashtextgreaterZn); metal export (Cd=Zn and probably Cu); responses to oxidative stress (CutextbackslashtextgreaterZntextbackslashtextgreaterCd); intracellular metal sequestration (Zn=Cu and probably Cd); hydrolysis of abnormally folded proteins (Cd=Cu), and the over-synthesis of proteins inhibited by metal (CdtextbackslashtextgreaterCutextbackslashtextgreaterZn). To the best of our knowledge, this is the first report showing that a marine P. fluorescens is able to acquire a metal-resistant phenotype, making the strain BA3SM1 a promising agent for bioremediation processes.
Habif Guillaume, Grasset Marie-France, Kieffer-Jaquinod Sylvie, Kuhn Lauriane, Mouchiroud Guy, Gobert-Gosse Stéphanie
Dans: Journal of proteomics, vol. 78, p. 231–244, 2013, ISSN: 1876-7737 1874-3919.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{habif_phosphoproteome_2013,
title = {Phosphoproteome analyses reveal specific implications of Hcls1, p21-activated kinase 1 and Ezrin in proliferation of a myeloid progenitor cell line downstream of wild-type and ITD mutant Fms-like tyrosine kinase 3 receptors.},
author = {Guillaume Habif and Marie-France Grasset and Sylvie Kieffer-Jaquinod and Lauriane Kuhn and Guy Mouchiroud and Stéphanie Gobert-Gosse},
doi = {10.1016/j.jprot.2012.09.009},
issn = {1876-7737 1874-3919},
year = {2013},
date = {2013-01-01},
journal = {Journal of proteomics},
volume = {78},
pages = {231--244},
abstract = {The tyrosine kinase receptor Flt3 (Fms-like tyrosine kinase 3) is almost always expressed in AML (acute myeloid leukemia) cells, and constitutive activation of Flt3 by ITD (internal tandem duplication) mutations is one of the most common molecular alterations known in AML, especially monocytic AML. Furthermore, Flt3-ligand (FL) was shown as an in vitro growth factor for monocytic precursors, pointing to the important role of Flt3 in the regulation of monocyte/macrophage production. To get a relevant model for studying the molecular mechanisms underlying the physiopathological role of Flt3 on monocytic lineage development, we used the IL-3 dependent murine myeloid progenitors FDC-P1 cell line to generate cells stably co-expressing murine Fms (M-CSF receptor) and human Flt3. Wild type (WT)-Flt3 expressing cells could proliferate in an FL-dependent manner, whereas those expressing Flt3-ITD all survived IL-3 deprivation and showed autonomous proliferation, whereas both types of cells could differentiate to monocytic cells in response to M-CSF. Next, by combining phosphoprotein detection or purification, comparative 2D-PAGE and mass spectrometry sequencing, we sought for downstream mediators of Flt3-WT or Flt3-ITD in FD/Fms cell proliferation. Amongst the differentially expressed and/or phosphorylated proteins, 3 showed a specific implication in FD/Fms cell proliferation: Hcls1 and the Pak1/2 in FL-dependent proliferation of Flt3-WT expressing cells and Ezrin in autonomous proliferation of Flt3-ITD expressing cells.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The tyrosine kinase receptor Flt3 (Fms-like tyrosine kinase 3) is almost always expressed in AML (acute myeloid leukemia) cells, and constitutive activation of Flt3 by ITD (internal tandem duplication) mutations is one of the most common molecular alterations known in AML, especially monocytic AML. Furthermore, Flt3-ligand (FL) was shown as an in vitro growth factor for monocytic precursors, pointing to the important role of Flt3 in the regulation of monocyte/macrophage production. To get a relevant model for studying the molecular mechanisms underlying the physiopathological role of Flt3 on monocytic lineage development, we used the IL-3 dependent murine myeloid progenitors FDC-P1 cell line to generate cells stably co-expressing murine Fms (M-CSF receptor) and human Flt3. Wild type (WT)-Flt3 expressing cells could proliferate in an FL-dependent manner, whereas those expressing Flt3-ITD all survived IL-3 deprivation and showed autonomous proliferation, whereas both types of cells could differentiate to monocytic cells in response to M-CSF. Next, by combining phosphoprotein detection or purification, comparative 2D-PAGE and mass spectrometry sequencing, we sought for downstream mediators of Flt3-WT or Flt3-ITD in FD/Fms cell proliferation. Amongst the differentially expressed and/or phosphorylated proteins, 3 showed a specific implication in FD/Fms cell proliferation: Hcls1 and the Pak1/2 in FL-dependent proliferation of Flt3-WT expressing cells and Ezrin in autonomous proliferation of Flt3-ITD expressing cells.
2012
Kabbage Maria, Trimeche Mounir, Nasr Hela Ben, Hammann Philippe, Kuhn Lauriane, Hamrita Bechr, Chaieb Anouar, Chouchane Lotfi, Chahed Karim
Dans: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, vol. 33, no. 6, p. 2279–2288, 2012, ISSN: 1423-0380 1010-4283.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{kabbage_expression_2012,
title = {Expression of the molecular chaperone αB-crystallin in infiltrating ductal breast carcinomas and the significance thereof: an immunohistochemical and proteomics-based strategy.},
author = {Maria Kabbage and Mounir Trimeche and Hela Ben Nasr and Philippe Hammann and Lauriane Kuhn and Bechr Hamrita and Anouar Chaieb and Lotfi Chouchane and Karim Chahed},
doi = {10.1007/s13277-012-0490-4},
issn = {1423-0380 1010-4283},
year = {2012},
date = {2012-12-01},
journal = {Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine},
volume = {33},
number = {6},
pages = {2279--2288},
abstract = {This study aims to evaluate αB-crystallin expression in infiltrating ductal breast carcinomas (IDCAs), as well as, its prognostic significance. Using a two-dimensional electrophoresis matrix-assisted laser desorption/ionisation-time of flight mass spectrometry investigation coupled to an immunohistochemical approach, we have assessed the expression of αB-crystallin in IDCAs, as well as, in other types of breast tumors (invasive lobular carcinomas, medullary carcinomas, and in situ ductal carcinomas). Correlation between αB-crystallin expression and clinicopathological parameters of breast cancer has also been investigated. Proteomic analyses revealed an increased expression of αB-crystallin in IDCA tumors compared to adjacent nontumor tissues. Overexpression of this molecular chaperone was further confirmed in 51 tumor specimens. Statistical analyses revealed, however, no significant correlations between αB-crystallin expression and clinicopathological parameters of the disease (tumor stage, patient age, hormone receptors, SBR grade, and lymph node metastases). This study demonstrates the upregulation of αB-crystallin in IDCA tissues which may highlight its possible involvement in breast cancer development. Our findings do not, however, support the involvement of this molecular chaperone in the progression of this disease.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
This study aims to evaluate αB-crystallin expression in infiltrating ductal breast carcinomas (IDCAs), as well as, its prognostic significance. Using a two-dimensional electrophoresis matrix-assisted laser desorption/ionisation-time of flight mass spectrometry investigation coupled to an immunohistochemical approach, we have assessed the expression of αB-crystallin in IDCAs, as well as, in other types of breast tumors (invasive lobular carcinomas, medullary carcinomas, and in situ ductal carcinomas). Correlation between αB-crystallin expression and clinicopathological parameters of breast cancer has also been investigated. Proteomic analyses revealed an increased expression of αB-crystallin in IDCA tumors compared to adjacent nontumor tissues. Overexpression of this molecular chaperone was further confirmed in 51 tumor specimens. Statistical analyses revealed, however, no significant correlations between αB-crystallin expression and clinicopathological parameters of the disease (tumor stage, patient age, hormone receptors, SBR grade, and lymph node metastases). This study demonstrates the upregulation of αB-crystallin in IDCA tissues which may highlight its possible involvement in breast cancer development. Our findings do not, however, support the involvement of this molecular chaperone in the progression of this disease.
Anes Amel Ben, Nasr Hela Ben, Hammann Philippe, Kuhn Lauriane, Trimeche Mounir, Hamrita Bechr, Bougmiza Iheb, Chaieb Anouar, Khairi Hedi, Chahed Karim
Assessment of the clinical significance of antigenic and functional levels of α1-proteinase inhibitor (α1-Pi) in infiltrating ductal breast carcinomas. Article de journal
Dans: Clinical biochemistry, vol. 45, no. 16-17, p. 1421–1431, 2012, ISSN: 1873-2933 0009-9120.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{anes_assessment_2012,
title = {Assessment of the clinical significance of antigenic and functional levels of α1-proteinase inhibitor (α1-Pi) in infiltrating ductal breast carcinomas.},
author = {Amel Ben Anes and Hela Ben Nasr and Philippe Hammann and Lauriane Kuhn and Mounir Trimeche and Bechr Hamrita and Iheb Bougmiza and Anouar Chaieb and Hedi Khairi and Karim Chahed},
doi = {10.1016/j.clinbiochem.2012.07.099},
issn = {1873-2933 0009-9120},
year = {2012},
date = {2012-11-01},
journal = {Clinical biochemistry},
volume = {45},
number = {16-17},
pages = {1421--1431},
abstract = {OBJECTIVES: To determine the clinical significance of α1-proteinase inhibitor (α1-Pi) in infiltrating ductal breast carcinoma patients. DESIGN AND METHODS: Serum levels of α1-Pi, tryptic specific inhibitory capacity and α1-Pi circulating immune complexes were determined using radial immunodiffusion, BAPNA assays and ELISA, respectively. 2-DE-MS and immunohistochemistry were performed to examine α1-Pi protein expression. RESULTS: A decreased serum level of α1-Pi was found among breast cancer patients in comparison to controls. In addition, we found a significantly decreased mean level of α1-Pi in the node metastatic group when compared to node negative patients. However, the functional activity of the inhibitor did not decrease proportionately. Through 2-DE analyses, a differential expression of α1-Pi isoforms according to tumor stage and node metastatic development was found. CONCLUSIONS: Both α1-Pi levels and specific activity could be a source of complementary clinical information and may provide useful information for a better understanding of the mechanisms of metastasis.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
OBJECTIVES: To determine the clinical significance of α1-proteinase inhibitor (α1-Pi) in infiltrating ductal breast carcinoma patients. DESIGN AND METHODS: Serum levels of α1-Pi, tryptic specific inhibitory capacity and α1-Pi circulating immune complexes were determined using radial immunodiffusion, BAPNA assays and ELISA, respectively. 2-DE-MS and immunohistochemistry were performed to examine α1-Pi protein expression. RESULTS: A decreased serum level of α1-Pi was found among breast cancer patients in comparison to controls. In addition, we found a significantly decreased mean level of α1-Pi in the node metastatic group when compared to node negative patients. However, the functional activity of the inhibitor did not decrease proportionately. Through 2-DE analyses, a differential expression of α1-Pi isoforms according to tumor stage and node metastatic development was found. CONCLUSIONS: Both α1-Pi levels and specific activity could be a source of complementary clinical information and may provide useful information for a better understanding of the mechanisms of metastasis.
Peltier Claire, Klein Elodie, Hleibieh Kamal, D'Alonzo Massimiliano, Hammann Philippe, Bouzoubaa Salah, Ratti Claudio, Gilmer David
Beet necrotic yellow vein virus subgenomic RNA3 is a cleavage product leading to stable non-coding RNA required for long-distance movement. Article de journal
Dans: The Journal of general virology, vol. 93, no. Pt 5, p. 1093–1102, 2012, ISSN: 1465-2099 0022-1317, (Place: England).
Résumé | Liens | BibTeX | Étiquettes: *Genome, Arabidopsis/virology, Beta vulgaris/virology, Plant Viruses/genetics/*pathogenicity, PPSE, RNA, RNA Stability, Tobacco/virology, Untranslated/*metabolism, Viral, Viral/genetics/*metabolism
@article{peltier_beet_2012,
title = {Beet necrotic yellow vein virus subgenomic RNA3 is a cleavage product leading to stable non-coding RNA required for long-distance movement.},
author = {Claire Peltier and Elodie Klein and Kamal Hleibieh and Massimiliano D'Alonzo and Philippe Hammann and Salah Bouzoubaa and Claudio Ratti and David Gilmer},
doi = {10.1099/vir.0.039685-0},
issn = {1465-2099 0022-1317},
year = {2012},
date = {2012-01-01},
journal = {The Journal of general virology},
volume = {93},
number = {Pt 5},
pages = {1093--1102},
abstract = {Beet necrotic yellow vein virus (BNYVV) is a multipartite RNA virus. BNYVV RNA3 does not accumulate in non-host transgenic Arabidopsis thaliana plants when expressed using a 35S promoter. However, a 3'-derivative species has been detected in transgenic plants and in transient expression assays conducted in Nicotiana benthamiana and Beta macrocarpa. The 3'-derivative species is similar to the previously reported subgenomic RNA3 produced during virus infection. 5' RACE revealed that the truncated forms had identical 5' ends. The 5' termini carried the coremin motif also present on BNYVV RNA5, beet soil-borne mosaic virus RNA3 and 4, and cucumber mosaic virus group 2 RNAs. This RNA3 species lacks a m(7)Gppp at the 5' end of the cleavage products, whether expressed transiently or virally. Mutagenesis revealed the importance of the coremin sequence for both long-distance movement and stabilization of the cleavage product in vivo and in vitro. The isolation of various RNA3 5'-end products suggests the existence of a cleavage between nt 212 and 1234 and subsequent exonucleolytic degradation, leading to the accumulation of a non-coding RNA. When RNA3 was incubated in wheatgerm extracts, truncated forms appeared rapidly and their appearance was protein- and divalent ion-dependent.},
note = {Place: England},
keywords = {*Genome, Arabidopsis/virology, Beta vulgaris/virology, Plant Viruses/genetics/*pathogenicity, PPSE, RNA, RNA Stability, Tobacco/virology, Untranslated/*metabolism, Viral, Viral/genetics/*metabolism},
pubstate = {published},
tppubtype = {article}
}
Beet necrotic yellow vein virus (BNYVV) is a multipartite RNA virus. BNYVV RNA3 does not accumulate in non-host transgenic Arabidopsis thaliana plants when expressed using a 35S promoter. However, a 3'-derivative species has been detected in transgenic plants and in transient expression assays conducted in Nicotiana benthamiana and Beta macrocarpa. The 3'-derivative species is similar to the previously reported subgenomic RNA3 produced during virus infection. 5' RACE revealed that the truncated forms had identical 5' ends. The 5' termini carried the coremin motif also present on BNYVV RNA5, beet soil-borne mosaic virus RNA3 and 4, and cucumber mosaic virus group 2 RNAs. This RNA3 species lacks a m(7)Gppp at the 5' end of the cleavage products, whether expressed transiently or virally. Mutagenesis revealed the importance of the coremin sequence for both long-distance movement and stabilization of the cleavage product in vivo and in vitro. The isolation of various RNA3 5'-end products suggests the existence of a cleavage between nt 212 and 1234 and subsequent exonucleolytic degradation, leading to the accumulation of a non-coding RNA. When RNA3 was incubated in wheatgerm extracts, truncated forms appeared rapidly and their appearance was protein- and divalent ion-dependent.
Hamrita Bechr, Nasr Hela Ben, Hammann Philippe, Kuhn Lauriane, Anes Amel Ben, Dimassi Saloua, Chaieb Anouar, Khairi Hedi, Chahed Karim
[Towards a better knowledge of breast cancer physiopathology: the proteomics approach]. Article de journal
Dans: Annales de biologie clinique, vol. 70, no. 5, p. 553–565, 2012, ISSN: 1950-6112 0003-3898.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{hamrita_towards_2012,
title = {[Towards a better knowledge of breast cancer physiopathology: the proteomics approach].},
author = {Bechr Hamrita and Hela Ben Nasr and Philippe Hammann and Lauriane Kuhn and Amel Ben Anes and Saloua Dimassi and Anouar Chaieb and Hedi Khairi and Karim Chahed},
doi = {10.1684/abc.2012.0741},
issn = {1950-6112 0003-3898},
year = {2012},
date = {2012-01-01},
journal = {Annales de biologie clinique},
volume = {70},
number = {5},
pages = {553--565},
abstract = {Breast cancer represents a major public health problem. Approximately one woman in ten is likely to develop a malignant tumor of the breast in their lifetime. The frequency of breast cancer is rising steadily for 20 years and the practical benefits in the diagnosis, prognosis and treatment of this disease are still too limited. Actually, there is no tumor marker with a specificity and sensitivity sufficient to have an utility in clinical and early diagnosis of breast cancer, although, carcinoembryonic antigen (CEA), MUC-1 and CA 15-3 were reported to be useful as markers for monitoring this disease. Thus, proteomics approaches are needed for the discovery and the identification of new protein biomarkers that may allow a better understanding of biological mechanisms of breast tumor development and serve as potential therapeutic targets. This article reviews advances in this field, as well as, the major contribution of these markers in breast pathology, with a focus on their biological characteristics and their clinical and therapeutic involvement.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Breast cancer represents a major public health problem. Approximately one woman in ten is likely to develop a malignant tumor of the breast in their lifetime. The frequency of breast cancer is rising steadily for 20 years and the practical benefits in the diagnosis, prognosis and treatment of this disease are still too limited. Actually, there is no tumor marker with a specificity and sensitivity sufficient to have an utility in clinical and early diagnosis of breast cancer, although, carcinoembryonic antigen (CEA), MUC-1 and CA 15-3 were reported to be useful as markers for monitoring this disease. Thus, proteomics approaches are needed for the discovery and the identification of new protein biomarkers that may allow a better understanding of biological mechanisms of breast tumor development and serve as potential therapeutic targets. This article reviews advances in this field, as well as, the major contribution of these markers in breast pathology, with a focus on their biological characteristics and their clinical and therapeutic involvement.
2011
Hamrita Bechr, Nasr Hela Ben, Hammann Philippe, Kuhn Lauriane, Guillier Christelle-Lemaitre, Chaieb Anouar, Khairi Hedi, Chahed Karim
An elongation factor-like protein (EF-Tu) elicits a humoral response in infiltrating ductal breast carcinomas: an immunoproteomics investigation. Article de journal
Dans: Clinical biochemistry, vol. 44, no. 13, p. 1097–1104, 2011, ISSN: 1873-2933 0009-9120.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{hamrita_elongation_2011,
title = {An elongation factor-like protein (EF-Tu) elicits a humoral response in infiltrating ductal breast carcinomas: an immunoproteomics investigation.},
author = {Bechr Hamrita and Hela Ben Nasr and Philippe Hammann and Lauriane Kuhn and Christelle-Lemaitre Guillier and Anouar Chaieb and Hedi Khairi and Karim Chahed},
doi = {10.1016/j.clinbiochem.2011.06.005},
issn = {1873-2933 0009-9120},
year = {2011},
date = {2011-09-01},
journal = {Clinical biochemistry},
volume = {44},
number = {13},
pages = {1097--1104},
abstract = {OBJECTIVES: In the current study, we have used an immunoproteomics approach to identify proteins that commonly elicit a humoral response in patients with infiltrating ductal carcinomas of the breast. DESIGN AND METHODS: Sera obtained at the time of diagnosis from 40 patients with invasive breast cancer and 42 healthy controls were screened for the presence of IgG antibodies to MCF-7 cell line proteins using a serological proteomics-based approach. RESULTS: An immunoreactive protein detected in sera from 21 of 40 patients was isolated and subsequently identified as elongation factor-Tu. CONCLUSIONS: The immunoproteomic approach implemented here offers a powerful tool for determining novel tumor antigens that induce a humoral immune response in cancer patients. From our findings, the immunoreactive EF-Tu protein and/or the related circulating antibodies may display clinical usefulness as potential diagnostic markers and provide a means for a better understanding of the molecular mechanisms underlying breast cancer development.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
OBJECTIVES: In the current study, we have used an immunoproteomics approach to identify proteins that commonly elicit a humoral response in patients with infiltrating ductal carcinomas of the breast. DESIGN AND METHODS: Sera obtained at the time of diagnosis from 40 patients with invasive breast cancer and 42 healthy controls were screened for the presence of IgG antibodies to MCF-7 cell line proteins using a serological proteomics-based approach. RESULTS: An immunoreactive protein detected in sera from 21 of 40 patients was isolated and subsequently identified as elongation factor-Tu. CONCLUSIONS: The immunoproteomic approach implemented here offers a powerful tool for determining novel tumor antigens that induce a humoral immune response in cancer patients. From our findings, the immunoreactive EF-Tu protein and/or the related circulating antibodies may display clinical usefulness as potential diagnostic markers and provide a means for a better understanding of the molecular mechanisms underlying breast cancer development.
Guillaumond Fabienne, Boyer Benedicte, Becquet Denis, Guillen Severine, Kuhn Lauriane, Garin Jerome, Belghazi Maya, Bosler Olivier, Franc Jean-Louis, François-Bellan Anne-Marie
Chromatin remodeling as a mechanism for circadian prolactin transcription: rhythmic NONO and SFPQ recruitment to HLTF. Article de journal
Dans: FASEB journal : official publication of the Federation of American Societies for Experimental Biology, vol. 25, no. 8, p. 2740–2756, 2011, ISSN: 1530-6860 0892-6638.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{guillaumond_chromatin_2011,
title = {Chromatin remodeling as a mechanism for circadian prolactin transcription: rhythmic NONO and SFPQ recruitment to HLTF.},
author = {Fabienne Guillaumond and Benedicte Boyer and Denis Becquet and Severine Guillen and Lauriane Kuhn and Jerome Garin and Maya Belghazi and Olivier Bosler and Jean-Louis Franc and Anne-Marie François-Bellan},
doi = {10.1096/fj.10-178616},
issn = {1530-6860 0892-6638},
year = {2011},
date = {2011-08-01},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {25},
number = {8},
pages = {2740--2756},
abstract = {Most clock-controlled genes (CCGs) lack the specific E-box response element necessary for direct circadian regulation. This is the case for the prolactin (Prl) gene, the expression of which oscillates in individual lactotrope pituitary cells. To characterize the processes underlying this oscillation, we used a lactotrope cell line (GH4C1 cells). In these cells, Prl gene expression fluctuated significantly during 24 h (P=0.0418). Circadian Prl transcription depended on an interaction between the pituitary-specific transcription factor, PIT-1, and the helicase-like transcription factor (HLTF), a SWI/SNF chromatin remodeler, shown here to bind the Prl promoter on an E-box that differs from the specific E-box preferentially bound by clock proteins. Circadian Prl transcription was further accompanied by marked daily chromatin transitions. While neither HLTF nor PIT-1 was rhythmically expressed, NONO and SFPQ, identified as HLTF-associated proteins by mass spectrometry, displayed a circadian pattern and bound rhythmically to the Prl promoter. Furthermore, NONO and SFPQ were functionally involved in circadian Prl transcription since overexpression of both proteins greatly reduced Prl promoter activity (Ptextbackslashtextless0.001) and disrupted its circadian pattern. A mechanism involving a rhythm in paraspeckle protein recruitment is proposed to explain how the core oscillator can generate a circadian pattern of CCGs lacking the specific E-box response element.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Most clock-controlled genes (CCGs) lack the specific E-box response element necessary for direct circadian regulation. This is the case for the prolactin (Prl) gene, the expression of which oscillates in individual lactotrope pituitary cells. To characterize the processes underlying this oscillation, we used a lactotrope cell line (GH4C1 cells). In these cells, Prl gene expression fluctuated significantly during 24 h (P=0.0418). Circadian Prl transcription depended on an interaction between the pituitary-specific transcription factor, PIT-1, and the helicase-like transcription factor (HLTF), a SWI/SNF chromatin remodeler, shown here to bind the Prl promoter on an E-box that differs from the specific E-box preferentially bound by clock proteins. Circadian Prl transcription was further accompanied by marked daily chromatin transitions. While neither HLTF nor PIT-1 was rhythmically expressed, NONO and SFPQ, identified as HLTF-associated proteins by mass spectrometry, displayed a circadian pattern and bound rhythmically to the Prl promoter. Furthermore, NONO and SFPQ were functionally involved in circadian Prl transcription since overexpression of both proteins greatly reduced Prl promoter activity (Ptextbackslashtextless0.001) and disrupted its circadian pattern. A mechanism involving a rhythm in paraspeckle protein recruitment is proposed to explain how the core oscillator can generate a circadian pattern of CCGs lacking the specific E-box response element.
2010
Nicolas Armel, Alazard-Dany Nathalie, Biollay Coline, Arata Loredana, Jolinon Nelly, Kuhn Lauriane, Ferro Myriam, Weller Sandra K, Epstein Alberto L, Salvetti Anna, Greco Anna
Identification of rep-associated factors in herpes simplex virus type 1-induced adeno-associated virus type 2 replication compartments. Article de journal
Dans: Journal of virology, vol. 84, no. 17, p. 8871–8887, 2010, ISSN: 1098-5514 0022-538X 0022-538X.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{nicolas_identification_2010,
title = {Identification of rep-associated factors in herpes simplex virus type 1-induced adeno-associated virus type 2 replication compartments.},
author = {Armel Nicolas and Nathalie Alazard-Dany and Coline Biollay and Loredana Arata and Nelly Jolinon and Lauriane Kuhn and Myriam Ferro and Sandra K Weller and Alberto L Epstein and Anna Salvetti and Anna Greco},
doi = {10.1128/JVI.00725-10},
issn = {1098-5514 0022-538X 0022-538X},
year = {2010},
date = {2010-09-01},
journal = {Journal of virology},
volume = {84},
number = {17},
pages = {8871--8887},
abstract = {Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase delta was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase delta was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.
Zhang Na, Fu Zhenxing, Linke Sarah, Chicher Johana, Gorman Jeffrey J, Visk DeeAnn, Haddad Gabriel G, Poellinger Lorenz, Peet Daniel J, Powell Frank, Johnson Randall S
The asparaginyl hydroxylase factor inhibiting HIF-1alpha is an essential regulator of metabolism. Article de journal
Dans: Cell metabolism, vol. 11, no. 5, p. 364–378, 2010, ISSN: 1932-7420 1550-4131 1550-4131.
Résumé | Liens | BibTeX | Étiquettes: alpha Subunit/*antagonists & inhibitors/genetics, Animals, Asparagine/genetics/metabolism, Dietary Fats/pharmacology, Fatty Liver/etiology, Glucose/metabolism, Hyperventilation/etiology, Hypoxia-Inducible Factor 1, Insulin/metabolism, Knockout, Lipid Metabolism, Mice, Mixed Function Oxygenases/deficiency/genetics/*metabolism, PPSE, Transcriptional Activation, Weight Gain
@article{zhang_asparaginyl_2010,
title = {The asparaginyl hydroxylase factor inhibiting HIF-1alpha is an essential regulator of metabolism.},
author = {Na Zhang and Zhenxing Fu and Sarah Linke and Johana Chicher and Jeffrey J Gorman and DeeAnn Visk and Gabriel G Haddad and Lorenz Poellinger and Daniel J Peet and Frank Powell and Randall S Johnson},
doi = {10.1016/j.cmet.2010.03.001},
issn = {1932-7420 1550-4131 1550-4131},
year = {2010},
date = {2010-01-01},
journal = {Cell metabolism},
volume = {11},
number = {5},
pages = {364--378},
abstract = {Factor inhibiting HIF-1alpha (FIH) is an asparaginyl hydroxylase. Hydroxylation of HIF-alpha proteins by FIH blocks association of HIFs with the transcriptional coactivators CBP/p300, thus inhibiting transcriptional activation. We have created mice with a null mutation in the FIH gene and found that it has little or no discernable role in mice in altering classical aspects of HIF function, e.g., angiogenesis, erythropoiesis, or development. Rather, it is an essential regulator of metabolism: mice lacking FIH exhibit reduced body weight, elevated metabolic rate, hyperventilation, and improved glucose and lipid homeostasis and are resistant to high-fat-diet-induced weight gain and hepatic steatosis. Neuron-specific loss of FIH phenocopied some of the major metabolic phenotypes of the global null animals: those mice have reduced body weight, increased metabolic rate, and enhanced insulin sensitivity and are also protected against high-fat-diet-induced weight gain. These results demonstrate that FIH acts to a significant degree through the nervous system to regulate metabolism.},
keywords = {alpha Subunit/*antagonists & inhibitors/genetics, Animals, Asparagine/genetics/metabolism, Dietary Fats/pharmacology, Fatty Liver/etiology, Glucose/metabolism, Hyperventilation/etiology, Hypoxia-Inducible Factor 1, Insulin/metabolism, Knockout, Lipid Metabolism, Mice, Mixed Function Oxygenases/deficiency/genetics/*metabolism, PPSE, Transcriptional Activation, Weight Gain},
pubstate = {published},
tppubtype = {article}
}
Factor inhibiting HIF-1alpha (FIH) is an asparaginyl hydroxylase. Hydroxylation of HIF-alpha proteins by FIH blocks association of HIFs with the transcriptional coactivators CBP/p300, thus inhibiting transcriptional activation. We have created mice with a null mutation in the FIH gene and found that it has little or no discernable role in mice in altering classical aspects of HIF function, e.g., angiogenesis, erythropoiesis, or development. Rather, it is an essential regulator of metabolism: mice lacking FIH exhibit reduced body weight, elevated metabolic rate, hyperventilation, and improved glucose and lipid homeostasis and are resistant to high-fat-diet-induced weight gain and hepatic steatosis. Neuron-specific loss of FIH phenocopied some of the major metabolic phenotypes of the global null animals: those mice have reduced body weight, increased metabolic rate, and enhanced insulin sensitivity and are also protected against high-fat-diet-induced weight gain. These results demonstrate that FIH acts to a significant degree through the nervous system to regulate metabolism.
2009
Nicolau Elodie, Kuhn Lauriane, Marchal Rémy, Jouanneau Yves
Proteomic investigation of enzymes involved in 2-ethylhexyl nitrate biodegradation in Mycobacterium austroafricanum IFP 2173. Article de journal
Dans: Research in microbiology, vol. 160, no. 10, p. 838–847, 2009, ISSN: 1769-7123 0923-2508.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{nicolau_proteomic_2009,
title = {Proteomic investigation of enzymes involved in 2-ethylhexyl nitrate biodegradation in Mycobacterium austroafricanum IFP 2173.},
author = {Elodie Nicolau and Lauriane Kuhn and Rémy Marchal and Yves Jouanneau},
doi = {10.1016/j.resmic.2009.09.017},
issn = {1769-7123 0923-2508},
year = {2009},
date = {2009-12-01},
journal = {Research in microbiology},
volume = {160},
number = {10},
pages = {838--847},
abstract = {2-Ethyhexyl nitrate (2-EHN) is a synthetic chemical used as a diesel fuel additive, which is recalcitrant to biodegradation. In this study, the enzymes involved in 2-EHN degradation were investigated in Mycobacterium austroafricanum IFP 2173. Using two-dimensional gel electrophoresis and a shotgun proteomic approach, a total of 398 proteins appeared to be more abundant in cells exposed to 2-EHN than in acetate-grown cells. This set of proteins includes multiple isoenzymes of the beta-oxidation pathway, two alcohol and one aldehyde dehydrogenase, as well as four cytochromes P450, including one CYP153 which functions as an alkane hydroxylase. Strain IFP 2173 was also found to contain two alkB-like genes encoding putative membrane-bound alkane hydroxylases. RT-PCR experiments showed that the gene encoding the CYP153 protein, as well as alkB genes, were expressed on 2-EHN. These findings are discussed in the light of a recently proposed 2-EHN degradation pathway involving an initial attack by an alkane hydroxylase and one turn of beta-oxidation, leading to the accumulation of a gamma-lactone as a dead-end product.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
2-Ethyhexyl nitrate (2-EHN) is a synthetic chemical used as a diesel fuel additive, which is recalcitrant to biodegradation. In this study, the enzymes involved in 2-EHN degradation were investigated in Mycobacterium austroafricanum IFP 2173. Using two-dimensional gel electrophoresis and a shotgun proteomic approach, a total of 398 proteins appeared to be more abundant in cells exposed to 2-EHN than in acetate-grown cells. This set of proteins includes multiple isoenzymes of the beta-oxidation pathway, two alcohol and one aldehyde dehydrogenase, as well as four cytochromes P450, including one CYP153 which functions as an alkane hydroxylase. Strain IFP 2173 was also found to contain two alkB-like genes encoding putative membrane-bound alkane hydroxylases. RT-PCR experiments showed that the gene encoding the CYP153 protein, as well as alkB genes, were expressed on 2-EHN. These findings are discussed in the light of a recently proposed 2-EHN degradation pathway involving an initial attack by an alkane hydroxylase and one turn of beta-oxidation, leading to the accumulation of a gamma-lactone as a dead-end product.
Atteia Ariane, Adrait Annie, Brugière Sabine, Tardif Marianne, van Lis Robert, Deusch Oliver, Dagan Tal, Kuhn Lauriane, Gontero Brigitte, Martin William, Garin Jérôme, Joyard Jacques, Rolland Norbert
Dans: Molecular biology and evolution, vol. 26, no. 7, p. 1533–1548, 2009, ISSN: 1537-1719 0737-4038.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{atteia_proteomic_2009,
title = {A proteomic survey of Chlamydomonas reinhardtii mitochondria sheds new light on the metabolic plasticity of the organelle and on the nature of the alpha-proteobacterial mitochondrial ancestor.},
author = {Ariane Atteia and Annie Adrait and Sabine Brugière and Marianne Tardif and Robert van Lis and Oliver Deusch and Tal Dagan and Lauriane Kuhn and Brigitte Gontero and William Martin and Jérôme Garin and Jacques Joyard and Norbert Rolland},
doi = {10.1093/molbev/msp068},
issn = {1537-1719 0737-4038},
year = {2009},
date = {2009-07-01},
journal = {Molecular biology and evolution},
volume = {26},
number = {7},
pages = {1533--1548},
abstract = {Mitochondria play a key role in the life and death of eukaryotic cells, yet the full spectrum of mitochondrial functions is far from being fully understood, especially in photosynthetic organisms. To advance our understanding of mitochondrial functions in a photosynthetic cell, an extensive proteomic survey of Percoll-purified mitochondria from the metabolically versatile, hydrogen-producing green alga Chlamydomonas reinhardtii was performed. Different fractions of purified mitochondria from Chlamydomonas cells grown under aerobic conditions were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry after protein separation on sodium dodecyl sulfate polyacrylamide gel electrophoresis or on blue-native polyacrylamide gel electrophoresis. Of the 496 nonredundant proteins identified, 149 are known or predicted to reside in other cellular compartments and were thus excluded from the molecular and evolutionary analyses of the Chlamydomonas proteome. The mitochondrial proteome of the photosynthetic alga reveals important lineage-specific differences with other mitochondrial proteomes, reflecting the high metabolic diversity of the organelle. Some mitochondrial metabolic pathways in Chlamydomonas appear to combine typical mitochondrial enzymes and bacterial-type ones, whereas others are unknown among mitochondriate eukaryotes. The comparison of the Chlamydomonas proteins to their identifiable homologs predicted from 354 sequenced genomes indicated that Arabidopsis is the most closely related nonalgal eukaryote. Furthermore, this phylogenomic analysis shows that free-living alpha-proteobacteria from the metabolically versatile orders Rhizobiales and Rhodobacterales better reflect the gene content of the ancestor of the chlorophyte mitochondria than parasitic alpha-proteobacteria with reduced and specialized genomes.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Mitochondria play a key role in the life and death of eukaryotic cells, yet the full spectrum of mitochondrial functions is far from being fully understood, especially in photosynthetic organisms. To advance our understanding of mitochondrial functions in a photosynthetic cell, an extensive proteomic survey of Percoll-purified mitochondria from the metabolically versatile, hydrogen-producing green alga Chlamydomonas reinhardtii was performed. Different fractions of purified mitochondria from Chlamydomonas cells grown under aerobic conditions were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry after protein separation on sodium dodecyl sulfate polyacrylamide gel electrophoresis or on blue-native polyacrylamide gel electrophoresis. Of the 496 nonredundant proteins identified, 149 are known or predicted to reside in other cellular compartments and were thus excluded from the molecular and evolutionary analyses of the Chlamydomonas proteome. The mitochondrial proteome of the photosynthetic alga reveals important lineage-specific differences with other mitochondrial proteomes, reflecting the high metabolic diversity of the organelle. Some mitochondrial metabolic pathways in Chlamydomonas appear to combine typical mitochondrial enzymes and bacterial-type ones, whereas others are unknown among mitochondriate eukaryotes. The comparison of the Chlamydomonas proteins to their identifiable homologs predicted from 354 sequenced genomes indicated that Arabidopsis is the most closely related nonalgal eukaryote. Furthermore, this phylogenomic analysis shows that free-living alpha-proteobacteria from the metabolically versatile orders Rhizobiales and Rhodobacterales better reflect the gene content of the ancestor of the chlorophyte mitochondria than parasitic alpha-proteobacteria with reduced and specialized genomes.
Kraut Alexandra, Marcellin Marlène, Adrait Annie, Kuhn Lauriane, Louwagie Mathilde, Kieffer-Jaquinod Sylvie, Lebert Dorothée, Masselon Christophe D, Dupuis Alain, Bruley Christophe, Jaquinod Michel, Garin Jérôme, Gallagher-Gambarelli Maighread
Peptide storage: are you getting the best return on your investment? Defining optimal storage conditions for proteomics samples. Article de journal
Dans: Journal of proteome research, vol. 8, no. 7, p. 3778–3785, 2009, ISSN: 1535-3893 1535-3893.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{kraut_peptide_2009,
title = {Peptide storage: are you getting the best return on your investment? Defining optimal storage conditions for proteomics samples.},
author = {Alexandra Kraut and Marlène Marcellin and Annie Adrait and Lauriane Kuhn and Mathilde Louwagie and Sylvie Kieffer-Jaquinod and Dorothée Lebert and Christophe D Masselon and Alain Dupuis and Christophe Bruley and Michel Jaquinod and Jérôme Garin and Maighread Gallagher-Gambarelli},
doi = {10.1021/pr900095u},
issn = {1535-3893 1535-3893},
year = {2009},
date = {2009-07-01},
journal = {Journal of proteome research},
volume = {8},
number = {7},
pages = {3778--3785},
abstract = {To comply with current proteomics guidelines, it is often necessary to analyze the same peptide samples several times. Between analyses, the sample must be stored in such a way as to conserve its intrinsic properties, without losing either peptides or signal intensity. This article describes two studies designed to define the optimal storage conditions for peptide samples between analyses. With the use of a label-free strategy, peptide conservation was compared over a 28-day period in three different recipients: standard plastic tubes, glass tubes, and low-adsorption plastic tubes. The results of this study showed that standard plastic tubes are unsuitable for peptide storage over the period studied. Glass tubes were found to perform better than standard plastic, but optimal peptide recovery was achieved using low-adsorption plastic tubes. The peptides showing poor recovery following storage were mainly hydrophobic in nature. The differences in peptide recovery between glass and low-adsorption plastic tubes were further studied using isotopically labeled proteins. This study allowed accurate comparison of peptide recovery between the two tube types within the same LC-MS run. The results of the label-free study were confirmed. Further, it was possible to demonstrate that peptide recovery in low-adsorption plastic tubes was optimal whatever the peptide concentration stored.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
To comply with current proteomics guidelines, it is often necessary to analyze the same peptide samples several times. Between analyses, the sample must be stored in such a way as to conserve its intrinsic properties, without losing either peptides or signal intensity. This article describes two studies designed to define the optimal storage conditions for peptide samples between analyses. With the use of a label-free strategy, peptide conservation was compared over a 28-day period in three different recipients: standard plastic tubes, glass tubes, and low-adsorption plastic tubes. The results of this study showed that standard plastic tubes are unsuitable for peptide storage over the period studied. Glass tubes were found to perform better than standard plastic, but optimal peptide recovery was achieved using low-adsorption plastic tubes. The peptides showing poor recovery following storage were mainly hydrophobic in nature. The differences in peptide recovery between glass and low-adsorption plastic tubes were further studied using isotopically labeled proteins. This study allowed accurate comparison of peptide recovery between the two tube types within the same LC-MS run. The results of the label-free study were confirmed. Further, it was possible to demonstrate that peptide recovery in low-adsorption plastic tubes was optimal whatever the peptide concentration stored.
Hamrita Bechr, Chahed Karim, Trimeche Mounir, Guillier Christelle Lemaitre, Hammann Philippe, Chaïeb Anouar, Korbi Sadok, Chouchane Lotfi
Proteomics-based identification of alpha1-antitrypsin and haptoglobin precursors as novel serum markers in infiltrating ductal breast carcinomas. Article de journal
Dans: Clinica chimica acta; international journal of clinical chemistry, vol. 404, no. 2, p. 111–118, 2009, ISSN: 1873-3492 0009-8981, (Place: Netherlands).
Résumé | Liens | BibTeX | Étiquettes: 80 and over, Adult, Aged, alpha 1-Antitrypsin/*blood, Amino Acid Sequence, Biomarkers, Breast Neoplasms/blood/*pathology, Carcinoma, Ductal/blood/*pathology, Electrophoresis, Female, Gel, Haptoglobins/*analysis, Humans, Mass, Matrix-Assisted Laser Desorption-Ionization, Middle Aged, Molecular Sequence Data, PPSE, Protein Isoforms/blood, proteomics, Spectrometry, Tumor/*blood, Two-Dimensional
@article{hamrita_proteomics-based_2009,
title = {Proteomics-based identification of alpha1-antitrypsin and haptoglobin precursors as novel serum markers in infiltrating ductal breast carcinomas.},
author = {Bechr Hamrita and Karim Chahed and Mounir Trimeche and Christelle Lemaitre Guillier and Philippe Hammann and Anouar Chaïeb and Sadok Korbi and Lotfi Chouchane},
doi = {10.1016/j.cca.2009.03.033},
issn = {1873-3492 0009-8981},
year = {2009},
date = {2009-06-01},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {404},
number = {2},
pages = {111--118},
abstract = {BACKGROUND: The identification of pathological markers of breast cancer for either diagnosis, treatment response or for survival is of critical importance. METHODS: Serum protein profiling using 2-DE separations coupled to matrix-assisted laser desorption ionization mass spectrometry has been used to explore protein alterations in patients with infiltrating ductal breast carcinomas (IDCA). Sera from 39 breast cancer patients and 40 healthy controls were selected for screening study using 2-DE combined with MS. The protein expression patterns obtained after the depletion of high abundance proteins was determined by coomassie blue G-250 stain after 2-DE electrophoresis. RESULTS: Six proteins that expressed differentially in the IDCA group were found. The expression levels of four isoforms corresponding to haptoglobin precursor and two isoforms of alpha1-antitrypsin precursor (alpha1-AT) were upregulated in sera from breast cancer patients. There was an increased expression of both proteins in the sera of patients with various tumor stages (I, II, III) in comparison to healthy women. Applying immunohistochemistry, we further validated alpha1-AT immunoreactivity in 51 formalin-fixed paraffin-embedded sections of breast tumors. Enhanced expression of alpha1-AT like activity has been found in IDCA breast tumors, as well as, in different histological types of breast cancer. No significant association has been found with lymph node occurrence, while in high tumor categories a tendency to an increased expression of alpha1-AT has been found, thereby suggesting a possible role of this protein in tumor growth. CONCLUSIONS: These proteins may constitute new and useful markers of breast cancer that offer a clue to a better understanding of inflammatory pathways and carcinogenesis events linked to breast cancer progression.},
note = {Place: Netherlands},
keywords = {80 and over, Adult, Aged, alpha 1-Antitrypsin/*blood, Amino Acid Sequence, Biomarkers, Breast Neoplasms/blood/*pathology, Carcinoma, Ductal/blood/*pathology, Electrophoresis, Female, Gel, Haptoglobins/*analysis, Humans, Mass, Matrix-Assisted Laser Desorption-Ionization, Middle Aged, Molecular Sequence Data, PPSE, Protein Isoforms/blood, proteomics, Spectrometry, Tumor/*blood, Two-Dimensional},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: The identification of pathological markers of breast cancer for either diagnosis, treatment response or for survival is of critical importance. METHODS: Serum protein profiling using 2-DE separations coupled to matrix-assisted laser desorption ionization mass spectrometry has been used to explore protein alterations in patients with infiltrating ductal breast carcinomas (IDCA). Sera from 39 breast cancer patients and 40 healthy controls were selected for screening study using 2-DE combined with MS. The protein expression patterns obtained after the depletion of high abundance proteins was determined by coomassie blue G-250 stain after 2-DE electrophoresis. RESULTS: Six proteins that expressed differentially in the IDCA group were found. The expression levels of four isoforms corresponding to haptoglobin precursor and two isoforms of alpha1-antitrypsin precursor (alpha1-AT) were upregulated in sera from breast cancer patients. There was an increased expression of both proteins in the sera of patients with various tumor stages (I, II, III) in comparison to healthy women. Applying immunohistochemistry, we further validated alpha1-AT immunoreactivity in 51 formalin-fixed paraffin-embedded sections of breast tumors. Enhanced expression of alpha1-AT like activity has been found in IDCA breast tumors, as well as, in different histological types of breast cancer. No significant association has been found with lymph node occurrence, while in high tumor categories a tendency to an increased expression of alpha1-AT has been found, thereby suggesting a possible role of this protein in tumor growth. CONCLUSIONS: These proteins may constitute new and useful markers of breast cancer that offer a clue to a better understanding of inflammatory pathways and carcinogenesis events linked to breast cancer progression.
Bernay Benoît, Gaillard Marie-Claude, Guryca Vilém, Emadali Anouk, Kuhn Lauriane, Bertrand Anne, Detraz Isabelle, Carcenac Carole, Savasta Marc, Brouillet Emmanuel, Garin Jérôme, Elalouf Jean-Marc
Discovering new bioactive neuropeptides in the striatum secretome using in vivo microdialysis and versatile proteomics. Article de journal
Dans: Molecular & cellular proteomics : MCP, vol. 8, no. 5, p. 946–958, 2009, ISSN: 1535-9484 1535-9476 1535-9476.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{bernay_discovering_2009,
title = {Discovering new bioactive neuropeptides in the striatum secretome using in vivo microdialysis and versatile proteomics.},
author = {Benoît Bernay and Marie-Claude Gaillard and Vilém Guryca and Anouk Emadali and Lauriane Kuhn and Anne Bertrand and Isabelle Detraz and Carole Carcenac and Marc Savasta and Emmanuel Brouillet and Jérôme Garin and Jean-Marc Elalouf},
doi = {10.1074/mcp.M800501-MCP200},
issn = {1535-9484 1535-9476 1535-9476},
year = {2009},
date = {2009-05-01},
journal = {Molecular & cellular proteomics : MCP},
volume = {8},
number = {5},
pages = {946--958},
abstract = {The striatum, a major component of the brain basal nuclei, is central for planning and executing voluntary movements and undergoes lesions in neurodegenerative disorders such as Huntington disease. To perform highly integrated tasks, the striatum relies on a complex network of communication within and between brain regions with a key role devoted to secreted molecules. To characterize the rat striatum secretome, we combined in vivo microdialysis together with proteomics analysis of trypsin digests and peptidomics studies of native fragments. This versatile approach, carried out using different microdialysis probes and mass spectrometer devices, allowed evidencing with high confidence the expression of 88 proteins and 100 processed peptides. Their secretory pathways were predicted by in silico analysis. Whereas high molecular weight proteins were mainly secreted by the classical mode (94%), low molecular weight proteins equally used classical and non-classical modes (53 and 47%, respectively). In addition, our results suggested alternative secretion mechanisms not predicted by bioinformatics tools. Based on spectrum counting, we performed a relative quantification of secreted proteins and peptides in both basal and neuronal depolarization conditions. This allowed detecting a series of neuropeptide precursors and a 6-fold increase for neurosecretory protein VGF and proenkephalin (PENK) levels. A focused investigation and a long peptide experiment led to the identification of new secreted non-opioid PENK peptides, referred to as PENK 114-133, PENK 239-260, and PENK 143-185. Moreover we showed that injecting synthetic PENK 114-133 and PENK 239-260 into the striatum robustly increased glutamate release in this region. Thus, the combination of microdialysis and versatile proteomics methods shed new light on the secreted protein repertoire and evidenced novel neuropeptide transmitters.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The striatum, a major component of the brain basal nuclei, is central for planning and executing voluntary movements and undergoes lesions in neurodegenerative disorders such as Huntington disease. To perform highly integrated tasks, the striatum relies on a complex network of communication within and between brain regions with a key role devoted to secreted molecules. To characterize the rat striatum secretome, we combined in vivo microdialysis together with proteomics analysis of trypsin digests and peptidomics studies of native fragments. This versatile approach, carried out using different microdialysis probes and mass spectrometer devices, allowed evidencing with high confidence the expression of 88 proteins and 100 processed peptides. Their secretory pathways were predicted by in silico analysis. Whereas high molecular weight proteins were mainly secreted by the classical mode (94%), low molecular weight proteins equally used classical and non-classical modes (53 and 47%, respectively). In addition, our results suggested alternative secretion mechanisms not predicted by bioinformatics tools. Based on spectrum counting, we performed a relative quantification of secreted proteins and peptides in both basal and neuronal depolarization conditions. This allowed detecting a series of neuropeptide precursors and a 6-fold increase for neurosecretory protein VGF and proenkephalin (PENK) levels. A focused investigation and a long peptide experiment led to the identification of new secreted non-opioid PENK peptides, referred to as PENK 114-133, PENK 239-260, and PENK 143-185. Moreover we showed that injecting synthetic PENK 114-133 and PENK 239-260 into the striatum robustly increased glutamate release in this region. Thus, the combination of microdialysis and versatile proteomics methods shed new light on the secreted protein repertoire and evidenced novel neuropeptide transmitters.
Wilkins Sarah E, Hyvärinen Jaana, Chicher Johana, Gorman Jeffrey J, Peet Daniel J, Bilton Rebecca L, Koivunen Peppi
Differences in hydroxylation and binding of Notch and HIF-1alpha demonstrate substrate selectivity for factor inhibiting HIF-1 (FIH-1). Article de journal
Dans: The international journal of biochemistry & cell biology, vol. 41, no. 7, p. 1563–1571, 2009, ISSN: 1878-5875 1357-2725, (Place: Netherlands).
Résumé | Liens | BibTeX | Étiquettes: alpha Subunit/*metabolism, Amino Acid Sequence, Animals, Asparagine/metabolism, Humans, Hydroxylation, Hypoxia-Inducible Factor 1, Kinetics, Mice, Mixed Function Oxygenases, Molecular Sequence Data, Notch/chemistry/*metabolism, Oxygen/metabolism, Peptides/chemistry/metabolism, PPSE, Protein Binding, Receptors, Recombinant Proteins/metabolism, Repressor Proteins/*metabolism, Substrate Specificity
@article{wilkins_differences_2009,
title = {Differences in hydroxylation and binding of Notch and HIF-1alpha demonstrate substrate selectivity for factor inhibiting HIF-1 (FIH-1).},
author = {Sarah E Wilkins and Jaana Hyvärinen and Johana Chicher and Jeffrey J Gorman and Daniel J Peet and Rebecca L Bilton and Peppi Koivunen},
doi = {10.1016/j.biocel.2009.01.005},
issn = {1878-5875 1357-2725},
year = {2009},
date = {2009-01-01},
journal = {The international journal of biochemistry & cell biology},
volume = {41},
number = {7},
pages = {1563--1571},
abstract = {FIH-1, factor inhibiting hypoxia-inducible factor-1 (HIF-1), regulates oxygen sensing by hydroxylating an asparagine within HIF-alpha. It also hydroxylates asparagines in many proteins containing ankyrin repeats, including Notch1-3, p105 and I?B?. Relative binding affinity and hydroxylation rate are crucial determinants of substrate selection and modification. We determined the contributions of substrate sequence composition and length and of oxygen concentration to the FIH-1-binding and/or hydroxylation of Notch1-4 and compared them with those for HIF-1alpha. We also demonstrated hydroxylation of two asparagines in Notch2 and 3, corresponding to Sites 1 and 2 of Notch1, by mass spectrometry for the first time. Our data demonstrate that substrate length has a much greater influence on FIH-1-dependent hydroxylation of Notch than of HIF-1alpha, predominantly through binding affinity rather than maximal reaction velocity. The K(m) value of FIH-1 for Notch1, textless 0.2 microM, is at least 250-fold lower than that of 50 microM for HIF-1alpha. Site 1 of Notch1-3 appeared the preferred site of FIH-1 hydroxylation in these substrates. Interestingly, binding of Notch4 to FIH-1 was observed with an affinity almost 10-fold lower than for Notch1-3, but no hydroxylation was detected. Importantly, we demonstrate that the K(m) of FIH-1 for oxygen at the preferred Site 1 of Notch1-3, 10-19 microM, is an order of magnitude lower than that for Site 2 or HIF-1alpha. Hence, at least during in vitro hydroxylation, Notch is likely to become efficiently hydroxylated by FIH-1 even under relatively severe hypoxic conditions, where HIF-1alpha hydroxylation would be reduced.},
note = {Place: Netherlands},
keywords = {alpha Subunit/*metabolism, Amino Acid Sequence, Animals, Asparagine/metabolism, Humans, Hydroxylation, Hypoxia-Inducible Factor 1, Kinetics, Mice, Mixed Function Oxygenases, Molecular Sequence Data, Notch/chemistry/*metabolism, Oxygen/metabolism, Peptides/chemistry/metabolism, PPSE, Protein Binding, Receptors, Recombinant Proteins/metabolism, Repressor Proteins/*metabolism, Substrate Specificity},
pubstate = {published},
tppubtype = {article}
}
FIH-1, factor inhibiting hypoxia-inducible factor-1 (HIF-1), regulates oxygen sensing by hydroxylating an asparagine within HIF-alpha. It also hydroxylates asparagines in many proteins containing ankyrin repeats, including Notch1-3, p105 and I?B?. Relative binding affinity and hydroxylation rate are crucial determinants of substrate selection and modification. We determined the contributions of substrate sequence composition and length and of oxygen concentration to the FIH-1-binding and/or hydroxylation of Notch1-4 and compared them with those for HIF-1alpha. We also demonstrated hydroxylation of two asparagines in Notch2 and 3, corresponding to Sites 1 and 2 of Notch1, by mass spectrometry for the first time. Our data demonstrate that substrate length has a much greater influence on FIH-1-dependent hydroxylation of Notch than of HIF-1alpha, predominantly through binding affinity rather than maximal reaction velocity. The K(m) value of FIH-1 for Notch1, textless 0.2 microM, is at least 250-fold lower than that of 50 microM for HIF-1alpha. Site 1 of Notch1-3 appeared the preferred site of FIH-1 hydroxylation in these substrates. Interestingly, binding of Notch4 to FIH-1 was observed with an affinity almost 10-fold lower than for Notch1-3, but no hydroxylation was detected. Importantly, we demonstrate that the K(m) of FIH-1 for oxygen at the preferred Site 1 of Notch1-3, 10-19 microM, is an order of magnitude lower than that for Site 2 or HIF-1alpha. Hence, at least during in vitro hydroxylation, Notch is likely to become efficiently hydroxylated by FIH-1 even under relatively severe hypoxic conditions, where HIF-1alpha hydroxylation would be reduced.
2008
Bringel Françoise, Hammann Philippe, Kugler Valérie, Arsène-Ploetze Florence
Dans: Microbiology (Reading, England), vol. 154, no. Pt 9, p. 2629–2640, 2008, ISSN: 1350-0872 1350-0872, (Place: England).
Résumé | Liens | BibTeX | Étiquettes: Arginine/*biosynthesis, Argininosuccinate Lyase/genetics, Bacterial, Bacterial Proteins/*genetics, Bacterial/genetics, Carbon Compounds, Carbon Dioxide/metabolism, Electrophoresis, Gel, Gene Expression Regulation, Genetic, IMP Dehydrogenase/genetics, Inorganic/*metabolism, Lactobacillus plantarum/enzymology/genetics/*metabolism, Mass, Matrix-Assisted Laser Desorption-Ionization, Nucleotides/*biosynthesis, Pentosyltransferases/*genetics, PPSE, proteomics, Repressor Proteins/*genetics, Reverse Transcriptase Polymerase Chain Reaction, RNA, Spectrometry, Transcription, Two-Dimensional
@article{bringel_lactobacillus_2008,
title = {Lactobacillus plantarum response to inorganic carbon concentrations: PyrR2-dependent and -independent transcription regulation of genes involved in arginine and nucleotide metabolism.},
author = {Françoise Bringel and Philippe Hammann and Valérie Kugler and Florence Arsène-Ploetze},
doi = {10.1099/mic.0.2008/018184-0},
issn = {1350-0872 1350-0872},
year = {2008},
date = {2008-09-01},
journal = {Microbiology (Reading, England)},
volume = {154},
number = {Pt 9},
pages = {2629--2640},
abstract = {Lactobacillus plantarum susbp. plantarum is a capnophilic Gram-positive heterotroph with optimal growth in 4 % CO(2)-enriched air. At low inorganic carbon (C(i)) concentrations, the pyr genes encoding the enzymes of the pyrimidine biosynthetic pathway were overexpressed, in agreement with a previous study showing that these genes are regulated at the transcription level in response to C(i) via a PyrR(2)-mediated mechanism. A previous study of high-CO(2)-requiring (HCR) mutants revealed an unknown genetic link between arginine regulation and C(i)-dependent nutritional needs. To better understand L. plantarum's adaptation to C(i) availability, additional C(i)-responsive genes were sought in the arginine biosynthetic pathway (arg and car genes) using slot-blot hybridization and a proteomic differential 2D gel electrophoresis (DIGE) global approach. Besides the nine pyr-encoded proteins, 16 new Icr (inorganic-carbon-regulated) proteins accumulated differentially in response to C(i) availability, suggesting that the C(i) response involves several metabolic pathways and adaptation processes. Among these Icr proteins only argininosuccinate lyase, encoded by argH, was involved in arginine biosynthesis. Three proteins involved in the purine biosynthetic pathway and nucleotide conversion, adenylate kinase (Adk), GMP synthase (GuaA), and IMP dehydrogenase (GuaB), accumulated differentially in response to changes in C(i) levels. Expression of the Icr protein-encoding genes argH and guaB was regulated at the transcription level or by RNA stability in response to C(i) availability, as previously demonstrated for the pyr genes. However, PyrR(2) was not essential for the C(i)-regulated transcription of argH and guaB, demonstrating that PyrR(2) modulates only a subset of C(i)-regulated genes. These results suggest that the C(i) response may involve at least two regulatory mechanisms in L. plantarum.},
note = {Place: England},
keywords = {Arginine/*biosynthesis, Argininosuccinate Lyase/genetics, Bacterial, Bacterial Proteins/*genetics, Bacterial/genetics, Carbon Compounds, Carbon Dioxide/metabolism, Electrophoresis, Gel, Gene Expression Regulation, Genetic, IMP Dehydrogenase/genetics, Inorganic/*metabolism, Lactobacillus plantarum/enzymology/genetics/*metabolism, Mass, Matrix-Assisted Laser Desorption-Ionization, Nucleotides/*biosynthesis, Pentosyltransferases/*genetics, PPSE, proteomics, Repressor Proteins/*genetics, Reverse Transcriptase Polymerase Chain Reaction, RNA, Spectrometry, Transcription, Two-Dimensional},
pubstate = {published},
tppubtype = {article}
}
Lactobacillus plantarum susbp. plantarum is a capnophilic Gram-positive heterotroph with optimal growth in 4 % CO(2)-enriched air. At low inorganic carbon (C(i)) concentrations, the pyr genes encoding the enzymes of the pyrimidine biosynthetic pathway were overexpressed, in agreement with a previous study showing that these genes are regulated at the transcription level in response to C(i) via a PyrR(2)-mediated mechanism. A previous study of high-CO(2)-requiring (HCR) mutants revealed an unknown genetic link between arginine regulation and C(i)-dependent nutritional needs. To better understand L. plantarum's adaptation to C(i) availability, additional C(i)-responsive genes were sought in the arginine biosynthetic pathway (arg and car genes) using slot-blot hybridization and a proteomic differential 2D gel electrophoresis (DIGE) global approach. Besides the nine pyr-encoded proteins, 16 new Icr (inorganic-carbon-regulated) proteins accumulated differentially in response to C(i) availability, suggesting that the C(i) response involves several metabolic pathways and adaptation processes. Among these Icr proteins only argininosuccinate lyase, encoded by argH, was involved in arginine biosynthesis. Three proteins involved in the purine biosynthetic pathway and nucleotide conversion, adenylate kinase (Adk), GMP synthase (GuaA), and IMP dehydrogenase (GuaB), accumulated differentially in response to changes in C(i) levels. Expression of the Icr protein-encoding genes argH and guaB was regulated at the transcription level or by RNA stability in response to C(i) availability, as previously demonstrated for the pyr genes. However, PyrR(2) was not essential for the C(i)-regulated transcription of argH and guaB, demonstrating that PyrR(2) modulates only a subset of C(i)-regulated genes. These results suggest that the C(i) response may involve at least two regulatory mechanisms in L. plantarum.
Shen Da-Kang, Quenee Lauriane, Bonnet Mariette, Kuhn Lauriane, Derouazi Madiha, Lamotte Daniele, Toussaint Bertrand, Polack Benoit
Orf1/SpcS chaperones ExoS for type three secretion by Pseudomonas aeruginosa. Article de journal
Dans: Biomedical and environmental sciences : BES, vol. 21, no. 2, p. 103–109, 2008, ISSN: 0895-3988 0895-3988.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{shen_orf1spcs_2008,
title = {Orf1/SpcS chaperones ExoS for type three secretion by Pseudomonas aeruginosa.},
author = {Da-Kang Shen and Lauriane Quenee and Mariette Bonnet and Lauriane Kuhn and Madiha Derouazi and Daniele Lamotte and Bertrand Toussaint and Benoit Polack},
doi = {10.1016/S0895-3988(08)60014-8},
issn = {0895-3988 0895-3988},
year = {2008},
date = {2008-04-01},
journal = {Biomedical and environmental sciences : BES},
volume = {21},
number = {2},
pages = {103--109},
abstract = {OBJECTIVE: Pseudomonas aeruginosa is a ubiquitous and opportunistic pathogen that uses the type III secretion system (TTSS) to inject effector proteins directly into the cytosol of target cells to subvert the host cell's functions. Specialized bacterial chaperones are required for effective secretion of some effectors. To identify the chaperone of ExoS, the representative effector secreted by the TTSS of P. aeruginosa, we analyzed the role of a postulated chaperone termed Orf1. METHODS: By allelic exchange, we constructed the mutant with the deletion of gene Orf1. Analysis of secreted and cell-associated fractions was performed by SDS-PAGE and Western blotting. Using strain expressing in trans Orf1, tagged by V5 polypeptide and histidine, protein-protein interaction was determined by affinity resin pull-down assay in combination with MALDI-TOF. The role of Orf1 in the expression of exoS was evaluated by gene reporter analysis. RESULTS: Pull-down assay showed that Orf1 binds to ExoS and ExoT. Secretion profile analysis showed that Orf1 was necessary for the optimal secretion of ExoS and ExoT. However, Orf1 had no effect on the expression of exoS. CONCLUSION: Orf1 is important for the secretion of ExoS probably by maintaining ExoS in a secretion-competent conformation. We propose to name Orf1 as SpcS for "specific Pseudomonas chaperone for ExoS".},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
OBJECTIVE: Pseudomonas aeruginosa is a ubiquitous and opportunistic pathogen that uses the type III secretion system (TTSS) to inject effector proteins directly into the cytosol of target cells to subvert the host cell's functions. Specialized bacterial chaperones are required for effective secretion of some effectors. To identify the chaperone of ExoS, the representative effector secreted by the TTSS of P. aeruginosa, we analyzed the role of a postulated chaperone termed Orf1. METHODS: By allelic exchange, we constructed the mutant with the deletion of gene Orf1. Analysis of secreted and cell-associated fractions was performed by SDS-PAGE and Western blotting. Using strain expressing in trans Orf1, tagged by V5 polypeptide and histidine, protein-protein interaction was determined by affinity resin pull-down assay in combination with MALDI-TOF. The role of Orf1 in the expression of exoS was evaluated by gene reporter analysis. RESULTS: Pull-down assay showed that Orf1 binds to ExoS and ExoT. Secretion profile analysis showed that Orf1 was necessary for the optimal secretion of ExoS and ExoT. However, Orf1 had no effect on the expression of exoS. CONCLUSION: Orf1 is important for the secretion of ExoS probably by maintaining ExoS in a secretion-competent conformation. We propose to name Orf1 as SpcS for "specific Pseudomonas chaperone for ExoS".
2007
Marmagne Anne, Ferro Myriam, Meinnel Thierry, Bruley Christophe, Kuhn Lauriane, Garin Jérome, Barbier-Brygoo Hélène, Ephritikhine Geneviève
A high content in lipid-modified peripheral proteins and integral receptor kinases features in the arabidopsis plasma membrane proteome. Article de journal
Dans: Molecular & cellular proteomics : MCP, vol. 6, no. 11, p. 1980–1996, 2007, ISSN: 1535-9476 1535-9476.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{marmagne_high_2007,
title = {A high content in lipid-modified peripheral proteins and integral receptor kinases features in the arabidopsis plasma membrane proteome.},
author = {Anne Marmagne and Myriam Ferro and Thierry Meinnel and Christophe Bruley and Lauriane Kuhn and Jérome Garin and Hélène Barbier-Brygoo and Geneviève Ephritikhine},
doi = {10.1074/mcp.M700099-MCP200},
issn = {1535-9476 1535-9476},
year = {2007},
date = {2007-11-01},
journal = {Molecular & cellular proteomics : MCP},
volume = {6},
number = {11},
pages = {1980--1996},
abstract = {The proteomics of plasma membrane has brought to date only scarce and partial information on the actual protein repertoire. In this work, the plant plasma membrane proteome of Arabidopsis thaliana was investigated. A highly purified plasma membrane fraction was washed by NaCl and Na2CO3 salts, and the insoluble fractions were further analyzed by nano-LC-MS/MS. With 446 proteins identified, we hereby describe the largest plasma membrane proteome diversity reported so far. Half of the proteins were predicted to display transmembrane domains and/or to be anchored to the membrane, validating a posteriori the pertinence of the approach. A fine analysis highlighted two main specific and novel features. First, the main functional category is represented by a majority of as yet unreported signaling proteins, including 11% receptor-like kinases. Second, 16% of the identified proteins are predicted to be lipid-modified, specifically involving double lipid linkage through N-terminal myristoylation, S-palmitoylation, C-terminal prenylation, or glycosylphosphatidylinositol anchors. Thus, our approach led for the first time to the identification of a large number of peripheral proteins as part of the plasma membrane and allowed the functionality of the plasma membrane in the cell context to be reconsidered.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The proteomics of plasma membrane has brought to date only scarce and partial information on the actual protein repertoire. In this work, the plant plasma membrane proteome of Arabidopsis thaliana was investigated. A highly purified plasma membrane fraction was washed by NaCl and Na2CO3 salts, and the insoluble fractions were further analyzed by nano-LC-MS/MS. With 446 proteins identified, we hereby describe the largest plasma membrane proteome diversity reported so far. Half of the proteins were predicted to display transmembrane domains and/or to be anchored to the membrane, validating a posteriori the pertinence of the approach. A fine analysis highlighted two main specific and novel features. First, the main functional category is represented by a majority of as yet unreported signaling proteins, including 11% receptor-like kinases. Second, 16% of the identified proteins are predicted to be lipid-modified, specifically involving double lipid linkage through N-terminal myristoylation, S-palmitoylation, C-terminal prenylation, or glycosylphosphatidylinositol anchors. Thus, our approach led for the first time to the identification of a large number of peripheral proteins as part of the plasma membrane and allowed the functionality of the plasma membrane in the cell context to be reconsidered.
Lelong Cécile, Aguiluz Kryssia, Luche Sylvie, Kuhn Lauriane, Garin Jérôme, Rabilloud Thierry, Geiselmann Johannes
The Crl-RpoS regulon of Escherichia coli. Article de journal
Dans: Molecular & cellular proteomics : MCP, vol. 6, no. 4, p. 648–659, 2007, ISSN: 1535-9476 1535-9476.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{lelong_crl-rpos_2007,
title = {The Crl-RpoS regulon of Escherichia coli.},
author = {Cécile Lelong and Kryssia Aguiluz and Sylvie Luche and Lauriane Kuhn and Jérôme Garin and Thierry Rabilloud and Johannes Geiselmann},
doi = {10.1074/mcp.M600191-MCP200},
issn = {1535-9476 1535-9476},
year = {2007},
date = {2007-04-01},
journal = {Molecular & cellular proteomics : MCP},
volume = {6},
number = {4},
pages = {648--659},
abstract = {The RpoS subunit of RNA polymerase controls the expression of numerous genes involved in stationary phase and in response to different stress conditions. The regulatory protein Crl increases the activity of RpoS by direct interaction with the RpoS holoenzyme. To define the extent of the Crl regulon, we used two-dimensional SDS-PAGE to measure the role of Crl in regulating the expression of the Escherichia coliproteome in stationary phase at 30 degrees C. By comparing the proteome of four strains (wild type, crl(-), rpoS(-), and crl(-)rpoS(-)), we observed that the intensity of 74 spots was modified in at least one mutant context. 62 spots were identified by mass spectrometry and correspond to 40 distinct proteins. They were classified in four main categories: DNA metabolism, central metabolism, response to environmental modifications, and miscellaneous. Three proteins were specifically involved in quorum sensing: TnaA (the tryptophanase that converts tryptophan to indole), WrbA (Trp repressor-binding protein), and YgaG (homologous to LuxS, autoinducer-2 synthase). Because little is known about the regulation of Crl expression, we investigated the influence of diffusible molecules on the expression of Crl. Using Western blotting experiments, we showed that, at 30 degrees C, a diffusible molecule(s) produced during the transition phase between the exponential and stationary phases induces a premature expression of Crl. Indole was tested as one of the potential candidates: at 37 degrees C, it is present in the extracellular medium at a constant concentration, but at 30 degrees C, its concentration peaks during the transition phase. When indole was added to the culture medium, it also induced prematurely the expression of Crl at both the transcriptional and translational levels in a Crl-dependent manner. Crl may thus be considered a new environmental sensor via the indole concentration.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The RpoS subunit of RNA polymerase controls the expression of numerous genes involved in stationary phase and in response to different stress conditions. The regulatory protein Crl increases the activity of RpoS by direct interaction with the RpoS holoenzyme. To define the extent of the Crl regulon, we used two-dimensional SDS-PAGE to measure the role of Crl in regulating the expression of the Escherichia coliproteome in stationary phase at 30 degrees C. By comparing the proteome of four strains (wild type, crl(-), rpoS(-), and crl(-)rpoS(-)), we observed that the intensity of 74 spots was modified in at least one mutant context. 62 spots were identified by mass spectrometry and correspond to 40 distinct proteins. They were classified in four main categories: DNA metabolism, central metabolism, response to environmental modifications, and miscellaneous. Three proteins were specifically involved in quorum sensing: TnaA (the tryptophanase that converts tryptophan to indole), WrbA (Trp repressor-binding protein), and YgaG (homologous to LuxS, autoinducer-2 synthase). Because little is known about the regulation of Crl expression, we investigated the influence of diffusible molecules on the expression of Crl. Using Western blotting experiments, we showed that, at 30 degrees C, a diffusible molecule(s) produced during the transition phase between the exponential and stationary phases induces a premature expression of Crl. Indole was tested as one of the potential candidates: at 37 degrees C, it is present in the extracellular medium at a constant concentration, but at 30 degrees C, its concentration peaks during the transition phase. When indole was added to the culture medium, it also induced prematurely the expression of Crl at both the transcriptional and translational levels in a Crl-dependent manner. Crl may thus be considered a new environmental sensor via the indole concentration.
Paclet Marie-Hélène, Berthier Sylvie, Kuhn Lauriane, Garin Jérôme, Morel Françoise
Regulation of phagocyte NADPH oxidase activity: identification of two cytochrome b558 activation states. Article de journal
Dans: FASEB journal : official publication of the Federation of American Societies for Experimental Biology, vol. 21, no. 4, p. 1244–1255, 2007, ISSN: 1530-6860 0892-6638.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{paclet_regulation_2007,
title = {Regulation of phagocyte NADPH oxidase activity: identification of two cytochrome b558 activation states.},
author = {Marie-Hélène Paclet and Sylvie Berthier and Lauriane Kuhn and Jérôme Garin and Françoise Morel},
doi = {10.1096/fj.06-6852com},
issn = {1530-6860 0892-6638},
year = {2007},
date = {2007-04-01},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {21},
number = {4},
pages = {1244--1255},
abstract = {Activation of the phagocyte NADPH oxidase (phox) requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac1/2) with the membrane cytochrome b558, leading to a hemoprotein conformation change. To clarify this mechanism, the phagocyte NADPH oxidase complex was isolated through cytochrome b558 purification after three chromatographic steps. The purified neutrophil complex was constitutively active in the absence of an amphiphile agent with a maximum turnover (125 mol O2(-) x s(-1) x mol heme b(-1)), indicating that cytochrome b558 has been activated by cytosolic proteins and is in an "open conformation," able to transfer a maximum rate of electrons. In contrast, the phox complex prepared with B lymphocyte cytosol shows a lower constitutive turnover (approximately 50 mol O2(-) x s(-1) x mol heme b(-1)). Analysis of phox complex components by Western blot and mass spectrometry showed the presence of cytosolic factors (especially p67-phox) and structural proteins (moesin, ezrin). To investigate the difference in activity of phox complexes, we evaluated the effect of MRP8 and MRP14, specifically expressed in neutrophils, on the activity of the B lymphocyte complex. MRPs induce the switch between the partially and the fully "open" cytochrome b558 conformation. Moreover, their effect was independent of p67-phox. Data point out two potential cytochrome b558 activation states.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Activation of the phagocyte NADPH oxidase (phox) requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac1/2) with the membrane cytochrome b558, leading to a hemoprotein conformation change. To clarify this mechanism, the phagocyte NADPH oxidase complex was isolated through cytochrome b558 purification after three chromatographic steps. The purified neutrophil complex was constitutively active in the absence of an amphiphile agent with a maximum turnover (125 mol O2(-) x s(-1) x mol heme b(-1)), indicating that cytochrome b558 has been activated by cytosolic proteins and is in an "open conformation," able to transfer a maximum rate of electrons. In contrast, the phox complex prepared with B lymphocyte cytosol shows a lower constitutive turnover (approximately 50 mol O2(-) x s(-1) x mol heme b(-1)). Analysis of phox complex components by Western blot and mass spectrometry showed the presence of cytosolic factors (especially p67-phox) and structural proteins (moesin, ezrin). To investigate the difference in activity of phox complexes, we evaluated the effect of MRP8 and MRP14, specifically expressed in neutrophils, on the activity of the B lymphocyte complex. MRPs induce the switch between the partially and the fully "open" cytochrome b558 conformation. Moreover, their effect was independent of p67-phox. Data point out two potential cytochrome b558 activation states.
Lanquar Viviane, Kuhn Lauriane, Lelièvre Françoise, Khafif Mehdi, Espagne Christelle, Bruley Christophe, Barbier-Brygoo Hélène, Garin Jérôme, Thomine Sébastien
15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells. Article de journal
Dans: Proteomics, vol. 7, no. 5, p. 750–754, 2007, ISSN: 1615-9853 1615-9853.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{lanquar_15n-metabolic_2007,
title = {15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells.},
author = {Viviane Lanquar and Lauriane Kuhn and Françoise Lelièvre and Mehdi Khafif and Christelle Espagne and Christophe Bruley and Hélène Barbier-Brygoo and Jérôme Garin and Sébastien Thomine},
doi = {10.1002/pmic.200600791},
issn = {1615-9853 1615-9853},
year = {2007},
date = {2007-03-01},
journal = {Proteomics},
volume = {7},
number = {5},
pages = {750--754},
abstract = {An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.
Govin Jérôme, Escoffier Emmanuelle, Rousseaux Sophie, Kuhn Lauriane, Ferro Myriam, Thévenon Julien, Catena Raffaella, Davidson Irwin, Garin Jérôme, Khochbin Saadi, Caron Cécile
Pericentric heterochromatin reprogramming by new histone variants during mouse spermiogenesis. Article de journal
Dans: The Journal of cell biology, vol. 176, no. 3, p. 283–294, 2007, ISSN: 0021-9525 1540-8140 0021-9525.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{govin_pericentric_2007,
title = {Pericentric heterochromatin reprogramming by new histone variants during mouse spermiogenesis.},
author = {Jérôme Govin and Emmanuelle Escoffier and Sophie Rousseaux and Lauriane Kuhn and Myriam Ferro and Julien Thévenon and Raffaella Catena and Irwin Davidson and Jérôme Garin and Saadi Khochbin and Cécile Caron},
doi = {10.1083/jcb.200604141},
issn = {0021-9525 1540-8140 0021-9525},
year = {2007},
date = {2007-01-01},
journal = {The Journal of cell biology},
volume = {176},
number = {3},
pages = {283--294},
abstract = {During male germ cell postmeiotic maturation, dramatic chromatin reorganization occurs, which is driven by completely unknown mechanisms. For the first time, we describe a specific reprogramming of mouse pericentric heterochromatin. Initiated when histones undergo global acetylation in early elongating spermatids, this process leads to the establishment of new DNA packaging structures organizing the pericentric regions in condensing spermatids. Five new histone variants were discovered, which are expressed in late spermiogenic cells. Two of them, which we named H2AL1 and H2AL2, specifically mark the pericentric regions in condensing spermatids and participate in the formation of new nucleoprotein structures. Moreover, our investigations also suggest that TH2B, an already identified testis-specific H2B variant of unknown function, could provide a platform for the structural transitions accompanying the incorporation of these new histone variants.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
During male germ cell postmeiotic maturation, dramatic chromatin reorganization occurs, which is driven by completely unknown mechanisms. For the first time, we describe a specific reprogramming of mouse pericentric heterochromatin. Initiated when histones undergo global acetylation in early elongating spermatids, this process leads to the establishment of new DNA packaging structures organizing the pericentric regions in condensing spermatids. Five new histone variants were discovered, which are expressed in late spermiogenic cells. Two of them, which we named H2AL1 and H2AL2, specifically mark the pericentric regions in condensing spermatids and participate in the formation of new nucleoprotein structures. Moreover, our investigations also suggest that TH2B, an already identified testis-specific H2B variant of unknown function, could provide a platform for the structural transitions accompanying the incorporation of these new histone variants.
2006
Govin Jérôme, Caron Cécile, Escoffier Emmanuelle, Ferro Myriam, Kuhn Lauriane, Rousseaux Sophie, Eddy Edward M, Garin Jérôme, Khochbin Saadi
Post-meiotic shifts in HSPA2/HSP70.2 chaperone activity during mouse spermatogenesis. Article de journal
Dans: The Journal of biological chemistry, vol. 281, no. 49, p. 37888–37892, 2006, ISSN: 0021-9258 1083-351X 0021-9258.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{govin_post-meiotic_2006,
title = {Post-meiotic shifts in HSPA2/HSP70.2 chaperone activity during mouse spermatogenesis.},
author = {Jérôme Govin and Cécile Caron and Emmanuelle Escoffier and Myriam Ferro and Lauriane Kuhn and Sophie Rousseaux and Edward M Eddy and Jérôme Garin and Saadi Khochbin},
doi = {10.1074/jbc.M608147200},
issn = {0021-9258 1083-351X 0021-9258},
year = {2006},
date = {2006-12-01},
journal = {The Journal of biological chemistry},
volume = {281},
number = {49},
pages = {37888--37892},
abstract = {HSPA2 (formerly HSP70.2) is a testis-specific member of the HSP70 family known to play a critical role in the completion of meiosis during male germ cell differentiation. Although abundantly present in post-meiotic cells, its function during spermiogenesis remained obscure. Here, using a global proteomic approach to identify genome-organizing proteins in condensing spermatids, we discovered an unexpected role for HSPA2, which acquires new functions and becomes tightly associated with major spermatid DNA-packaging proteins, transition proteins 1 and 2. Hence, HSPA2 is identified here as the first transition protein chaperone, and these data shed a new light on the yet totally unknown process of genome-condensing structure assembly in spermatids.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
HSPA2 (formerly HSP70.2) is a testis-specific member of the HSP70 family known to play a critical role in the completion of meiosis during male germ cell differentiation. Although abundantly present in post-meiotic cells, its function during spermiogenesis remained obscure. Here, using a global proteomic approach to identify genome-organizing proteins in condensing spermatids, we discovered an unexpected role for HSPA2, which acquires new functions and becomes tightly associated with major spermatid DNA-packaging proteins, transition proteins 1 and 2. Hence, HSPA2 is identified here as the first transition protein chaperone, and these data shed a new light on the yet totally unknown process of genome-condensing structure assembly in spermatids.
Brosson Damien, Kuhn Lauriane, Delbac Frédéric, Garin Jérôme, Vivarès Christian P, Texier Catherine
Proteomic analysis of the eukaryotic parasite Encephalitozoon cuniculi (microsporidia): a reference map for proteins expressed in late sporogonial stages. Article de journal
Dans: Proteomics, vol. 6, no. 12, p. 3625–3635, 2006, ISSN: 1615-9853 1615-9853.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{brosson_proteomic_2006,
title = {Proteomic analysis of the eukaryotic parasite Encephalitozoon cuniculi (microsporidia): a reference map for proteins expressed in late sporogonial stages.},
author = {Damien Brosson and Lauriane Kuhn and Frédéric Delbac and Jérôme Garin and Christian P Vivarès and Catherine Texier},
doi = {10.1002/pmic.200500796},
issn = {1615-9853 1615-9853},
year = {2006},
date = {2006-06-01},
journal = {Proteomics},
volume = {6},
number = {12},
pages = {3625--3635},
abstract = {The microsporidian Encephalitozoon cuniculi is a unicellular obligate intracellular parasite considered as an emerging opportunistic human pathogen. The differentiation phase of its life cycle leads to the formation of stress-resistant spores. The E. cuniculi genome (2.9 Mbp) having been sequenced, we undertook a descriptive proteomic study of a spore-rich cell population isolated from culture supernatants. A combination of 2-DE and 2-DE-free techniques was applied to whole-cell protein extracts. Protein identification was performed using an automated MALDI-TOF-MS platform and a nanoLC-MS/MS instrument. A reference 2-DE map of about 350 major spots with multiple isoforms was obtained, and for the first time in microsporidia, a large set of unique proteins (177) including proteins with unknown function in a proportion of 25.6% was identified. The data are mainly discussed with reference to secretion and spore structural features, energy and carbohydrate metabolism, cell cycle control and parasite survival in the environment.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The microsporidian Encephalitozoon cuniculi is a unicellular obligate intracellular parasite considered as an emerging opportunistic human pathogen. The differentiation phase of its life cycle leads to the formation of stress-resistant spores. The E. cuniculi genome (2.9 Mbp) having been sequenced, we undertook a descriptive proteomic study of a spore-rich cell population isolated from culture supernatants. A combination of 2-DE and 2-DE-free techniques was applied to whole-cell protein extracts. Protein identification was performed using an automated MALDI-TOF-MS platform and a nanoLC-MS/MS instrument. A reference 2-DE map of about 350 major spots with multiple isoforms was obtained, and for the first time in microsporidia, a large set of unique proteins (177) including proteins with unknown function in a proportion of 25.6% was identified. The data are mainly discussed with reference to secretion and spore structural features, energy and carbohydrate metabolism, cell cycle control and parasite survival in the environment.
Sarry Jean-Emmanuel, Kuhn Lauriane, Ducruix Céline, Lafaye Alexandra, Junot Christophe, Hugouvieux Véronique, Jourdain Agnès, Bastien Olivier, Fievet Julie B, Vailhen Dominique, Amekraz Badia, Moulin Christophe, Ezan Eric, Garin Jérôme, Bourguignon Jacques
The early responses of Arabidopsis thaliana cells to cadmium exposure explored by protein and metabolite profiling analyses. Article de journal
Dans: Proteomics, vol. 6, no. 7, p. 2180–2198, 2006, ISSN: 1615-9853 1615-9853.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{sarry_early_2006,
title = {The early responses of Arabidopsis thaliana cells to cadmium exposure explored by protein and metabolite profiling analyses.},
author = {Jean-Emmanuel Sarry and Lauriane Kuhn and Céline Ducruix and Alexandra Lafaye and Christophe Junot and Véronique Hugouvieux and Agnès Jourdain and Olivier Bastien and Julie B Fievet and Dominique Vailhen and Badia Amekraz and Christophe Moulin and Eric Ezan and Jérôme Garin and Jacques Bourguignon},
doi = {10.1002/pmic.200500543},
issn = {1615-9853 1615-9853},
year = {2006},
date = {2006-04-01},
journal = {Proteomics},
volume = {6},
number = {7},
pages = {2180--2198},
abstract = {To get more insight into plant cell response to cadmium (Cd) stress, both proteomic and metabolomic "differential display" analyses were performed on Arabidopsis thaliana cells exposed to different concentrations of the toxic chemical. After a 24 h treatment, soluble proteins extracted from untreated and treated cells were separated by 2-D-PAGE and image analyses were performed to quantify and compare protein levels. Proteins up- and down-regulated in response to Cd were identified by MS and mapped into specific metabolic pathways and cellular processes, highlighting probable activation of the carbon, nitrogen, and sulfur metabolic pathways. For some of these proteins, Northern blot and RT-PCR analyses were performed to test transcript accumulation in response to Cd. In parallel, metabolite profiling analyses by LC coupled to ESI MS were initiated to better characterize the metabolic adaptation to the chemical stress. This study revealed that the main variation at the metabolite level came from the presence of six different families of phytochelatins, in A. thaliana cells treated with Cd, whose accumulation increases with Cd concentrations. Taken together these data provide an overview of the molecular and cellular changes elicited by Cd exposure.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
To get more insight into plant cell response to cadmium (Cd) stress, both proteomic and metabolomic "differential display" analyses were performed on Arabidopsis thaliana cells exposed to different concentrations of the toxic chemical. After a 24 h treatment, soluble proteins extracted from untreated and treated cells were separated by 2-D-PAGE and image analyses were performed to quantify and compare protein levels. Proteins up- and down-regulated in response to Cd were identified by MS and mapped into specific metabolic pathways and cellular processes, highlighting probable activation of the carbon, nitrogen, and sulfur metabolic pathways. For some of these proteins, Northern blot and RT-PCR analyses were performed to test transcript accumulation in response to Cd. In parallel, metabolite profiling analyses by LC coupled to ESI MS were initiated to better characterize the metabolic adaptation to the chemical stress. This study revealed that the main variation at the metabolite level came from the presence of six different families of phytochelatins, in A. thaliana cells treated with Cd, whose accumulation increases with Cd concentrations. Taken together these data provide an overview of the molecular and cellular changes elicited by Cd exposure.
Lévine Alain, Vannier Françoise, Absalon Cédric, Kuhn Lauriane, Jackson Peter, Scrivener Elaine, Labas Valérie, Vinh Joëlle, Courtney Patrick, Garin Jérôme, Séror Simone J
Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes. Article de journal
Dans: Proteomics, vol. 6, no. 7, p. 2157–2173, 2006, ISSN: 1615-9853 1615-9853.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{levine_analysis_2006,
title = {Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes.},
author = {Alain Lévine and Françoise Vannier and Cédric Absalon and Lauriane Kuhn and Peter Jackson and Elaine Scrivener and Valérie Labas and Joëlle Vinh and Patrick Courtney and Jérôme Garin and Simone J Séror},
doi = {10.1002/pmic.200500352},
issn = {1615-9853 1615-9853},
year = {2006},
date = {2006-04-01},
journal = {Proteomics},
volume = {6},
number = {7},
pages = {2157--2173},
abstract = {The physiological role of proteins phosphorylated on serine/threonine/tyrosine (Ser/Thr/Tyr) residues or the identity of the corresponding kinases and phosphatases is generally poorly understood in bacteria. As a first step in analysing the importance of such phosphorylation, we sought to establish the nature of the Ser/Thr/Tyr phosphoproteome in Bacillus subtilis, using in vivo labelling with [(32)P]-orthophosphate, one-unit pH 2-DE, combined with MS. Highly reproducible 2-D profiles of phosphoproteins were obtained with early stationary-phase cells. The 2-D profiles contained at least 80 clearly labelled spots in the pH range 4-7. Forty-six spots were analysed by MS (confirmed in most cases by LC-MS/MS), identifying a total of 29 different proteins, with 19 identified for the first time as bacterial phosphoproteins. These phosphoproteins are implicated in a wide variety of cellular processes, including carbon and energy metabolism, transport, stress and development. Significant changes to the profiles were obtained as a result of cold, heat or osmotic shock, demonstrating that, in stationary-phase cells, the phosphoproteome is dynamic. An initial comparative study indicated that at least 25 [(32)P]-labelled spots were also stained by Pro-Q Diamond, with apparently six additional phosphoproteins uniquely detected by Pro-Q.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The physiological role of proteins phosphorylated on serine/threonine/tyrosine (Ser/Thr/Tyr) residues or the identity of the corresponding kinases and phosphatases is generally poorly understood in bacteria. As a first step in analysing the importance of such phosphorylation, we sought to establish the nature of the Ser/Thr/Tyr phosphoproteome in Bacillus subtilis, using in vivo labelling with [(32)P]-orthophosphate, one-unit pH 2-DE, combined with MS. Highly reproducible 2-D profiles of phosphoproteins were obtained with early stationary-phase cells. The 2-D profiles contained at least 80 clearly labelled spots in the pH range 4-7. Forty-six spots were analysed by MS (confirmed in most cases by LC-MS/MS), identifying a total of 29 different proteins, with 19 identified for the first time as bacterial phosphoproteins. These phosphoproteins are implicated in a wide variety of cellular processes, including carbon and energy metabolism, transport, stress and development. Significant changes to the profiles were obtained as a result of cold, heat or osmotic shock, demonstrating that, in stationary-phase cells, the phosphoproteome is dynamic. An initial comparative study indicated that at least 25 [(32)P]-labelled spots were also stained by Pro-Q Diamond, with apparently six additional phosphoproteins uniquely detected by Pro-Q.
Peuvel-Fanget Isabelle, Polonais Valérie, Brosson Damien, Texier Catherine, Kuhn Lauriane, Peyret Pierre, Vivarès Christian, Delbac Frédéric
EnP1 and EnP2, two proteins associated with the Encephalitozoon cuniculi endospore, the chitin-rich inner layer of the microsporidian spore wall. Article de journal
Dans: International journal for parasitology, vol. 36, no. 3, p. 309–318, 2006, ISSN: 0020-7519 0020-7519.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{peuvel-fanget_enp1_2006,
title = {EnP1 and EnP2, two proteins associated with the Encephalitozoon cuniculi endospore, the chitin-rich inner layer of the microsporidian spore wall.},
author = {Isabelle Peuvel-Fanget and Valérie Polonais and Damien Brosson and Catherine Texier and Lauriane Kuhn and Pierre Peyret and Christian Vivarès and Frédéric Delbac},
doi = {10.1016/j.ijpara.2005.10.005},
issn = {0020-7519 0020-7519},
year = {2006},
date = {2006-03-01},
journal = {International journal for parasitology},
volume = {36},
number = {3},
pages = {309--318},
abstract = {Microsporidia are obligate intracellular parasites forming environmentally resistant spores that harbour a rigid cell wall. This wall comprises an outer layer or exospore and a chitin-rich inner layer or endospore. So far, only a chitin deacetylase-like protein has been shown to localize to the Encephalitozoon cuniculi endospore and either one or two proteins have been clearly assigned to the exospore in two Encephalitozoon species: SWP1 in E. cuniculi, SWP1 and SWP2 in Encephalitozoon intestinalis. Here, we report the identification of two new spore wall proteins in E. cuniculi, EnP1 and EnP2, the genes of which are both located on chromosome I (ECU01_0820 and ECU01_1270, respectively) and have no known homologue. Detected by immunoscreening of an E. cuniculi cDNA library, enp1 is characterized by small-sized 5' and 3' untranslated regions and is highly expressed throughout the whole intracellular cycle. The encoded basic 40 kDa antigen displays a high proportion of cysteine residues, arguing for a significant role of disulfide bridges in spore wall assembly. EnP2 is a 22 kDa serine-rich protein that is predicted to be O-glycosylated and glycosylated phosphatidyl inositol-anchored. Although having been identified by mass spectrometry of a dithiothreitol-soluble fraction, this protein contains only two cysteine residues. Mouse polyclonal antibodies were raised against EnP1 and EnP2 recombinant proteins produced in Escherichia coli Our immunolocalisation data indicate that EnP1 and EnP2 are targeted to the cell surface as early as the onset of sporogony and are finally associated with the chitin-rich layer of the wall in mature spores.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Microsporidia are obligate intracellular parasites forming environmentally resistant spores that harbour a rigid cell wall. This wall comprises an outer layer or exospore and a chitin-rich inner layer or endospore. So far, only a chitin deacetylase-like protein has been shown to localize to the Encephalitozoon cuniculi endospore and either one or two proteins have been clearly assigned to the exospore in two Encephalitozoon species: SWP1 in E. cuniculi, SWP1 and SWP2 in Encephalitozoon intestinalis. Here, we report the identification of two new spore wall proteins in E. cuniculi, EnP1 and EnP2, the genes of which are both located on chromosome I (ECU01_0820 and ECU01_1270, respectively) and have no known homologue. Detected by immunoscreening of an E. cuniculi cDNA library, enp1 is characterized by small-sized 5' and 3' untranslated regions and is highly expressed throughout the whole intracellular cycle. The encoded basic 40 kDa antigen displays a high proportion of cysteine residues, arguing for a significant role of disulfide bridges in spore wall assembly. EnP2 is a 22 kDa serine-rich protein that is predicted to be O-glycosylated and glycosylated phosphatidyl inositol-anchored. Although having been identified by mass spectrometry of a dithiothreitol-soluble fraction, this protein contains only two cysteine residues. Mouse polyclonal antibodies were raised against EnP1 and EnP2 recombinant proteins produced in Escherichia coli Our immunolocalisation data indicate that EnP1 and EnP2 are targeted to the cell surface as early as the onset of sporogony and are finally associated with the chitin-rich layer of the wall in mature spores.
Sarry Jean-Emmanuel, Kuhn Lauriane, Lay Pascaline Le, Garin Jérôme, Bourguignon Jacques
Dynamics of Arabidopsis thaliana soluble proteome in response to different nutrient culture conditions. Article de journal
Dans: Electrophoresis, vol. 27, no. 2, p. 495–507, 2006, ISSN: 0173-0835 0173-0835.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{sarry_dynamics_2006,
title = {Dynamics of Arabidopsis thaliana soluble proteome in response to different nutrient culture conditions.},
author = {Jean-Emmanuel Sarry and Lauriane Kuhn and Pascaline Le Lay and Jérôme Garin and Jacques Bourguignon},
doi = {10.1002/elps.200500561},
issn = {0173-0835 0173-0835},
year = {2006},
date = {2006-02-01},
journal = {Electrophoresis},
volume = {27},
number = {2},
pages = {495--507},
abstract = {In an effort to determine the best extraction procedure compatible with the high-reproducible 2-DE, different methods of soluble protein extraction from Arabidopsis cell culture suspensions grown in Gamborg B5 medium were tested. A reference 2-DE map was established for this soluble extract revealing 1184 spots. The most abundant protein spots were excised, trypsin-digested, and mass spectra obtained via MALDI-TOF and/or LC coupled to ESI-MS. Three hundred and thirty one proteins were identified and their functions were defined based on sequence comparisons and classified in different protein families. In order to analyze the impact of culture medium on the Arabidopsis proteome, we performed the 2-DE map from Arabidopsis cell suspensions cultured in another growth medium Murashige and Skoog (M-S) and 327 major spots were identified. Using PDQuest imaging analysis, significant increases in the amount of several housekeeping enzymes, stress/defense proteins, and heat shock proteins were found in M-S medium. Modified expression of certain proteins and detection of new isoforms involved in nitrate assimilation, nitrogen, and sulfur metabolism were also observed in the M-S medium. This study provides the first 2-DE maps of the soluble proteome of Arabidopsis cell suspensions. The comparative analysis of the Arabidopsis proteome in respect to different nutrient supplies shows that the culture medium may significantly influence the expression pattern of major soluble proteins in Arabidopsis cells. This work also constitutes an important step for further proteomic analysis concerning cell responses to abiotic or biotic stresses.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
In an effort to determine the best extraction procedure compatible with the high-reproducible 2-DE, different methods of soluble protein extraction from Arabidopsis cell culture suspensions grown in Gamborg B5 medium were tested. A reference 2-DE map was established for this soluble extract revealing 1184 spots. The most abundant protein spots were excised, trypsin-digested, and mass spectra obtained via MALDI-TOF and/or LC coupled to ESI-MS. Three hundred and thirty one proteins were identified and their functions were defined based on sequence comparisons and classified in different protein families. In order to analyze the impact of culture medium on the Arabidopsis proteome, we performed the 2-DE map from Arabidopsis cell suspensions cultured in another growth medium Murashige and Skoog (M-S) and 327 major spots were identified. Using PDQuest imaging analysis, significant increases in the amount of several housekeeping enzymes, stress/defense proteins, and heat shock proteins were found in M-S medium. Modified expression of certain proteins and detection of new isoforms involved in nitrate assimilation, nitrogen, and sulfur metabolism were also observed in the M-S medium. This study provides the first 2-DE maps of the soluble proteome of Arabidopsis cell suspensions. The comparative analysis of the Arabidopsis proteome in respect to different nutrient supplies shows that the culture medium may significantly influence the expression pattern of major soluble proteins in Arabidopsis cells. This work also constitutes an important step for further proteomic analysis concerning cell responses to abiotic or biotic stresses.
2005
Brosson Damien, Kuhn Lauriane, Prensier Gérard, Vivarès Christian P, Texier Catherine
The putative chitin deacetylase of Encephalitozoon cuniculi: a surface protein implicated in microsporidian spore-wall formation. Article de journal
Dans: FEMS microbiology letters, vol. 247, no. 1, p. 81–90, 2005, ISSN: 0378-1097 0378-1097.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{brosson_putative_2005,
title = {The putative chitin deacetylase of Encephalitozoon cuniculi: a surface protein implicated in microsporidian spore-wall formation.},
author = {Damien Brosson and Lauriane Kuhn and Gérard Prensier and Christian P Vivarès and Catherine Texier},
doi = {10.1016/j.femsle.2005.04.031},
issn = {0378-1097 0378-1097},
year = {2005},
date = {2005-06-01},
journal = {FEMS microbiology letters},
volume = {247},
number = {1},
pages = {81--90},
abstract = {Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide deacetylase family with a suspected chitin deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide deacetylase family with a suspected chitin deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.
1999
Otten L, Salomone J Y, Helfer A, Schmidt J, Hammann P, Ruffray P De
Sequence and functional analysis of the left-hand part of the Ŧ-region from the nopaline-type Ti plasmid, pTiC58. Article de journal
Dans: Plant molecular biology, vol. 41, no. 6, p. 765–776, 1999, ISSN: 0167-4412 0167-4412, (Place: Netherlands).
Résumé | Liens | BibTeX | Étiquettes: Agrobacterium tumefaciens/*genetics/pathogenicity, Bacterial/chemistry/*genetics, Bacterial/genetics, Chromosome Mapping, DNA, Gene Deletion, Genes, Genetic Complementation Test, Lycopersicon esculentum/genetics/microbiology, Medicinal/genetics/microbiology, Molecular Sequence Data, Mutation, Phylogeny, Plant Tumors/genetics/microbiology, Plants, Plasmids/chemistry/*genetics, PPSE, Sequence Analysis, Species Specificity, Tobacco/genetics/microbiology, Toxic, Virulence/genetics
@article{otten_sequence_1999,
title = {Sequence and functional analysis of the left-hand part of the Ŧ-region from the nopaline-type Ti plasmid, pTiC58.},
author = {L Otten and J Y Salomone and A Helfer and J Schmidt and P Hammann and P De Ruffray},
doi = {10.1023/a:1006370207379},
issn = {0167-4412 0167-4412},
year = {1999},
date = {1999-12-01},
journal = {Plant molecular biology},
volume = {41},
number = {6},
pages = {765--776},
abstract = {The Agrobacterium tumefaciens nopaline strain C58 transfers a large, 29 kb T-DNA into plant cells during infection. Part of this DNA (the 'common DNA') is also found on the T-DNA of octopine strains, the remaining DNA is nopaline strain-specific. Up to now, only parts of the C58 T-DNA and related T37 T-DNA have been sequenced. We have sequenced the remainder of the nopaline-specific T-DNA (containing genes a to d) and acs to iaaM. Gene c codes for a new unknown T-DNA protein. Gene a is homologous to the agrocinopine synthase gene. Genes b, c', d and e are part of a larger family: they are related to the T-DNA genes 5, rolB, lso and 3'. Genes 5, rolB and lso induce or modify plant growth and have been called T-DNA oncogenes. Our studies show that gene 3' (located on the TR-DNA of octopine strains) is also oncogenic. Although the b-e T-DNA fragment from C58 and its individual genes lack growth-inducing activity, an a-acs deletion mutant was distinctly less virulent on Kalanchoe daigremontiana and showed reduced shoot formation on Kalanchoe tubiflora. Shoot formation could be restored by genes c and c' in co-infection experiments. Contrary to an earlier report, a C58 e gene deletion mutant was fully virulent on all plants tested.},
note = {Place: Netherlands},
keywords = {Agrobacterium tumefaciens/*genetics/pathogenicity, Bacterial/chemistry/*genetics, Bacterial/genetics, Chromosome Mapping, DNA, Gene Deletion, Genes, Genetic Complementation Test, Lycopersicon esculentum/genetics/microbiology, Medicinal/genetics/microbiology, Molecular Sequence Data, Mutation, Phylogeny, Plant Tumors/genetics/microbiology, Plants, Plasmids/chemistry/*genetics, PPSE, Sequence Analysis, Species Specificity, Tobacco/genetics/microbiology, Toxic, Virulence/genetics},
pubstate = {published},
tppubtype = {article}
}
The Agrobacterium tumefaciens nopaline strain C58 transfers a large, 29 kb T-DNA into plant cells during infection. Part of this DNA (the 'common DNA') is also found on the T-DNA of octopine strains, the remaining DNA is nopaline strain-specific. Up to now, only parts of the C58 T-DNA and related T37 T-DNA have been sequenced. We have sequenced the remainder of the nopaline-specific T-DNA (containing genes a to d) and acs to iaaM. Gene c codes for a new unknown T-DNA protein. Gene a is homologous to the agrocinopine synthase gene. Genes b, c', d and e are part of a larger family: they are related to the T-DNA genes 5, rolB, lso and 3'. Genes 5, rolB and lso induce or modify plant growth and have been called T-DNA oncogenes. Our studies show that gene 3' (located on the TR-DNA of octopine strains) is also oncogenic. Although the b-e T-DNA fragment from C58 and its individual genes lack growth-inducing activity, an a-acs deletion mutant was distinctly less virulent on Kalanchoe daigremontiana and showed reduced shoot formation on Kalanchoe tubiflora. Shoot formation could be restored by genes c and c' in co-infection experiments. Contrary to an earlier report, a C58 e gene deletion mutant was fully virulent on all plants tested.
