In an attempt to identify proteins that may regulate ai1-fetoprotein (AFP) gene expression, we sc... more In an attempt to identify proteins that may regulate ai1-fetoprotein (AFP) gene expression, we screened a cDNA expression library from neonatal rat liver with two essential cis-elements of the AFP promoter and enhancer. We isolated two cDNAs which were found to correspond to leucine zipper proteins of the CC-AAT/enhancer binding protein (C/EBP) family: C/EBP3 and C/EBPy. The three related proteins C/EBP a, 3 and y bind with indistinguishable specificity to multiple DNA sites in the promoter and the enhancer of the AFP gene. In addition, C/EBPj and C/EBPy readily heterodimerize with each other as well as with C/EBPa. The mRNAs coding for C/EBPO and C/EBPy are expressed in a wider variety of rat tissues than C/EBPa mRNA, including yolk sac and fetal liver. The steady-state levels of C/EBP a, 13 and ey mRNAs increase during liver development, in parallel with their respective gene transcriptional rates. The high levels of C/EBP 3 and-y mRNAs in rat yolk sac and fetal liver, where C/EBPa is poorly expressed, suggest that C/EBP 1 and/or-y could be preponderant or early regulators of the AFP gene in these tissues.
When the green unicellular alga Chlamydomonas eugametos is grown under light/dark regimes, nuclea... more When the green unicellular alga Chlamydomonas eugametos is grown under light/dark regimes, nuclear genes are periodically activated in response to the changes in light conditions. These genetic responses are dependent upon the activation of genes associated with photosynthesis (LI616 and LI637), nonphotosynthetic photoreceptors (LI410 and LI818) and the biological clock (LI818). We report here that the LI410 and LI637 genes are part of a small gene family encoding hemoglobins (Hbs) related to those from two unicellular eukaryotes, the ciliated protozoa Paramecium caudatum and Tetrahymena pyriformis, and from the cyanobacterium Nostoc commune. Investigations of the intracellular localization of C. eugametos Hbs by means of immunogold electron microscopy indicate that these proteins are predominantly located in the chloroplast, particularly in the pyrenoid and the thylakoid region. To our knowledge, this constitutes the first evidence for the presence of Hbs in chloroplasts. Alignment of the LI637 cDNA nucleotide sequence with its corresponding genomic sequence indicates that the LI637 gene contains three introns, the positions of which are compared with those in the Hb genes of plants, animals and the ciliate P. caudatum. Although the LI637 gene possesses a three-intron/four-exon pattern similar to that of plant leghemoglobin genes, introns are inserted at different positions. Similarly the position of the single intron in the P. caudatum gene differs from the intron sites in the LI637 gene. The latter observations argue against the current view that all eukaryotic Hbs have evolved from a common ancestor having a gene structure identical to that of plant or animal Hbs.
Macrophage-generated oxygen-and nitrogen-reactive species control the development of Mycobacteriu... more Macrophage-generated oxygen-and nitrogen-reactive species control the development of Mycobacterium tuberculosis infection in the host. Mycobacterium tuberculosis`truncated hemoglobin' N (trHbN) has been related to nitric oxide (NO) detoxi®cation, in response to macrophage nitrosative stress, during the bacterium latent infection stage. The three-dimensional structure of oxygenated trHbN, solved at 1.9 A Ê resolution, displays the two-over-two a-helical sandwich fold recently characterized in two homologous truncated hemoglobins, featuring an extra N-terminal a-helix and homodimeric assembly. In the absence of a polar distal E7 residue, the O 2 heme ligand is stabilized by two hydrogen bonds to TyrB10(33). Strikingly, ligand diffusion to the heme in trHbN may occur via an apolar tunnel/cavity system extending for 28 A Ê through the protein matrix, connecting the heme distal cavity to two distinct protein surface sites. This unique structural feature appears to be conserved in several homologous truncated hemoglobins. It is proposed that in trHbN, heme Fe/O 2 stereochemistry and the protein matrix tunnel may promote O 2 /NO chemistry in vivo, as a M.tuberculosis defense mechanism against macrophage nitrosative stress.
Truncated hemoglobins (trHbs) 1 (1) constitute a family of small oxygen-binding heme proteins dis... more Truncated hemoglobins (trHbs) 1 (1) constitute a family of small oxygen-binding heme proteins distributed in eubacteria, cyanobacteria, protozoa, and plants (Table I, Supplemental Material) forming a distinct group within the hemoglobin (Hb) superfamily (2). They are nearly ubiquitous in the plant kingdom, occur in many aggressively pathogenic bacteria, and are held to be of very ancient origin. None have been detected in the genomes of archaea or metazoa. Characteristically, trHbs occur at nano-to micromolar intracellular concentration, hinting at a possible role as catalytic proteins. Many trHbs display amino acid sequences 20-40 residues shorter than non-vertebrate hemoglobins to which they are scarcely related by sequence similarity. Crystal structures (1, 3) show that trHb tertiary structure is based on a 2-on-2 ␣-helical sandwich, which represents an unprecedented editing of the highly conserved globin fold. Moreover, an almost continuous hydrophobic tunnel, traversing the protein matrix from the molecular surface to the heme distal site, may provide a path for ligand diffusion to the heme. trHbs Are Phylogenetically Distinct within the Hb Superfamily More than 40 putative trHb genes have been identified (Table I, Supplemental Material). A phylogenetic analysis (Fig. 1) indicates that trHbs form a distinct family separate from bacterial flavohemoglobins, Vitreoscilla Hb, plant symbiotic and non-symbiotic hemoglobins, and animal hemoglobins. Notably, trHb genes and flavohemoglobin genes frequently coexist in the same bacterium (Table I, Supplemental Material), suggesting distinct functions for each. Three distinct groups (groups I, II, and III) can be distinguished within the trHb family with four clusters within group II. The extent of amino acid identity between members of the different groups, e.g. Mycobacterium tuberculosis trHbO (group II) and trHbN (group I), can be low (18%) (Fig. 1, Supplemental Material). However, identity rises dramatically when the M. tuberculosis trHbO sequence is compared with the orthologue sequences from Mycobacterium avium (84%), Mycobacterium leprae (83%), Cornybacterium diphterae (64%), and Streptomyces coelicolor (66%) or when the M. tuberculosis trHbN is compared with its M. avium orthologue (79%). The presence of trHb genes from groups I, II, and III in M. avium (Gram-positive) and in Methylococcus capsulatus (proteobacteria) may indicate that the different trHb lineages diverged from their last common ancestor before the separation of the main prokaryotic lineages (Fig. 1). An interesting progression is found in the genus Mycobacterium. The genome of the opportunistic pathogen, M. avium, contains one trHb from each group of the family, trHbP (group III), trHbO (group II), and trHbN (group I) (Fig. 1). The facultative intracellular pathogen, M. tuberculosis, that infects man, has two, trHbN and trHbO, and the obligate intracellular pathogen, M. leprae, which has undergone extensive reductive evolution and is thought to have only a minimal gene set for a pathogenic mycobacterium (4), retains solely trHbO, which accordingly may play an essential role. * This minireview will be reprinted in the 2002 Minireview Compendium, which will be available in December, 2002.
Mutations were introduced in 7 kilobases of 5'-flanking rat ac-fetoprotein (AFP) genomic DNA, lin... more Mutations were introduced in 7 kilobases of 5'-flanking rat ac-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at-3.7 kilobases and a liver developmental stage-specific site at-2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at-65 base pairs, PEII at-120 base pairs, and DE at-160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.
During liver development, the tandem ac-fetoprotein (AFP)/albumin locus is triggered at the AFP e... more During liver development, the tandem ac-fetoprotein (AFP)/albumin locus is triggered at the AFP end and then asymmetrically enhanced; this is followed by autonomous repression of the AFP-encoding gene. To understand this regulation better, we characterized the two early developmental stage-specific DNase I-hypersensitive (DH) sites so far identified in rat liver AFP/albumin chromatic: an intergenic DH-enhancer site and the AFP DH-promoter site. Mutation-transfection analyses circumscribed the DH-enhancer domain to a 200-bp DNA segment stringently conserved among species. Targeted mutations, DNA-protein-binding assays, and coexpression experiments pinpointed C/EBP as the major activator component of the intergenic enhancer. Structure-function relationships at the AFP DH-promoter site defined a discrete glucocorticoid-regulated domain activated cooperatively by HNF1 and a highly specific AFP transcription factor, F1F, which binds to a steroid receptor recognition motif. The HNF1/FTF/DNA complex is deactivated by glucocorticoid receptors or by the ubiquitous factor NF1, which eliminates HNF1 by competition at an overlapping, high-affinity binding site. We propose that the HNF1-NF1 site might serve as a developmental switch to direct autonomous AFP gene repression in late liver development. We also conclude that the intergenic enhancer is driven by C/EBPat primarily to fulfill albumin gene activation functions at early developmental stages. Factor FTF seems to be the key regulator of AFP gene-specific functions in carcinoembryonic states.
Cloned cDNA fragments spanning nearly the entire coding regions of rat AFP and albumin genes were... more Cloned cDNA fragments spanning nearly the entire coding regions of rat AFP and albumin genes were used in liver nuclear runoff assays. Under standard assay conditions, transcription signals detected with 5' probes were systematically stronger than with 3' probes. Heparin eliminated this phenomenon, which suggests that nuclear runoff assays are subject to in vitro reinitiation occurring preferentially in promoter gene regions. Transcription in the presence of heparin indicates that very few polymerases are engaged on the AFP gene in adult rat liver. Dexamethasone treatment of developing rat liver results in the loss of transcribing polymerases from all regions of the AFP gene. Albumin gene transcription is unaffected. Inhibition of liver protein synthesis with cycloheximide does not modify the AFP gene suppressive action of dexamethasone. Glucocorticoid hormone receptors may thus directly interact with the AFP locus, blocking polymerase initiation. We also report the sequence analysis of rat AFP mRNA, which reveals the existence of two potential initiation codons on this molecule.
Truncated hemoglobins (trHbs) build a separate subfamily within the hemoglobin superfamily; they ... more Truncated hemoglobins (trHbs) build a separate subfamily within the hemoglobin superfamily; they are scarcely related by sequence similarity to (non-)vertebrate hemoglobins, displaying amino acid sequences in the 115-130 residue range. The trHb tertiary structure is based on a 2-on-2 a-helical sandwich, which hosts a unique hydrophobic cavity/tunnel system, traversing the protein matrix, from the molecular surface to the heme distal site. Such a protein matrix system may provide a path for diffusion of ligands to the heme. In Mycobacterium tuberculosis trHbN the heme-bound oxygen molecule is part of an extended hydrogen bond network including the heme distal residues TyrB10 and GlnE11. In vitro experiments have shown that M. tuberculosis trHbN supports efficiently nitric oxide dioxygenation, yielding nitrate. Such a reaction would provide a defense barrier against the nitrosative stress raised by host macrophages during lung infection. It is proposed that the whole protein architecture, the heme distal site hydrogen bonded network, and the unique protein matrix tunnel, are optimally designed to support the pseudo-catalytic role of trHbN in converting the reactive NO species into the harmless NO 3 7 .
Our relationship as friends and colleagues began when we met at a Gordon conference in 1994. It m... more Our relationship as friends and colleagues began when we met at a Gordon conference in 1994. It marked the beginning of animated, challenging and respectful scientific discussions that have greatly benefited my development as a scientist. The work sessions in our respective laboratories, often very intensive, have also been privileged moments for me and my graduate students. Jonathan, your unique
Three major regions of DNase I hypersensitivity (DH) were found in al-fetoprotein (AFP) chromatin... more Three major regions of DNase I hypersensitivity (DH) were found in al-fetoprotein (AFP) chromatin of rat liver. DH site I is located at the transcription initiation site and associated with ongoing AFP transcription. DH site II is located 2.5 kb upstream from the cap site: it is developmental stage-dependent but dissociable from ongoing AFP transcription. DH site III, 3.7 kb upstream from the cap site, behaves as hepatocyte-constitutive. DH sites are present in similar regions of liver albumin chromatin. Dexamethasone-induced AFP gene repression is accompanied by the selective loss of AFP DH site I, a likely result of glucocorticoid receptors binding to a DNA recognition sequence located 5'-adjacent to DH site I. SI nucleasehypersensitive sites were found on naked superhelical AFP and albumin DNA, but do not appear to contribute DH sites in liver chromatin. The extent of hypomethylation of HpaII sites at the 5'-end of the AFP gene correlates positively with the level of potential and actual expression of the gene. We conclude that developmental and hormonal regulation of the AFP gene is confined within =4 kb of 5'-flanking DNA, and we discuss possible hierarchical interactions among DH sites, in relation to DNA methylation and replication.
Infections caused by bacteria belonging to genus Mycobacterium are among the most challenging thr... more Infections caused by bacteria belonging to genus Mycobacterium are among the most challenging threats for human health. The ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of effective detoxification mechanisms. Mycobacterial truncated hemoglobins (trHbs) have recently been implicated in scavenging of reactive nitrogen species. Individual members from each trHb family (N, O, and P) can be present in the same mycobacterial species. The distinct features of the heme active site structure combined with different ligand binding properties and in vivo expression patterns of mycobacterial trHbs suggest that these globins may accomplish diverse functions. Here, recent genomic, structural and biochemical information on mycobacterial trHbs is reviewed, with the aim of providing further insights into the role of these globins in mycobacterial physiology.
The administration of glumrticosteroid hormones to newborn rats interrupts selectively (and rever... more The administration of glumrticosteroid hormones to newborn rats interrupts selectively (and reversibly, if the hormone is withdrawn) the hepatic production of cY1-fetoprotein (AFP). This results from a decreased concentration of AFP mRNA in the liver [
Truncated hemoglobin O (trHbO) is one of two trHbs in Mycobacterium tuberculosis. Remarkably, trH... more Truncated hemoglobin O (trHbO) is one of two trHbs in Mycobacterium tuberculosis. Remarkably, trHbO possesses two novel distal residues, in addition to the B10 tyrosine, that may be important in ligand binding. These are the CD1 tyrosine and G8 tryptophan. Here we investigate the reactions of trHbO and mutants using stopped-flow spectrometry, flash photolysis, and UV-enhanced resonance Raman spectroscopy. A biphasic kinetic behavior is observed for combination and dissociation of O 2 and CO that is controlled by the B10 and CD1 residues. The rate constants for combination (<1.0 µM-1 s-1) and dissociation (<0.006 s-1) of O 2 are among the slowest known, precluding transport or diffusion of O 2 as a major function. Mutation of CD1 tyrosine to phenylalanine shows that this group controls ligand binding, as evidenced by 25-and 77-fold increases in the combination rate constants for O 2 and CO, respectively. In support of a functional role for G8 tryptophan, UV resonance Raman indicates that the (2,1) dihedral angle for the indole ring increases progressively from approximately 93°to at least 100°in going sequentially from the deoxy to CO to O 2 derivative, demonstrating a significant conformational change in the G8 tryptophan with ligation. Remarkably, protein modeling predicts a network of hydrogen bonds between B10 tyrosine, CD1 tyrosine, and G8 tryptophan, with the latter residues being within hydrogen bonding distance of the heme-bound ligand. Such a rigid hydrogen bonding network may thus represent a considerable barrier to ligand entrance and escape. In accord with this model, we found that changing CD1 or B10 tyrosine for phenylalanine causes only small changes in the rate of O 2 dissociation, suggesting that more than one hydrogen bond must be broken at a time to promote ligand escape. Furthermore, trHbO-CO cannot be photodissociated under conditions where the CO derivative of myoglobin is extensively photodissociated, indicating that CO is constrained near the heme by the hydrogen bonding network.
KCNE1 (also known as MinK) is a membrane-bound protein with a single transmembrane section consis... more KCNE1 (also known as MinK) is a membrane-bound protein with a single transmembrane section consisting of 129 amino acids with a molecular weight of 15.7 kD. The main function of KCNE1 is to modulate the activity of the KCNQ1 voltage-gated potassium ion channel in the human heart. This potassium modulation is critical for repolarization in the heart, specifically in the left ventricle. Mutations in the KCNE1 gene can result in genetic disorders such as long QT syndrome, sudden infant death syndrome (SIDS), as well as deafness. However, most current KCNE1 studies are from a functional standpoint, which leaves the structural and dynamic information of the protein unknown. EPR spectroscopy is a very powerful structural biology tool that can be used to determine the structural topology of membrane proteins with respect to its membrane environment. In this study, we use the EPR power saturation and EPR membrane alignment techniques coupled with site-directed spin-labeling (SDSL) to study the structural topology of KCNE1 with respect to the lipid bilayer.
A singular aspect of the 2-on-2 hemoglobin structures of groups I and II is the presence of tunne... more A singular aspect of the 2-on-2 hemoglobin structures of groups I and II is the presence of tunnels linking the protein surface to the distal heme pocket, supporting the storage and the diffusion of small apolar ligands to/from the buried active site. As the solubility of apolar ligands is greater in biological membranes than in solution, the association of these proteins with biological membranes may improve the efficiency of ligand capture. As very little is known on this subject, we have investigated the interactions between hemoglobin N (HbN), a group I 2-on-2 hemoglobin from the pathogenic Mycobacterium tuberculosis (Mtb), and biological membranes using both experimental techniques and MD simulations. HbN has a potent nitric oxide dioxygenase activity (HbN-Fe(2+)-O2 + •NO + H2O → HbN-Fe(3+)-OH2 + NO3(-)) that is thought to protect the aerobic respiration of Mtb from inhibition by •NO. Three different membrane compositions were chosen for the studies, representative of the mycobacterial plasma membrane and the mammalian cell membranes. Both the experimental and the modeling results agreed with each other and allow for a detailed molecular description of HbN in association with membranes of different compositions. The results indicated that HbN is a peripheral protein, and the association with the membranes occurred via the pre-A, G, and H helices. In addition, HbN would be allowed to modulate the binding to the membranes via electrostatic interactions between the lipid membranes and the Asp100 residue. In its membrane-bound form the short tunnel of HbN is oriented toward the membrane interior and the other tunnels point toward the solvent. Such protein orientation would facilitate the uptake of nonpolar substrates from the membrane and the release of products to the solvent. It is interesting to note that the pre-A, G, and H helices are conserved among HbN from a few other Mycobacteria.
In an attempt to identify proteins that may regulate ai1-fetoprotein (AFP) gene expression, we sc... more In an attempt to identify proteins that may regulate ai1-fetoprotein (AFP) gene expression, we screened a cDNA expression library from neonatal rat liver with two essential cis-elements of the AFP promoter and enhancer. We isolated two cDNAs which were found to correspond to leucine zipper proteins of the CC-AAT/enhancer binding protein (C/EBP) family: C/EBP3 and C/EBPy. The three related proteins C/EBP a, 3 and y bind with indistinguishable specificity to multiple DNA sites in the promoter and the enhancer of the AFP gene. In addition, C/EBPj and C/EBPy readily heterodimerize with each other as well as with C/EBPa. The mRNAs coding for C/EBPO and C/EBPy are expressed in a wider variety of rat tissues than C/EBPa mRNA, including yolk sac and fetal liver. The steady-state levels of C/EBP a, 13 and ey mRNAs increase during liver development, in parallel with their respective gene transcriptional rates. The high levels of C/EBP 3 and-y mRNAs in rat yolk sac and fetal liver, where C/EBPa is poorly expressed, suggest that C/EBP 1 and/or-y could be preponderant or early regulators of the AFP gene in these tissues.
When the green unicellular alga Chlamydomonas eugametos is grown under light/dark regimes, nuclea... more When the green unicellular alga Chlamydomonas eugametos is grown under light/dark regimes, nuclear genes are periodically activated in response to the changes in light conditions. These genetic responses are dependent upon the activation of genes associated with photosynthesis (LI616 and LI637), nonphotosynthetic photoreceptors (LI410 and LI818) and the biological clock (LI818). We report here that the LI410 and LI637 genes are part of a small gene family encoding hemoglobins (Hbs) related to those from two unicellular eukaryotes, the ciliated protozoa Paramecium caudatum and Tetrahymena pyriformis, and from the cyanobacterium Nostoc commune. Investigations of the intracellular localization of C. eugametos Hbs by means of immunogold electron microscopy indicate that these proteins are predominantly located in the chloroplast, particularly in the pyrenoid and the thylakoid region. To our knowledge, this constitutes the first evidence for the presence of Hbs in chloroplasts. Alignment of the LI637 cDNA nucleotide sequence with its corresponding genomic sequence indicates that the LI637 gene contains three introns, the positions of which are compared with those in the Hb genes of plants, animals and the ciliate P. caudatum. Although the LI637 gene possesses a three-intron/four-exon pattern similar to that of plant leghemoglobin genes, introns are inserted at different positions. Similarly the position of the single intron in the P. caudatum gene differs from the intron sites in the LI637 gene. The latter observations argue against the current view that all eukaryotic Hbs have evolved from a common ancestor having a gene structure identical to that of plant or animal Hbs.
Macrophage-generated oxygen-and nitrogen-reactive species control the development of Mycobacteriu... more Macrophage-generated oxygen-and nitrogen-reactive species control the development of Mycobacterium tuberculosis infection in the host. Mycobacterium tuberculosis`truncated hemoglobin' N (trHbN) has been related to nitric oxide (NO) detoxi®cation, in response to macrophage nitrosative stress, during the bacterium latent infection stage. The three-dimensional structure of oxygenated trHbN, solved at 1.9 A Ê resolution, displays the two-over-two a-helical sandwich fold recently characterized in two homologous truncated hemoglobins, featuring an extra N-terminal a-helix and homodimeric assembly. In the absence of a polar distal E7 residue, the O 2 heme ligand is stabilized by two hydrogen bonds to TyrB10(33). Strikingly, ligand diffusion to the heme in trHbN may occur via an apolar tunnel/cavity system extending for 28 A Ê through the protein matrix, connecting the heme distal cavity to two distinct protein surface sites. This unique structural feature appears to be conserved in several homologous truncated hemoglobins. It is proposed that in trHbN, heme Fe/O 2 stereochemistry and the protein matrix tunnel may promote O 2 /NO chemistry in vivo, as a M.tuberculosis defense mechanism against macrophage nitrosative stress.
Truncated hemoglobins (trHbs) 1 (1) constitute a family of small oxygen-binding heme proteins dis... more Truncated hemoglobins (trHbs) 1 (1) constitute a family of small oxygen-binding heme proteins distributed in eubacteria, cyanobacteria, protozoa, and plants (Table I, Supplemental Material) forming a distinct group within the hemoglobin (Hb) superfamily (2). They are nearly ubiquitous in the plant kingdom, occur in many aggressively pathogenic bacteria, and are held to be of very ancient origin. None have been detected in the genomes of archaea or metazoa. Characteristically, trHbs occur at nano-to micromolar intracellular concentration, hinting at a possible role as catalytic proteins. Many trHbs display amino acid sequences 20-40 residues shorter than non-vertebrate hemoglobins to which they are scarcely related by sequence similarity. Crystal structures (1, 3) show that trHb tertiary structure is based on a 2-on-2 ␣-helical sandwich, which represents an unprecedented editing of the highly conserved globin fold. Moreover, an almost continuous hydrophobic tunnel, traversing the protein matrix from the molecular surface to the heme distal site, may provide a path for ligand diffusion to the heme. trHbs Are Phylogenetically Distinct within the Hb Superfamily More than 40 putative trHb genes have been identified (Table I, Supplemental Material). A phylogenetic analysis (Fig. 1) indicates that trHbs form a distinct family separate from bacterial flavohemoglobins, Vitreoscilla Hb, plant symbiotic and non-symbiotic hemoglobins, and animal hemoglobins. Notably, trHb genes and flavohemoglobin genes frequently coexist in the same bacterium (Table I, Supplemental Material), suggesting distinct functions for each. Three distinct groups (groups I, II, and III) can be distinguished within the trHb family with four clusters within group II. The extent of amino acid identity between members of the different groups, e.g. Mycobacterium tuberculosis trHbO (group II) and trHbN (group I), can be low (18%) (Fig. 1, Supplemental Material). However, identity rises dramatically when the M. tuberculosis trHbO sequence is compared with the orthologue sequences from Mycobacterium avium (84%), Mycobacterium leprae (83%), Cornybacterium diphterae (64%), and Streptomyces coelicolor (66%) or when the M. tuberculosis trHbN is compared with its M. avium orthologue (79%). The presence of trHb genes from groups I, II, and III in M. avium (Gram-positive) and in Methylococcus capsulatus (proteobacteria) may indicate that the different trHb lineages diverged from their last common ancestor before the separation of the main prokaryotic lineages (Fig. 1). An interesting progression is found in the genus Mycobacterium. The genome of the opportunistic pathogen, M. avium, contains one trHb from each group of the family, trHbP (group III), trHbO (group II), and trHbN (group I) (Fig. 1). The facultative intracellular pathogen, M. tuberculosis, that infects man, has two, trHbN and trHbO, and the obligate intracellular pathogen, M. leprae, which has undergone extensive reductive evolution and is thought to have only a minimal gene set for a pathogenic mycobacterium (4), retains solely trHbO, which accordingly may play an essential role. * This minireview will be reprinted in the 2002 Minireview Compendium, which will be available in December, 2002.
Mutations were introduced in 7 kilobases of 5'-flanking rat ac-fetoprotein (AFP) genomic DNA, lin... more Mutations were introduced in 7 kilobases of 5'-flanking rat ac-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at-3.7 kilobases and a liver developmental stage-specific site at-2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at-65 base pairs, PEII at-120 base pairs, and DE at-160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.
During liver development, the tandem ac-fetoprotein (AFP)/albumin locus is triggered at the AFP e... more During liver development, the tandem ac-fetoprotein (AFP)/albumin locus is triggered at the AFP end and then asymmetrically enhanced; this is followed by autonomous repression of the AFP-encoding gene. To understand this regulation better, we characterized the two early developmental stage-specific DNase I-hypersensitive (DH) sites so far identified in rat liver AFP/albumin chromatic: an intergenic DH-enhancer site and the AFP DH-promoter site. Mutation-transfection analyses circumscribed the DH-enhancer domain to a 200-bp DNA segment stringently conserved among species. Targeted mutations, DNA-protein-binding assays, and coexpression experiments pinpointed C/EBP as the major activator component of the intergenic enhancer. Structure-function relationships at the AFP DH-promoter site defined a discrete glucocorticoid-regulated domain activated cooperatively by HNF1 and a highly specific AFP transcription factor, F1F, which binds to a steroid receptor recognition motif. The HNF1/FTF/DNA complex is deactivated by glucocorticoid receptors or by the ubiquitous factor NF1, which eliminates HNF1 by competition at an overlapping, high-affinity binding site. We propose that the HNF1-NF1 site might serve as a developmental switch to direct autonomous AFP gene repression in late liver development. We also conclude that the intergenic enhancer is driven by C/EBPat primarily to fulfill albumin gene activation functions at early developmental stages. Factor FTF seems to be the key regulator of AFP gene-specific functions in carcinoembryonic states.
Cloned cDNA fragments spanning nearly the entire coding regions of rat AFP and albumin genes were... more Cloned cDNA fragments spanning nearly the entire coding regions of rat AFP and albumin genes were used in liver nuclear runoff assays. Under standard assay conditions, transcription signals detected with 5' probes were systematically stronger than with 3' probes. Heparin eliminated this phenomenon, which suggests that nuclear runoff assays are subject to in vitro reinitiation occurring preferentially in promoter gene regions. Transcription in the presence of heparin indicates that very few polymerases are engaged on the AFP gene in adult rat liver. Dexamethasone treatment of developing rat liver results in the loss of transcribing polymerases from all regions of the AFP gene. Albumin gene transcription is unaffected. Inhibition of liver protein synthesis with cycloheximide does not modify the AFP gene suppressive action of dexamethasone. Glucocorticoid hormone receptors may thus directly interact with the AFP locus, blocking polymerase initiation. We also report the sequence analysis of rat AFP mRNA, which reveals the existence of two potential initiation codons on this molecule.
Truncated hemoglobins (trHbs) build a separate subfamily within the hemoglobin superfamily; they ... more Truncated hemoglobins (trHbs) build a separate subfamily within the hemoglobin superfamily; they are scarcely related by sequence similarity to (non-)vertebrate hemoglobins, displaying amino acid sequences in the 115-130 residue range. The trHb tertiary structure is based on a 2-on-2 a-helical sandwich, which hosts a unique hydrophobic cavity/tunnel system, traversing the protein matrix, from the molecular surface to the heme distal site. Such a protein matrix system may provide a path for diffusion of ligands to the heme. In Mycobacterium tuberculosis trHbN the heme-bound oxygen molecule is part of an extended hydrogen bond network including the heme distal residues TyrB10 and GlnE11. In vitro experiments have shown that M. tuberculosis trHbN supports efficiently nitric oxide dioxygenation, yielding nitrate. Such a reaction would provide a defense barrier against the nitrosative stress raised by host macrophages during lung infection. It is proposed that the whole protein architecture, the heme distal site hydrogen bonded network, and the unique protein matrix tunnel, are optimally designed to support the pseudo-catalytic role of trHbN in converting the reactive NO species into the harmless NO 3 7 .
Our relationship as friends and colleagues began when we met at a Gordon conference in 1994. It m... more Our relationship as friends and colleagues began when we met at a Gordon conference in 1994. It marked the beginning of animated, challenging and respectful scientific discussions that have greatly benefited my development as a scientist. The work sessions in our respective laboratories, often very intensive, have also been privileged moments for me and my graduate students. Jonathan, your unique
Three major regions of DNase I hypersensitivity (DH) were found in al-fetoprotein (AFP) chromatin... more Three major regions of DNase I hypersensitivity (DH) were found in al-fetoprotein (AFP) chromatin of rat liver. DH site I is located at the transcription initiation site and associated with ongoing AFP transcription. DH site II is located 2.5 kb upstream from the cap site: it is developmental stage-dependent but dissociable from ongoing AFP transcription. DH site III, 3.7 kb upstream from the cap site, behaves as hepatocyte-constitutive. DH sites are present in similar regions of liver albumin chromatin. Dexamethasone-induced AFP gene repression is accompanied by the selective loss of AFP DH site I, a likely result of glucocorticoid receptors binding to a DNA recognition sequence located 5'-adjacent to DH site I. SI nucleasehypersensitive sites were found on naked superhelical AFP and albumin DNA, but do not appear to contribute DH sites in liver chromatin. The extent of hypomethylation of HpaII sites at the 5'-end of the AFP gene correlates positively with the level of potential and actual expression of the gene. We conclude that developmental and hormonal regulation of the AFP gene is confined within =4 kb of 5'-flanking DNA, and we discuss possible hierarchical interactions among DH sites, in relation to DNA methylation and replication.
Infections caused by bacteria belonging to genus Mycobacterium are among the most challenging thr... more Infections caused by bacteria belonging to genus Mycobacterium are among the most challenging threats for human health. The ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of effective detoxification mechanisms. Mycobacterial truncated hemoglobins (trHbs) have recently been implicated in scavenging of reactive nitrogen species. Individual members from each trHb family (N, O, and P) can be present in the same mycobacterial species. The distinct features of the heme active site structure combined with different ligand binding properties and in vivo expression patterns of mycobacterial trHbs suggest that these globins may accomplish diverse functions. Here, recent genomic, structural and biochemical information on mycobacterial trHbs is reviewed, with the aim of providing further insights into the role of these globins in mycobacterial physiology.
The administration of glumrticosteroid hormones to newborn rats interrupts selectively (and rever... more The administration of glumrticosteroid hormones to newborn rats interrupts selectively (and reversibly, if the hormone is withdrawn) the hepatic production of cY1-fetoprotein (AFP). This results from a decreased concentration of AFP mRNA in the liver [
Truncated hemoglobin O (trHbO) is one of two trHbs in Mycobacterium tuberculosis. Remarkably, trH... more Truncated hemoglobin O (trHbO) is one of two trHbs in Mycobacterium tuberculosis. Remarkably, trHbO possesses two novel distal residues, in addition to the B10 tyrosine, that may be important in ligand binding. These are the CD1 tyrosine and G8 tryptophan. Here we investigate the reactions of trHbO and mutants using stopped-flow spectrometry, flash photolysis, and UV-enhanced resonance Raman spectroscopy. A biphasic kinetic behavior is observed for combination and dissociation of O 2 and CO that is controlled by the B10 and CD1 residues. The rate constants for combination (<1.0 µM-1 s-1) and dissociation (<0.006 s-1) of O 2 are among the slowest known, precluding transport or diffusion of O 2 as a major function. Mutation of CD1 tyrosine to phenylalanine shows that this group controls ligand binding, as evidenced by 25-and 77-fold increases in the combination rate constants for O 2 and CO, respectively. In support of a functional role for G8 tryptophan, UV resonance Raman indicates that the (2,1) dihedral angle for the indole ring increases progressively from approximately 93°to at least 100°in going sequentially from the deoxy to CO to O 2 derivative, demonstrating a significant conformational change in the G8 tryptophan with ligation. Remarkably, protein modeling predicts a network of hydrogen bonds between B10 tyrosine, CD1 tyrosine, and G8 tryptophan, with the latter residues being within hydrogen bonding distance of the heme-bound ligand. Such a rigid hydrogen bonding network may thus represent a considerable barrier to ligand entrance and escape. In accord with this model, we found that changing CD1 or B10 tyrosine for phenylalanine causes only small changes in the rate of O 2 dissociation, suggesting that more than one hydrogen bond must be broken at a time to promote ligand escape. Furthermore, trHbO-CO cannot be photodissociated under conditions where the CO derivative of myoglobin is extensively photodissociated, indicating that CO is constrained near the heme by the hydrogen bonding network.
KCNE1 (also known as MinK) is a membrane-bound protein with a single transmembrane section consis... more KCNE1 (also known as MinK) is a membrane-bound protein with a single transmembrane section consisting of 129 amino acids with a molecular weight of 15.7 kD. The main function of KCNE1 is to modulate the activity of the KCNQ1 voltage-gated potassium ion channel in the human heart. This potassium modulation is critical for repolarization in the heart, specifically in the left ventricle. Mutations in the KCNE1 gene can result in genetic disorders such as long QT syndrome, sudden infant death syndrome (SIDS), as well as deafness. However, most current KCNE1 studies are from a functional standpoint, which leaves the structural and dynamic information of the protein unknown. EPR spectroscopy is a very powerful structural biology tool that can be used to determine the structural topology of membrane proteins with respect to its membrane environment. In this study, we use the EPR power saturation and EPR membrane alignment techniques coupled with site-directed spin-labeling (SDSL) to study the structural topology of KCNE1 with respect to the lipid bilayer.
A singular aspect of the 2-on-2 hemoglobin structures of groups I and II is the presence of tunne... more A singular aspect of the 2-on-2 hemoglobin structures of groups I and II is the presence of tunnels linking the protein surface to the distal heme pocket, supporting the storage and the diffusion of small apolar ligands to/from the buried active site. As the solubility of apolar ligands is greater in biological membranes than in solution, the association of these proteins with biological membranes may improve the efficiency of ligand capture. As very little is known on this subject, we have investigated the interactions between hemoglobin N (HbN), a group I 2-on-2 hemoglobin from the pathogenic Mycobacterium tuberculosis (Mtb), and biological membranes using both experimental techniques and MD simulations. HbN has a potent nitric oxide dioxygenase activity (HbN-Fe(2+)-O2 + •NO + H2O → HbN-Fe(3+)-OH2 + NO3(-)) that is thought to protect the aerobic respiration of Mtb from inhibition by •NO. Three different membrane compositions were chosen for the studies, representative of the mycobacterial plasma membrane and the mammalian cell membranes. Both the experimental and the modeling results agreed with each other and allow for a detailed molecular description of HbN in association with membranes of different compositions. The results indicated that HbN is a peripheral protein, and the association with the membranes occurred via the pre-A, G, and H helices. In addition, HbN would be allowed to modulate the binding to the membranes via electrostatic interactions between the lipid membranes and the Asp100 residue. In its membrane-bound form the short tunnel of HbN is oriented toward the membrane interior and the other tunnels point toward the solvent. Such protein orientation would facilitate the uptake of nonpolar substrates from the membrane and the release of products to the solvent. It is interesting to note that the pre-A, G, and H helices are conserved among HbN from a few other Mycobacteria.
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Papers by michel guertin