Jean-marc Moulis - Academia.edu (original) (raw)
Papers by Jean-marc Moulis
JBIC Journal of Biological Inorganic Chemistry, 2009
The crystal structures of the C57A and V13G molecular variants of Allochromatium vinosum 2[4Fe-4S... more The crystal structures of the C57A and V13G molecular variants of Allochromatium vinosum 2[4Fe-4S] ferredoxin (AlvinFd) and that of the homologous ferredoxin from Escherichia coli (EcFd) have been determined at 1.05-, 1.48-, and 1.65-A resolution, respectively. The present structures combined with cyclic voltammetry studies establish clear effects of the degree of exposure of the cluster with the lowest reduction potential (cluster I) towards less negative reduction potentials (E degrees ). This is better illustrated by V13G AlvinFd (high exposure, E degrees = -594 mV) and EcFd (low exposure, E degrees = -675 mV). In C57A AlvinFd, the movement of the protein backbone, as a result of replacing the noncoordinating Cys57 by Ala, leads to a +50-mV upshift of the potential of the nearby cluster I, by removal of polar interactions involving the thiolate group and adjustment of the hydrogen-bond network involving the cluster atoms. In addition, the present structures and other previously reported accurate structures of this family of ferredoxins indicate that polar interactions of side chains and water molecules with cluster II sulfur atoms, which are absent in the environment of cluster I, are correlated to the approximately 180-250 mV difference between the reduction potentials of clusters I and II. These findings provide insight into the significant effects of subtle structural differences of the protein and solvent environment around the clusters of [4Fe-4S] ferredoxins on their electrochemical properties.
![Research paper thumbnail of A bacteria-specific 2[4Fe-4S] ferredoxin is essential in Pseudomonas aeruginosa](https://attachments.academia-assets.com/41386878/thumbnails/1.jpg)
](BMC Microbiology, 2010
Background: Ferredoxins are small iron-sulfur proteins belonging to all domains of life. A sub-gr... more Background: Ferredoxins are small iron-sulfur proteins belonging to all domains of life. A sub-group binds two [4Fe-4S] clusters with unequal and extremely low values of the reduction potentials. These unusual properties are associated with two specific fragments of sequence. The functional importance of the very low potential ferredoxins is unknown.
![Research paper thumbnail of Characterization of [4Fe-4Se]2+/3+ high-potential iron-sulfur protein from Chromatium vinosum](https://attachments.academia-assets.com/40847527/thumbnails/1.jpg)
](Biochemistry, 1988
Selenium atoms have been introduced into Chromatium vinosum high-potential ironsulfur protein (Hi... more Selenium atoms have been introduced into Chromatium vinosum high-potential ironsulfur protein (HiPIP) in place of inorganic sulfur at the [4Fe-4S] active site. The substitution induces a decrease of the redox potential by ca. 65 mV (from 350 to 285 mV vs N H E , 25 O C , p H 7.8) and results in changes of the optical and EPR spectra. Compared to the corresponding properties of native HiPIP, some of the charge-transfer transitions of the S e derivative shift to lower energies, and the almost axial S = EPR signal occurs at lower field with a larger anisotropy. As in the case of clostridial ferredoxins, the resonance Raman spectra of reduced HiPIP are very sensitive to S*/Se substitution. The bridging stretching modes of the inorganic core shift to lower frequencies upon replacement of S* by S e as expected, but the local D2d symmetry assumed by the active site remains. The availability of the Se derivative has further allowed us to define the vibrational properties of the oxidized cluster. The bridging modes are only marginally affected by electron removal, and thus, the relevant symmetry point group for the inorganic core is the same as at the reduced level (DZd). In contrast, the Fe-Scys stretching modes occur a t higher frequencies with larger splittings, implying that the major effect of oxidation is to strengthen and distort the Fe-Scys bonds. These data strongly suggest that one or two of the latter bonds play a crucial role in the electron exchange reaction between the active site and the redox partners of HiPIP.
Electronic Proceedings in Theoretical Computer Science, 2012
This paper presents a novel framework for the modeling of biological networks. It makes use of re... more This paper presents a novel framework for the modeling of biological networks. It makes use of recent tools analyzing the robust satisfaction of properties of (hybrid) dynamical systems. The main challenge of this approach as applied to biological systems is to get access to the relevant parameter sets despite gaps in the available knowledge. An initial estimate of useful parameters was sought by formalizing the known behavior of the biological network in the STL logic using the tool Breach. Then, once a set of parameter values consistent with known biological properties was found, we tried to locally expand it into the largest possible valid region. We applied this methodology in an effort to model and better understand the complex network regulating iron homeostasis in mammalian cells. This system plays an important role in many biological functions, including erythropoiesis, resistance against infections, and proliferation of cancer cells.
The international journal of biochemistry & cell biology, 2008
The two mammalian iron regulatory proteins, IRP1 and IRP2, are post-transcriptional regulators of... more The two mammalian iron regulatory proteins, IRP1 and IRP2, are post-transcriptional regulators of cellular iron homeostasis. These cytosolic RNA-binding proteins control the synthesis of proteins involved in storage, transport, and utilization of iron. Whereas IRP1 levels remain nearly constant, IRP2 is rapidly degraded by the proteasome in iron-replete cells. In non iron-loaded H1299 human lung cancer cells, the decay of transfected hemagglutinin-tagged IRP2 was significantly antagonized by addition of not only proteasomal, but also lysosomal inhibitors. Similar results were obtained with IRP2(-Ins5), a molecular form lacking the specific IRP2 domain of 73 amino acids that is absent from IRP1. These data uncover an alternative, iron independent, mechanism of IRP2 degradation via the lysosomal pathway. Transfected IRP1 decayed slowly over several days and, in contrast to IRP2, was not further stabilized by proteasomal or lysosomal inhibitors. Experiments with an IRP1/IRP2 hybrid mol...
European Journal of Biochemistry, 1996
The iron ion of rubredoxins efficiently exchanges one electron between the Fe(I1) and Fe(II1) oxi... more The iron ion of rubredoxins efficiently exchanges one electron between the Fe(I1) and Fe(II1) oxidation states in mixtures of oxidized and reduced protein. The conditions under which the relaxation properties of the NMR signals can provide information about this exchange process have been worked out. The rate constant for the rubredoxin electron self-exchange ranges between 1.5XIO5 M -I s K 1 at 12°C and 3x10' M-' S K I at 30°C with an activation energy of the order of 24-30 kJ mo1-I in 50 mM potassium phosphate, pH 7. The increase of the electron self-exchange rate constant with ionic strength suggests that neutralizing electrostatic repulsion between the active sites of two molecules further accelerates the already fast electron exchange.
Iron is an essential yet potentially toxic element for all animal cells. Its absorption, traffic,... more Iron is an essential yet potentially toxic element for all animal cells. Its absorption, traffic, and use are tightly regulated since no efficient excretion system exists in mammals. This control occurs at two levels, a systemic one dependent on the hormone hepcidin, and a cellular one in which Iron Regulatory Proteins take a major part. These proteins are often considered as mammalian iron sensors, but the actual mechanisms of iron detection remain obscure. They have been investigated here, and in recently published experiments, by combining approaches using recombinant proteins and cellular data obtained in the yeast physiological heterologous context and in human cell lines. The search for accessory proteins modulating the function of these proteins has not returned any positive identification of candidates, but indirect data strongly suggest that these sensors must respond to their cellular environment through molecular interactions. The actual iron species that is/are sensed by...
As with virtually all biologically essential transition metals, but probably in a more acute way ... more As with virtually all biologically essential transition metals, but probably in a more acute way than most, iron excess and deficiency underlie a range of pathological conditions in animals. Accordingly, regulatory systems maintain the proper iron amount to fulfill the needs of the whole body and of each individual cell, while avoiding deleterious effects. The latter may be due to lack of iron availability, e.g. at the active site of iron enzymes, or to It now appears that the complex interactions among the networks influencing iron and redox homeostasis should be treated with new integrated data and modeling tools, with the aim to provide a global view of the functional differences between normal and pathological hematopoiesis in particular. The outcomes of the currently on-going efforts in this area are presented herein.
As with virtually all biologically essential transition metals, but probably in a more acute way ... more As with virtually all biologically essential transition metals, but probably in a more acute way than most, iron excess and deficiency underlie a range of pathological conditions in animals. Accordingly, regulatory systems maintain the proper iron amount to fulfill the needs of the whole body and of each individual cell, while avoiding deleterious effects. The latter may be due to lack of iron availability, e.g. at the active site of iron enzymes, or to reductive catalysis promoted by uncontrolled ferrous ions leading to the formation of reactive species such as the hydroxyl radical. Two major regulators maintain metazoan iron homeostasis, a systemic one relying on the circulating hormone hepcidin, and a ubiquitous cellular one organized 2 around the Iron Regulatory Proteins. These central nodes of iron homeostasis are themselves regulated by numerous effectors beyond iron availability, and they impact other biological processes not directly connected to the use of iron by animal cells. Further, the use of iron resources and conditions impacting it, such as variations of the redox balance, regulate cell fate, e.g. self-renewal of stem cells and differentiation in hematopoiesis. Iron and redox homeostasis are grounded on a series of identified molecular events, but it is not clear how changes of the associated biological parameters may favor proliferation of leukemic clones detrimental to maturation, in acute myeloid leukemia for instance. It now appears that the complex interactions among the networks influencing iron and redox homeostasis should be treated with new integrated data and modeling tools, with the aim to provide a global view of the functional differences between normal and pathological hematopoiesis in particular.
Electronic Proceedings in Theoretical Computer Science, 2013
Handbook of Metalloproteins, 2006
Handbook of Metalloproteins, 2006
Toxicology and Applied Pharmacology, 2008
A human epithelial cell line (HZR) growing with high zinc concentrations has been analyzed for it... more A human epithelial cell line (HZR) growing with high zinc concentrations has been analyzed for its ability to sustain high cadmium concentrations. Exposure to up to 200 microM of cadmium acetate for 24 h hardly impacted viability, whereas most of parental HeLa cells were killed by less than 10 microM of cadmium. Upon challenge by 35 fold higher cadmium concentrations than HeLa cells, HZR cells did not display increased DNA damage, increased protein oxidation, or changed intracellular cadmium localization. Rather, the main cause of resistance against cadmium was by avoiding cadmium entry into cells, which differs from that against zinc as the latter accumulates inside cells. The zinc-resistant phenotype of these cells was shown to also impair extracellular manganese uptake. Manganese and cadmium competed for entry into HeLa cells. Probing formerly identified cadmium or manganese transport systems in different animal cells did not evidence any significant change between HeLa and HZR cells. These results reveal zinc adaptation influences manganese and cadmium cellular traffic and they highlight previously unknown connections among homeostasis of divalent metals.
Theoretical Chemistry Accounts: Theory, Computation, and Modeling (Theoretica Chimica Acta), 1999
ABSTRACT Rubredoxins are small electron transfer proteins containing one iron atom at their activ... more ABSTRACT Rubredoxins are small electron transfer proteins containing one iron atom at their active site. The rubredoxin from the anaerobic bacterium Clostridium pasteurianum has been subjected to molecular dynamics studies starting from the minimized solvated structure. The results of the simulations have been compared with identical ones carried out with selected mutated forms of the protein obtained by molecular modeling. Surface residues, which are highly conserved among rubredoxins and close to the cysteine ligands, can be replaced by glutamates, i.e. long chain carboxylates. The main structural consequence is a shift of the protein backbone bearing conserved aromatic residues. Reciprocally, substitution of the aromatic residue closest to the iron atom shifts the cysteine-containing peptide fragments. These observations have been related to the changes in electron transfer and redox properties previously measured for this set of rubredoxin molecular variants.
FEBS Journal, 2007
Aconitases are found in a wide range of living organisms, from bacteria to higher eukaryotes [1].... more Aconitases are found in a wide range of living organisms, from bacteria to higher eukaryotes [1]. They are metalloproteins containing a [4Fe)4S] cluster that binds citrate or isocitrate and acts as a Lewis acid to isomerize these substrates. Aconitases catalyse one reaction of the citric acid cycle and they participate in supplying the precursors of essential nutrients such as glutamate. In eukaryotic cells, proteins with aconitase activity can be found in different compartments, including mitochondria, for the enzymes of the citric acid cycle, and the cytosol [2]. Deletion of ACO1, YLR304C, the gene encoding mitochondrial aconitase, Abbreviations hAco2, human mitochondrial aconitase; (h)IRP, (human) iron regulatory protein(s); KGD1, a-ketoglutarate dehydrogenase complex component 1; m-and c-, location for protein production, mitochondria and cytosol, respectively; yAco1, yeast mitochondrial aconitase.
Structure, 2006
Iron regulatory proteins (IRPs) control the translation of proteins involved in iron uptake, stor... more Iron regulatory proteins (IRPs) control the translation of proteins involved in iron uptake, storage and utilization by binding to specific noncoding sequences of the corresponding mRNAs known as iron-responsive elements (IREs). This strong interaction assures proper iron homeostasis in animal cells under iron shortage. Conversely, under iron-replete conditions, IRP1 binds a [4Fe-4S] cluster and functions as cytosolic aconitase. Regulation of the balance between the two IRP1 activities is complex, and it does not depend only on iron availability. Here, we report the crystal structure of human IRP1 in its aconitase form. Comparison with known structures of homologous enzymes reveals well-conserved folds and active site environments with significantly different surface shapes and charge distributions. The specific features of human IRP1 allow us to propose a tentative model of an IRP1-IRE complex that agrees with a range of previously obtained data.
"Protein Engineering, Design and Selection", 1994
The widespread occurrence of Pro residues adjacent to Cys ligands in the sequences of [4Fe-4S] el... more The widespread occurrence of Pro residues adjacent to Cys ligands in the sequences of [4Fe-4S] electron transfer proteins has not yet found a functional basis. The two such Pro of Clostridium pasteurianum 2[4Fe-4S] ferredoxin have been probed by site-directed mutagenesis. Any one of them, but not both simultaneously, can be substituted without impairing the proper folding of the protein. The reduction potentials of the ferredoxin variants fall in a narrow range of < 20 mV above the potential of the native protein. The biological activities with C. pasteurianum hydrogenase and pyruvate-ferredoxin oxidoreductase do not change significantly, except when Lys replaces Pro. In these cases, the data suggest that the two clusters of 2[4Fe-4S] ferredoxin may not always be equivalent in the interaction with the redox partners. Destabilization of the structure has been observed as the consequence of the Pro19 or Pro48 substitutions. Using 2-D NMR, this effect has been associated with perturbations of both the hydrogen bond network and one amino acid side chain around the [4Fe-4S] clusters. Thus, the conserved Pro found in the binding motif of [4Fe-4S] clusters in proteins strongly stabilizes the active site but does not play an essential role in the mechanism of electron transfer.
Proceedings of the National Academy of Sciences, 1996
The Zn(S,ys)4 unit is present in numerous proteins, where it assumes structural, regulatory, or c... more The Zn(S,ys)4 unit is present in numerous proteins, where it assumes structural, regulatory, or catalytic roles. The same coordination is found naturally around iron in rubredoxins, several structures of which have been refined at resolutions of, or near to, 1 A. The fold of the small protein rubredoxin around its metal ion is an excellent model for many zinc finger proteins. Zn-substituted rubredoxin and its Fe-containing counterpart were both obtained as the products of the expression in Escherichia coli of the rubredoxinencoding gene from Clostridium pasteurianum. The structures of both proteins have been refined with an anisotropic model at atomic resolution (1.1 A, R = 8.3% for Fe-rubredoxin, and 1.2 A, R = 9.6% for Zn-rubredoxin) and are very similar. The most significant differences are increased lengths of the M-S
JBIC Journal of Biological Inorganic Chemistry, 2009
The crystal structures of the C57A and V13G molecular variants of Allochromatium vinosum 2[4Fe-4S... more The crystal structures of the C57A and V13G molecular variants of Allochromatium vinosum 2[4Fe-4S] ferredoxin (AlvinFd) and that of the homologous ferredoxin from Escherichia coli (EcFd) have been determined at 1.05-, 1.48-, and 1.65-A resolution, respectively. The present structures combined with cyclic voltammetry studies establish clear effects of the degree of exposure of the cluster with the lowest reduction potential (cluster I) towards less negative reduction potentials (E degrees ). This is better illustrated by V13G AlvinFd (high exposure, E degrees = -594 mV) and EcFd (low exposure, E degrees = -675 mV). In C57A AlvinFd, the movement of the protein backbone, as a result of replacing the noncoordinating Cys57 by Ala, leads to a +50-mV upshift of the potential of the nearby cluster I, by removal of polar interactions involving the thiolate group and adjustment of the hydrogen-bond network involving the cluster atoms. In addition, the present structures and other previously reported accurate structures of this family of ferredoxins indicate that polar interactions of side chains and water molecules with cluster II sulfur atoms, which are absent in the environment of cluster I, are correlated to the approximately 180-250 mV difference between the reduction potentials of clusters I and II. These findings provide insight into the significant effects of subtle structural differences of the protein and solvent environment around the clusters of [4Fe-4S] ferredoxins on their electrochemical properties.
BMC Microbiology, 2010
Background: Ferredoxins are small iron-sulfur proteins belonging to all domains of life. A sub-gr... more Background: Ferredoxins are small iron-sulfur proteins belonging to all domains of life. A sub-group binds two [4Fe-4S] clusters with unequal and extremely low values of the reduction potentials. These unusual properties are associated with two specific fragments of sequence. The functional importance of the very low potential ferredoxins is unknown.
Biochemistry, 1988
Selenium atoms have been introduced into Chromatium vinosum high-potential ironsulfur protein (Hi... more Selenium atoms have been introduced into Chromatium vinosum high-potential ironsulfur protein (HiPIP) in place of inorganic sulfur at the [4Fe-4S] active site. The substitution induces a decrease of the redox potential by ca. 65 mV (from 350 to 285 mV vs N H E , 25 O C , p H 7.8) and results in changes of the optical and EPR spectra. Compared to the corresponding properties of native HiPIP, some of the charge-transfer transitions of the S e derivative shift to lower energies, and the almost axial S = EPR signal occurs at lower field with a larger anisotropy. As in the case of clostridial ferredoxins, the resonance Raman spectra of reduced HiPIP are very sensitive to S*/Se substitution. The bridging stretching modes of the inorganic core shift to lower frequencies upon replacement of S* by S e as expected, but the local D2d symmetry assumed by the active site remains. The availability of the Se derivative has further allowed us to define the vibrational properties of the oxidized cluster. The bridging modes are only marginally affected by electron removal, and thus, the relevant symmetry point group for the inorganic core is the same as at the reduced level (DZd). In contrast, the Fe-Scys stretching modes occur a t higher frequencies with larger splittings, implying that the major effect of oxidation is to strengthen and distort the Fe-Scys bonds. These data strongly suggest that one or two of the latter bonds play a crucial role in the electron exchange reaction between the active site and the redox partners of HiPIP.
Electronic Proceedings in Theoretical Computer Science, 2012
This paper presents a novel framework for the modeling of biological networks. It makes use of re... more This paper presents a novel framework for the modeling of biological networks. It makes use of recent tools analyzing the robust satisfaction of properties of (hybrid) dynamical systems. The main challenge of this approach as applied to biological systems is to get access to the relevant parameter sets despite gaps in the available knowledge. An initial estimate of useful parameters was sought by formalizing the known behavior of the biological network in the STL logic using the tool Breach. Then, once a set of parameter values consistent with known biological properties was found, we tried to locally expand it into the largest possible valid region. We applied this methodology in an effort to model and better understand the complex network regulating iron homeostasis in mammalian cells. This system plays an important role in many biological functions, including erythropoiesis, resistance against infections, and proliferation of cancer cells.
The international journal of biochemistry & cell biology, 2008
The two mammalian iron regulatory proteins, IRP1 and IRP2, are post-transcriptional regulators of... more The two mammalian iron regulatory proteins, IRP1 and IRP2, are post-transcriptional regulators of cellular iron homeostasis. These cytosolic RNA-binding proteins control the synthesis of proteins involved in storage, transport, and utilization of iron. Whereas IRP1 levels remain nearly constant, IRP2 is rapidly degraded by the proteasome in iron-replete cells. In non iron-loaded H1299 human lung cancer cells, the decay of transfected hemagglutinin-tagged IRP2 was significantly antagonized by addition of not only proteasomal, but also lysosomal inhibitors. Similar results were obtained with IRP2(-Ins5), a molecular form lacking the specific IRP2 domain of 73 amino acids that is absent from IRP1. These data uncover an alternative, iron independent, mechanism of IRP2 degradation via the lysosomal pathway. Transfected IRP1 decayed slowly over several days and, in contrast to IRP2, was not further stabilized by proteasomal or lysosomal inhibitors. Experiments with an IRP1/IRP2 hybrid mol...
European Journal of Biochemistry, 1996
The iron ion of rubredoxins efficiently exchanges one electron between the Fe(I1) and Fe(II1) oxi... more The iron ion of rubredoxins efficiently exchanges one electron between the Fe(I1) and Fe(II1) oxidation states in mixtures of oxidized and reduced protein. The conditions under which the relaxation properties of the NMR signals can provide information about this exchange process have been worked out. The rate constant for the rubredoxin electron self-exchange ranges between 1.5XIO5 M -I s K 1 at 12°C and 3x10' M-' S K I at 30°C with an activation energy of the order of 24-30 kJ mo1-I in 50 mM potassium phosphate, pH 7. The increase of the electron self-exchange rate constant with ionic strength suggests that neutralizing electrostatic repulsion between the active sites of two molecules further accelerates the already fast electron exchange.
Iron is an essential yet potentially toxic element for all animal cells. Its absorption, traffic,... more Iron is an essential yet potentially toxic element for all animal cells. Its absorption, traffic, and use are tightly regulated since no efficient excretion system exists in mammals. This control occurs at two levels, a systemic one dependent on the hormone hepcidin, and a cellular one in which Iron Regulatory Proteins take a major part. These proteins are often considered as mammalian iron sensors, but the actual mechanisms of iron detection remain obscure. They have been investigated here, and in recently published experiments, by combining approaches using recombinant proteins and cellular data obtained in the yeast physiological heterologous context and in human cell lines. The search for accessory proteins modulating the function of these proteins has not returned any positive identification of candidates, but indirect data strongly suggest that these sensors must respond to their cellular environment through molecular interactions. The actual iron species that is/are sensed by...
As with virtually all biologically essential transition metals, but probably in a more acute way ... more As with virtually all biologically essential transition metals, but probably in a more acute way than most, iron excess and deficiency underlie a range of pathological conditions in animals. Accordingly, regulatory systems maintain the proper iron amount to fulfill the needs of the whole body and of each individual cell, while avoiding deleterious effects. The latter may be due to lack of iron availability, e.g. at the active site of iron enzymes, or to It now appears that the complex interactions among the networks influencing iron and redox homeostasis should be treated with new integrated data and modeling tools, with the aim to provide a global view of the functional differences between normal and pathological hematopoiesis in particular. The outcomes of the currently on-going efforts in this area are presented herein.
As with virtually all biologically essential transition metals, but probably in a more acute way ... more As with virtually all biologically essential transition metals, but probably in a more acute way than most, iron excess and deficiency underlie a range of pathological conditions in animals. Accordingly, regulatory systems maintain the proper iron amount to fulfill the needs of the whole body and of each individual cell, while avoiding deleterious effects. The latter may be due to lack of iron availability, e.g. at the active site of iron enzymes, or to reductive catalysis promoted by uncontrolled ferrous ions leading to the formation of reactive species such as the hydroxyl radical. Two major regulators maintain metazoan iron homeostasis, a systemic one relying on the circulating hormone hepcidin, and a ubiquitous cellular one organized 2 around the Iron Regulatory Proteins. These central nodes of iron homeostasis are themselves regulated by numerous effectors beyond iron availability, and they impact other biological processes not directly connected to the use of iron by animal cells. Further, the use of iron resources and conditions impacting it, such as variations of the redox balance, regulate cell fate, e.g. self-renewal of stem cells and differentiation in hematopoiesis. Iron and redox homeostasis are grounded on a series of identified molecular events, but it is not clear how changes of the associated biological parameters may favor proliferation of leukemic clones detrimental to maturation, in acute myeloid leukemia for instance. It now appears that the complex interactions among the networks influencing iron and redox homeostasis should be treated with new integrated data and modeling tools, with the aim to provide a global view of the functional differences between normal and pathological hematopoiesis in particular.
Electronic Proceedings in Theoretical Computer Science, 2013
Handbook of Metalloproteins, 2006
Handbook of Metalloproteins, 2006
Toxicology and Applied Pharmacology, 2008
A human epithelial cell line (HZR) growing with high zinc concentrations has been analyzed for it... more A human epithelial cell line (HZR) growing with high zinc concentrations has been analyzed for its ability to sustain high cadmium concentrations. Exposure to up to 200 microM of cadmium acetate for 24 h hardly impacted viability, whereas most of parental HeLa cells were killed by less than 10 microM of cadmium. Upon challenge by 35 fold higher cadmium concentrations than HeLa cells, HZR cells did not display increased DNA damage, increased protein oxidation, or changed intracellular cadmium localization. Rather, the main cause of resistance against cadmium was by avoiding cadmium entry into cells, which differs from that against zinc as the latter accumulates inside cells. The zinc-resistant phenotype of these cells was shown to also impair extracellular manganese uptake. Manganese and cadmium competed for entry into HeLa cells. Probing formerly identified cadmium or manganese transport systems in different animal cells did not evidence any significant change between HeLa and HZR cells. These results reveal zinc adaptation influences manganese and cadmium cellular traffic and they highlight previously unknown connections among homeostasis of divalent metals.
Theoretical Chemistry Accounts: Theory, Computation, and Modeling (Theoretica Chimica Acta), 1999
ABSTRACT Rubredoxins are small electron transfer proteins containing one iron atom at their activ... more ABSTRACT Rubredoxins are small electron transfer proteins containing one iron atom at their active site. The rubredoxin from the anaerobic bacterium Clostridium pasteurianum has been subjected to molecular dynamics studies starting from the minimized solvated structure. The results of the simulations have been compared with identical ones carried out with selected mutated forms of the protein obtained by molecular modeling. Surface residues, which are highly conserved among rubredoxins and close to the cysteine ligands, can be replaced by glutamates, i.e. long chain carboxylates. The main structural consequence is a shift of the protein backbone bearing conserved aromatic residues. Reciprocally, substitution of the aromatic residue closest to the iron atom shifts the cysteine-containing peptide fragments. These observations have been related to the changes in electron transfer and redox properties previously measured for this set of rubredoxin molecular variants.
FEBS Journal, 2007
Aconitases are found in a wide range of living organisms, from bacteria to higher eukaryotes [1].... more Aconitases are found in a wide range of living organisms, from bacteria to higher eukaryotes [1]. They are metalloproteins containing a [4Fe)4S] cluster that binds citrate or isocitrate and acts as a Lewis acid to isomerize these substrates. Aconitases catalyse one reaction of the citric acid cycle and they participate in supplying the precursors of essential nutrients such as glutamate. In eukaryotic cells, proteins with aconitase activity can be found in different compartments, including mitochondria, for the enzymes of the citric acid cycle, and the cytosol [2]. Deletion of ACO1, YLR304C, the gene encoding mitochondrial aconitase, Abbreviations hAco2, human mitochondrial aconitase; (h)IRP, (human) iron regulatory protein(s); KGD1, a-ketoglutarate dehydrogenase complex component 1; m-and c-, location for protein production, mitochondria and cytosol, respectively; yAco1, yeast mitochondrial aconitase.
Structure, 2006
Iron regulatory proteins (IRPs) control the translation of proteins involved in iron uptake, stor... more Iron regulatory proteins (IRPs) control the translation of proteins involved in iron uptake, storage and utilization by binding to specific noncoding sequences of the corresponding mRNAs known as iron-responsive elements (IREs). This strong interaction assures proper iron homeostasis in animal cells under iron shortage. Conversely, under iron-replete conditions, IRP1 binds a [4Fe-4S] cluster and functions as cytosolic aconitase. Regulation of the balance between the two IRP1 activities is complex, and it does not depend only on iron availability. Here, we report the crystal structure of human IRP1 in its aconitase form. Comparison with known structures of homologous enzymes reveals well-conserved folds and active site environments with significantly different surface shapes and charge distributions. The specific features of human IRP1 allow us to propose a tentative model of an IRP1-IRE complex that agrees with a range of previously obtained data.
"Protein Engineering, Design and Selection", 1994
The widespread occurrence of Pro residues adjacent to Cys ligands in the sequences of [4Fe-4S] el... more The widespread occurrence of Pro residues adjacent to Cys ligands in the sequences of [4Fe-4S] electron transfer proteins has not yet found a functional basis. The two such Pro of Clostridium pasteurianum 2[4Fe-4S] ferredoxin have been probed by site-directed mutagenesis. Any one of them, but not both simultaneously, can be substituted without impairing the proper folding of the protein. The reduction potentials of the ferredoxin variants fall in a narrow range of < 20 mV above the potential of the native protein. The biological activities with C. pasteurianum hydrogenase and pyruvate-ferredoxin oxidoreductase do not change significantly, except when Lys replaces Pro. In these cases, the data suggest that the two clusters of 2[4Fe-4S] ferredoxin may not always be equivalent in the interaction with the redox partners. Destabilization of the structure has been observed as the consequence of the Pro19 or Pro48 substitutions. Using 2-D NMR, this effect has been associated with perturbations of both the hydrogen bond network and one amino acid side chain around the [4Fe-4S] clusters. Thus, the conserved Pro found in the binding motif of [4Fe-4S] clusters in proteins strongly stabilizes the active site but does not play an essential role in the mechanism of electron transfer.
Proceedings of the National Academy of Sciences, 1996
The Zn(S,ys)4 unit is present in numerous proteins, where it assumes structural, regulatory, or c... more The Zn(S,ys)4 unit is present in numerous proteins, where it assumes structural, regulatory, or catalytic roles. The same coordination is found naturally around iron in rubredoxins, several structures of which have been refined at resolutions of, or near to, 1 A. The fold of the small protein rubredoxin around its metal ion is an excellent model for many zinc finger proteins. Zn-substituted rubredoxin and its Fe-containing counterpart were both obtained as the products of the expression in Escherichia coli of the rubredoxinencoding gene from Clostridium pasteurianum. The structures of both proteins have been refined with an anisotropic model at atomic resolution (1.1 A, R = 8.3% for Fe-rubredoxin, and 1.2 A, R = 9.6% for Zn-rubredoxin) and are very similar. The most significant differences are increased lengths of the M-S