Partenaires

CNRS


Rechercher


Accueil > Annuaire

Gabant Guillaume


email

Ingénieur d’études

Groupe thématique : Spectrométrie de masse fonctionnelle des métastases

Publications

2016   Références trouvées : 2

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.


2014   Références trouvées : 2

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.

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.


2012   Références trouvées : 2

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.


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.


2008   Références trouvées : 3

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

Gabant G. , Cadene M.   (2008)

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

Source:Methods, 46, 54-61.


2006   Références trouvées : 1

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