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Papers by Bertrand Cahuzac

Research paper thumbnail of The phosphatidyl-myo-inositol anchor of the lipoarabinomannans from Mycobacterium bovis Bacillus Calmette Guérin

Journal of Biological …, 1997

Lipoarabinomannans are major mycobacterial antigens capable of modulating the host immune respons... more Lipoarabinomannans are major mycobacterial antigens capable of modulating the host immune response; however, the molecular basis underlying the diversity of their immunological properties remain an open question. In this study a new extraction and purification approach was successfully applied to isolate ManLAMs (lipoarabinomannans with mannosyl extensions) from bacillus Calmette Gué rin leading to the obtention of two types of ManLAMs namely parietal and cellular. Structurally, they were found to differ by the percentage of mannooligosaccharide caps, 76 and 48%, respectively, and also, thanks to a new analytical method, by the structure of the phosphatidyl-myo-inositol anchor lipid moiety. A novel fatty acid in the mycobacterium genus assigned to a 12-O-(methoxypropanoyl)-12-hydroxystearic acid was the only fatty acid esterifying C-1 of the glycerol residue of the parietal ManLAMs, while the phosphatidyl unit of the cellular ManLAMs showed a large heterogeneity due to a combination of palmitic and tuberculostearic acid. Finally, parietal and cellular ManLAMs were found to differentially affect interleukin-8 and tumor necrosis factor-␣ secretion from human dendritic cells. We show that parietal but not cellular ManLAMs were able to stimulate tumor necrosis factor-␣ secretion from dendritic cells. From these studies we propose that the 1-[12-O-(methoxypropanoyl)-12-hydroxystearoyl]-sn-glycerol part is the major cytokineregulating component of the ManLAMs. It seems likely that modification of the ManLAM lipid part, which may occur in hostile environments, could regulate macrophagic mycobacterial survival by altering cytokine stimulation.

Research paper thumbnail of Structure par RMN d'un complexe AlcR(1-60)-ADN: Reconnaissance du petit sillon par la partie N-terminale

Journal de Chimie Physique et de Physico-Chimie Biologique, 1999

Research paper thumbnail of A recurrent RNA-binding domain is appended to eukaryotic aminoacyl-tRNA synthetases

Research paper thumbnail of Differential chemical labeling of the AlcR DNA-binding domain from Aspergillas nidulans versus its complex with a 16-mer DNA target: Identification of an essential tryptophan involved in the recognition and the interaction with the nucleic acid

Protein Science, 2001

DNA binding of the ethanol regulon transcription factor AlcR from Aspergillus nidulans was shown ... more DNA binding of the ethanol regulon transcription factor AlcR from Aspergillus nidulans was shown to involve a consensus basic region as in the other zinc cluster proteins. However, additional interactions between some residues and DNA were suspected, among which were a hypothetic hydrophobic interaction between Trp45 and the T residue of the consensus TGCGG sequence. In the present study, the differential chemical labeling of both the free protein and the protein/DNA complex showed significantly different behaviors of the three tryptophan residues comprised in the AlcR sequence toward the Koshland reagent. The spectacular decreased reaction rate for Trp45 within the complex confirmed the location of this residue at the protein/DNA interface. A similar result obtained with Trp53, an amino acid present at the C-terminal side of AlcR, also indicated its involvement in the DNA recognition. In contrast, the formation of the complex accompanied by an allosteric rearrangement allowed the Trp36 to be much more exposed to the solvent than in the free protein. These data provide additional evidence that the unique specificity of AlcR among the zinc binuclear cluster family results in new types of interactions between AlcR and its cognate targets. From a methodological point of view, the approach of differential chemical labeling combined with mass spectrometric analyses proved to be an interesting tool for the recognition of hydrophobic interactions between the tryptophan residues of a protein and its macromolecular target.

Research paper thumbnail of Structural study of the LipoMannans from Mycobacterium bovis BCG: characterisation of multiacylated forms of the phosphatidyl-myo-inositol anchor

Journal of Molecular Biology, 1999

A biosynthetic ®liation is postulated between the mycobacterial phosphatidyl-myo-inositol mannosi... more A biosynthetic ®liation is postulated between the mycobacterial phosphatidyl-myo-inositol mannosides (PIMs), the lipomannans (LMs) and the lipoarabinomannans (LAMs), the major antigens of the envelopes. Moreover, as the PI anchor is thought to play a role in the biological functions of the LAMs, we characterized the lipid moiety of the PI anchor from Mycobacterium bovis BCG cellular LMs. Their structure was investigated along with that of a puri®ed tetra-acylated form of PIM 2 (Ac 4 PIM 2). A two-dimensional 1 H-31 P heteronuclear multiple quantum correlation homonuclear Hartmann-Hahn spectroscopy study of Ac 4 PIM 2 unambiguously localised a fourth fatty acid on the C3 of the myo-Ins beside the fatty acids already described on the C1 and C2 position of the glycerol and on the C6 position of the mannose. This analytical strategy was extended to the structural study of the cellular LM anchor. Using an appropriate solvent system, the one dimensional 31 P NMR spectrum exhibited four major resonances typifying the LM populations. These populations differed in number and location of the fatty acids. For one of these populations, we established the presence of an extra fatty acid on the C3 of the myo-Ins of the LM anchor. The fact that both types of molecules have an elaborated anchor in common, indicates that cellular LMs are multimannosylated forms of PIMs. In addition, the LM mannan core structure was analysed by two-dimensional NMR, pointing to a high level of branching by single a1 3 2 Manp side-chains.

Research paper thumbnail of NMR solution structure of AlcR (1-60) provides insight in the unusual DNA binding properties of this zinc binuclear cluster protein1

Journal of molecular biology, 2000

The three-dimensional structure of the DNA-binding domain (residues 1-60) of the ethanol regulon ... more The three-dimensional structure of the DNA-binding domain (residues 1-60) of the ethanol regulon transcription factor AlcR from Aspergillus nidulans has been solved by NMR. This domain belongs to the zinc binuclear cluster class. Although the core of the protein is similar to previously characterized structures, consisting of two helices organized around a Zn 2 Cys 6 motif, the present structure presents important variations, among them the presence of two supplementary helices. This structure gives new insight into the understanding of the AlcR speci®cities in DNA binding such as longer consensus half-sites, in vitro monomeric binding but in vivo multiple repeat transcriptional activation, either in direct or inverse orientations. The presence of additional contacts of the protein with its DNA target can be predicted from a model proposed for the interaction with the consensus DNA target. The clustering of accessible negative charges on helix 2 delineates a possible interaction site for other determinants of the transcriptional machinery, responsible for the ®ne tuning of the selection of the AlcR cognate sites.

Research paper thumbnail of The Solution Structure of an AlcR-DNA Complex Sheds Light onto the Unique Tight and Monomeric DNA Binding of a Zn2Cys6 Protein

Structure, 2001

In Aspergillus nidulans, the transcription activator AlcR mediates specific induction of a number... more In Aspergillus nidulans, the transcription activator AlcR mediates specific induction of a number of the genes of the alc cluster. This cluster includes genes involved in the oxidation of ethanol and other alcohols to acetate. The pattern of binding and of transactivation of AlcR is unique within the Zn(2)Cys(6) family. The structural bases for these specificities have not been analyzed at the atomic level until now. We have used NMR spectroscopy and restrained molecular dynamics to determine a set of structures of the AlcR DNA binding domain [AlcR(1-60)] in complex with a 10-mer DNA duplex. Analysis of the structures reveals specific interactions between AlcR and DNA common to the other known zinc clusters. In addition, the involvement of the N-terminal residues upstream of the AlcR zinc cluster in DNA binding is clearly highlighted, and the pivotal role of R6 is confirmed. Totally unprecedented specific and nonspecific contacts of two additional regions of the protein with the DNA are demonstrated. The differences with the available crystallographic structures of other zinc binuclear cluster proteins-DNA complexes are analyzed. The structures of the AlcR(1-60)-DNA complex provide the basis for a better understanding of some of the specificities of the AlcR system: the DNA consensus recognition sequence--usually the triplet CGG--is extended to five base pairs, AlcR acts as a monomer, and additional contacts inside and outside the DNA binding domain in the major and minor groove are observed. These extensive interactions stabilize the AlcR monomer to its cognate DNA site.

Research paper thumbnail of The phosphatidyl-myo-inositol anchor of the lipoarabinomannans from Mycobacterium bovis Bacillus Calmette Guérin

Journal of Biological …, 1997

Lipoarabinomannans are major mycobacterial antigens capable of modulating the host immune respons... more Lipoarabinomannans are major mycobacterial antigens capable of modulating the host immune response; however, the molecular basis underlying the diversity of their immunological properties remain an open question. In this study a new extraction and purification approach was successfully applied to isolate ManLAMs (lipoarabinomannans with mannosyl extensions) from bacillus Calmette Gué rin leading to the obtention of two types of ManLAMs namely parietal and cellular. Structurally, they were found to differ by the percentage of mannooligosaccharide caps, 76 and 48%, respectively, and also, thanks to a new analytical method, by the structure of the phosphatidyl-myo-inositol anchor lipid moiety. A novel fatty acid in the mycobacterium genus assigned to a 12-O-(methoxypropanoyl)-12-hydroxystearic acid was the only fatty acid esterifying C-1 of the glycerol residue of the parietal ManLAMs, while the phosphatidyl unit of the cellular ManLAMs showed a large heterogeneity due to a combination of palmitic and tuberculostearic acid. Finally, parietal and cellular ManLAMs were found to differentially affect interleukin-8 and tumor necrosis factor-␣ secretion from human dendritic cells. We show that parietal but not cellular ManLAMs were able to stimulate tumor necrosis factor-␣ secretion from dendritic cells. From these studies we propose that the 1-[12-O-(methoxypropanoyl)-12-hydroxystearoyl]-sn-glycerol part is the major cytokineregulating component of the ManLAMs. It seems likely that modification of the ManLAM lipid part, which may occur in hostile environments, could regulate macrophagic mycobacterial survival by altering cytokine stimulation.

Research paper thumbnail of Structure par RMN d'un complexe AlcR(1-60)-ADN: Reconnaissance du petit sillon par la partie N-terminale

Journal de Chimie Physique et de Physico-Chimie Biologique, 1999

Research paper thumbnail of A recurrent RNA-binding domain is appended to eukaryotic aminoacyl-tRNA synthetases

Research paper thumbnail of Differential chemical labeling of the AlcR DNA-binding domain from Aspergillas nidulans versus its complex with a 16-mer DNA target: Identification of an essential tryptophan involved in the recognition and the interaction with the nucleic acid

Protein Science, 2001

DNA binding of the ethanol regulon transcription factor AlcR from Aspergillus nidulans was shown ... more DNA binding of the ethanol regulon transcription factor AlcR from Aspergillus nidulans was shown to involve a consensus basic region as in the other zinc cluster proteins. However, additional interactions between some residues and DNA were suspected, among which were a hypothetic hydrophobic interaction between Trp45 and the T residue of the consensus TGCGG sequence. In the present study, the differential chemical labeling of both the free protein and the protein/DNA complex showed significantly different behaviors of the three tryptophan residues comprised in the AlcR sequence toward the Koshland reagent. The spectacular decreased reaction rate for Trp45 within the complex confirmed the location of this residue at the protein/DNA interface. A similar result obtained with Trp53, an amino acid present at the C-terminal side of AlcR, also indicated its involvement in the DNA recognition. In contrast, the formation of the complex accompanied by an allosteric rearrangement allowed the Trp36 to be much more exposed to the solvent than in the free protein. These data provide additional evidence that the unique specificity of AlcR among the zinc binuclear cluster family results in new types of interactions between AlcR and its cognate targets. From a methodological point of view, the approach of differential chemical labeling combined with mass spectrometric analyses proved to be an interesting tool for the recognition of hydrophobic interactions between the tryptophan residues of a protein and its macromolecular target.

Research paper thumbnail of Structural study of the LipoMannans from Mycobacterium bovis BCG: characterisation of multiacylated forms of the phosphatidyl-myo-inositol anchor

Journal of Molecular Biology, 1999

A biosynthetic ®liation is postulated between the mycobacterial phosphatidyl-myo-inositol mannosi... more A biosynthetic ®liation is postulated between the mycobacterial phosphatidyl-myo-inositol mannosides (PIMs), the lipomannans (LMs) and the lipoarabinomannans (LAMs), the major antigens of the envelopes. Moreover, as the PI anchor is thought to play a role in the biological functions of the LAMs, we characterized the lipid moiety of the PI anchor from Mycobacterium bovis BCG cellular LMs. Their structure was investigated along with that of a puri®ed tetra-acylated form of PIM 2 (Ac 4 PIM 2). A two-dimensional 1 H-31 P heteronuclear multiple quantum correlation homonuclear Hartmann-Hahn spectroscopy study of Ac 4 PIM 2 unambiguously localised a fourth fatty acid on the C3 of the myo-Ins beside the fatty acids already described on the C1 and C2 position of the glycerol and on the C6 position of the mannose. This analytical strategy was extended to the structural study of the cellular LM anchor. Using an appropriate solvent system, the one dimensional 31 P NMR spectrum exhibited four major resonances typifying the LM populations. These populations differed in number and location of the fatty acids. For one of these populations, we established the presence of an extra fatty acid on the C3 of the myo-Ins of the LM anchor. The fact that both types of molecules have an elaborated anchor in common, indicates that cellular LMs are multimannosylated forms of PIMs. In addition, the LM mannan core structure was analysed by two-dimensional NMR, pointing to a high level of branching by single a1 3 2 Manp side-chains.

Research paper thumbnail of NMR solution structure of AlcR (1-60) provides insight in the unusual DNA binding properties of this zinc binuclear cluster protein1

Journal of molecular biology, 2000

The three-dimensional structure of the DNA-binding domain (residues 1-60) of the ethanol regulon ... more The three-dimensional structure of the DNA-binding domain (residues 1-60) of the ethanol regulon transcription factor AlcR from Aspergillus nidulans has been solved by NMR. This domain belongs to the zinc binuclear cluster class. Although the core of the protein is similar to previously characterized structures, consisting of two helices organized around a Zn 2 Cys 6 motif, the present structure presents important variations, among them the presence of two supplementary helices. This structure gives new insight into the understanding of the AlcR speci®cities in DNA binding such as longer consensus half-sites, in vitro monomeric binding but in vivo multiple repeat transcriptional activation, either in direct or inverse orientations. The presence of additional contacts of the protein with its DNA target can be predicted from a model proposed for the interaction with the consensus DNA target. The clustering of accessible negative charges on helix 2 delineates a possible interaction site for other determinants of the transcriptional machinery, responsible for the ®ne tuning of the selection of the AlcR cognate sites.

Research paper thumbnail of The Solution Structure of an AlcR-DNA Complex Sheds Light onto the Unique Tight and Monomeric DNA Binding of a Zn2Cys6 Protein

Structure, 2001

In Aspergillus nidulans, the transcription activator AlcR mediates specific induction of a number... more In Aspergillus nidulans, the transcription activator AlcR mediates specific induction of a number of the genes of the alc cluster. This cluster includes genes involved in the oxidation of ethanol and other alcohols to acetate. The pattern of binding and of transactivation of AlcR is unique within the Zn(2)Cys(6) family. The structural bases for these specificities have not been analyzed at the atomic level until now. We have used NMR spectroscopy and restrained molecular dynamics to determine a set of structures of the AlcR DNA binding domain [AlcR(1-60)] in complex with a 10-mer DNA duplex. Analysis of the structures reveals specific interactions between AlcR and DNA common to the other known zinc clusters. In addition, the involvement of the N-terminal residues upstream of the AlcR zinc cluster in DNA binding is clearly highlighted, and the pivotal role of R6 is confirmed. Totally unprecedented specific and nonspecific contacts of two additional regions of the protein with the DNA are demonstrated. The differences with the available crystallographic structures of other zinc binuclear cluster proteins-DNA complexes are analyzed. The structures of the AlcR(1-60)-DNA complex provide the basis for a better understanding of some of the specificities of the AlcR system: the DNA consensus recognition sequence--usually the triplet CGG--is extended to five base pairs, AlcR acts as a monomer, and additional contacts inside and outside the DNA binding domain in the major and minor groove are observed. These extensive interactions stabilize the AlcR monomer to its cognate DNA site.