A Globin in the Nucleus!
2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês
10.1074/jbc.c300203200
ISSN1083-351X
AutoresEva Geuens, Inge Brouns, Daisy Flamez, Sylvia Dewilde, Jean‐Pierre Timmermans, Luc Moëns,
Tópico(s)Neonatal Health and Biochemistry
ResumoCytoglobin and neuroglobin are recently discovered members of the globin family. In situ hybridization localized neuroglobin mainly in brain and retina, while cytoglobin was expressed ubiquitously in all analyzed tissues. In the present study, polyclonal antibodies were raised against both proteins and the distribution of them was studied by immunocytochemistry at tissue and subcellular level. Cytoglobin immunoreactivity was uniformly distributed and found in all tissues studied. At the subcellular level, cytoglobin immunoreactivity was exclusively detected in the cell nucleus. In contrast, neuroglobin immunoreactivity was detected in specific brain regions with varying intensities and in the islet of Langerhans in the pancreas. The immunoreactivity was restricted to the cytoplasm of neurons and endocrine beta cells. The nuclear localization of cytoglobin opens new perspectives for possible function(s) of globin-folded proteins as transcriptional regulators. Cytoglobin and neuroglobin are recently discovered members of the globin family. In situ hybridization localized neuroglobin mainly in brain and retina, while cytoglobin was expressed ubiquitously in all analyzed tissues. In the present study, polyclonal antibodies were raised against both proteins and the distribution of them was studied by immunocytochemistry at tissue and subcellular level. Cytoglobin immunoreactivity was uniformly distributed and found in all tissues studied. At the subcellular level, cytoglobin immunoreactivity was exclusively detected in the cell nucleus. In contrast, neuroglobin immunoreactivity was detected in specific brain regions with varying intensities and in the islet of Langerhans in the pancreas. The immunoreactivity was restricted to the cytoplasm of neurons and endocrine beta cells. The nuclear localization of cytoglobin opens new perspectives for possible function(s) of globin-folded proteins as transcriptional regulators. The widespread occurrence of hemoglobins (Hbs) 1The abbreviations used are: Hb, hemoglobin; Mb, myoglobin; Ngb, neuroglobin; mNgb, mouse neuroglobin; Cygb, cytoglobin; mCygb, mouse cytoglobin; hCYGB, human cytoglobin; PBS, phosphate-buffered salin; FITC, fluororescein isothiocyanate; H+L, heavy and light chains; GFAP, glial fibrillary acidic protein; DAPI, 4′-6-diamidino-2-phenylindole; EcDos, E. coli direct oxygen sensor; PAS, sensory domain named after the eukaryotic proteins Period, Arnt, and Simple-minded; NPAS2, neuronal PAS protein 2; ELISA, enzyme-linked immunosorbent assay.1The abbreviations used are: Hb, hemoglobin; Mb, myoglobin; Ngb, neuroglobin; mNgb, mouse neuroglobin; Cygb, cytoglobin; mCygb, mouse cytoglobin; hCYGB, human cytoglobin; PBS, phosphate-buffered salin; FITC, fluororescein isothiocyanate; H+L, heavy and light chains; GFAP, glial fibrillary acidic protein; DAPI, 4′-6-diamidino-2-phenylindole; EcDos, E. coli direct oxygen sensor; PAS, sensory domain named after the eukaryotic proteins Period, Arnt, and Simple-minded; NPAS2, neuronal PAS protein 2; ELISA, enzyme-linked immunosorbent assay. in virtually all kingdoms indicates that the gene for Hb is very ancient and that Hbs may serve functions other than simple O2 carriers (1Hardison R. 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Dewilde S. Blaxter M.L. Vanfleteren J.R. Moens L. J. Biol. Chem. 2000; 275: 4810-4815Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) have been suggested and may illustrate the coincidental use of a stable protein fold during evolution.Recently, two new members of the vertebrate globin family, namely neuroglobin (Ngb) and cytoglobin (Cygb), have been discovered (12Burmester T. Weich B. Reinhardt S. Hankeln T. Nature. 2000; 407: 520-523Crossref PubMed Scopus (887) Google Scholar, 13Burmester T. Ebner B. Weich B. Hankeln T. Mol. Biol. Evol. 2002; 19: 416-421Crossref PubMed Scopus (430) Google Scholar, 14Trent III, J.T. Watts R.A. Hargrove M.S. J. Biol. Chem. 2001; 276: 30106-30110Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 15Trent III, J.T. Hargrove M.S. J. Biol. Chem. 2002; 277: 19538-19545Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 16Pesce A. Bolognesi M. Bocedi A. Ascenzi P. Dewilde S. Moens L. Hankeln T. Burmester T. EMBO Rep. 2002; 3: 1146-1151Crossref PubMed Scopus (253) Google Scholar). Both are monomeric (151 and 190 amino acid residues, respectively), intracellular proteins, displaying all determinants of the globin fold. Sequence analyses reveal low sequence identity with vertebrate Hb and Mb (20–25%), as well as a very ancient origin, i.e. much older than Mb (16Pesce A. Bolognesi M. Bocedi A. Ascenzi P. Dewilde S. Moens L. Hankeln T. Burmester T. EMBO Rep. 2002; 3: 1146-1151Crossref PubMed Scopus (253) Google Scholar). The heme-iron atom of Ngb and Cygb is hexacoordinated, showing a His-Fe-His binding scheme, both histidines being the proximal and the distal histidine, respectively (13Burmester T. Ebner B. Weich B. Hankeln T. Mol. Biol. Evol. 2002; 19: 416-421Crossref PubMed Scopus (430) Google Scholar, 15Trent III, J.T. Hargrove M.S. J. Biol. Chem. 2002; 277: 19538-19545Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 17Dewilde S. Kiger L. Burmester T. Hankeln T. Baudin-Creuza V. Aerts T. Marden M.C. Caubergs R. Moens L. J. Biol. Chem. 2001; 276: 38949-38955Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). Ngb has a high recombination rate (k on) and a slow dissociation rate (k off), indicating a high intrinsic affinity for the ligands (O2/CO). Before binding, the external ligands must compete with the internal sixth ligand, resulting in an observed O2 affinity of the recombinant proteins similar to that of Mb (1 torr at 37 °C).The function of Ngb and Cygb is a matter of debate. In response to hypoxia, Ngb is up-regulated in vivo and in vitro and protects the neurons against hypoxic damage (18Sun Y. Jin K. Mao X.O. Zhu Y. Greenberg D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15306-15311Crossref PubMed Scopus (454) Google Scholar). High concentrations (∼100 μm) of Ngb are observed in the retina and its subcellular distribution correlates with the localization of the mitochondria (19Schmidt M. Giessl A. Laufs T. Hankeln T. Wolfrum U. Burmester T. J. Biol. Chem. 2003; 278: 1932-1935Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Both observations suggest a role in intracellular O2 supply.A protein, identical to Cygb and described in liver tissue as stellate cell activation-associated protein, was found to have a peroxidase activity in vitro and to be up-regulated upon the development of hepatic inflammation and fibrosis (20Kawada N. Kristensen D.B. Asahina K. Nakatani K. Minamiyama Y. Seki S. Yoshizato K. J. Biol. Chem. 2001; 276: 25318-25323Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 21Asahina K. Kawada N. Kristensen D.B. Nakatani K. Seki S. Shiokawa M. Tateno C. Obara M. Yoshizato K. Biochim. Biophys. Acta. 2002; 1577: 471-475Crossref PubMed Scopus (35) Google Scholar). Other potential functions of Cygb have not yet been documented.The present study aimed at elucidating the immunocytochemical localization of Ngb and Cygb in different mice tissues and the localization of Cygb in the cell nucleus.EXPERIMENTAL PROCEDURESExpression Cloning and Purification of Recombinant Ngb and Cygb—Mouse Ngb (mNgb) expression and purification were done as described previously (17Dewilde S. Kiger L. Burmester T. Hankeln T. Baudin-Creuza V. Aerts T. Marden M.C. Caubergs R. Moens L. J. Biol. Chem. 2001; 276: 38949-38955Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). Expression and purification of human cytoglobin (hCYGB) was done as follows. The expression plasmid containing hCYGB in pET3a (13Burmester T. Ebner B. Weich B. Hankeln T. Mol. Biol. Evol. 2002; 19: 416-421Crossref PubMed Scopus (430) Google Scholar) was transformed into Escherichia coli strain BL21(DE3)pLysS. Except for the omission of 5-aminolevulinic acid hydrochloride, cells were grown and harvested as described for mNgb (17Dewilde S. Kiger L. Burmester T. Hankeln T. Baudin-Creuza V. Aerts T. Marden M.C. Caubergs R. Moens L. J. Biol. Chem. 2001; 276: 38949-38955Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). Cells were suspended in 50 mm Tris-HCl, pH 7.5, 0.5 m NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride and 5 mm dithiotreitol. After suspension, 0.10 volume of 10% Triton X-100, 10% deoxycholic acid, 500 mm Tris-HCl, pH 7.5, 20 mm EDTA was added, and the cells were exposed to three freeze-thaw cycles and sonication until complete lysis. Inclusion bodies were isolated by centrifugation at 3,300 × g for 10 min, and the pellet was suspended in and washed three times with 1% Triton X-100, 1 mm EDTA, and 50 mm Tris-HCl, pH 7.5. Inclusion bodies were solubilized in 6 m guanidinium hydrochloride, 50 mm Tris-HCl, pH 7.5, and 1% 2-mercaptoethanol during 1 h at 0 °C. After elimination of the insoluble material by centrifugation (10 min 10,000 × g), hCYGB was reconstructed by adding a 1.4 m excess of hemin and dialyzed against 5 mm Tris-HCl, pH 8.5, at 4 °C. The reconstructed hCYGB was further purified as described for mNgb.Immunohistochemistry—Polyclonal antibodies against purified recombinant mNgb and hCYGB were raised in rabbits and monitored by ELISA using standard protocols. The anti-mNgb and anti-hCYGB antibodies were purified from sera by ammonium sulfate precipitation and affinity chromatography using the corresponding immobilized antigen coupled to cyanogen bromide-activated Sepharose™ 4b (Amersham Biosciences) as matrix (22Gonyea L.M. Clin. Chem. 1977; 23: 234-236Crossref PubMed Scopus (31) Google Scholar).Adult Swiss mice (n = 6) were used for immunohistochemistry. All procedures were approved by the local ethics committee of the University of Antwerp and conformed to European Community regulations. After lethal injection with Nembutal (sodium pentobarbital) mice were transcardially perfused with physiological saline, followed by Zamboni's fixative (30 ml for 10 min). Tissues (brain, liver, heart, striated muscle, lung, kidney, and small bowel) were removed and post-fixed in Zamboni's fixative for 30 min at room temperature, after which they were treated for improving the immunocytochemical conditions according to Llewellyn-Smith et al. (23Llewellyn-Smith I.J. Costa M. Furness J.B. J. Histochem. Cytochem. 1985; 33: 857-866Crossref PubMed Scopus (95) Google Scholar). After being stored overnight in 20% sucrose in phosphate-buffered saline (PBS) at 4 °C, tissues were mounted in Tissue Tek (Sakura Finettek). Twenty-μm-thick cryostat sections were thaw-mounted on poly-l-lysine-coated microscope slides and dried for 2 h at 37 °C. All primary and secondary antisera were diluted in PBS containing 10% normal goat serum (Dako X 0907, Carpinteria, CA) 0.1% bovine serum albumin, 0.05% thimerosal, and 0.01% NaN3 (PBS). All incubations were carried out at room temperature. After preincubation with PBS containing 1% Triton X-100, sections were incubated overnight with rabbit polyclonal antibodies against mNgb (diluted 1:2,000) or hCYGB (diluted 1:1,000), followed by incubation with fluororescein isothiocyanate (FITC)-conjugated Fab fragments of goat anti-rabbit IgG heavy and light chains (H+L) (diluted 1:200; 1 h; Jackson ImmunoResearch number 111-097-003, West Grove, PA). Double labeling studies for these globins and specific markers were performed to determine the nature of mNgb- or hCYGB-immunoreactive cells. Since all antibodies had been raised in rabbits, the double labeling procedure initially described by Negoescu et al. (24Negoescu A. Labat-Moleur F. Lorimier P. Lamarcq L. Guillermet C. Chambaz E. Brambilla E. J. Histochem. Cytochem. 1994; 42: 433-437Crossref PubMed Scopus (155) Google Scholar) and modified by Brouns et al. (25Brouns I. Van Nassauw L. Van Genechten J. Majewski M. Scheuermann D.W. Timmermans J.P. Adriaensen D. J. Histochem. Cytochem. 2002; 50: 575-582Crossref PubMed Scopus (69) Google Scholar) had to be applied. Briefly, an incubation with primary antibodies raised against mNgb or hCYGB was performed overnight, after which the sections were incubated with FITC-conjugated Fab fragments of goat anti-rabbit IgG (H+L) (diluted 1:200) for 6 h, followed by a 2-h incubation with unlabeled Fab fragments of goat anti-rabbit IgG (Jackson ImmunoResearch number 111-007-003) to block all possible remaining binding sites of the first primary antibody. The sections were then incubated overnight with the second primary antibody, which was directed against glial fibrillary acidic protein (anti-GFAP, rabbit polyclonal, 1:200, Dako Z334), calretinin (rabbit polyclonal, 1:5,000, Swant 7696, Bellinzona, Switzerland) or calbindin (rabbit polyclonal, 1:2,000, Swant CB-38). These antibodies were detected with a Cy3-labeled goat anti-rabbit IgG (H+L) (GAR-Cy3; diluted 1:4,000, 1 h, Jackson ImmunoResearch number 111-165-144). Negative controls were performed by substitution of non-immune sera for the primary or secondary antisera and antibody specificity was checked by preabsorption of the mNgb and hCYGB antisera with the antigen. To this end, antibodies were diluted 1:1,000 in PBS and incubated overnight at room temperature with 1–10 μg of protein/100 μl.For the detection of Ngb and Cygb in mouse pancreas, the tissue was fixed in buffered 4% paraformaldehyde for 4 h and embedded in paraffin. Sections of 4 μm were immunostained with anti-mNgb (1:1,000) or anti-hCYGB (1:1,000) following the same procedures as described above. To determine the immunoreactive endocrine cells co-localizing with Ngb expression, the following markers were used: insulin (guinea pig polyclonal, 1:10,000, Dako A564), somatostatin (rat monoclonal, 1:100, Biogenesis 8330-0009, Poole, UK), and glucagon (rabbit polyclonal, 1:200, Affiniti GA 1181, Exeter, UK). The antibodies were detected with Cy3-labeled donkey anti guinea pig IgG (H+L) (diluted 1:500, Jackson ImmunoResearch number 706-165-148), a Cy3-labeled donkey anti rat IgG (H+L) (diluted 1:500, Jackson ImmunoResearch number 712-165-153) or GAR-Cy3, respectively.Sections were cross-explored using fluorescence (Olympus BX50 or Zeiss Axiophot) microscopy. Fluorescence micrographs of 4′-6-diamidino-2-phenylindole (DAPI)-stained tissues were made on the Olympus Microscope with a Sony 9100 VID CAM and processed with AnalySIS software. To obtain detailed images of labeled neurons, a confocal laser scanning microscope (Zeiss LSM 410) equipped with image reconstruction facilities (Imaris 2.7 software; Bitplane AG, Zürich, Switzerland; Silicon Graphics Indigo 2 work station) was used. Excitation of the FITC-fluorophore and the Cy3-fluorophore was achieved with an argon laser (488 nm) and a helium/neon laser (543 nm), respectively.Western Blotting—mCygb was isolated from a nuclear extract of mouse liver. The nuclei were isolated according to the method of Blobbel and Potter as cited by Tata (26Tata J.R. Methods Enzymol. 1974; 31: 253-262Crossref PubMed Scopus (72) Google Scholar), and mCygb was isolated by affinity chromatography on a matrix-immobilized anti-hCYGB antibody column. Extracts of the nuclear and the cytosolic fraction were analyzed by SDS-PAGE. For immunodetection the anti-hCYGB antibody (1:1,000) was used.RESULTS AND DISCUSSIONELISA and Western blotting confirmed the specificity of the antibodies against mNgb and hCYGB, which did not cross-react or produce any reaction against recombinant Mb and Hb, and no cross-reaction was seen between mNGB and hCYGB (see Supplemental Material).Immunoreactivity of mCygb was detected in all studied tissues (Fig. 1). Within the brain, mCygb immunoreactivity was uniformly distributed over the different brain regions. No co-localization of mCygb and GFAP immunoreactivity was observed but some mCygb immunoreactivity-positive cells also displayed immunoreactive for calbindin, a calcium-binding protein known to be present in neurons within the central nervous system (Fig. 1), and calretinin, corroborating that, mCygb immunoreactivity is limited to neurons within the brain. In all mCygb-positive cells the immunoreactivity was specifically confined to the cell nucleus, as evidenced by combined detection of mCygb-immunoreactivity and DAPI, a specific nuclear staining (Fig. 2).Fig. 2Fluorescence micrograph of mouse liver stained with mCygb (green FITC fluorescence) and DAPI, a marker for cell nuclei. A, immunocytochemical staining for mCygb B, DAPI staining of the same position as in A conforming the presence of mCygb in nuclei of hepatocytes. c, cytoplasm; n, nucleusView Large Image Figure ViewerDownload Hi-res image Download (PPT)The nuclear localization of mCygb was independently confirmed by isolating mCygb from a nuclear liver extract by affinity chromatography on a matrix-immobilized anti-hCYGB antibody column, followed by Western blot analysis (Fig. 3).Fig. 3Western blot analysis of mouse liver subcellular fractions. Detection was done with anti-hCYGB antibodies. In lanes 2 and 3 similar protein concentrations (10 μg) of an affinity-purified nuclear extract (lane 2) and a cytosolic extract (lane 3) were analyzed, whereas in lane 1 recombinant mCygb is used as a positive control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In contrast, mNgb was expressed in distinct tissues: no immunoreactivity was detected in liver, heart, striated muscle, lung, small bowel, or kidney. In pancreas, mNgb immunoreactivity was restricted to the islets of Langerhans. It was visible in the brain, as such confirming the results of Mammen et al. (27Mammen P.P. Shelton J.M. Goetsch S.C. Williams S.C. Richardson J.A. Garry M.G. Garry D.J. J. Histochem. Cytochem. 2002; 50: 1591-1598Crossref PubMed Scopus (127) Google Scholar) and Reuss et al. (28Reuss S. Saaler-Reinhardt S. Weich B. Wystub S. Reuss M.H. Burmester T. Hankeln T. Neuroscience. 2002; 115: 645-656Crossref PubMed Scopus (165) Google Scholar). Within the brain, mNgb immunoreactivity was found focally having different staining intensities. The most intense mNgb immunoreactivity was found in the medial vestibular nucleus (Fig. 4) and the paraolivary nucleus, and less intense reactivity was seen in the thalamic and subthalamic regions and in the cortex, whereas none was detected in the hippocampus and corpus callosum. These results mainly confirm previous in situ hybridization data (27Mammen P.P. Shelton J.M. Goetsch S.C. Williams S.C. Richardson J.A. Garry M.G. Garry D.J. J. Histochem. Cytochem. 2002; 50: 1591-1598Crossref PubMed Scopus (127) Google Scholar, 28Reuss S. Saaler-Reinhardt S. Weich B. Wystub S. Reuss M.H. Burmester T. Hankeln T. Neuroscience. 2002; 115: 645-656Crossref PubMed Scopus (165) Google Scholar). However, in contrast to Reuss et al. (28Reuss S. Saaler-Reinhardt S. Weich B. Wystub S. Reuss M.H. Burmester T. Hankeln T. Neuroscience. 2002; 115: 645-656Crossref PubMed Scopus (165) Google Scholar) who claimed a more uniform distribution of mNgb throughout the distinct brain regions, found a more focal distribution pattern of mNgb. This difference could be the result of the used techniques: with in situ hybridization the distribution of the mRNA was studied, whereas the present study detected the distribution of the protein. Combined detection of mNgb and GFAP and calretinin and calbindin revealed the absence of GFAP and calretinin immunoreactivity from mNgb-immunopositive cells. These mNgb immunoreactive cells clearly resembled a neuronal morphology. Single staining (Fig. 4) as well as double labeling with an antibody raised against calbindin revealed the cytosolic localization of mNgb both at the level of the perikaryon and neurites.Fig. 4Maximum value projections of confocal optical sections of mNgb-containing cells in mouse brain. mNgb-containing neurons in the medial ventricular nuclei of the mouse brain are shown. Immunoreactivity is present in the cytoplasm of the neuronal cell body and in the neuronal extensions (asterisks). The nucleus remains unstained. c, cytoplasm; n, nucleus; *, neuronal extension.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The observation that mNgb and mCygb occur in neurons the same regions suggests that both molecules might be expressed in the same cells. This hypothesis was substantiated the presence of both mNgb and mCygb immunoreactivity cultured HN33 cells (data not shown) and by PCR experiments on neuronal cells in vitro. 2E. Fordel, E. Geuens, S. Dewilde, P. Rottier, P. Carmeliet, J. Grooten, and L. Moens, manuscript in preparation.Despite their different embryological origin, pancreatic β-cells and neuronal cells share the expression of similar proteins such as glutamic acid decarboxylase, tyrosine hydroxylase, and neurofilament proteins. The expression of the same genes in two different cell types could be explained either by the presence of specific transcriptional activators (Islet-1, Pax-6, Beta-2, etc.) or by the absence of specific transcriptional repressors (example NRSF = neuron-restrictive silencing factor). mNgb was shown to be expressed in the endocrine pancreatic islets of Langerhans. This was confirmed by double labeling experiments with specific markers for the different types of islet cells: insulin, which is only present in β-cells; glucagon, an α-cell specific protein; and somatostatin, a δ-cell marker (29Baetens D. Malaisse-Lagae F. Perrelet A. Orci L. Science. 1979; 206: 1323-1325Crossref PubMed Scopus (193) Google Scholar). No specific co-localization was observed with glucagon or somatostatin; mNgb immunoreactivity was present in the β-cells in the center of the islet (Fig. 5). However, the immunoreactivity intensities of mNgb in the β-cells was quite variable, especially when comparing with that of insulin. Nevertheless, mNGB immunoreactivity was observed in each islet of the studied sections.Fig. 5Single confocal optical sections of mNgb (green, FITC fluorescence) immunoreactivity in mouse pancreas. Double staining of mNgb (green) with glucagon (A) and insulin (B) (both red, Cy-3 fluorescence) showing the mNgb immunoreactivity in cells of the islets of Langerhans.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The presence of a globin in the nucleus of animal cells is totally unexpected. A precedent has been described in plants, i.e. in cultured alfalfa cells, where a hypoxia-inducible non-symbiotic Hb was localized in the nucleus by immuno-electron microscopy methods (30Seregelyes C. Mustardy L. Ayaydin F. Sass L. Kovacs L. Endre G. Lukacs N. Kovacs I. Vass I. Kiss G.B. Horvath G.V. Dudits D. FEBS Lett. 2000; 482: 125-130Crossref PubMed Scopus (57) Google Scholar).How globin-like molecules enter the nucleus is unclear, since no known nuclear transport signal could be detected in Cygb or the non-symbiotic alfalfa Hb. However, given the small size and mass of these molecules (44 × 44 × 25 Å and M r ∼17,000–21,000), simple diffusion cannot be excluded (31Quadrini K.J. Bieker J.J. J. Biol. Chem. 2002; 277: 32243-32252Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 32Pandya K. Townes T.M. J. Biol. 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A. 1997; 94: 713-718Crossref PubMed Scopus (161) Google Scholar)).With the exception of FixL, which is a high spin and penta-coordinated protein, the heme-iron atom occurring in these proteins is always in the hexacoordinated low spin state, displaying cytochrome-like spectra.The overall structure of these molecules is variable. They may exist as a single molecule containing a heme-based sensor domain and an effector domain with enzymatic activity. This is the case for FixL and EcDos, where a PAS-folded heme-based sensor domain is covalently linked to a kinase and a phosphodiesterase domain, respectively (38Gilles-Gonzalez M.A. IUBMB Life. 2001; 51: 165-173Crossref PubMed Scopus (45) Google Scholar). CooA, a homodimer, consists of a heme-containing cAMP receptor protein linked to a small DNA-binding domain with a typical helix-turn-helix motif (42Lanzilotta W.N. Schuller D.J. Thorsteinsson M.V. Kerby R.L. Roberts G.P. Poulos T.L. Nat. Struct. Biol. 2000; 7: 876-880Crossref PubMed Scopus (234) Google Scholar). In contrast, NPAS2 is part of a non-covalent tetrameric complex, containing two NPAS2 subunits and two BMAL1 subunits, the latter allowing specific DNA binding (39Dioum E.M. Rutter J. Tuckerman J.R. Gonzalez G. Gilles-Gonzalez M.A. McKnight S.L. Science. 2002; 298: 2385-2387Crossref PubMed Scopus (374) Google Scholar, 43Rutter J. Reick M. Wu L.C. McKnight S.L. Science. 2001; 293: 510-514Crossref PubMed Scopus (762) Google Scholar).All these molecules are, upon activation, involved in different signal transduction pathways: FixL will control the expression of N2 fixation genes in Rhizobia; EcDos will regulate the aerobic respiration in E. coli; CooA, a bacterial CO sensor, will regulate the transcription of two operons encoding a CO oxidizing system; and NPAS2 is a gas-responsive transcription factor involved in the regulation of the circadian rhythm (38Gilles-Gonzalez M.A. IUBMB Life. 2001; 51: 165-173Crossref PubMed Scopus (45) Google Scholar, 39Dioum E.M. Rutter J. Tuckerman J.R. Gonzalez G. Gilles-Gonzalez M.A. McKnight S.L. Science. 2002; 298: 2385-2387Crossref PubMed Scopus (374) Google Scholar).In bacterial flavohemoglobins, globin-folded domains occur linked to a FAD/NADP binding dehydrogenase domain and are used in the detoxification of NO as a dioxygenase
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