Axelle RENODON-CORNIERE
Chargée de recherche CNRS
| Équipe : |
Publications
1 publication
Renodon-Corniere, Axelle; Mikawa, Tsutomu; Kuwabara, Naoyuki; Ito, Kentaro; Levitsky, Dmitri; Iwasaki, Hiroshi; Takahashi, Masayuki
Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-Loop Formation than for Oligonucleotide Strand Exchange Article de journal
Dans: International Journal of Molecular Sciences, vol. 25, no. 7, 2024, ISSN: 1422-0067.
@article{ijms25073633,
title = {Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-Loop Formation than for Oligonucleotide Strand Exchange},
author = {Axelle Renodon-Corniere and Tsutomu Mikawa and Naoyuki Kuwabara and Kentaro Ito and Dmitri Levitsky and Hiroshi Iwasaki and Masayuki Takahashi},
url = {https://www.mdpi.com/1422-0067/25/7/3633},
doi = {10.3390/ijms25073633},
issn = {1422-0067},
year = {2024},
date = {2024-01-01},
urldate = {2024-01-01},
journal = {International Journal of Molecular Sciences},
volume = {25},
number = {7},
abstract = {Human Rad51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. Both the activator protein Swi5/Sfr1 and Ca2+ ions stimulate different reaction steps and induce perpendicular DNA base alignment in the presynaptic complex. To investigate the role of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) required Ca2+ concentrations greater than 5 mM, whereas 1 mM Ca2+ was sufficient for strand exchange between two oligonucleotides. Structural changes indicated by increased fluorescence intensity of poly(dεA) (a poly(dA) analog) reached a plateau at 1 mM Ca2+. Ca2+ > 2 mM was required for saturation of linear dichroism signal intensity at 260 nm, associated with rigid perpendicular DNA base orientation, suggesting a correlation with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. Our results indicate parallels between the Mg2+ activation of RecA and the Ca2+ activation of HsRad51.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Human Rad51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. Both the activator protein Swi5/Sfr1 and Ca2+ ions stimulate different reaction steps and induce perpendicular DNA base alignment in the presynaptic complex. To investigate the role of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) required Ca2+ concentrations greater than 5 mM, whereas 1 mM Ca2+ was sufficient for strand exchange between two oligonucleotides. Structural changes indicated by increased fluorescence intensity of poly(dεA) (a poly(dA) analog) reached a plateau at 1 mM Ca2+. Ca2+ > 2 mM was required for saturation of linear dichroism signal intensity at 260 nm, associated with rigid perpendicular DNA base orientation, suggesting a correlation with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. Our results indicate parallels between the Mg2+ activation of RecA and the Ca2+ activation of HsRad51.
2 publications
Dubois, Nolwenn; Muñoz-Garcia, Javier; Heymann, Dominique; Renodon-Cornière, Axelle
High glucose exposure drives intestinal barrier dysfunction by altering its morphological, structural and functional properties. Article de journal
Dans: Biochemical pharmacology, vol. 216, p. 115765, 2023, ISSN: 1873-2968 0006-2952, (Place: England).
@article{dubois_high_2023,
title = {High glucose exposure drives intestinal barrier dysfunction by altering its morphological, structural and functional properties.},
author = {Nolwenn Dubois and Javier Muñoz-Garcia and Dominique Heymann and Axelle Renodon-Cornière},
url = {hal-04210189v1 },
doi = {10.1016/j.bcp.2023.115765},
issn = {1873-2968 0006-2952},
year = {2023},
date = {2023-10-01},
urldate = {2023-10-01},
journal = {Biochemical pharmacology},
volume = {216},
pages = {115765},
abstract = {High dietary glucose consumption and hyperglycemia can result in chronic complications. Several studies suggest that high glucose (HG) induces dysfunction of the intestinal barrier. However, the precise changes remain unclear. In our study, we used in vitro models composed of Caco-2 and/or HT29-MTX cells in both monoculture and co-culture to assess the effects of long-term HG exposure on the morphological, structural, and functional properties of the intestinal barrier. Cells were grown in medium containing normal physiologic glucose (NG, 5.5 mM) or a clinically relevant HG (25 mM) concentration until 21 days. Results demonstrated that HG induced morphological changes, with the layers appearing denser and less organized than under physiological conditions, which is in accordance with the increased migration capacity of Caco-2 cells and proliferation properties of HT29-MTX cells. Although we mostly observed a small decrease in mRNA and protein expressions of three junction proteins (ZO-1, OCLN and E-cad) in both Caco-2 and HT29-MTX cells cultured in HG medium, confocal microscopy showed that HG induced a remarkable reduction in their immunofluorescence intensity, triggering disruption of their associated structural network. In addition, we highlighted that HG affected different functionalities (permeability, mucus production and alkaline phosphatase activity) of monolayers with Caco-2 and HT29-MTX cells. Interestingly, these alterations were stronger in co-culture than in monoculture, suggesting a cross-relationship between enterocytes and goblet cells. Controlling hyperglycemia remains a major therapeutical method for reducing damage to the intestinal barrier and improving therapies.},
note = {Place: England},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
High dietary glucose consumption and hyperglycemia can result in chronic complications. Several studies suggest that high glucose (HG) induces dysfunction of the intestinal barrier. However, the precise changes remain unclear. In our study, we used in vitro models composed of Caco-2 and/or HT29-MTX cells in both monoculture and co-culture to assess the effects of long-term HG exposure on the morphological, structural, and functional properties of the intestinal barrier. Cells were grown in medium containing normal physiologic glucose (NG, 5.5 mM) or a clinically relevant HG (25 mM) concentration until 21 days. Results demonstrated that HG induced morphological changes, with the layers appearing denser and less organized than under physiological conditions, which is in accordance with the increased migration capacity of Caco-2 cells and proliferation properties of HT29-MTX cells. Although we mostly observed a small decrease in mRNA and protein expressions of three junction proteins (ZO-1, OCLN and E-cad) in both Caco-2 and HT29-MTX cells cultured in HG medium, confocal microscopy showed that HG induced a remarkable reduction in their immunofluorescence intensity, triggering disruption of their associated structural network. In addition, we highlighted that HG affected different functionalities (permeability, mucus production and alkaline phosphatase activity) of monolayers with Caco-2 and HT29-MTX cells. Interestingly, these alterations were stronger in co-culture than in monoculture, suggesting a cross-relationship between enterocytes and goblet cells. Controlling hyperglycemia remains a major therapeutical method for reducing damage to the intestinal barrier and improving therapies.
Panez-Toro, Isidora; Muñoz-García, Javier; Vargas-Franco, Jorge W.; Renodon-Cornière, Axelle; Heymann, Marie-Françoise; Lézot, Frédéric; Heymann, Dominique
Advances in Osteosarcoma Article de journal
Dans: Current Osteoporosis Reports, 2023, ISSN: 1544-1873, 1544-2241.
@article{panez-toro_advances_2023,
title = {Advances in Osteosarcoma},
author = {Isidora Panez-Toro and Javier Muñoz-García and Jorge W. Vargas-Franco and Axelle Renodon-Cornière and Marie-Françoise Heymann and Frédéric Lézot and Dominique Heymann},
url = {https://link.springer.com/10.1007/s11914-023-00803-9
inserm-04119793v1 },
doi = {10.1007/s11914-023-00803-9},
issn = {1544-1873, 1544-2241},
year = {2023},
date = {2023-06-01},
urldate = {2023-06-01},
journal = {Current Osteoporosis Reports},
abstract = {Purpose of Review
This article gives a brief overview of the most recent developments in osteosarcoma treatment, including targeting of signaling pathways, immune checkpoint inhibitors, drug delivery strategies as single or combined approaches, and the identification of new therapeutic targets to face this highly heterogeneous disease.
Recent Findings
Osteosarcoma is one of the most common primary malignant bone tumors in children and young adults, with a high risk of bone and lung metastases and a 5-year survival rate around 70% in the absence of metastases and 30% if metastases are detected at the time of diagnosis. Despite the novel advances in neoadjuvant chemotherapy, the effective treatment for osteosarcoma has not improved in the last 4 decades. The emergence of immunotherapy has transformed the paradigm of treatment, focusing therapeutic strategies on the potential of immune checkpoint inhibitors. However, the most recent clinical trials show a slight improvement over the conventional polychemotherapy scheme.
Summary
The tumor microenvironment plays a crucial role in the pathogenesis of osteosarcoma by controlling the tumor growth, the metastatic process and the drug resistance and paved the way of new therapeutic options that must be validated by accurate pre-clinical studies and clinical trials.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Purpose of Review
This article gives a brief overview of the most recent developments in osteosarcoma treatment, including targeting of signaling pathways, immune checkpoint inhibitors, drug delivery strategies as single or combined approaches, and the identification of new therapeutic targets to face this highly heterogeneous disease.
Recent Findings
Osteosarcoma is one of the most common primary malignant bone tumors in children and young adults, with a high risk of bone and lung metastases and a 5-year survival rate around 70% in the absence of metastases and 30% if metastases are detected at the time of diagnosis. Despite the novel advances in neoadjuvant chemotherapy, the effective treatment for osteosarcoma has not improved in the last 4 decades. The emergence of immunotherapy has transformed the paradigm of treatment, focusing therapeutic strategies on the potential of immune checkpoint inhibitors. However, the most recent clinical trials show a slight improvement over the conventional polychemotherapy scheme.
Summary
The tumor microenvironment plays a crucial role in the pathogenesis of osteosarcoma by controlling the tumor growth, the metastatic process and the drug resistance and paved the way of new therapeutic options that must be validated by accurate pre-clinical studies and clinical trials.
This article gives a brief overview of the most recent developments in osteosarcoma treatment, including targeting of signaling pathways, immune checkpoint inhibitors, drug delivery strategies as single or combined approaches, and the identification of new therapeutic targets to face this highly heterogeneous disease.
Recent Findings
Osteosarcoma is one of the most common primary malignant bone tumors in children and young adults, with a high risk of bone and lung metastases and a 5-year survival rate around 70% in the absence of metastases and 30% if metastases are detected at the time of diagnosis. Despite the novel advances in neoadjuvant chemotherapy, the effective treatment for osteosarcoma has not improved in the last 4 decades. The emergence of immunotherapy has transformed the paradigm of treatment, focusing therapeutic strategies on the potential of immune checkpoint inhibitors. However, the most recent clinical trials show a slight improvement over the conventional polychemotherapy scheme.
Summary
The tumor microenvironment plays a crucial role in the pathogenesis of osteosarcoma by controlling the tumor growth, the metastatic process and the drug resistance and paved the way of new therapeutic options that must be validated by accurate pre-clinical studies and clinical trials.
1 publication
Chabot, Thomas; Defontaine, Alain; Marquis, Damien; Renodon-Corniere, Axelle; Courtois, Emmanuelle; Fleury, Fabrice; Cheraud, Yvonnick
New phosphorylation sites of rad51 by c-met modulates presynaptic filament stability Article de journal
Dans: Cancers, vol. 11, no. 3, 2019, ISSN: 20726694.
@article{Chabot2019a,
title = {New phosphorylation sites of rad51 by c-met modulates presynaptic filament stability},
author = {Thomas Chabot and Alain Defontaine and Damien Marquis and Axelle Renodon-Corniere and Emmanuelle Courtois and Fabrice Fleury and Yvonnick Cheraud},
doi = {10.3390/cancers11030413},
issn = {20726694},
year = {2019},
date = {2019-01-01},
journal = {Cancers},
volume = {11},
number = {3},
abstract = {Genomic instability through deregulation of DNA repair pathways can initiate cancer and subsequently result in resistance to chemo and radiotherapy. Understanding these biological mechanisms is therefore essential to overcome cancer. RAD51 is the central protein of the Homologous Recombination (HR) DNA repair pathway, which leads to faithful DNA repair of DSBs. The recombinase activity of RAD51 requires nucleofilament formation and is regulated by post-translational modifications such as phosphorylation. In the last decade, studies have suggested the existence of a relationship between receptor tyrosine kinases (RTK) and Homologous Recombination DNA repair. Among these RTK the c-MET receptor is often overexpressed or constitutively activated in many cancer types and its inhibition induces the decrease of HR. In this study, we show for the first time that c-MET is able to phosphorylate the RAD51 protein. We demonstrate in vitro that c-MET phosphorylates four tyrosine residues localized mainly in the subunit-subunit interface of RAD51. Whereas these post-translational modifications do not affect the presynaptic filament formation, they strengthen its stability against the inhibitor effect of the BRC peptide obtained from BRCA2. Taken together, these results confirm the role of these modifications in the regulation of the BRCA2-RAD51 interaction and underline the importance of c-MET in DNA damage response.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Genomic instability through deregulation of DNA repair pathways can initiate cancer and subsequently result in resistance to chemo and radiotherapy. Understanding these biological mechanisms is therefore essential to overcome cancer. RAD51 is the central protein of the Homologous Recombination (HR) DNA repair pathway, which leads to faithful DNA repair of DSBs. The recombinase activity of RAD51 requires nucleofilament formation and is regulated by post-translational modifications such as phosphorylation. In the last decade, studies have suggested the existence of a relationship between receptor tyrosine kinases (RTK) and Homologous Recombination DNA repair. Among these RTK the c-MET receptor is often overexpressed or constitutively activated in many cancer types and its inhibition induces the decrease of HR. In this study, we show for the first time that c-MET is able to phosphorylate the RAD51 protein. We demonstrate in vitro that c-MET phosphorylates four tyrosine residues localized mainly in the subunit-subunit interface of RAD51. Whereas these post-translational modifications do not affect the presynaptic filament formation, they strengthen its stability against the inhibitor effect of the BRC peptide obtained from BRCA2. Taken together, these results confirm the role of these modifications in the regulation of the BRCA2-RAD51 interaction and underline the importance of c-MET in DNA damage response.
2021
Muñoz-Garcia, Javier; Jubelin, Camille; Loussouarn, Aurélie; Goumard, Matisse; Griscom, Laurent; Renodon-Cornière, Axelle; Heymann, Marie-Françoise; Heymann, Dominique
In vitro three-dimensional cell cultures for bone sarcomas Article de journal
Dans: Journal of Bone Oncology, vol. 30, p. 100379, 2021, ISSN: 2212-1374.
@article{munoz2021vitro,
title = {In vitro three-dimensional cell cultures for bone sarcomas},
author = {Javier Muñoz-Garcia and Camille Jubelin and Aurélie Loussouarn and Matisse Goumard and Laurent Griscom and Axelle Renodon-Cornière and Marie-Françoise Heymann and Dominique Heymann},
editor = {Elsevier},
doi = {10.1016/j.jbo.2021.100379},
issn = {2212-1374},
year = {2021},
date = {2021-10-01},
urldate = {2021-10-01},
journal = {Journal of Bone Oncology},
volume = {30},
pages = {100379},
publisher = {Elsevier},
abstract = {Bone sarcomas are rare tumour entities that arise from the mesenchyme most of which are highly heterogeneous at the cellular, genetic and epigenetic levels. The three main types are osteosarcoma, Ewing sarcoma, and chondrosarcoma. These oncological entities are characterised by high morbidity and mortality and an absence of significant therapeutic improvement in the last four decades. In the field of oncology, in vitro cultures of cancer cells have been extensively used for drug screening unfortunately with limited success. Indeed, despite the massive knowledge acquired from conventional 2D culture methods, scientific community has been challenged by the loss of efficacy of drugs when moved to clinical trials. The recent explosion of new 3D culture methods is paving the way to more relevant in vitro models mimicking the in vivo tumour environment (e.g. bone structure) with biological responses close to the in vivo context. The present review gives a brief overview of the latest advances of the 3D culture methods used for studying primary bone sarcomas.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Bone sarcomas are rare tumour entities that arise from the mesenchyme most of which are highly heterogeneous at the cellular, genetic and epigenetic levels. The three main types are osteosarcoma, Ewing sarcoma, and chondrosarcoma. These oncological entities are characterised by high morbidity and mortality and an absence of significant therapeutic improvement in the last four decades. In the field of oncology, in vitro cultures of cancer cells have been extensively used for drug screening unfortunately with limited success. Indeed, despite the massive knowledge acquired from conventional 2D culture methods, scientific community has been challenged by the loss of efficacy of drugs when moved to clinical trials. The recent explosion of new 3D culture methods is paving the way to more relevant in vitro models mimicking the in vivo tumour environment (e.g. bone structure) with biological responses close to the in vivo context. The present review gives a brief overview of the latest advances of the 3D culture methods used for studying primary bone sarcomas.
2020
Loussouarn, Aurélie; Béhar, Ghislaine; Pecorari, Frédéric; Croyal, Mikael; Renodon-Cornière, Axelle
Characterization of Affitin proteolytic digestion in biorelevant media and improvement of their stabilities via protein engineering Article de journal
Dans: Sci Rep, vol. 10, no. 1, p. 19703, 2020, ISSN: 2045-2322.
@article{pmid33184451,
title = {Characterization of Affitin proteolytic digestion in biorelevant media and improvement of their stabilities via protein engineering},
author = {Aurélie Loussouarn and Ghislaine Béhar and Frédéric Pecorari and Mikael Croyal and Axelle Renodon-Cornière},
doi = {10.1038/s41598-020-76855-z},
issn = {2045-2322},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Sci Rep},
volume = {10},
number = {1},
pages = {19703},
abstract = {Affitins are a novel class of small 7 kDa artificial proteins which can be used as antibody substitutes in therapeutic, diagnostic and biotechnological applications. One challenge for this type of protein agent is their behaviour in the context of oral administration. The digestive system is central, and biorelevant media have fast emerged as relevant and reliable tools for evaluating the bioavailability of drugs. This study describes, for the first time, the stability of Affitins under simulated gastric and intestinal digestion conditions. Affitins appear to be degraded into stable fragments in in vitro gastric medium. We identified cleavage sites generated by pepsin that were silenced by site-directed mutagenesis. This protein engineering allowed us to enhance Affitin properties. We showed that a mutant M1 containing a double mutation of amino acid residues 6 and 7 in H4 and C3 Affitins acquired a resistance against proteolytic digestion. In addition, these mutations were beneficial for target affinity, as well as for production yield. Finally, we found that the mutated residues kept or increased the important pH and temperature stabilities of Affitins. These improvements are particularly sought after in the development of engineered binding proteins for research tools, preclinical studies and clinical applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Affitins are a novel class of small 7 kDa artificial proteins which can be used as antibody substitutes in therapeutic, diagnostic and biotechnological applications. One challenge for this type of protein agent is their behaviour in the context of oral administration. The digestive system is central, and biorelevant media have fast emerged as relevant and reliable tools for evaluating the bioavailability of drugs. This study describes, for the first time, the stability of Affitins under simulated gastric and intestinal digestion conditions. Affitins appear to be degraded into stable fragments in in vitro gastric medium. We identified cleavage sites generated by pepsin that were silenced by site-directed mutagenesis. This protein engineering allowed us to enhance Affitin properties. We showed that a mutant M1 containing a double mutation of amino acid residues 6 and 7 in H4 and C3 Affitins acquired a resistance against proteolytic digestion. In addition, these mutations were beneficial for target affinity, as well as for production yield. Finally, we found that the mutated residues kept or increased the important pH and temperature stabilities of Affitins. These improvements are particularly sought after in the development of engineered binding proteins for research tools, preclinical studies and clinical applications.
2019
Kalichuk, Valentina; Kambarev, Stanimir; Béhar, Ghislaine; Chalopin, Benjamin; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric
Affitins: Ribosome Display for Selection of Aho7c-Based Affinity Proteins Chapitre d'ouvrage
Dans: S, Zielonka; S, Krah (Ed.): vol. 2070, p. 19–41, Methods in Molecular Biology, 2019, ISBN: 978-1-4939-9853-1.
@inbook{pmid31625088,
title = {Affitins: Ribosome Display for Selection of Aho7c-Based Affinity Proteins},
author = {Valentina Kalichuk and Stanimir Kambarev and Ghislaine Béhar and Benjamin Chalopin and Axelle Renodon-Cornière and Barbara Mouratou and Frédéric Pecorari},
editor = {Zielonka S and Krah S},
doi = {10.1007/978-1-4939-9853-1_2},
isbn = {978-1-4939-9853-1},
year = {2019},
date = {2019-10-18},
urldate = {2019-10-18},
journal = {Methods Mol Biol},
volume = {2070},
pages = {19--41},
edition = {Methods in Molecular Biology},
series = {Genotype Phenotype Coupling},
abstract = {Engineered protein scaffolds have made a tremendous contribution to the panel of affinity tools owing to their favorable biophysical properties that make them useful for many applications. In 2007, our group paved the way for using archaeal Sul7d proteins for the design of artificial affinity ligands, so-called Affitins. For many years, Sac7d and Sso7d have been used as molecular basis to obtain binders for various targets. Recently, we characterized their old gifted protein family and identified Aho7c, originating from Acidianus hospitalis, as the shortest member (60 amino-acids) with impressive stability (96.5 °C, pH 0-12). Here, we describe the construction of Aho7c combinatorial libraries and their use for selection of binders by ribosome display.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}
Engineered protein scaffolds have made a tremendous contribution to the panel of affinity tools owing to their favorable biophysical properties that make them useful for many applications. In 2007, our group paved the way for using archaeal Sul7d proteins for the design of artificial affinity ligands, so-called Affitins. For many years, Sac7d and Sso7d have been used as molecular basis to obtain binders for various targets. Recently, we characterized their old gifted protein family and identified Aho7c, originating from Acidianus hospitalis, as the shortest member (60 amino-acids) with impressive stability (96.5 °C, pH 0-12). Here, we describe the construction of Aho7c combinatorial libraries and their use for selection of binders by ribosome display.
Béhar, Ghislaine; Renodon-Cornière, Axelle; Kambarev, Stanimir; Vukojicic, Petar; Caroff, Nathalie; Corvec, Stéphane; Mouratou, Barbara; Pecorari, Frédéric
Whole-bacterium ribosome display selection for isolation of highly specific anti-Staphyloccocus aureus Affitins for detection- and capture-based biomedical applications Article de journal
Dans: Biotechnol Bioeng, vol. 116, no. 8, p. 1844–1855, 2019, ISSN: 1097-0290.
@article{pmid30982947,
title = {Whole-bacterium ribosome display selection for isolation of highly specific anti-Staphyloccocus aureus Affitins for detection- and capture-based biomedical applications},
author = {Ghislaine Béhar and Axelle Renodon-Cornière and Stanimir Kambarev and Petar Vukojicic and Nathalie Caroff and Stéphane Corvec and Barbara Mouratou and Frédéric Pecorari},
doi = {10.1002/bit.26989},
issn = {1097-0290},
year = {2019},
date = {2019-05-15},
urldate = {2019-01-01},
journal = {Biotechnol Bioeng},
volume = {116},
number = {8},
pages = {1844--1855},
abstract = {Detection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole-living-cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K of 108 ± 2 nM and has a high thermostability (T = 77.0°C). Anti-S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead-fishing, and fluorescence imaging. Thus, novel anti-bacteria Affitins which are cost-effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface-exposed marker. This class of reagents can be useful in diagnostic and biomedical applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Detection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole-living-cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K of 108 ± 2 nM and has a high thermostability (T = 77.0°C). Anti-S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead-fishing, and fluorescence imaging. Thus, novel anti-bacteria Affitins which are cost-effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface-exposed marker. This class of reagents can be useful in diagnostic and biomedical applications.
2018
Kalichuk, Valentina; Renodon-Cornière, Axelle; Béhar, Ghislaine; Carrión, Federico; Obal, Gonzalo; Maillasson, Mike; Mouratou, Barbara; Préat, Véronique; Pecorari, Frédéric
A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM Article de journal
Dans: Biotechnol Bioeng, vol. 115, no. 2, p. 290–299, 2018, ISSN: 1097-0290.
@article{pmid28976545,
title = {A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM},
author = {Valentina Kalichuk and Axelle Renodon-Cornière and Ghislaine Béhar and Federico Carrión and Gonzalo Obal and Mike Maillasson and Barbara Mouratou and Véronique Préat and Frédéric Pecorari},
doi = {10.1002/bit.26463},
issn = {1097-0290},
year = {2018},
date = {2018-01-01},
urldate = {2018-01-01},
journal = {Biotechnol Bioeng},
volume = {115},
number = {2},
pages = {290--299},
abstract = {Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from Sulfolobus genera. Their efficiency as reagents for intracellular targeting, enzyme inhibition, affinity purification, immunolocalization, and various other applications has been demonstrated. Recently, we have characterized the 7 kDa DNA-binding family, and Aho7c originating from Acidianus hospitalis was shown to be its smallest member with thermostability comparable to those of Sac7d and Sso7d. Here, after four rounds of selection by ribosome display against the human recombinant Epithelial Cell Adhesion Molecule (hrEpCAM), we obtained novel Aho7c-based Affitins. The binders were expressed in soluble form in Escherichia coli, displayed high stability (up to 74°C; pH 0-12) and were shown to be specific for the hrEpCAM extracellular domain with picomolar affinities (K = 110 pM). Thus, we propose Aho7c as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d-based ones (60 vs. 66 amino acids).},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from Sulfolobus genera. Their efficiency as reagents for intracellular targeting, enzyme inhibition, affinity purification, immunolocalization, and various other applications has been demonstrated. Recently, we have characterized the 7 kDa DNA-binding family, and Aho7c originating from Acidianus hospitalis was shown to be its smallest member with thermostability comparable to those of Sac7d and Sso7d. Here, after four rounds of selection by ribosome display against the human recombinant Epithelial Cell Adhesion Molecule (hrEpCAM), we obtained novel Aho7c-based Affitins. The binders were expressed in soluble form in Escherichia coli, displayed high stability (up to 74°C; pH 0-12) and were shown to be specific for the hrEpCAM extracellular domain with picomolar affinities (K = 110 pM). Thus, we propose Aho7c as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d-based ones (60 vs. 66 amino acids).
2016
Kalichuk, Valentina; Béhar, Ghislaine; Renodon-Cornière, Axelle; Danovski, Georgi; Obal, Gonzalo; Barbet, Jacques; Mouratou, Barbara; Pecorari, Frédéric
The archaeal 7 kDa DNA-binding proteins: extended characterization of an old gifted family Article de journal
Dans: Sci Rep, vol. 6, p. 37274, 2016, ISSN: 2045-2322.
@article{pmid27853299,
title = {The archaeal 7 kDa DNA-binding proteins: extended characterization of an old gifted family},
author = {Valentina Kalichuk and Ghislaine Béhar and Axelle Renodon-Cornière and Georgi Danovski and Gonzalo Obal and Jacques Barbet and Barbara Mouratou and Frédéric Pecorari},
doi = {10.1038/srep37274},
issn = {2045-2322},
year = {2016},
date = {2016-11-17},
urldate = {2016-11-17},
journal = {Sci Rep},
volume = {6},
pages = {37274},
abstract = {The "7 kDa DNA-binding" family, also known as the Sul7d family, is composed of chromatin proteins from the Sulfolobales archaeal order. Among them, Sac7d and Sso7d have been the focus of several studies with some characterization of their properties. Here, we studied eleven other proteins alongside Sac7d and Sso7d under the same conditions. The dissociation constants of the purified proteins for binding to double-stranded DNA (dsDNA) were determined in phosphate-buffered saline at 25 °C and were in the range from 11 μM to 22 μM with a preference for G/C rich sequences. In accordance with the extremophilic origin of their hosts, the proteins were found highly stable from pH 0 to pH 12 and at temperatures from 85.5 °C to 100 °C. Thus, these results validate eight putative "7 kDa DNA-binding" family proteins and show that they behave similarly regarding both their function and their stability among various genera and species. As Sac7d and Sso7d have found numerous uses as molecular biology reagents and artificial affinity proteins, this study also sheds light on even more attractive proteins that will facilitate engineering of novel highly robust reagents.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The "7 kDa DNA-binding" family, also known as the Sul7d family, is composed of chromatin proteins from the Sulfolobales archaeal order. Among them, Sac7d and Sso7d have been the focus of several studies with some characterization of their properties. Here, we studied eleven other proteins alongside Sac7d and Sso7d under the same conditions. The dissociation constants of the purified proteins for binding to double-stranded DNA (dsDNA) were determined in phosphate-buffered saline at 25 °C and were in the range from 11 μM to 22 μM with a preference for G/C rich sequences. In accordance with the extremophilic origin of their hosts, the proteins were found highly stable from pH 0 to pH 12 and at temperatures from 85.5 °C to 100 °C. Thus, these results validate eight putative "7 kDa DNA-binding" family proteins and show that they behave similarly regarding both their function and their stability among various genera and species. As Sac7d and Sso7d have found numerous uses as molecular biology reagents and artificial affinity proteins, this study also sheds light on even more attractive proteins that will facilitate engineering of novel highly robust reagents.
Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric
Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins Article de journal
Dans: J Chromatogr A, vol. 1441, p. 44–51, 2016, ISSN: 1873-3778.
@article{pmid26952369,
title = {Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins},
author = {Ghislaine Béhar and Axelle Renodon-Cornière and Barbara Mouratou and Frédéric Pecorari},
doi = {10.1016/j.chroma.2016.02.068},
issn = {1873-3778},
year = {2016},
date = {2016-04-08},
urldate = {2016-04-01},
journal = {J Chromatogr A},
volume = {1441},
pages = {44--51},
abstract = {Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals.
2014
Esnault, Charles; Renodon-Cornière, Axelle; Takahashi, Masayuki; Casse, Nathalie; Delorme, Nicolas; Louarn, Guy; Fleury, Fabrice; Pilard, Jean-François; Chénais, Benoît
Assessment of DNA binding to human Rad51 protein by using quartz crystal microbalance and atomic force microscopy: effects of ADP and BRC4-28 peptide inhibitor Article de journal
Dans: Chemphyschem, vol. 15, no. 17, p. 3753–3760, 2014, ISSN: 1439-7641.
@article{pmid25208912,
title = {Assessment of DNA binding to human Rad51 protein by using quartz crystal microbalance and atomic force microscopy: effects of ADP and BRC4-28 peptide inhibitor},
author = {Charles Esnault and Axelle Renodon-Cornière and Masayuki Takahashi and Nathalie Casse and Nicolas Delorme and Guy Louarn and Fabrice Fleury and Jean-François Pilard and Benoît Chénais},
doi = {10.1002/cphc.201402451},
issn = {1439-7641},
year = {2014},
date = {2014-12-01},
urldate = {2014-12-01},
journal = {Chemphyschem},
volume = {15},
number = {17},
pages = {3753--3760},
abstract = {The interaction of human Rad51 protein (HsRad51) with single-stranded deoxyribonucleic acid (ssDNA) was investigated by using quartz crystal microbalance (QCM) monitoring and atomic force microscopy (AFM) visualization. Gold surfaces for QCM and AFM were modified by electrografting of the in situ generated aryldiazonium salt from the sulfanilic acid to obtain the organic layer Au-ArSO3 H. The Au-ArSO3 H layer was activated by using a solution of PCl5 in CH2 Cl2 to give a Au-ArSO2 Cl layer. The modified surface was then used to immobilize long ssDNA molecules. The results obtained showed that the presence of adenosine diphosphate promotes the protein autoassociation rather than nucleation around DNA. In addition, when the BRC4-28 peptide inhibitor was used, both QCM and AFM confirmed the inhibitory effect of BRC4-28 toward HsRad51 autoassociation. Altogether these results show the suitability of this modified surface to investigate the kinetics and structure of DNA-protein interactions and for the screening of inhibitors. },
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The interaction of human Rad51 protein (HsRad51) with single-stranded deoxyribonucleic acid (ssDNA) was investigated by using quartz crystal microbalance (QCM) monitoring and atomic force microscopy (AFM) visualization. Gold surfaces for QCM and AFM were modified by electrografting of the in situ generated aryldiazonium salt from the sulfanilic acid to obtain the organic layer Au-ArSO3 H. The Au-ArSO3 H layer was activated by using a solution of PCl5 in CH2 Cl2 to give a Au-ArSO2 Cl layer. The modified surface was then used to immobilize long ssDNA molecules. The results obtained showed that the presence of adenosine diphosphate promotes the protein autoassociation rather than nucleation around DNA. In addition, when the BRC4-28 peptide inhibitor was used, both QCM and AFM confirmed the inhibitory effect of BRC4-28 toward HsRad51 autoassociation. Altogether these results show the suitability of this modified surface to investigate the kinetics and structure of DNA-protein interactions and for the screening of inhibitors.
Normand, Anaïs; Rivière, Emmanuelle; Renodon-Cornière, Axelle
Identification and characterization of human Rad51 inhibitors by screening of an existing drug library Article de journal
Dans: Biochemical Pharmacology, vol. 91, no. 3, p. 293-300, 2014, ISSN: 0006-2952.
@article{NORMAND2014293,
title = {Identification and characterization of human Rad51 inhibitors by screening of an existing drug library},
author = {Anaïs Normand and Emmanuelle Rivière and Axelle Renodon-Cornière},
url = {https://www.sciencedirect.com/science/article/pii/S0006295214004547},
doi = {https://doi.org/10.1016/j.bcp.2014.07.033},
issn = {0006-2952},
year = {2014},
date = {2014-01-01},
urldate = {2014-01-01},
journal = {Biochemical Pharmacology},
volume = {91},
number = {3},
pages = {293-300},
abstract = {Homologous Recombination (HR) plays an essential role in cellular proliferation and in maintaining genomic stability by repairing DNA double-stranded breaks that appear during replication. Rad51, a key protein of HR in eukaryotes, can have an elevated expression level in tumor cells, which correlates with their resistance to anticancer therapies. Therefore, targeted inhibition of Rad51 through inhibitor may improve the tumor response to these therapies. In order to identify small molecules that inhibit Rad51 activity, we screened the Prestwick Library (1120 molecules) for their effect on the strand exchange reaction catalyzed by Rad51. We found that Chicago Sky Blue (CSB) is a potent inhibitor of Rad51, showing IC50 values in the low nanomolar range (400nM). Biochemical analysis demonstrated that the inhibitory mechanism probably occurs by disrupting the Rad51 association with the single-stranded DNA, which prevents the nucleoprotein filament formation, the first step of the protein activity. Structure Activity Relationship analysis with a number of compounds that shared structure homology with CSB was also performed. The sensitivity of Rad51 inhibition to CSB modifications suggests specific interactions between the molecule and Rad51 nucleofilament. CSB and some of its analogs open up new perspectives in the search for agents capable of potentiating chemo- and radio-therapy treatments for cancer. Moreover, these compounds may be excellent tools to analyze Rad51 cellular functions. Our study also highlights how CSB and its analogs, which are frequently used in colorants, stains and markers, could be responsible of unwanted side effects by perturbing the DNA repair process.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Homologous Recombination (HR) plays an essential role in cellular proliferation and in maintaining genomic stability by repairing DNA double-stranded breaks that appear during replication. Rad51, a key protein of HR in eukaryotes, can have an elevated expression level in tumor cells, which correlates with their resistance to anticancer therapies. Therefore, targeted inhibition of Rad51 through inhibitor may improve the tumor response to these therapies. In order to identify small molecules that inhibit Rad51 activity, we screened the Prestwick Library (1120 molecules) for their effect on the strand exchange reaction catalyzed by Rad51. We found that Chicago Sky Blue (CSB) is a potent inhibitor of Rad51, showing IC50 values in the low nanomolar range (400nM). Biochemical analysis demonstrated that the inhibitory mechanism probably occurs by disrupting the Rad51 association with the single-stranded DNA, which prevents the nucleoprotein filament formation, the first step of the protein activity. Structure Activity Relationship analysis with a number of compounds that shared structure homology with CSB was also performed. The sensitivity of Rad51 inhibition to CSB modifications suggests specific interactions between the molecule and Rad51 nucleofilament. CSB and some of its analogs open up new perspectives in the search for agents capable of potentiating chemo- and radio-therapy treatments for cancer. Moreover, these compounds may be excellent tools to analyze Rad51 cellular functions. Our study also highlights how CSB and its analogs, which are frequently used in colorants, stains and markers, could be responsible of unwanted side effects by perturbing the DNA repair process.