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Accueil > Thèmes de recherche > Aspects moléculaires du vivant > Spectrométrie de Masse Fonctionnelle des Métastases

Functional Mass Spectrometry in Metastasis

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RESEARCH THEME

While the world of tumor suppressors and promoters has been active and productive for decades, to date less than ten genes have been identified as metastasis suppressors, and much about their function mechanism is unknown. This may explain why their therapeutic potential is still untapped. Since metastasis, in most types of cancers, is what puts patients at risk, the search for molecules that can help understand the mechanism of natural suppressors and take advantage of their activity for diagnosis and treatment is both timely and desirable.

Since most biological functions ultimately rest on the activity of proteins, the elucidation of the molecular mechanisms underlying metastasis at the protein level has naturally become a major step towards this goal. Mass spectrometry (MS) working hand in hand with structural and cell biology methods, is in a unique position to provide accurate tools for both structural and functional studies of metastasis suppressor gene products.

Our group develops methods at the interface of biochemistry and mass spectrometry for the characterization of protein-ligand and protein-protein interactions. Recent years have seen the emergence of high-resolution, quantitative approaches which put MS squarely in the game of structure-function elucidation. We developed tools to quantify protein-ligand interactions for both noncovalent drug candidates (MS method for KD measurements that accurately reflect solution-phase affinity) and covalent drug candidates (localization by quantification, SSPaQ method and software).

This last method can also be applied to the localization and quantification of protein modifications. The modifications we characterize can be post-translational (PTMs), or induced voluntarily or adventitiously, through exposure to chemical agents or radiation.

We currently focus on elucidating the mechanism of action of metastasis suppressor proteins involved in signaling and proteinase inhibition. In parallel, we look at inhibitor ligands of metastasis promoter ion channels. To the end, we build on our expertise in the characterization of integral membrane proteins to gather structural information that is preliminary and/or complementary to protein complexes structure elucidation.

Besides research activities, part of the team also pilots CBM’s mass spectrometry facility, offering analysis of peptides, whole proteins, oligonucleotides, hybrid biomolecules and small molecules (see link below).

More information about the platform and mass spectrometry

ILLUSTRATIONS

Selected publications

  • Chait B. T., Cadene M., Olinares P. D., Rout M. and Shi Y.
    Revealing higher order protein structure using mass spectrometry
    J. Am. Soc. Mass Spec. (2016) 27, 952-65.
  • Gabant G., Boyer A. and Cadene M.*
    SSPaQ : A subtractive segmentation approach for the exhaustive parallel quantification of the extent of protein modification at every possible site
    J. Am. Soc. Mass Spec. (2016) 27, 1328-1343.
  • Bouchet§ N., Jaillet§ J., Gabant§ G., Brillet B., Briseno Roa L., Cadene M. and Augé-Gouillou C.
    cAMP protein kinase phosphorylates the Mos1 transposase and regulates its activity : evidences from mass spectrometry and biochemical analyses
    Nucleic Acids Res. (2014) 42, 1117-28. §equal contributors
  • Beaufour M., Godin F., Vallee B., Cadene* M. and Benedetti H.
    Interaction proteomics suggests a new role for the Tfs1 protein in yeast
    J. Proteome Res. (2012) 11, 3211-3218.
  • Jaquillard L., Saab F., Schoentgen F. and Cadene* M.
    Improved accuracy of low-affinity protein-ligand equilibrium dissociation constants determined by ESI-MS
    J. Am. Soc. Mass Spec. (2012) 23, 908-922.
  • Marques-Carvalho M. J., Sahoo N., Muskett F. W., Vieira-Pires R. S., Gabant G., Cadene M., Schönherr R. and Morais-Cabral J. H.
    Structural, biochemical and functional characterization of the cyclic nucleotide binding homology domain from the mouse EAG1 potassium channel
    J. Mol. Biol. (2012) 423, 34-46.
  • Gabant G. and Cadene* M.
    Mass spectrometry of full-length integral membrane proteins to define functionally relevant structural features
    Methods (2008) 46, 54-61.

Available equipment

Our mass spectrometry instruments consists of a MALDI TOF/TOF MS (Ultraflex, Bruker) and an ESI-Ion Trap MS (with ETD, Bruker). We also share, within FR2708, two UHR Q/TOF MS (with CID or ETD fragmentation), equipped with ESI / nano ESI sources. Two nano UHPLC, an analytical UHPLC (U3000) and a micro-HPLC (CapLC, Waters) can be coupled to our instruments for LC–MS and LC–MS/MS online analysis. Additionally, a laminar flow hood is available for the sterile and dust-free manipulation of electrophoresis gels.


Group members

CADENE Martine Senior CNRS researcher, Thematic group leader   @

BEAUFOUR Martine Lecturer, University of Orleans   @

COLAS Cyril CNRS Research engineer (50% FR2708)   @

GABANT Guillaume CNRS Engineer   @

BIJOT Teddy Phd student   @


Former members

Pr Kevin Mark, CUNY, New York, USA
Dr Lucie Jaquillard, Société Smartox, Grenoble, France
Emmanuelle Mebold, Plateforme de Spectrométrie de Masse et Chromatographie de l’Institut des Molécules et Matériaux, Le Mans, France
Rémy Puppo, Plateforme Protéomique de l’Institut de Microbiologie de la Méditerranée, Marseille, France
Pawel Szczesniak, Doctorant, Université de Guiessen, Allemagne
Nicolas Gayot, SNECMA, Levallois, France
Corinne Buré,
Christophe Février, Ingénieur Clients Waters, France
Sonya Gebczynski, Seratec, France
David Ginguené


Publications

2016   Références trouvées : 4

Marques-Carvalho M. J., Oppermann J., Muñoz E., Fernandes A. S., Gabant G., Cadene M., Heinemann S. H., Schönherr R. and Morais-Cabral J. H.  (2016)

Molecular insights into the mechanism of calmodulin inhibition of the EAG1 potassium channel

Structure (2016) 24 (10) 1742-1754 - doi : 10.1016/j.str.2016.07.020
The human EAG1 potassium channel belongs to the superfamily of KCNH voltage-gated potassium channels that have roles in cardiac repolarization and neuronal excitability. EAG1 is strongly inhibited by Ca2+/calmodulin (CaM) through a mechanism that is not understood. We determined the binding properties of CaM with each one of three previously identified binding sites (BDN, BDC1, and BDC2), analyzed binding to protein stretches that include more than one site, and determined the effect of neighboring globular domains on the binding properties. The determination of the crystal structure of CaM bound to BDC2 shows the channel fragment interacting with only the C lobe of calmodulin and adopting an unusual bent conformation. Based on this structure and on a functional and biochemical analysis of mutants, we propose a model for the mechanism of inhibition whereby the local conformational change induced by CaM binding at BDC2 lies at the basis of channel modulation.

The human EAG1 potassium channel belongs to the superfamily of KCNH voltage-gated potassium channels that have roles in cardiac repolarization and neuronal excitability. EAG1 is strongly inhibited by Ca2+/calmodulin (CaM) through a mechanism that is not understood. We determined the binding properties of CaM with each one of three previously identified binding sites (BDN, BDC1, and BDC2), analyzed binding to protein stretches that include more than one site, and determined the effect of neighboring globular domains on the binding properties. The determination of the crystal structure of CaM bound to BDC2 shows the channel fragment interacting with only the C lobe of calmodulin and adopting an unusual bent conformation. Based on this structure and on a functional and biochemical analysis of mutants, we propose a model for the mechanism of inhibition whereby the local conformational change induced by CaM binding at BDC2 lies at the basis of channel modulation.

Gabant G., Boyer A. and Cadène M.  (2016)

SSPaQ : a subtractive segmentation approach for the exhaustive parallel quantification of the extent of protein modification at a very possible site

Journal of the American Society for Mass Spectrometry (2016) 27 (8) 1328-1343 - doi : 10.1007/s13361-016-1416-y
Protein modifications, whether chemically induced or post-translational (PTMs), play an essential role for the biological activity of proteins. Understanding biological processes and alterations thereof will rely on the quantification of these modifications on individual residues. Here we present SSPaQ, a subtractive method for the parallel quantification of the extent of modification at each possible site of a protein. The method combines uniform isotopic labeling and proteolysis with MS, followed by a segmentation approach, a powerful tool to refine the quantification of the degree of modification of a peptide to a segment containing a single modifiable amino acid. The strength of this strategy resides in : (1) quantification of all modifiable sites in a protein without prior knowledge of the type(s) of modified residues ; (2) insensitivity to changes in the solubility and ionization efficiency of peptides upon modification ; and (3) detection of missed cleavages caused by the modification for mitigation. The SSPaQ method was applied to quantify modifications resulting from the interaction of human phosphatidyl ethanolamine binding protein 1 (hPEBP1), a metastasis suppressor gene product, with locostatin, a covalent ligand and antimigratory compound with demonstrated activity towards hPEBP1. Locostatin is shown to react with several residues of the protein. SSPaQ can more generally be applied to induced modification in the context of drugs that covalently bind their target protein. With an alternate front-end protocol, it could also be applied to the quantification of protein PTMs, provided a removal tool is available for that PTM.

Protein modifications, whether chemically induced or post-translational (PTMs), play an essential role for the biological activity of proteins. Understanding biological processes and alterations thereof will rely on the quantification of these modifications on individual residues. Here we present SSPaQ, a subtractive method for the parallel quantification of the extent of modification at each possible site of a protein. The method combines uniform isotopic labeling and proteolysis with MS, followed by a segmentation approach, a powerful tool to refine the quantification of the degree of modification of a peptide to a segment containing a single modifiable amino acid. The strength of this strategy resides in : (1) quantification of all modifiable sites in a protein without prior knowledge of the type(s) of modified residues ; (2) insensitivity to changes in the solubility and ionization efficiency of peptides upon modification ; and (3) detection of missed cleavages caused by the modification for mitigation. The SSPaQ method was applied to quantify modifications resulting from the interaction of human phosphatidyl ethanolamine binding protein 1 (hPEBP1), a metastasis suppressor gene product, with locostatin, a covalent ligand and antimigratory compound with demonstrated activity towards hPEBP1. Locostatin is shown to react with several residues of the protein. SSPaQ can more generally be applied to induced modification in the context of drugs that covalently bind their target protein. With an alternate front-end protocol, it could also be applied to the quantification of protein PTMs, provided a removal tool is available for that PTM.

Chait B. T., Cadène M., Olinares P. D., Rout M., Shi Y.  (2016)

Revealing Higher Order Protein Structure Using Mass Spectrometry

Journal of the American Society of Mass Spectrometry (2016) 27 (6) 952-965 - doi : 10.1007/s13361-016-1385-1.
The development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope.

The development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope.

Rivera-Torres I. O., Jin T. B., Cadène M., Chait B. T., Poget S. F.  (2016)

Discovery and characterization of a novel toxin from Dendroaspis angusticeps, named Tx7335, with an activating effect on the potassium channel KcsA

Scientific Reports 6,23904.
Due to their central role in essential physiological processes, potassium channels are common targets for animal toxins. These toxins in turn are of great value as tools for studying channel function and as lead compounds for drug development. Here, we used a direct toxin pull-down assay with immobilised KcsA potassium channel to isolate a novel KcsA-binding toxin (called Tx7335) from eastern green mamba snake (Dendroaspis angusticeps) venom. Sequencing of the toxin by Edman degradation and mass spectrometry revealed a 63 amino acid residue peptide with 4 disulphide bonds that belongs to the three-finger toxin family, but with a unique modification of its disulphide-bridge scaffold. The toxin induces a dose-dependent increase in both open probabilities and mean open times on KcsA in artificial bilayers. Thus, it unexpectedly behaves as a channel activator rather than an inhibitor. A charybdotoxin-sensitive mutant of KcsA exhibits similar susceptibility to Tx7335 as wild-type, indicating that the binding site for Tx7335 is distinct from that of canonical pore-blocker toxins. Based on the extracellular location of the toxin binding site (far away from the intracellular pH gate), we propose that Tx7335 increases potassium flow through KcsA by allosterically reducing inactivation of the channel.

Due to their central role in essential physiological processes, potassium channels are common targets for animal toxins. These toxins in turn are of great value as tools for studying channel function and as lead compounds for drug development. Here, we used a direct toxin pull-down assay with immobilised KcsA potassium channel to isolate a novel KcsA-binding toxin (called Tx7335) from eastern green mamba snake (Dendroaspis angusticeps) venom. Sequencing of the toxin by Edman degradation and mass spectrometry revealed a 63 amino acid residue peptide with 4 disulphide bonds that belongs to the three-finger toxin family, but with a unique modification of its disulphide-bridge scaffold. The toxin induces a dose-dependent increase in both open probabilities and mean open times on KcsA in artificial bilayers. Thus, it unexpectedly behaves as a channel activator rather than an inhibitor. A charybdotoxin-sensitive mutant of KcsA exhibits similar susceptibility to Tx7335 as wild-type, indicating that the binding site for Tx7335 is distinct from that of canonical pore-blocker toxins. Based on the extracellular location of the toxin binding site (far away from the intracellular pH gate), we propose that Tx7335 increases potassium flow through KcsA by allosterically reducing inactivation of the channel.


2015   Références trouvées : 1

Bertrand, M., Chabin, A., Colas, C., Cadène, M., Chaput, D., Brack, A., Cottin, H. and Westall,F.  (2015)

The AMINO experiment : exposure of amino acids in the EXPOSE-R experiment on the International Space Station and in laboratory

International Journal of Astrobiology (2015) 14 (1) 89-97 - doi : 10.1017/S1473550414000354
In order to confirm the results of previous experiments concerning the chemical behaviour of organic molecules in the space environment, organic molecules (amino acids and a dipeptide) in pure form and embedded in meteorite powder were exposed in the AMINO experiment in the EXPOSE-R facility onboard the International Space Station. After exposure to space conditions for 24 months (2843 h of irradiation), the samples were returned to the Earth and analysed in the laboratory for reactions caused by solar ultraviolet (UV) and other electromagnetic radiation. Laboratory UV exposure was carried out in parallel in the Cologne DLR Center (Deutsches Zentrum für Luft und Raumfahrt). The molecules were extracted from the sample holder and then (1) derivatized by silylation and analysed by gas chromatography coupled to a mass spectrometer (GC–MS) in order to quantify the rate of degradation of the compounds and (2) analysed by high-resolution mass spectrometry (HRMS) in order to understand the chemical reactions that occurred. The GC–MS results confirm that resistance to irradiation is a function of the chemical nature of the exposed molecules and of the wavelengths of the UV light. They also confirm the protective effect of a coating of meteorite powder. The most altered compounds were the dipeptides and aspartic acid while the most robust were compounds with a hydrocarbon chain. The MS analyses document the products of reactions, such as decarboxylation and decarbonylation of aspartic acid, taking place after UV exposure. Given the universality of chemistry in space, our results have a broader implication for the fate of organic molecules that seeded the planets as soon as they became habitable as well as for the effects of UV radiation on exposed molecules at the surface of Mars, for example.

In order to confirm the results of previous experiments concerning the chemical behaviour of organic molecules in the space environment, organic molecules (amino acids and a dipeptide) in pure form and embedded in meteorite powder were exposed in the AMINO experiment in the EXPOSE-R facility onboard the International Space Station. After exposure to space conditions for 24 months (2843 h of irradiation), the samples were returned to the Earth and analysed in the laboratory for reactions caused by solar ultraviolet (UV) and other electromagnetic radiation. Laboratory UV exposure was carried out in parallel in the Cologne DLR Center (Deutsches Zentrum für Luft und Raumfahrt). The molecules were extracted from the sample holder and then (1) derivatized by silylation and analysed by gas chromatography coupled to a mass spectrometer (GC–MS) in order to quantify the rate of degradation of the compounds and (2) analysed by high-resolution mass spectrometry (HRMS) in order to understand the chemical reactions that occurred. The GC–MS results confirm that resistance to irradiation is a function of the chemical nature of the exposed molecules and of the wavelengths of the UV light. They also confirm the protective effect of a coating of meteorite powder. The most altered compounds were the dipeptides and aspartic acid while the most robust were compounds with a hydrocarbon chain. The MS analyses document the products of reactions, such as decarboxylation and decarbonylation of aspartic acid, taking place after UV exposure. Given the universality of chemistry in space, our results have a broader implication for the fate of organic molecules that seeded the planets as soon as they became habitable as well as for the effects of UV radiation on exposed molecules at the surface of Mars, for example.


2014   Références trouvées : 5

Guarino C, Legowska M, Epinette C, Kellenberger C, Dallet-Choisy S, Sienczyk M, Gabant G, Cadene M, Zoikadis J, Vlahou A, Wysocka M, Marchand-Adam S, Jenne DE, Lesner A, Gauthier F and Korkmaz B.  (2014)

New selective peptidyl di(chlorophenyl)-phosphonate esters to visualize and block neutrophil proteinase 3 in human diseases

Journal of Biological Chemistry (2014) - doi : 10.1074/jbc.M114.591339
The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure : Ac-peptidylP(O-C6H4-4-Cl)2. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl-phosphonates to identify PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils, despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect and control PR3 activity in a variety of inflammatory diseases.

The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure : Ac-peptidylP(O-C6H4-4-Cl)2. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl-phosphonates to identify PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils, despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect and control PR3 activity in a variety of inflammatory diseases.

Morel A, Trontin JF, Corbineau F, Lomenech AM, Beaufour M, Reymond I, Le Metté C, Ader K, Harvengt L, Cadene M, Label P, Teyssier C, Lelu-Walter MA.   (2014)

Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh, maturing cotyledonary zygotic embryos : biological, carbohydrate and proteomic analyses

Planta (2014) 240 (5) 1075-1095 - doi : 10.1007/s00425-014-2125-z
Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

Bouchet N., Jaillet J., Gabant G., Brillet B., Briseno Roa L., Cadene M. and Augé-Gouillou C.  (2014)

cAMP protein kinase phosphorylates the Mos1 transposase and regulates its activity : evidences from mass spectrometry and biochemical analyses

Nucleic Acids Research 42 (2) 1117-1128 - doi : 10.1093/nar/gkt874
Genomic plasticity mediated by transposable elements can have a dramatic impact on genome integrity. In order to minimize its genotoxic effects, it is tightly regulated either by intrinsic mechanisms (linked to the element itself) or by host-mediated mechanisms. Using mass spectrometry, we show here for the first time that MOS1, the transposase driving the mobility of the mariner Mos1 element, is phosphorylated. We also show that the transposition activity of MOS1 is down regulated by PKA phosphorylation at S170, which renders the transposase unable to promote Mos1 transposition. One step in the transposition cycle, the assembly of the paired-end complex, is specifically inhibited. At the cellular level, we provide evidence that phosphorylation at S170 prevents the active transport of the transposase into the nucleus. Our data suggest that PKA phosphorylation may play a double role in the early stages of genome invasion by mariner elements.

Genomic plasticity mediated by transposable elements can have a dramatic impact on genome integrity. In order to minimize its genotoxic effects, it is tightly regulated either by intrinsic mechanisms (linked to the element itself) or by host-mediated mechanisms. Using mass spectrometry, we show here for the first time that MOS1, the transposase driving the mobility of the mariner Mos1 element, is phosphorylated. We also show that the transposition activity of MOS1 is down regulated by PKA phosphorylation at S170, which renders the transposase unable to promote Mos1 transposition. One step in the transposition cycle, the assembly of the paired-end complex, is specifically inhibited. At the cellular level, we provide evidence that phosphorylation at S170 prevents the active transport of the transposase into the nucleus. Our data suggest that PKA phosphorylation may play a double role in the early stages of genome invasion by mariner elements.

Teyssier C., Maury S., Beaufour, M., Grondin C., Delaunay A., Le Mette C., Ader K., Cadene M., Label P. and Lelu-Walter M. A.  (2014)

In search of markers for somatic embryo maturation in hybrid larch (Larix x eurolepis) : global DNA methylation and proteomic analyses

Physiologia plantarum (2014) 150 (2) 271-291 - doi : 10.1111/ppl.12081
A global DNA methylation and proteomics approach was used to investigate somatic embryo maturation in hybrid larch. Each developmental step during somatic embryogenesis was associated with a distinct and significantly different global DNA methylation level : from 45.8% mC for undifferentiated somatic embryos (1-week proliferation) to 61.5% mC for immature somatic embryos (1-week maturation), while maturation was associated with a decrease in DNA methylation to 53.4% for mature cotyledonary somatic embryos (8-weeks maturation). The presence of 5-azacytidine (hypo-methylating agent) or hydroxyurea (hyper-methylating agent) in the maturation medium altered the global DNA methylation status of the embryogenic cultures, and significantly reduced both their relative growth rate and embryogenic potential, suggesting an important role for DNA methylation in embryogenesis. Maturation was also assessed by examining changes in the total protein profile. Storage proteins, identified as legumin- and vicilin-like, appeared at the precotyledonary stage. In the proteomic study, total soluble proteins were extracted from embryos after 1 and 8 weeks of maturation, and separated by two-dimensional gel electrophoresis. There were 147 spots which showed significant differences between the stages of maturation ; they were found to be involved mainly in primary metabolism and the stabilization of the resulting metabolites. This indicated that the somatic embryo was still metabolically active at 8 weeks of maturation. This is the first report of analyses of global DNA methylation (including the effects of hyper- and hypo-treatments) and proteome during somatic embryogenesis in hybrid larch, and thus provides novel insights into maturation of conifer somatic embryos.

A global DNA methylation and proteomics approach was used to investigate somatic embryo maturation in hybrid larch. Each developmental step during somatic embryogenesis was associated with a distinct and significantly different global DNA methylation level : from 45.8% mC for undifferentiated somatic embryos (1-week proliferation) to 61.5% mC for immature somatic embryos (1-week maturation), while maturation was associated with a decrease in DNA methylation to 53.4% for mature cotyledonary somatic embryos (8-weeks maturation). The presence of 5-azacytidine (hypo-methylating agent) or hydroxyurea (hyper-methylating agent) in the maturation medium altered the global DNA methylation status of the embryogenic cultures, and significantly reduced both their relative growth rate and embryogenic potential, suggesting an important role for DNA methylation in embryogenesis. Maturation was also assessed by examining changes in the total protein profile. Storage proteins, identified as legumin- and vicilin-like, appeared at the precotyledonary stage. In the proteomic study, total soluble proteins were extracted from embryos after 1 and 8 weeks of maturation, and separated by two-dimensional gel electrophoresis. There were 147 spots which showed significant differences between the stages of maturation ; they were found to be involved mainly in primary metabolism and the stabilization of the resulting metabolites. This indicated that the somatic embryo was still metabolically active at 8 weeks of maturation. This is the first report of analyses of global DNA methylation (including the effects of hyper- and hypo-treatments) and proteome during somatic embryogenesis in hybrid larch, and thus provides novel insights into maturation of conifer somatic embryos.

Morel A., Teyssier C., Trontin J.F., Eliasova K., Pesek B., Beaufour M., Morabito D., Boizot N., Le Metté C., Belal-Bessai, L., Reymond I, Harvengt L., Cadène M., Corbineau F., Vágner M., Label P and Lelu-Walter M.A.  (2014)

Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability : transcriptomic and proteomic analyses

Physiologia Plantarum (2014) 152 (1) 184-120 - doi : 10.1111/ppl.12158
Maritime pine somatic embryos require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome (2D-SDS-PAGE with MS identification) of immature somatic embryos, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous ABA (GC-MS), soluble sugars (HPLC), starch, and confocal laser microscope observations. This multi-scale, integrated analysis was used to unravel early molecular and physiological events involved in somatic embryo development. Under unfavourable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation which may be antagonistic to somatic embryo development. Under favourable conditions (9G), somatic embryos adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin like protein and ubiquitin-protein ligase could be used as predictive markers of somatic embryo development whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in development of pine somatic embryos following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets.

Maritime pine somatic embryos require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome (2D-SDS-PAGE with MS identification) of immature somatic embryos, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous ABA (GC-MS), soluble sugars (HPLC), starch, and confocal laser microscope observations. This multi-scale, integrated analysis was used to unravel early molecular and physiological events involved in somatic embryo development. Under unfavourable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation which may be antagonistic to somatic embryo development. Under favourable conditions (9G), somatic embryos adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin like protein and ubiquitin-protein ligase could be used as predictive markers of somatic embryo development whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in development of pine somatic embryos following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets.


2012   Références trouvées : 7

Derache, C., Epinette, C., Roussel, A., Gabant, G., Cadene, M., Korkmaz, B., Gauthier, F. and Kellenberger, C.  (2012)

Crystal structure of greglin, a novel non-classical Kazal inhibitor, in complex with subtilisin

FEBS Journal 279 (24) 4466-4478
Greglin is an 83-residue serine protease inhibitor purified from the ovaries of the locust Schistocerca gregaria. Greglin is a strong inhibitor of subtilisin and human neutrophil elastase, acting at sub-nanomolar and nanomolar concentrations, respectively ; it also inhibits neutrophil cathepsin G, α-chymotrypsin and porcine pancreatic elastase, but to a lesser extent. In the present study, we show that greglin resists denaturation at high temperature (95 °C) and after exposure to acetonitrile and acidic or basic pH. Greglin is composed of two domains consisting of residues 1–20 and 21–83. Mass spectrometry indicates that the N-terminal domain (1–20) is post-translationally modified by phosphorylations at three sites and probably contains a glycosylation site. The crystal structure of the region of greglin comprising residues 21–78 in complex with subtilisin was determined at 1.75 Å resolution. Greglin represents a novel member of the non-classical Kazal inhibitors, as it has a unique additional C-terminal region (70–83) connected to the core of the molecule via a supplementary disulfide bond. The stability of greglin was compared with that of an ovomucoid inhibitor. The thermostability and inhibitory specificity of greglin are discussed in light of its structure. In particular, we propose that the C-terminal region is responsible for non-favourable interactions with the autolysis loop (140-loop) of serine proteases of the chymotrypsin family, and thus governs specificity.

Greglin is an 83-residue serine protease inhibitor purified from the ovaries of the locust Schistocerca gregaria. Greglin is a strong inhibitor of subtilisin and human neutrophil elastase, acting at sub-nanomolar and nanomolar concentrations, respectively ; it also inhibits neutrophil cathepsin G, α-chymotrypsin and porcine pancreatic elastase, but to a lesser extent. In the present study, we show that greglin resists denaturation at high temperature (95 °C) and after exposure to acetonitrile and acidic or basic pH. Greglin is composed of two domains consisting of residues 1–20 and 21–83. Mass spectrometry indicates that the N-terminal domain (1–20) is post-translationally modified by phosphorylations at three sites and probably contains a glycosylation site. The crystal structure of the region of greglin comprising residues 21–78 in complex with subtilisin was determined at 1.75 Å resolution. Greglin represents a novel member of the non-classical Kazal inhibitors, as it has a unique additional C-terminal region (70–83) connected to the core of the molecule via a supplementary disulfide bond. The stability of greglin was compared with that of an ovomucoid inhibitor. The thermostability and inhibitory specificity of greglin are discussed in light of its structure. In particular, we propose that the C-terminal region is responsible for non-favourable interactions with the autolysis loop (140-loop) of serine proteases of the chymotrypsin family, and thus governs specificity.

Marques-Carvalho, M.J., Sahoo, N., Muskett, F.W., Vieira-Pires, R.S., Gabant, G., Cadene, M., Schönherr, R. and Morais-Cabral, J.H.  (2012)

Structural, Biochemical, and Functional Characterization of the Cyclic Nucleotide Binding Homology Domain from the Mouse EAG1 Potassium Channel

Journal of Molecular Biology 423 (1) 34-46
KCNH channels are voltage-gated potassium channels with important physiological functions. In these channels, a C-terminal cytoplasmic region, known as the cyclic nucleotide binding homology (CNB-homology) domain displays strong sequence similarity to cyclic nucleotide binding (CNB) domains. However, the isolated domain does not bind cyclic nucleotides. Here, we report the X-ray structure of the CNB-homology domain from the mouse EAG1 channel. Through comparison with the recently determined structure of the CNB-homology domain from the zebrafish ELK (eag‐like K+) channel and the CNB domains from the MlotiK1 and HCN (hyperpolarization‐activated cyclic nucleotide‐gated) potassium channels, we establish the structural features of CNB-homology domains that explain the low affinity for cyclic nucleotides. Our structure establishes that the “self-liganded” conformation, where two residues of the C-terminus of the domain are bound in an equivalent position to cyclic nucleotides in CNB domains, is a conserved feature of CNB-homology domains. Importantly, we provide biochemical evidence that suggests that there is also an unliganded conformation where the C-terminus of the domain peels away from its bound position. A functional characterization of this unliganded conformation reveals a role of the CNB-homology domain in channel gating.

KCNH channels are voltage-gated potassium channels with important physiological functions. In these channels, a C-terminal cytoplasmic region, known as the cyclic nucleotide binding homology (CNB-homology) domain displays strong sequence similarity to cyclic nucleotide binding (CNB) domains. However, the isolated domain does not bind cyclic nucleotides. Here, we report the X-ray structure of the CNB-homology domain from the mouse EAG1 channel. Through comparison with the recently determined structure of the CNB-homology domain from the zebrafish ELK (eag‐like K+) channel and the CNB domains from the MlotiK1 and HCN (hyperpolarization‐activated cyclic nucleotide‐gated) potassium channels, we establish the structural features of CNB-homology domains that explain the low affinity for cyclic nucleotides. Our structure establishes that the “self-liganded” conformation, where two residues of the C-terminus of the domain are bound in an equivalent position to cyclic nucleotides in CNB domains, is a conserved feature of CNB-homology domains. Importantly, we provide biochemical evidence that suggests that there is also an unliganded conformation where the C-terminus of the domain peels away from its bound position. A functional characterization of this unliganded conformation reveals a role of the CNB-homology domain in channel gating.

Tavel, L., Jaquillard, L., Karsisiotis, A.I., Saab, F., Jouvensal, L., Brans, A., Delmas, A., Schoentgen, F., Cadene, M. and Damblon, C.  (2012)

Ligand binding study of human PEBP1/RKIP : interaction with nucleotides and Raf-1 peptides evidenced by NMR and Mass Spectrometry

PLoS ONE 7 (4) e36187
In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands : GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.

In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands : GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.

Beaufour, M, Godin, F, Vallée, B, Cadene, M. and Bénédetti, H.  (2012)

Interaction proteomics suggests a new role for the Tfs1 protein in yeast

Journal of Proteome Research (2012) 11 (6) 3211-3218
The PEBP (phosphatidylethanolamine-binding protein) family is a large group of proteins whose human member, hPEBP1, has been shown to play multiple functions, influencing intracellular signaling cascades, cell cycle regulation, neurodegenerative processes, and reproduction. It also acts, by an unknown mechanism, as a metastasis suppressor in a number of cancers. A more complete understanding of its biological role is thus necessary. As the yeast Saccharomyces cerevisiae is a powerful and easy to handle model organism, we focused on Tfs1p, the yeast ortholog of hPEBP1. In a previous study based on a two-hybrid approach, we showed that Tfs1p interacts and inhibits Ira2p, a GTPase Activating Protein (GAP) of the small GTPase Ras. In order to further characterize the molecular functions of Tfs1p, we undertook the identification of protein complexes formed around Tfs1p using a targeted proteomics approach. Complexed proteins were purified by tandem-affinity, cleaved with trypsin, and identified by nanoflow liquid chromatography coupled with tandem mass spectrometry. Overall, 14 new interactors were identified, including several proteins involved in intermediate metabolism. We confirmed by co-immunoprecipitation that Tfs1p interacts with Glo3p, a GAP for Arf GTPases belonging to the Ras superfamily of small GTPases, indicating that Tfs1p may be involved in the regulation of another GAP. We similarly confirmed the binding of Tfs1p with the metabolic enzymes Idp1p and Pro1p. Integration of these results with known functional partners of Tfs1p shows that two subnetworks meet through the Tfs1p node, suggesting that it may act as a bridge between cell signaling and intermediate metabolism in yeast.

The PEBP (phosphatidylethanolamine-binding protein) family is a large group of proteins whose human member, hPEBP1, has been shown to play multiple functions, influencing intracellular signaling cascades, cell cycle regulation, neurodegenerative processes, and reproduction. It also acts, by an unknown mechanism, as a metastasis suppressor in a number of cancers. A more complete understanding of its biological role is thus necessary. As the yeast Saccharomyces cerevisiae is a powerful and easy to handle model organism, we focused on Tfs1p, the yeast ortholog of hPEBP1. In a previous study based on a two-hybrid approach, we showed that Tfs1p interacts and inhibits Ira2p, a GTPase Activating Protein (GAP) of the small GTPase Ras. In order to further characterize the molecular functions of Tfs1p, we undertook the identification of protein complexes formed around Tfs1p using a targeted proteomics approach. Complexed proteins were purified by tandem-affinity, cleaved with trypsin, and identified by nanoflow liquid chromatography coupled with tandem mass spectrometry. Overall, 14 new interactors were identified, including several proteins involved in intermediate metabolism. We confirmed by co-immunoprecipitation that Tfs1p interacts with Glo3p, a GAP for Arf GTPases belonging to the Ras superfamily of small GTPases, indicating that Tfs1p may be involved in the regulation of another GAP. We similarly confirmed the binding of Tfs1p with the metabolic enzymes Idp1p and Pro1p. Integration of these results with known functional partners of Tfs1p shows that two subnetworks meet through the Tfs1p node, suggesting that it may act as a bridge between cell signaling and intermediate metabolism in yeast.

Jaquillard, L., Saab, F., Schoentgen, F. and Cadene, M.  (2012)

Improved accuracy of low-affinity protein-ligand equilibrium dissociation constants determined by ESI-MS

Journal of The American Society for Mass Spectrometry 23 (5) 908-922
There is continued interest in the determination by ESI-MS of equilibrium dissociation constants (K(D)) that accurately reflect the affinity of a protein-ligand complex in solution. Issues in the measurement of K(D) are compounded in the case of low affinity complexes. Here we present a K(D) measurement method and corresponding mathematical model dealing with both gas-phase dissociation (GPD) and aggregation. To this end, a rational mathematical correction of GPD (f(sat)) is combined with the development of an experimental protocol to deal with gas-phase aggregation. A guide to apply the method to noncovalent protein-ligand systems according to their kinetic behavior is provided. The approach is validated by comparing the K(D) values determined by this method with in-solution K(D) literature values. The influence of the type of molecular interactions and instrumental setup on f(sat) is examined as a first step towards a fine dissection of factors affecting GPD. The method can be reliably applied to a wide array of low affinity systems without the need for a reference ligand or protein.

There is continued interest in the determination by ESI-MS of equilibrium dissociation constants (K(D)) that accurately reflect the affinity of a protein-ligand complex in solution. Issues in the measurement of K(D) are compounded in the case of low affinity complexes. Here we present a K(D) measurement method and corresponding mathematical model dealing with both gas-phase dissociation (GPD) and aggregation. To this end, a rational mathematical correction of GPD (f(sat)) is combined with the development of an experimental protocol to deal with gas-phase aggregation. A guide to apply the method to noncovalent protein-ligand systems according to their kinetic behavior is provided. The approach is validated by comparing the K(D) values determined by this method with in-solution K(D) literature values. The influence of the type of molecular interactions and instrumental setup on f(sat) is examined as a first step towards a fine dissection of factors affecting GPD. The method can be reliably applied to a wide array of low affinity systems without the need for a reference ligand or protein.

Epinette, C., Croix, C., Jaquillard, L., Marchand-Adam, S., Kellenberger, C., Lalmanach, G., Cadene, M., Viaud-Massuard, M.C., Gauthier, F. and Korkmaz, B.  (2012)

A selective reversible azapeptide inhibitor of human neutrophil proteinase 3 derived from a high affinity FRET substrate

Biochemical Pharmacology 83 (6) 788-796
The biological functions of human neutrophil proteinase 3 (PR3) remain unclear because of its close structural resemblance to neutrophil elastase and its apparent functional redundancy with the latter. Thus, all natural inhibitors of PR3 preferentially target neutrophil elastase. We have designed a selective PR3 inhibitor based on the sequence of one of its specific, sensitive FRET substrates. This azapeptide, azapro-3, inhibits free PR3 in solution, PR3 bound to neutrophil membranes, and the PR3 found in crude lung secretions from patients with chronic inflammatory pulmonary diseases. But it does not inhibit significantly neutrophil elastase or cathepsin G. Unlike most of azapeptides, this inhibitor does not form a stable acyl-enzyme complex ; it is a reversible competitive inhibitor with a K-i comparable to the K-m of the parent substrate. Low concentrations (60 mu M) of azapro-3 totally inhibited the PR3 secreted by triggered human neutrophils (200,000 cells/100 mu L) and the PR3 in neutrophil homogenates and in lung secretions of patients with lung inflammation for hours. Azapro-3 also resisted proteolysis by all proteases contained in these samples for at least 2 h.

The biological functions of human neutrophil proteinase 3 (PR3) remain unclear because of its close structural resemblance to neutrophil elastase and its apparent functional redundancy with the latter. Thus, all natural inhibitors of PR3 preferentially target neutrophil elastase. We have designed a selective PR3 inhibitor based on the sequence of one of its specific, sensitive FRET substrates. This azapeptide, azapro-3, inhibits free PR3 in solution, PR3 bound to neutrophil membranes, and the PR3 found in crude lung secretions from patients with chronic inflammatory pulmonary diseases. But it does not inhibit significantly neutrophil elastase or cathepsin G. Unlike most of azapeptides, this inhibitor does not form a stable acyl-enzyme complex ; it is a reversible competitive inhibitor with a K-i comparable to the K-m of the parent substrate. Low concentrations (60 mu M) of azapro-3 totally inhibited the PR3 secreted by triggered human neutrophils (200,000 cells/100 mu L) and the PR3 in neutrophil homogenates and in lung secretions of patients with lung inflammation for hours. Azapro-3 also resisted proteolysis by all proteases contained in these samples for at least 2 h.

Derache, C., Meudal, H., Aucagne, V., Mark, K.J., Cadène, M., Delmas, A.F., Lalmanach, A.C. and Landon, C.  (2012)

Initial insights into the structure-activity relationships of avian defensins

Journal of Biological Chemistry 287 (10) 7746-7755
Numerous β-defensins have been identified in birds and the potential use of these peptides as alternatives to antibiotics has been proposed, in particular to fight antibiotic-resistant and zoonotic bacterial species. Little is known about the mechanism of antibacterial activity of avian β-defensins (AvBDs), and the present work was carried out to obtain initial insights into the involvement of structural features or specific residues in the antimicrobial activity of chicken AvBD2. Chicken AvBD2 and its enantiomeric counterpart were chemically synthesized. Peptide elongation and oxidative folding were both optimized. The similar antimicrobial activity measured for both L- and D- proteins clearly indicates that there is no chiral partner. Therefore the bacterial membrane is in all likelihood the primary target. Moreover, this work evidences that the three-dimensional fold is required for an optimal antimicrobial activity, in particular for Gram-positive bacterial strains. The three-dimensional NMR structure of chicken AvBD2 defensin displays the structural 3-stranded antiparallel β-sheet characteristic of β-defensins. The surface of the molecule does not display any amphipathic character. In light of this new structure and of the king penguin AvBD103b defensin structure, the consensus sequence of avian β-defensin′s family was analyzed. Well conserved residues were highlighted and the potential strategic role of the lysine 31 residue of AvBD2 emphasized. The synthetic AvBD2-K31A variant displayed substantial N-terminal structural modifications and a dramatic decrease in activity. Taken together, these results demonstrate the structural as well as the functional role of the critical lysine 31 residue in antimicrobial activity.

Numerous β-defensins have been identified in birds and the potential use of these peptides as alternatives to antibiotics has been proposed, in particular to fight antibiotic-resistant and zoonotic bacterial species. Little is known about the mechanism of antibacterial activity of avian β-defensins (AvBDs), and the present work was carried out to obtain initial insights into the involvement of structural features or specific residues in the antimicrobial activity of chicken AvBD2. Chicken AvBD2 and its enantiomeric counterpart were chemically synthesized. Peptide elongation and oxidative folding were both optimized. The similar antimicrobial activity measured for both L- and D- proteins clearly indicates that there is no chiral partner. Therefore the bacterial membrane is in all likelihood the primary target. Moreover, this work evidences that the three-dimensional fold is required for an optimal antimicrobial activity, in particular for Gram-positive bacterial strains. The three-dimensional NMR structure of chicken AvBD2 defensin displays the structural 3-stranded antiparallel β-sheet characteristic of β-defensins. The surface of the molecule does not display any amphipathic character. In light of this new structure and of the king penguin AvBD103b defensin structure, the consensus sequence of avian β-defensin′s family was analyzed. Well conserved residues were highlighted and the potential strategic role of the lysine 31 residue of AvBD2 emphasized. The synthetic AvBD2-K31A variant displayed substantial N-terminal structural modifications and a dramatic decrease in activity. Taken together, these results demonstrate the structural as well as the functional role of the critical lysine 31 residue in antimicrobial activity.


2010   Références trouvées : 1

Vergne, P., Maene, M., Gabant, G., Chauvet, A., Debener, T. and Bendahmane, M.  (2010)

Somatic embryogenesis and transformation of the diploid Rosa chinensis cv Old Blush

Plant Cell Tissue and Organ Culture 100 (1) 73-81
Somatic embryogenesis was induced from in vitro-derived leaf explants of Rosa chinensis cultivar (cv) Old Blush. Calli producing embryos with expanded cotyledons (RcOBType1 embryos) were obtained. Further refinements of the callus maintenance medium generated a more typical rose embryogenic callus (RcOBType2) displaying high levels of secondary embryogenesis and embryos with limited cotyledon expansion Agrobacterium tumefaciens-mediated transformation assays using β-glucuronidase (GUS) reporter gene showed that both types of embryos were competent for transformation. Under selection conditions, transformed RcOBType1 explants produced non chimaeric transformed embryos, from which shoots could be adventitiously regenerated. In contrast to RcOBType1, transformed RcOBType2 embryos directly yielded transformed shoots when repeatedly cultured in selective regeneration conditions. Transformation efficiency ranged between three to nine percent and shoots suitable for rooting were obtained within 6–8 months. Transgenic plants were transferred into the greenhouse and molecularly confirmed. The availability of transformation methods in a diploid rose, R. chinensis cv. Old Blush, will be useful for gene functional studies.

Somatic embryogenesis was induced from in vitro-derived leaf explants of Rosa chinensis cultivar (cv) Old Blush. Calli producing embryos with expanded cotyledons (RcOBType1 embryos) were obtained. Further refinements of the callus maintenance medium generated a more typical rose embryogenic callus (RcOBType2) displaying high levels of secondary embryogenesis and embryos with limited cotyledon expansion Agrobacterium tumefaciens-mediated transformation assays using β-glucuronidase (GUS) reporter gene showed that both types of embryos were competent for transformation. Under selection conditions, transformed RcOBType1 explants produced non chimaeric transformed embryos, from which shoots could be adventitiously regenerated. In contrast to RcOBType1, transformed RcOBType2 embryos directly yielded transformed shoots when repeatedly cultured in selective regeneration conditions. Transformation efficiency ranged between three to nine percent and shoots suitable for rooting were obtained within 6–8 months. Transgenic plants were transferred into the greenhouse and molecularly confirmed. The availability of transformation methods in a diploid rose, R. chinensis cv. Old Blush, will be useful for gene functional studies.


2009   Références trouvées : 1

Bure, C., Pichon, C. & Midoux, P.  (2009)

Involvement of histidines 11, 15 and 19 in the binding of zinc to the fusogenic H5WYG peptide

(2009) J Mass Spectrom 44, 1163-1170.


2008   Références trouvées : 4

Gabant, G., Lorphelin, A., Nozerand, N., Marchetti, C., Bellanger, L., Dedieu, A., Quéméneur, E., Alpha-Bazin, B.   (2008)

Autophosphorylation residues involved in the regulation of human Chk2 in vitro

(2008) J. Mol. Biol. 380, 489-503.

Gabant, G ; Augier, J ; Armengaud, J  (2008)

Assessment of solvent residues accessibility using a combination of three Sulfo-NHS-biotin reagents : application to delineate contacts of a methyltransferase with its substrate

Journal of Mass Spectrometry 43 (3) 360-370

Bure, C ; Goffinont, S ; Delmas, AF ; Cadene, M ; Culard, F  (2008)

Oxidation-sensitive residues mediate the DNA bending abilities of the architectural MC1 protein

Journal of Molecular Biology 376 (1) 120-130
The Methanosarcina thermophila MC1 protein is a small basic protein that is able to bend DNA sharply. When this protein is submitted to oxidative stress through gamma irradiation, it loses its original DNA interaction properties. The protein can still bind DNA but its ability to bend DNA is decreased dramatically. Here, we used different approaches to determine the oxidations that are responsible for this inactivation. Through a combination of proteolysis and mass spectrometry we have identified the three residues that are oxidized preferentially.

The Methanosarcina thermophila MC1 protein is a small basic protein that is able to bend DNA sharply. When this protein is submitted to oxidative stress through gamma irradiation, it loses its original DNA interaction properties. The protein can still bind DNA but its ability to bend DNA is decreased dramatically. Here, we used different approaches to determine the oxidations that are responsible for this inactivation. Through a combination of proteolysis and mass spectrometry we have identified the three residues that are oxidized preferentially.

Gabant G. , Cadene M.   (2008)

Mass spectrometry of full-length integral membrane proteins to define functionally relevant features.

Source:Methods, 46, 54-61.


2007   Références trouvées : 4

Gillard, N ; Goffinont, S ; Bure, C ; Davidkova, M ; Maurizot, JC ; Cadene, M ; Spotheim-Maurizot, M  (2007)

Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor

Biochemical Journal 403 463-472 Part 3
Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with gamma-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (CH radicals)which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece.

Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with gamma-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (CH radicals)which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece.

Bourgeaux, V ; Cadene, M ; Piller, F ; Piller, V  (2007)

Efficient enzymatic glycosylation of peptides and oligosaccharides from GalNAc and UTP

Chembiochem 8 (1) 37-40

Fenyo, D ; Wang, QJ ; DeGrasse, JA ; Padovan, JC ; Cadene, M ; Chait BT  (2007)

MALDI Sample Preparation : the Ultra Thin Layer Method (Videopublication)

Journal of Visualized Experiments, Cellular Biology Issue 3 (http://www.jove.com/index/details.stp?ID=192)
This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) [1, 2]. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) [1, 2]. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity.

Nishida, M ; Cadene, M ; Chait, BT ; MacKinnon, R  (2007)

Crystal structure of a Kir3.1-prokaryotic Kir channel chimera

Embo Journal 26 4005-4015
The Kir3.1 K+ channel participates in heart rate control and neuronal excitability through G-protein and lipid signaling pathways. Expression in Escherichia coli has been achieved by replacing three fourths of the transmembrane pore with the pore of a prokaryotic Kir channel, leaving the cytoplasmic pore and membrane interfacial regions of Kir3.1 origin. Two structures were determined at 2.2A. The selectivity filter is identical to the Streptomyces lividans K+ channel within error of measurement (r.m.s.d.

The Kir3.1 K+ channel participates in heart rate control and neuronal excitability through G-protein and lipid signaling pathways. Expression in Escherichia coli has been achieved by replacing three fourths of the transmembrane pore with the pore of a prokaryotic Kir channel, leaving the cytoplasmic pore and membrane interfacial regions of Kir3.1 origin. Two structures were determined at 2.2A. The selectivity filter is identical to the Streptomyces lividans K+ channel within error of measurement (r.m.s.d.


2006   Références trouvées : 2

Beaufour, M ; Morin, P ; Ribet, JP ; Maurizot, JC  (2006)

HPLC quantitation of the four stereoisorners of benzoxathiepin derivatives with cellulose phenyl type chiral stationary phase and circular dichroism detection

Journal of Pharmaceutical and Biomedical Analysis 41 (2) 544-548
The chiral separation of a new antianginal agent has been investigated on a chiral cellulose column with UV and circular dichroism (CD) detection. This benzoxathiepin derivative under development has two stereogenic centers whose (R,S) stereoisomer shows an interesting, antianginal activity. After optimisation of the mobile phase composition, a baseline-resolved separation of the four stereoisomers was achieved on a Chiralcel OJ-H chiral column by using methanol-ethanol-diethylamine (25:75:0.1, v/v/v) as mobile phase. The CD detection system allowed quantitation and a linear response was observed within a 10-200 mu g mL(-1) concentration range (r(2) = 0.9966) and limit of quantification down to 2 jig mL(-1) was achieved. (c) 2005 Elsevier B.V. All rights reserved.

The chiral separation of a new antianginal agent has been investigated on a chiral cellulose column with UV and circular dichroism (CD) detection. This benzoxathiepin derivative under development has two stereogenic centers whose (R,S) stereoisomer shows an interesting, antianginal activity. After optimisation of the mobile phase composition, a baseline-resolved separation of the four stereoisomers was achieved on a Chiralcel OJ-H chiral column by using methanol-ethanol-diethylamine (25:75:0.1, v/v/v) as mobile phase. The CD detection system allowed quantitation and a linear response was observed within a 10-200 mu g mL(-1) concentration range (r(2) = 0.9966) and limit of quantification down to 2 jig mL(-1) was achieved. (c) 2005 Elsevier B.V. All rights reserved.

Gabant, G ; Auxilien, S ; Tuszynska, I ; Locard, M ; Gajda, MJ ; Chaussinand, G ; Fernandez, B ; Urbonavicius, J ; Dedieu, A ; Grosjean, H ; Golinelli-Pimpaneau , B ; Bujnicki, JM ; Armengaud, J  (2006)

THUMP from tRNA:m²2G10 dimethyltransferase, a genuine autonomously folding domain

Nucleic Acids Research 34 (9) 2483-2494


2005   Références trouvées : 1

Beaufour, M ; Ribet, JP ; Morin, P  (2005)

Chiral separation of the four stereoisomers of a novel antianginal agent using a dual cyclodextrin system in capillary electrophoresis

Journal of Separation Science 28 (6) 529-533
Reported here is an analytical method enabling the stereochemical resolution of a new antianginal compound possessing two stereogenic centers, leading to four stereoisomers. Only one of these isomers is currently under development as a novel antianginal agent and consequently, the other three isomers are considered as unwanted chiral impurities. Therefore, an enantioselective method is required in order to check its enantiomeric purity. This paper presents a method exploiting the high efficiency of capillary electrophoresis and the complexing properties of cyclodextrins to achieve the separation of the four stereoisomers of this weakly basic compound (pK(a) = 7.4).

Reported here is an analytical method enabling the stereochemical resolution of a new antianginal compound possessing two stereogenic centers, leading to four stereoisomers. Only one of these isomers is currently under development as a novel antianginal agent and consequently, the other three isomers are considered as unwanted chiral impurities. Therefore, an enantioselective method is required in order to check its enantiomeric purity. This paper presents a method exploiting the high efficiency of capillary electrophoresis and the complexing properties of cyclodextrins to achieve the separation of the four stereoisomers of this weakly basic compound (pK(a) = 7.4).


2004   Références trouvées : 1

Cadene, M ; Chait, BT  (2004)

Mass spectrometry and structural biology of ion channels.

Abstracts of Papers of The American Chemical Society 227 U264-U264 Art No. 127-Phys Part 2


2003   Références trouvées : 2

Jiang, YX ; Lee, A ; Chen, JY ; Ruta, V ; Cadene, M ; Chait, BT ; MacKinnon, R  (2003)

X-ray structure of a voltage-dependent K+ channel

Nature 423 (6935) 33-41
Voltage-dependent K+ channels are members of the family of voltage-dependent cation (K+, Na+ and Ca2+) channels that open and allow ion conduction in response to changes in cell membrane voltage. This form of gating underlies the generation of nerve and muscle action potentials, among other processes. Here we present the structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix. We have determined a crystal structure of the full-length channel at a resolution of 3.2 Angstrom, and of the isolated voltage-sensor domain at 1.9 Angstrom, both in complex with monoclonal Fab fragments. The channel contains a central ion-conduction pore surrounded by voltage sensors, which form what we call 'voltage-sensor paddles'-hydrophobic, cationic, helix-turn-helix structures on the channel's outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.

Voltage-dependent K+ channels are members of the family of voltage-dependent cation (K+, Na+ and Ca2+) channels that open and allow ion conduction in response to changes in cell membrane voltage. This form of gating underlies the generation of nerve and muscle action potentials, among other processes. Here we present the structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix. We have determined a crystal structure of the full-length channel at a resolution of 3.2 Angstrom, and of the isolated voltage-sensor domain at 1.9 Angstrom, both in complex with monoclonal Fab fragments. The channel contains a central ion-conduction pore surrounded by voltage sensors, which form what we call ’voltage-sensor paddles’-hydrophobic, cationic, helix-turn-helix structures on the channel’s outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.

Beaufour, M ; Merelli, B ; Menguy, L ; Cherton, JC  (2003)

Determination of the enantiomeric composition of chiral delta-2-oxazolines-1,3 by H-1 and F-19 NMR spectroscopy using chiral solvating agents

Chirality 15 (5) 382-390
Studies of the perturbing effect of chiral solvating agents (CSAs) 5a and mostly of 5c upon the NMR spectra of chiral Delta(2)-oxazoline 1 demonstrated the ability of these fluoroalcohols to afford diastereomeric solvates from these solutes. Thus, for all tested Delta(2)-oxazolines 1Aa-d, 1Ba, and 1e there is at least one possibility to proceed to their enantiomeric discrimination either by H-1 or F-19 NMR using these CSAs (see Fig. 1). NMR results are discussed from substrate and CSA structure standpoints and a solvation model is proposed on the basis of the inequivalence senses generally observed. Then the method was applied to extracts of incubated locust tissues obtained by solid phase extraction (SPE) after a partial unmasking of the substrate 1.

Studies of the perturbing effect of chiral solvating agents (CSAs) 5a and mostly of 5c upon the NMR spectra of chiral Delta(2)-oxazoline 1 demonstrated the ability of these fluoroalcohols to afford diastereomeric solvates from these solutes. Thus, for all tested Delta(2)-oxazolines 1Aa-d, 1Ba, and 1e there is at least one possibility to proceed to their enantiomeric discrimination either by H-1 or F-19 NMR using these CSAs (see Fig. 1). NMR results are discussed from substrate and CSA structure standpoints and a solvation model is proposed on the basis of the inequivalence senses generally observed. Then the method was applied to extracts of incubated locust tissues obtained by solid phase extraction (SPE) after a partial unmasking of the substrate 1.


2002   Références trouvées : 6

Seibert C., Cadene M., Sanfiz A., Chait B.T., Sakmar T.P  (2002)

Tyrosine sulfation of CCR5 N-terminal peptide by tyrosylprotein sulfotransferases 1 and 2 follows a discrete pattern and temporal sequence

Proceedings of the National Academy of Sciences, 99, 11031-6
The CC-chemokine receptor 5 (CCR5) is the major coreceptor for the entry of macrophage-tropic (R5) HIV-1 strains into target cells. Posttranslational sulfation of tyrosine residues in the N-terminal tail of CCR5 is critical for high affinity interaction of the receptor with the HIV-1 envelope glycoprotein gp120 in complex with CD4. Here, we focused on defining precisely the sulfation pattern of the N terminus of CCR5 by using recombinant human tyrosylprotein sulfotransferases TPST-1 and TPST-2 to modify a synthetic peptide that corresponds to amino acids 2-18 of the receptor (CCR5 2-18). Analysis of the reaction products was made with a combination of reversed-phase HPLC, proteolytic cleavage, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). We found that CCR5 2-18 is sulfated by both TPST isoenzymes leading to a final product with four sulfotyrosine residues. Sulfates were added stepwise to the peptide producing specific intermediates with one, two, or three sulfotyrosines. The pattern of sulfation in these intermediates suggests that Tyr-14 and Tyr-15 are sulfated first, followed by Tyr-10, and finally Tyr-3. These results represent a detailed analysis of the multiple sulfation reaction of a peptide substrate by TPSTs and provide a structural basis for understanding the role of tyrosine sulfation of CCR5 in HIV-1 coreceptor and chemokine receptor function.

The CC-chemokine receptor 5 (CCR5) is the major coreceptor for the entry of macrophage-tropic (R5) HIV-1 strains into target cells. Posttranslational sulfation of tyrosine residues in the N-terminal tail of CCR5 is critical for high affinity interaction of the receptor with the HIV-1 envelope glycoprotein gp120 in complex with CD4. Here, we focused on defining precisely the sulfation pattern of the N terminus of CCR5 by using recombinant human tyrosylprotein sulfotransferases TPST-1 and TPST-2 to modify a synthetic peptide that corresponds to amino acids 2-18 of the receptor (CCR5 2-18). Analysis of the reaction products was made with a combination of reversed-phase HPLC, proteolytic cleavage, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). We found that CCR5 2-18 is sulfated by both TPST isoenzymes leading to a final product with four sulfotyrosine residues. Sulfates were added stepwise to the peptide producing specific intermediates with one, two, or three sulfotyrosines. The pattern of sulfation in these intermediates suggests that Tyr-14 and Tyr-15 are sulfated first, followed by Tyr-10, and finally Tyr-3. These results represent a detailed analysis of the multiple sulfation reaction of a peptide substrate by TPSTs and provide a structural basis for understanding the role of tyrosine sulfation of CCR5 in HIV-1 coreceptor and chemokine receptor function.

Dutzler, R ; Campbell, EB ; Cadene, M ; Chait, BT ; MacKinnon, R  (2002)

X-ray structure of a CIC chloride channel at 3.0 angstrom reveals the molecular basis of anion selectivity

Nature 415 (6869) 287-294
The ClC chloride channels catalyse the selective flow of Cl- ions across cell membranes, thereby regulating electrical excitation in skeletal muscle and the flow of salt and water across epithelial barriers. Genetic defects in ClC Cl- channels underlie several familial muscle and kidney diseases. Here we present the X-ray structures of two prokaryotic ClC Cl- channels from Salmonella enterica serovar typhimurium and Escherichia coli at 3.0 and 3.5 Angstrom, respectively. Both structures reveal two identical pores, each pore being formed by a separate subunit contained within a homodimeric membrane protein. Individual subunits are composed of two roughly repeated halves that span the membrane with opposite orientations. This antiparallel architecture defines a selectivity filter in which a Cl- ion is stabilized by electrostatic interactions with a-helix dipoles and by chemical coordination with nitrogen atoms and hydroxyl groups. These findings provide a structural basis for further understanding the function of ClC Cl- channels, and establish the physical and chemical basis of their anion selectivity.

The ClC chloride channels catalyse the selective flow of Cl- ions across cell membranes, thereby regulating electrical excitation in skeletal muscle and the flow of salt and water across epithelial barriers. Genetic defects in ClC Cl- channels underlie several familial muscle and kidney diseases. Here we present the X-ray structures of two prokaryotic ClC Cl- channels from Salmonella enterica serovar typhimurium and Escherichia coli at 3.0 and 3.5 Angstrom, respectively. Both structures reveal two identical pores, each pore being formed by a separate subunit contained within a homodimeric membrane protein. Individual subunits are composed of two roughly repeated halves that span the membrane with opposite orientations. This antiparallel architecture defines a selectivity filter in which a Cl- ion is stabilized by electrostatic interactions with a-helix dipoles and by chemical coordination with nitrogen atoms and hydroxyl groups. These findings provide a structural basis for further understanding the function of ClC Cl- channels, and establish the physical and chemical basis of their anion selectivity.

Jiang, YX ; Lee, A ; Chen, JY ; Cadene, M ; Chait, BT ; MacKinnon, R  (2002)

The open pore conformation of potassium channels

Nature 417 (6888) 523-526
Living cells regulate the activity of their ion channels through a process known as gating. To open the pore, protein conformational changes must occur within a channel's membrane-spanning ion pathway. KcsA and MthK, closed and opened K+ channels, respectively, reveal how such gating transitions occur. Pore-lining 'inner' helices contain a 'gating hinge' that bends by approximately 30degrees. In a straight conformation four inner helices form a bundle, closing the pore near its intracellular surface. In a bent configuration the inner helices splay open creating a wide (12 Angstrom) entryway. Amino-acid sequence conservation suggests a common structural basis for gating in a wide range of K+ channels, both ligand- and voltage-gated. The open conformation favours high conduction by compressing the membrane field to the selectivity filter, and also permits large organic cations and inactivation peptides to enter the pore from the intracellular solution.

Living cells regulate the activity of their ion channels through a process known as gating. To open the pore, protein conformational changes must occur within a channel’s membrane-spanning ion pathway. KcsA and MthK, closed and opened K+ channels, respectively, reveal how such gating transitions occur. Pore-lining ’inner’ helices contain a ’gating hinge’ that bends by approximately 30degrees. In a straight conformation four inner helices form a bundle, closing the pore near its intracellular surface. In a bent configuration the inner helices splay open creating a wide (12 Angstrom) entryway. Amino-acid sequence conservation suggests a common structural basis for gating in a wide range of K+ channels, both ligand- and voltage-gated. The open conformation favours high conduction by compressing the membrane field to the selectivity filter, and also permits large organic cations and inactivation peptides to enter the pore from the intracellular solution.

Werner, MH ; Lambert, LJ ; Schirf, V ; Demmeler, B ; Cadene, M ; Ebright, RH  (2002)

Structural consequences of activator engagement with RNA polymerase

Faseb Journal 16 (4) A167-A167 Part 1

Jiang, YX ; Lee, A ; Chen, JY ; Cadene, M ; Chait, BT ; MacKinnon, R  (2002)

Crystal structure and mechanism of a calcium-gated potassium channel

Nature 417 (6888) 515-522
Ion channels exhibit two essential biophysical properties ; that is, selective ion conduction, and the ability to gate-open in response to an appropriate stimulus. Two general categories of ion channel gating are defined by the initiating stimulus : ligand binding (neurotransmitter- or second-messenger-gated channels) or membrane voltage (voltage-gated channels). Here we present the structural basis of ligand gating in a K(+) channel that opens in response to intracellular Ca(2+). We have cloned, expressed, analysed electrical properties, and determined the crystal structure of a K(+) channel (MthK) from Methanobacterium thermoautotrophicum in the Ca(2+)-bound, opened state. Eight RCK domains (regulators of K(+) conductance) form a gating ring at the intracellular membrane surface. The gating ring uses the free energy of Ca(2+) binding in a simple manner to perform mechanical work to open the pore.

Ion channels exhibit two essential biophysical properties ; that is, selective ion conduction, and the ability to gate-open in response to an appropriate stimulus. Two general categories of ion channel gating are defined by the initiating stimulus : ligand binding (neurotransmitter- or second-messenger-gated channels) or membrane voltage (voltage-gated channels). Here we present the structural basis of ligand gating in a K(+) channel that opens in response to intracellular Ca(2+). We have cloned, expressed, analysed electrical properties, and determined the crystal structure of a K(+) channel (MthK) from Methanobacterium thermoautotrophicum in the Ca(2+)-bound, opened state. Eight RCK domains (regulators of K(+) conductance) form a gating ring at the intracellular membrane surface. The gating ring uses the free energy of Ca(2+) binding in a simple manner to perform mechanical work to open the pore.

Cherton, JC ; Beaufour, M ; Menguy, L ; Merelli, B  (2002)

Determination of the enantiomeric composition of chiral Delta-2-oxazolines-1,3 by H-1 and F-19 NMR spectroscopy using chiral lanthanide-shift reagents

Spectroscopy Letters 35 (3) 415-437
The 300 MHz H-1 NMR spectra of 2-ethyl Delta(2) -oxazoline 1m have been studied in CCl4, CD3CN and C6D6 solutions, in the presence of the achiral lanthanide shift reagent (LSR), tris (6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionato)-europium III, 4 known as Eu(fod)(3), (see Sch. 1). All the protons of 1 were deshielded at various extent, and the sequence observed for their Deltadelta suggested a major complexation at the basic N(3) center of the heterocycle. Then the chiral monosubstituted oxazoline 1e and the disubstituted oxazolines 1Aa-d and 1Ba, were studied in the presence of chiral LSR, tris-[D, D dicampholylmethanato] europium III Eu(dcM)(3) 5 and tris-[3-(heptafluoropropylhydroxy-methylene)-d-camphorato] praseodym III Pr(hfC)(3) 6. H-1 NMR, and eventually F-19[H-1] NMR spectra were mostly recorded in C6D6 solution.

The 300 MHz H-1 NMR spectra of 2-ethyl Delta(2) -oxazoline 1m have been studied in CCl4, CD3CN and C6D6 solutions, in the presence of the achiral lanthanide shift reagent (LSR), tris (6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionato)-europium III, 4 known as Eu(fod)(3), (see Sch. 1). All the protons of 1 were deshielded at various extent, and the sequence observed for their Deltadelta suggested a major complexation at the basic N(3) center of the heterocycle. Then the chiral monosubstituted oxazoline 1e and the disubstituted oxazolines 1Aa-d and 1Ba, were studied in the presence of chiral LSR, tris-[D, D dicampholylmethanato] europium III Eu(dcM)(3) 5 and tris-[3-(heptafluoropropylhydroxy-methylene)-d-camphorato] praseodym III Pr(hfC)(3) 6. H-1 NMR, and eventually F-19[H-1] NMR spectra were mostly recorded in C6D6 solution.


2001   Références trouvées : 6

Jiang, YX ; Pico, A ; Cadene, M ; Chait, BT ; MacKinnon, R  (2001)

Structure of the RCK domain from the E. coli K+ channel and demonstration of its presence in the human BK channel

Neuron 29 (3) 593-601
The intracellular C-terminal domain structure of a six-transmembrane K+ channel from Escherichia coli has been solved by X-ray crystallography at 2.4 Angstrom resolution. The structure is representative of a broad class of domains/proteins that regulate the conductance of K+ there referred to as RCK domains) in prokaryotic K+ transporters and K+ channels. The RCK domain has a Rossmann-fold topology with unique positions, not commonly conserved among Rossmann-fold proteins, composing a well-conserved salt bridge and a hydrophobic dimer interface. Structure-based amino acid sequence alignments and mutational analysis are used to demonstrate that an RCK domain is also present and is an important component of the gating machinery in eukaryotic large-conductance Ca2+-activated K+ channels.

The intracellular C-terminal domain structure of a six-transmembrane K+ channel from Escherichia coli has been solved by X-ray crystallography at 2.4 Angstrom resolution. The structure is representative of a broad class of domains/proteins that regulate the conductance of K+ there referred to as RCK domains) in prokaryotic K+ transporters and K+ channels. The RCK domain has a Rossmann-fold topology with unique positions, not commonly conserved among Rossmann-fold proteins, composing a well-conserved salt bridge and a hydrophobic dimer interface. Structure-based amino acid sequence alignments and mutational analysis are used to demonstrate that an RCK domain is also present and is an important component of the gating machinery in eukaryotic large-conductance Ca2+-activated K+ channels.

Lambert, LJ ; Schirf, V ; Demeler, B ; Cadene, M ; Werner, MH  (2001)

Flipping a genetic switch by subunit exchange

Embo Journal 20 (24) 7149-7159
The bacteriophage T4 AsiA protein is a multifunctional protein that simultaneously acts as both a repressor and activator of gene expression during the phage life cycle. These dual roles with opposing transcriptional consequences are achieved by modification of the host RNA polymerase in which AsiA binds to conserved region 4 (SR4) of sigma (70), altering the pathway of promoter selection by the holoenzyme. The mechanism by which AsiA flips this genetic switch has now been revealed, in part, from the three-dimensional structure of AsiA and the elucidation of its interaction with SR4. The structure of AsiA is that of a novel homodimer in which each monomer is constructed as a seven-helix bundle arranged in four overlapping helix-loop-helix elements. Identification of the protein interfaces for both the AsiA homodimer and the AsiA-sigma (70) complex reveals that these interfaces are coincident. Thus, the AsiA interaction with sigma (70) necessitates that the AsiA homodimer dissociate to form an AsiA-SR4 heterodimer, exchanging one protein subunit for another to alter promoter choice by RNA polymerase.

The bacteriophage T4 AsiA protein is a multifunctional protein that simultaneously acts as both a repressor and activator of gene expression during the phage life cycle. These dual roles with opposing transcriptional consequences are achieved by modification of the host RNA polymerase in which AsiA binds to conserved region 4 (SR4) of sigma (70), altering the pathway of promoter selection by the holoenzyme. The mechanism by which AsiA flips this genetic switch has now been revealed, in part, from the three-dimensional structure of AsiA and the elucidation of its interaction with SR4. The structure of AsiA is that of a novel homodimer in which each monomer is constructed as a seven-helix bundle arranged in four overlapping helix-loop-helix elements. Identification of the protein interfaces for both the AsiA homodimer and the AsiA-sigma (70) complex reveals that these interfaces are coincident. Thus, the AsiA interaction with sigma (70) necessitates that the AsiA homodimer dissociate to form an AsiA-SR4 heterodimer, exchanging one protein subunit for another to alter promoter choice by RNA polymerase.

Linderoth, NA ; Simon, MN ; Rodionova, NA ; Cadene, M ; Laws, WR ; Chait, BT ; Sastry, S  (2001)

Biophysical analysis of the endoplasmic reticulum-resident chaperone/heat shock protein gp96/GRP94 and its complex with peptide antigen

Biochemistry 40 (5) 1483-1495
Animals Vaccinated with heat shock protein (HSP)-peptide complexes develop specific protective immunity against cancers from which the HSPs were originally isolated. This autologous specific immunity has been demonstrated using a number of HSP-peptide antigen complexes. A prototypical HSP-based cancer vaccine is the gp96-peptide antigen complex, which is currently undergoing human clinical trials. Here, we analyzed the structure of a recombinant wild-type and a mutant gp96 protein and their peptide complexes using a number of biophysical techniques, Gel filtration chromatography, dynamic light scattering, and equilibrium analytical ultracentrifugation demonstrated that both a wild-type gp96 and a gp96 mutant lacking a dimerization domain formed higher order structures. More detailed analysis using scanning transmission electron microscopy indicated that both the wild-type and dimerization deletion mutant gp96 protein were organized, unexpectedly, into large aggregates, Size distributions ranged from dimers to octamers and higher.

Animals Vaccinated with heat shock protein (HSP)-peptide complexes develop specific protective immunity against cancers from which the HSPs were originally isolated. This autologous specific immunity has been demonstrated using a number of HSP-peptide antigen complexes. A prototypical HSP-based cancer vaccine is the gp96-peptide antigen complex, which is currently undergoing human clinical trials. Here, we analyzed the structure of a recombinant wild-type and a mutant gp96 protein and their peptide complexes using a number of biophysical techniques, Gel filtration chromatography, dynamic light scattering, and equilibrium analytical ultracentrifugation demonstrated that both a wild-type gp96 and a gp96 mutant lacking a dimerization domain formed higher order structures. More detailed analysis using scanning transmission electron microscopy indicated that both the wild-type and dimerization deletion mutant gp96 protein were organized, unexpectedly, into large aggregates, Size distributions ranged from dimers to octamers and higher.

Cherton, JC ; Hamm, S ; Halle, JC ; Menguy, L ; Beaufour, M  (2001)

Potential proinsecticides of fluorinated carboxylic acids and beta-ethanolamines IV. Evaluation of the Delta(2)-oxazoline-1,3 structure by F-19 NMR monitoring of the in vitro metabolism in locust tissues

Journal of Fluorine Chemistry 107 (2) 387-395

Beaufour, M ; Cherton, JC ; Carlin-Sinclair, A ; Hamm, S  (2001)

Delta(2)-oxazolines-1,3 and N-acylaziridines as potential proinsecticides of carboxylic acids - V. Direct thin-layer chromatography monitoring of the metabolism in locust tissues

Journal of Chromatography B-Analytical Technologies in The Biomedical and Life Sciences 761 (1) 35-45
Modern thin-layer chromatography (TLC) was used for the evaluation of Delta (2) -oxazolines-1,3 I and N-acylaziridine VII structures, as potential proinsecticides of carboxylic acids III. Thus the unmasking(2) of the active principles III from Delta (2) -oxazolines-1,3 Ia-c and N-acylaziridine VIIc was monitored by spotting aliquots directly onto RP-18 TLC plates, without any sample pretreatment during in vitro assays performed in concentrated locust tissues. To achieve a good separation of carboxylate IIIa from endogenous components of the tissues, a short preliminary development with methanol or ion-pairing was necessary. From UV-TLC chromatograms (densitograms) it appeared that in a phosphate buffer at pH 7.4, the oxazoline Ia with a C-2 substituent devoid of alpha -ramification or alpha,beta -in saturation hydrolysed slowly into the corresponding beta -hydroxylamide VIa and intermediate aminoester Va.

Modern thin-layer chromatography (TLC) was used for the evaluation of Delta (2) -oxazolines-1,3 I and N-acylaziridine VII structures, as potential proinsecticides of carboxylic acids III. Thus the unmasking(2) of the active principles III from Delta (2) -oxazolines-1,3 Ia-c and N-acylaziridine VIIc was monitored by spotting aliquots directly onto RP-18 TLC plates, without any sample pretreatment during in vitro assays performed in concentrated locust tissues. To achieve a good separation of carboxylate IIIa from endogenous components of the tissues, a short preliminary development with methanol or ion-pairing was necessary. From UV-TLC chromatograms (densitograms) it appeared that in a phosphate buffer at pH 7.4, the oxazoline Ia with a C-2 substituent devoid of alpha -ramification or alpha,beta -in saturation hydrolysed slowly into the corresponding beta -hydroxylamide VIa and intermediate aminoester Va.

Terrier, F ; Beaufour, M ; Halle, JC ; Cherton, JC ; Buncel, E  (2001)

A remarkably facile oxygen transfer in a nitrobenzofuroxan structure activated through sigma-complex formation

Tetrahedron Letters 42 (27) 4 499-4501
Treatment of 4,6-dinitrobetlzofuroxan (DNBF) with the imidazoline 1-NRf is found to afford a zwitterionic nitrogen-bonded complex (2-NRf) which, in the presence of base (Et3N), undergoes a slow but quantitative transformation to give 7-hydroxy-4,6-dinitrobmzofurazan (5) as the final product. Overall, an oxygen transfer has thus occurred from the N-oxide function to the carbocyclic moiety of DNBF. The key point in this transformation is shown to be a facile abstraction of the sp(3) hydrogen bonded at C-7 of 2-NRf, providing important new evidence that the parent DNBF structure is extremely electron-withdrawing ('super-electrophile'). The overall conversion is also an unusual case of a catalytic process in which the catalysts (both 1-NRf and Et3N) partake to form covalent reaction intermediates and thereby lower the activation energy, resulting in a facile reaction. (C) 2001 Elsevier Science Ltd. All rights reserved.

Treatment of 4,6-dinitrobetlzofuroxan (DNBF) with the imidazoline 1-NRf is found to afford a zwitterionic nitrogen-bonded complex (2-NRf) which, in the presence of base (Et3N), undergoes a slow but quantitative transformation to give 7-hydroxy-4,6-dinitrobmzofurazan (5) as the final product. Overall, an oxygen transfer has thus occurred from the N-oxide function to the carbocyclic moiety of DNBF. The key point in this transformation is shown to be a facile abstraction of the sp(3) hydrogen bonded at C-7 of 2-NRf, providing important new evidence that the parent DNBF structure is extremely electron-withdrawing (’super-electrophile’). The overall conversion is also an unusual case of a catalytic process in which the catalysts (both 1-NRf and Et3N) partake to form covalent reaction intermediates and thereby lower the activation energy, resulting in a facile reaction. (C) 2001 Elsevier Science Ltd. All rights reserved.


2000   Références trouvées : 2

Rudd, PM ; Downing, AK ; Cadene, M ; Harvey, DJ ; Wormald, MR ; Weir, I ; Dwek, RA ; Rifkin, DB ; Gleizes, PE  (2000)

Hybrid and complex glycans are linked to the conserved N-glycosylation site of the third eight-cysteine domain of LTBP-1 in insect cells

Biochemistry 39 (7) 1596-1603
Covalent association of LTBP-1 (latent TGF-beta binding protein-1) to latent TGF-beta is mediated by the third eight-cysteine (also referred to as TB) module of LTBP-1, a domain designated as CR3. Spodoptera frugiperda (Sf9) cells have proved a suitable cell system in which to study this association and to produce recombinant CR3, and we show here that another lepidopteran cell line, Trichoplusia ni TN-SB 1-4 (High-Five) cells, allows the recovery of large amounts of functional recombinant CR3. CR3 contains an N-glycosylation site, which is conserved in all forms of LTBP known to date. When we examined the status of this N-glycosylation using MALDI-TOF mass spectrometry and enzymatic analysis, we found that CR3 is one of the rare recombinant peptides modified with complex glycans in insect cells.

Covalent association of LTBP-1 (latent TGF-beta binding protein-1) to latent TGF-beta is mediated by the third eight-cysteine (also referred to as TB) module of LTBP-1, a domain designated as CR3. Spodoptera frugiperda (Sf9) cells have proved a suitable cell system in which to study this association and to produce recombinant CR3, and we show here that another lepidopteran cell line, Trichoplusia ni TN-SB 1-4 (High-Five) cells, allows the recovery of large amounts of functional recombinant CR3. CR3 contains an N-glycosylation site, which is conserved in all forms of LTBP known to date. When we examined the status of this N-glycosylation using MALDI-TOF mass spectrometry and enzymatic analysis, we found that CR3 is one of the rare recombinant peptides modified with complex glycans in insect cells.

Cadene, M ; Chait, BT  (2000)

A robust, detergent-friendly method for mass spectrometric analysis of integral membrane proteins

Analytical Chemistry 72 (22) 5655-5658
Recent breakthroughs in the high-resolution structural elucidation of ion channels and transporters are prompting a growing interest in methods for characterizing integral membrane proteins. These methods are proving extremely valuable in facilitating the production of X-ray diffraction-grade crystals. Here we present a robust and straightforward mass spectrometric procedure that utilizes matrix-assisted laser desorption/ionization to analyze integral membrane proteins in the presence of detergents. The utility of this method is illustrated with examples of high-quality mass spectral data obtained from membrane proteins for which atomic resolution structural studies are ongoing.

Recent breakthroughs in the high-resolution structural elucidation of ion channels and transporters are prompting a growing interest in methods for characterizing integral membrane proteins. These methods are proving extremely valuable in facilitating the production of X-ray diffraction-grade crystals. Here we present a robust and straightforward mass spectrometric procedure that utilizes matrix-assisted laser desorption/ionization to analyze integral membrane proteins in the presence of detergents. The utility of this method is illustrated with examples of high-quality mass spectral data obtained from membrane proteins for which atomic resolution structural studies are ongoing.