Kostas Pantopoulos - Profile on Academia.edu (original) (raw)

Papers by Kostas Pantopoulos

Nitric oxide (NO) produced from L-arginine by NO synthases (NOS) is a transmitter known to be inv... more Nitric oxide (NO) produced from L-arginine by NO synthases (NOS) is a transmitter known to be involved in diverse biological processes, including immunomodulation, neurotransmission and blood vessel dilatation. We describe a novel role of NO as a signaling molecule in post-transcriptional gene regulation. We demonstrate that induction of NOS in macrophage and non-macrophage cell lines activates RNA binding by iron regulatory factor (LRFs), the central trans regulator of mRNAs involved in cellular iron metabolism. NO-induced binding of IRF to iron-responsive elements (IRE) specifically represses the translation of transfected IRE-containing indicator mRNAs as well as the biosynthesis of the cellular iron storage protein ferritin. These findings define a new biological function of NO and identify a regulatory connection between the NO/NOS pathway and cellular iron metabolism.

Motivation: The untranslated regions (UTRs) of mRNA upstream (5′UTR) and downstream (3′UTR) of th... more Motivation: The untranslated regions (UTRs) of mRNA upstream (5′UTR) and downstream (3′UTR) of the open reading frame, as well as the mRNA precursor, carry important regulatory sequences. To reveal unidentified regulatory signals, we combine information from experiments with computational approaches. Depending on available knowledge, three different strategies are employed. Results: Searching with a consensus template, new RNAs with regulatory RNA elements can be identified in genomic screens. By this approach, we identify new candidate regulatory motifs resembling iron-responsive elements in the 5′UTRs of HemA, FepB and FrdB mRNA from Escherichia coli. If an RNA element is not yet defined, it may be analyzed by combining results from SELEX (selective enrichment of ligands by exponential amplification) and a search of databases from RNA or genomic sequences. A cleavage stimulating factor (CstF) binding element 3 of the polyadenylation site in the mRNA precursor serves as a test example. Alternatively, the regulatory RNA element may be found by studying different RNA foldings and their correlation with simple experimental tests. We delineate a novel instability element in the 3′UTR of the estrogen receptor mRNA in this way. Availability: Strategy, methods and programs are available on request from T.Dandekar.

generated in various cells by NO synthases. Several important biological functions are controlled... more generated in various cells by NO synthases. Several important biological functions are controlled by this messenger, and recent data suggest a novel direct role for NO in post-transcriptional gene regulation mediated by iron regulatory protein (IRP). IRP is a cytoplasmic protein that coordinates cellular iron traffic by binding to iron.responsive elements in mRNAs encoding proteins involved in iron uptake, storage and utilization. NO activates the RNA. binding activit 7 of this protein and in this regard mimics the consequences of iron starvation. Cell biological and biochemical data on the function,s of NO and IRP suggest a mechanistic basis for these findings and raise the question of thetr biological implications. [I I1~ Mill Kostas Pantopoulos and Matthias Hentze are at the Gene Expression Programme, European

Reactive oxygen intermediates (ROIs), including superoxide anion (02-) and hydrogen peroxide (H20... more Reactive oxygen intermediates (ROIs), including superoxide anion (02-) and hydrogen peroxide (H202), translation of ferritin and eALAS mRNAs and degradation of TfR mRNA. Thus cellular iron uptake (TfR), storage (ferritin) and utilization (eALAS) are coordinately regulated by IRE-IRP interactions. The connection of iron biology with free radical biochemistry prompted us to investigate the effects of oxidative stress on cellular iron metabolism.

A salt extract of rabbit brain nuclei contains three endoribonucleases, designated RNases Y, A an... more A salt extract of rabbit brain nuclei contains three endoribonucleases, designated RNases Y, A and R, which produce acid-soluble products when incubated at near-neutral pH in the absence of metal ions. RNases Y and A yield products with the monoesterified phosphate at the 3' position, through 2',3'-(cyc1ic)phosphate intermediates. Oligonucleotides terminating with a 2',3'-(cyc1ic)phosphate are the end-products of the action of RNase R. Double-stranded substrates are highly resistant to the action of all enzymes. On the basis of limited hydrolysis of end-labelled 5s RNA, the three enzymes differ in their preference for the susceptible phosphodiester bond. Thus, RNase Y hydrolyses preferentially the YpN bond, RNase A the ApN bond and RNase R the RpU bond where R is guanosine in most cases. The advantages and disadvantages of using homopolyribonucleotides and dephosphorylated dinucleotides and trinucleotides in determining various aspects of the specificity of RNases are discussed.

Several cellular mRNAs are regulated posttranscriptionally by iron-responsive elements (IREs) and... more Several cellular mRNAs are regulated posttranscriptionally by iron-responsive elements (IREs) and the cytosolic IRE-binding proteins IRP-1 and IRP-2. Three different signals are known to elicit IRP-1 activity and thus regulate IRE-containing mRNAs: iron deficiency, nitric oxide (NO), and the reactive oxygen intermediate hydrogen peroxide (H 2 O 2 ). In this report, we characterize the pathways for IRP-1 regulation by NO and H 2 O 2 and examine their effects on IRP-2. We show that the responses of IRP-1 and IRP-2 to NO remarkably resemble those elicited by iron deficiency: IRP-1 induction by NO and by iron deficiency is slow and posttranslational, while IRP-2 induction by these inductive signals is slow and requires de novo protein synthesis. In contrast, H 2 O 2 induces a rapid posttranslational activation which is limited to IRP-1. Removal of the inductive signal H 2 O 2 after <15 min of treatment (induction phase) permits a complete IRP-1 activation within 60 min (execution phase) which is sustained for several hours. This contrasts with the IRP-1 activation pathway by NO and iron depletion, in which NO-releasing drugs or iron chelators need to be present during the entire activation phase. Finally, we demonstrate that biologically synthesized NO regulates the expression of IREcontaining mRNAs in target cells by passive diffusion and that oxidative stress endogenously generated by pharmacological modulation of the mitochondrial respiratory chain activates IRP-1, underscoring the physiological significance of NO and reactive oxygen intermediates as regulators of cellular iron metabolism. We discuss models to explain the activation pathways of IRP-1 and IRP-2. In particular, we suggest the possibility that NO affects iron availability rather than the iron-sulfur cluster of IRP-1.

The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive eleme... more The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. We have identified and characterized IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5 untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant

We have studied the responses of iron regulatory protein-1 (IRP-1) to extra-and intracellular sou... more We have studied the responses of iron regulatory protein-1 (IRP-1) to extra-and intracellular sources of reactive oxygen intermediates (ROIs). IRP-1 is a cytoplasmic RNA-binding protein that regulates iron metabolism following its activation by iron deficiency, nitric oxide, and administration of H 2 O 2 or antimycin A, an inhibitor of the respiratory chain (Hentze, M. W., and Kü hn, L. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8175-8182). We show that 10 M H 2 O 2 suffice for complete IRP-1 activation within 60 min when H 2 O 2 is generated extracellularly at steady-state. By contrast, rapid cellular H 2 O 2 degradation necessitates a 5-10-fold higher bolus dose. To study IRP-1 responses to intracellular oxidative stress, mitochondrial respiration was inhibited with antimycin A (to generate oxidative stress by leakage of ROIs from complex III), or catalase was blocked with 3-amino-1,2,4-triazole (to diminish H 2 O 2 degradation); in parallel, 2,7-dichlorodihydrofluorescein diacetate was used as a redox-sensitive probe to monitor intracellular H 2 O 2 levels by fluorescence-activated cell sorting. Catalase inhibition elevates intracellular H 2 O 2 , but surprisingly does not cause concomitant IRP-1 activation. Following antimycin A treatment, IRP-1 is activated, but the activation kinetics lag behind the rapid increase in detectable intracellular H 2 O 2 . IRP-1 is thus activated both by extra-and intracellular generation of ROIs.

Iron regulatory protein-1 (IRP-1), a central cytoplasmic regulator of cellular iron metabolism, i... more Iron regulatory protein-1 (IRP-1), a central cytoplasmic regulator of cellular iron metabolism, is rapidly activated by oxidative stress to bind to mRNA iron-responsive elements. We have reconstituted the response of IRP-1 to extracellular H 2 O 2 in a system derived from murine B6 fibroblasts permeabilized with streptolysin-O. This procedure allows separation of the cytosol from the remainder of the cells (cell pellet). IRP-1 in the cytosolic fraction fails to be directly activated by addition of H 2 O 2 . IRP-1 activation requires the presence of a nonsoluble, possibly membrane-associated component in the cell pellet. The streptolysin-O-based in vitro system faithfully recapitulates characteristic hallmarks of

Two ferritin cDNAs were cloned from the liver and spinal cord of the sanguivore lamprey Lampetra ... more Two ferritin cDNAs were cloned from the liver and spinal cord of the sanguivore lamprey Lampetra fluviatilis, an extant representative of the ancient agnathan (jawless) stage in vertebrate evolution. The deduced proteins of 20.2 kDa (H-subunit) and 20.1 kDa (M-subunit) display 73% sequence identity, and both contain the ferroxidase center characteristic of animal H-ferritin. A highly conserved iron-responsive element (IRE) was identified in the 5′ untranslated region of lamprey H-ferritin. Lamprey ferritin IRE forms a specific complex with crude lamprey and rat liver extracts, and with recombinant human ironregulatory protein (IRP-1) in an electrophoretic mobility shift assay. Furthermore, lamprey ferritin IRE competes with labeled human ferritin IRE for binding to IRP in lamprey and mammalian extracts. Two liver cDNA sequences encoding 323 residues and 101 residues of two genetically distinct lamprey IRP were amplified by PCR. Lamprey IRP-1 and IRP-2, which are 72% identical, display about 74% sequence identity to their presumed homologues in mammals. Northern blot analysis shows that two IRP transcripts of 3.6 kb and 5.8 kb are expressed in lamprey liver. Given the ancient lineage of lampreys, the results indicate that the IRE/IRP regulatory system has remained highly conserved during the evolution of vertebrates.

Iron regulatory protein-1 (IRP-1) controls the expression of several mRNAs by binding to iron-res... more Iron regulatory protein-1 (IRP-1) controls the expression of several mRNAs by binding to iron-responsive elements (IREs) in their untranslated regions. In ironreplete cells, a 4Fe-4S cluster converts IRP-1 to cytoplasmic aconitase. IRE binding activity is restored by cluster loss in response to iron starvation, NO, or extracellular H 2 O 2 . Here, we study the effects of intracellular quinone-induced oxidative stress on IRP-1.

Iron regulatory protein 1 (IRP1) regulates the synthesis of proteins involved in iron homeostasis... more Iron regulatory protein 1 (IRP1) regulates the synthesis of proteins involved in iron homeostasis by binding to iron-responsive elements (IREs) of messenger RNA. IRP1 is a cytoplasmic aconitase when it contains a [4Fe-4S] cluster and an RNA-binding protein after complete removal of the metal center by an unknown mechanism. Human IRP1, obtained as the pure recombinant [4Fe-4S] form, is an enzyme as efficient toward cis-aconitate as the homologous mitochondrial aconitase. The aconitase activity of IRP1 is rapidly lost by reaction with hydrogen peroxide as the [4Fe-4S] cluster is quantitatively converted into the [3Fe-4S] form with release of a single ferrous ion per molecule. The IRE binding capacity of IRP1 is not elicited with H 2 O 2 . Ferrous sulfate (but not other more tightly coordinated ferrous ions, such as the complex with ethylenediamine tetraacetic acid) counteracts the inhibitory action of hydrogen peroxide on cytoplasmic aconitase, probably by replenishing iron at the active site. These results cast doubt on the ability of reactive oxygen species to directly increase IRP1 binding to IRE and support a signaling role for hydrogen peroxide in the posttranscriptional control of proteins involved in iron homeostasis in vivo.

The molecular basis for the regulation of cellular iron metabolism in metazoans has been elucidat... more The molecular basis for the regulation of cellular iron metabolism in metazoans has been elucidated in the last years. Iron uptake, storage and utilization are coordinately regulated at the post-transcriptional level by mRNA-protein interactions. An increasing number of mRNAs encoding primarily proteins involved in cellular iron turnover, contain iron-responsive elements (IREs), conserved stem-loop structures in their untranslated regions. IREs provide the binding site for two homologous cytoplasmic iron-regulatory proteins, IRPl and IRP2. The iron-regulated IRE/IRP interactions control mRNA translation or stability. Thus, the IRE/IRP system has emerged as a major paradigm of post-transcriptional gene regulation in higher eukaryotes. The studies on IRP1, which appears to be the most abundant iron regulatory protein, have revealed an unexpected role of iron-sulfur clusters as posttranslational regulatory sites and have highlighted the versatility of iron-sulfur chemistry as a determinant of protein function. 132 8 Regulation of iron metabolism in higher eukaryotes acute or chronic iron overload poses a major threat for cells, especially under conditions of 'oxidative stress', where the concentrations of ROS are increased. All living cells, from prokaryotes to multicellular organisms, have adapted to the need for a balanced iron supply by developing elaborate regulatory systems. The regulatory circuit which controls iron homeostasis in higher eukaryotic cells is the main topic of this chapter and will be described below.

electrophoretic mobility shift assay; PMA, phorbol myristate acetate; TfR, transferrin receptor; ... more electrophoretic mobility shift assay; PMA, phorbol myristate acetate; TfR, transferrin receptor; GOX, glucose oxidase.

Cellular iron uptake and storage are coordinately controlled by binding of iron-regulatory protei... more Cellular iron uptake and storage are coordinately controlled by binding of iron-regulatory proteins (IRP), IRP1 and IRP2, to iron-responsive elements (IREs) within the mRNAs encoding transferrin receptor (TfR) and ferritin. Under conditions of iron starvation, both IRP1 and IRP2 bind with high affinity to cognate IREs, thus stabilizing TfR and inhibiting translation of ferritin mRNAs. The IRE/IRP regulatory system receives additional input by oxidative stress in the form of H 2 O 2 that leads to rapid activation of IRP1. Here we show that treating murine B6 fibroblasts with a pulse of 100 M H 2 O 2 for 1 h is sufficient to alter critical parameters of iron homeostasis in a time-dependent manner. First, this stimulus inhibits ferritin synthesis for at least 8 h, leading to a significant (50%) reduction of cellular ferritin content. Second, treatment with H 2 O 2 induces a ϳ4fold increase in TfR mRNA levels within 2-6 h, and subsequent accumulation of newly synthesized protein after 4 h. This is associated with a profound increase in the cell surface expression of TfR, enhanced binding to fluorescein-tagged transferrin, and stimulation of transferrin-mediated iron uptake into cells. Under these conditions, no significant alterations are observed in the levels of mitochondrial aconitase and the Divalent Metal Transporter DMT1, although both are encoded by two as yet lesser characterized IRE-containing mRNAs. Finally, H 2 O 2 -treated cells display an increased capacity to sequester 59 Fe in ferritin, despite a reduction in the ferritin pool, which results in a rearrangement of 59 Fe intracellular distribution. Our data suggest that H 2 O 2 regulates cellular iron acquisition and intracellular iron distribution by both IRP1-dependent and -independent mechanisms. . The abbreviations used are: Tf, transferrin; IRP1, iron regulatory protein 1; IRE, iron-responsive element; UTR, untranslated region; TfR, transferrin receptor; m-, mitochondrial and c-, cytosolic aconitase; DMT1, divalent metal transporter 1; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; DFO, desferrioxamine.

Ethyl-3,4-dihydroxybenzoate (EDHB) is commonly utilized as a substrate analog and competitive inh... more Ethyl-3,4-dihydroxybenzoate (EDHB) is commonly utilized as a substrate analog and competitive inhibitor of prolyl 4-hydroxylases. These iron-dependent enzymes have received a lot of attention for their involvement in crucial biochemical pathways such as collagen maturation and oxygen sensing. Since EDHB is also capable of chelating the enzyme-bound iron, we study here its function as a chelator. We show that the a⁄nity of EDHB for ferric iron is signi¢cantly lower than that of desferrioxamine. Nevertheless, EDHB is su⁄cient to promote e¡ective iron de¢ciency in cells, re£ected in the activation of the iron-responsive element/iron regulatory protein regulatory network. Thus, treatment of B6 ¢broblasts with EDHB results in slow activation of iron regulatory protein 1 accompanied by an increase in transferrin receptor levels and reduction of the ferritin pool. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Iron is an essential cellular constituent but, on the other hand, possesses an enormous toxic cap... more Iron is an essential cellular constituent but, on the other hand, possesses an enormous toxic capacity when present in excess. This is largely due to Fenton/Haber-Weiss chemistry, e.g. the aerobic iron-catalyzed generation of aggressive radicals, which readily attack and damage cell membranes, proteins and nucleic acids. 1 Thus, the regulation of iron homeostasis poses a challenge not only to satisfy the metabolic needs of cells and organisms for iron, but also to minimize the risk of iron-induced injury.

32] acids by the techniques of molecular biologyJ 5 The finding that the conserved carboxyl-termi... more 32] acids by the techniques of molecular biologyJ 5 The finding that the conserved carboxyl-terminal domain of human Alrp can functionally replace the yeast domain in vivo 17 extends the molecular studies to the homologous genes of higher eukaryotes. Currently, detailed site-directed mutagenesis experiments are on the way 18 to determine the functional involvement of the different cysteine residues of the protein in the catalytic reaction, in FAD binding, or in the process of dimer formation. summarizes the experimental approaches for molecular biology techniques that are possible with the yeast genetic system. 19 The value of our genetic approach is proven by the recent identification of the first target molecules for sulfhydryl oxidase functions in yeast mitochondria. 2° A conditional mutant for ERV112 identified the yeast Ervlp sulfhydryl oxidase as an essential constituent of the mitochondrial export machinery for iron/sulfur cluster.

Iron regulatory protein 1 (IRP1), a major posttranscriptional regulator of cellular iron and ener... more Iron regulatory protein 1 (IRP1), a major posttranscriptional regulator of cellular iron and energy metabolism, is controlled by an iron-sulfur cluster switch. Cysteine-437 is critical for coordinating the cluster, and its replacement yields mutants that do not respond to iron perturbations and constitutively bind to cognate mRNA iron-responsive elements (IREs). The expression of IRP1 C437S in cells has been associated with aberrations in iron homeostasis and toxicity. We have established clones of human lung (H1299) and breast (MCF7) cancer cells that express high levels of IRP1 C437S in a tetracycline-inducible manner. As expected, IRP1 C437S stabilizes transferrin receptor mRNA and inhibits translation of ferritin mRNA in both cell types by binding to their respective IREs. However, H1299 transfectants grown at high densities are able to overcome the IRP1 C437S -mediated inhibition in ferritin synthesis. The mechanism involves neither alteration in ferritin mRNA levels nor utilization of alternative transcription start sites to eliminate the IRE or relocate it in less inhibitory downstream positions. The derepression of ferritin mRNA translation occurs under conditions where global protein synthesis appears to be impaired, as judged by a significant enrichment in the expression of the underphosphorylated form of the translational regulator 4E-BP1. Collectively, these data document an example where ferritin mRNA translation evades control of the IRE-IRP system. The physiological implications of this response are reflected in protection against iron-mediated toxicity, oxidative stress, and apoptosis.

Iron regulatory proteins, IRP1 and IRP2 are post-transcriptional regulators of iron metabolism. I... more Iron regulatory proteins, IRP1 and IRP2 are post-transcriptional regulators of iron metabolism. In iron starved cells, they bind to iron responsive elements (IREs) within mRNAs and thereby control their translation or stability. The IRE/IRP system accounts for the coordinated reciprocal regulation in the expression of the transferrin receptor and ferritin, which are involved in cellular iron uptake and storage, respectively. The activities of IRP1 and IRP2 are regulated by distinct post-translational mechanisms in response to iron levels. Thus, in ironreplete cells, IRP1 assembles a cubane iron-sulfur cluster, which prevents IRE-binding, while IRP2 undergoes proteasomal degradation. IRP1 and IRP2 also respond, albeit differentially, to iron-independent signals, such H 2 O 2 , hypoxia or nitric oxide. These findings provide regulatory links between iron metabolism and oxidative stress.