Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

Jewish General Hospital

McGill University

Iron regulatory proteins, IRP1 and IRP2, are cytoplasmic proteins of the iron-sulfur cluster isomerase family and serve as major post-transcriptional regulators of cellular iron metabolism. They bind to 'iron responsive elements' (IREs) of several mRNAs and thereby control their translation or stability. IRP1 and IRP2 respond to alterations in intracellular iron levels, but also to other signals such as nitric oxide (NO) and reactive oxygen species (ROS). The redox regulation of IRP1 and IRP2 provides direct links between the control of iron homeostasis and oxidative stress.

Redox control of iron regulatory proteins.

Iron regulatory protein 1 (IRP1) binds to mRNA iron-responsive elements (IREs) and thereby controls the expression of IRE-containing mRNAs. In iron-replete cells, assembly of a cubane [4Fe-4S] cluster inhibits IRE-binding activity and converts IRP1 to a cytosolic aconitase. Earlier experiments with Saccharomyces cerevisiae suggested that phosphomimetic mutations of Ser-138 negatively affect the stability of the cluster (N. M. Brown, S. A. Anderson, D. W. Steffen, T. B. Carpenter, M. C. Kennedy, W. E. Walden, and R. S. Eisenstein, Proc. Natl. Acad. Sci. USA 95:15235-15240, 1998). Along these lines, we show here that a highly purified preparation of recombinant human IRP1 bearing a phosphomimetic S138E substitution (IRP1(S138E)) lacks aconitase activity, which is a hallmark of [4Fe-4S] cluster integrity. Similarly, IRP1(S138E) expressed in mammalian cells fails to function as aconitase. Furthermore, we demonstrate that the impairment of [4Fe-4S] cluster assembly in mammalian cells sensitizes IRP1(S138E) to iron-dependent degradation. This effect can be completely blocked by the iron chelator desferrioxamine or by the proteasome inhibitors MG132 and lactacystin. As expected, the stability of wild-type or phosphorylation-deficient IRP1(S138A) is not affected by iron manipulations. Ser-138 and flanking sequences appear to be highly conserved in the IRP1s of vertebrates, whereas insect IRP1 orthologues and nonvertebrate IRP1-like molecules contain an S138A substitution. Our data suggest that phosphorylation of Ser-138 may provide a basis for an additional mechanism for the control of vertebrate IRP1 activity at the level of protein stability.

A phosphomimetic mutation at Ser-138 renders iron regulatory protein 1 sensitive to iron-dependent degradation.

In iron-starved cells, IRP1 (iron regulatory protein 1) binds to mRNA iron-responsive elements and controls their translation or stability. In response to increased iron levels, RNA-binding is inhibited on assembly of a cubane [4Fe-4S] cluster, which renders IRP1 to a cytosolic aconitase. Phosphorylation at conserved serine residues may also regulate the activities of IRP1. We demonstrate that Ser-711 is a phosphorylation site in HEK-293 cells (human embryonic kidney 293 cells) treated with PMA, and we study the effects of the S711E (Ser-711-->Glu) mutation on IRP1 functions. A highly purified preparation of recombinant IRP1(S711E) displays negligible IRE-binding and aconitase activities. It appears that the first step in the aconitase reaction (conversion of citrate into the intermediate cis-aconitate) is more severely affected, as recombinant IRP1(S711E) retains approx. 45% of its capacity to catalyse the conversion of cis-aconitate into the end-product isocitrate. When expressed in mammalian cells, IRP1(S711E) completely fails to bind to RNA and to generate isocitrate from citrate. We demonstrate that the apparent inactivation of IRP1(S711E) is not related to mutation-associated protein misfolding or to alterations in its stability. Sequence analysis of IRP1 from all species currently deposited in protein databases shows that Ser-711 and flanking sequences are highly conserved in the evolutionary scale. Our results suggest that Ser-711 is a critical residue for the control of IRP1 activities.

IRP1 Ser-711 is a phosphorylation site, critical for regulation of RNA-binding and aconitase activities.

Clinical data suggest that iron is a negative factor in chronic hepatitis C; however, the molecular mechanisms by which iron modulates the infectious cycle of hepatitis C virus (HCV) remain elusive. To explore this, we utilized cells expressing a HCV replicon as a well-established model for viral replication. We demonstrate that iron administration dramatically inhibits the expression of viral proteins and RNA, without significantly affecting its translation or stability. Experiments with purified recombinant HCV RNA polymerase (NS5B) revealed that iron binds specifically and with high affinity (apparent Kd: 6 and 60 microM for Fe2+ and Fe3+, respectively) to the protein's Mg2+-binding pocket, thereby inhibiting its enzymatic activity. We propose that iron impairs HCV replication by inactivating NS5B and that its negative effects in chronic hepatitis C may be primarily due to attenuation of antiviral immune responses. Our data provide a direct molecular link between iron and HCV replication.

Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C Virus.

In iron-replete cells the posttranscriptional regulator IRP2 undergoes ubiquitination and proteasomal degradation. A similar response occurs in cells exposed to sodium nitroprusside (SNP), an NO-releasing drug. It has been proposed that nitroprusside ([Fe(CN)5NO]2-) fails to donate iron into cells and that it promotes IRP2 degradation via S nitrosylation at C178. This residue is located within a stretch of 73 amino acids, earlier proposed to define an iron-dependent degradation domain. Surprisingly, we show that IRP2 bearing a C178S mutation or a Delta73 deletion is sensitive to degradation not only by ferric ammonium citrate (FAC) but also by SNP. Moreover, FAC and SNP attenuate the RNA-binding activities of IRP2 and its homologue IRP1 with similar kinetics. Actinomycin D, cycloheximide, succinylacetone, and dimethyl-oxalylglycine antagonize IRP2 degradation in response to both FAC and SNP, suggesting a common mechanistic basis. IRP2 is not only sensitive to fresh, but also to photodegraded SNP and remains unaffected by S-nitrosoglutathione (GSNO), an established nitrosation agent. Importantly, both fresh and photodegraded SNP, but not GSNO, promote a >4-fold increase in the calcein-accessible labile iron pool. Collectively, these results suggest that IRP2 degradation by SNP does not require S nitrosylation but rather represents a response to iron loading.

Sodium nitroprusside promotes IRP2 degradation via an increase in intracellular iron and in the absence of S nitrosylation at C178.

Iron is essential for proliferation of normal and neoplastic cells. Cellular iron uptake, utilization and storage are regulated by transcriptional and post-transcriptional mechanisms. We hypothesized that the disruption of iron homeostasis may modulate the growth properties of cancer cells. To address this, we employed H1299 lung cancer cells engineered for tetracycline-inducible overexpression of the post-transcriptional regulator iron regulatory protein 1 (IRP1). The induction of IRP1 (wild-type or the constitutive IRP1(C437S) mutant) did not affect the proliferation of the cells in culture, and only modestly reduced their efficiency to form colonies in soft agar. However, IRP1 dramatically impaired the capacity of the cells to form solid tumor xenografts in nude mice. Tumors derived from IRP1-transfectants were <20% in size compared to those from parent cells. IRP1 coordinately controls the expression of transferrin receptor 1 (TfR1) and ferritin by binding to iron-responsive elements (IREs) within their mRNAs. Biochemical analysis revealed high expression of epitope-tagged IRP1 in tumor tissue, which was associated with a profound increase in IRE-binding activity. As expected, this response misregulated iron metabolism by increasing TfR1 levels. Surprisingly, IRP1 failed to suppress ferritin expression and did not affect the levels of the iron transporter ferroportin. Our results show that the overexpression of IRP1 is associated with an apparent tumor suppressor phenotype and provide a direct regulatory link between the IRE/IRP system and cancer.

Overexpression of iron regulatory protein 1 suppresses growth of tumor xenografts.

Iron regulatory protein 1 (IRP1) controls the translation or stability of several mRNAs by binding to "iron-responsive elements" within their untranslated regions. In iron-replete cells, IRP1 assembles a cubane iron-sulfur cluster (ISC) that inhibits RNA-binding activity and converts the protein to cytosolic aconitase. We show that the constitutive IRP1(C437S) mutant, which fails to form an ISC, is destabilized by iron. Thus, exposure of H1299 cells to ferric ammonium citrate reduced the half-life of transfected IRP1(C437S) from approximately 24 h to approximately 10 h. The iron-dependent degradation of IRP1(C437S) involved ubiquitination, required ongoing transcription and translation, and could be efficiently blocked by the proteasomal inhibitors MG132 and lactacystin. Similar results were obtained with overexpressed wild-type IRP1, which predominated in the apo-form even in iron-loaded H1299 cells, possibly due to saturation of the ISC assembly machinery. Importantly, inhibition of ISC biogenesis in HeLa cells by small interfering RNA knockdown of the cysteine desulfurase Nfs1 sensitized endogenous IRP1 for iron-dependent degradation. Collectively, these data uncover a mechanism for the regulation of IRP1 abundance as a means to control its RNA-binding activity, when the ISC assembly pathway is impaired.

Iron-dependent degradation of apo-IRP1 by the ubiquitin-proteasome pathway.

Infection with hepatitis C virus (HCV) is associated with alterations in body iron homeostasis by poorly defined mechanisms. To seek for molecular links, we employed an established cell culture model for viral replication, and assessed how the expression of an HCV subgenomic replicon affects iron metabolism in host Huh7 hepatoma cells.

Expression of the subgenomic hepatitis C virus replicon alters iron homeostasis in Huh7 cells.

Oxidative stress, deposition of non-transferrin iron, and mitochondrial insufficiency occur in the brains of patients with Alzheimer disease (AD) and Parkinson disease (PD). We previously demonstrated that heme oxygenase-1 (HO-1) is up-regulated in AD and PD brain and promotes the accumulation of non-transferrin iron in astroglial mitochondria. Herein, dynamic secondary ion mass spectrometry (SIMS) and other techniques were employed to ascertain (i) the impact of HO-1 over-expression on astroglial mitochondrial morphology in vitro, (ii) the topography of aberrant iron sequestration in astrocytes over-expressing HO-1, and (iii) the role of iron regulatory proteins (IRP) in HO-1-mediated iron deposition. Astroglial hHO-1 over-expression induced cytoplasmic vacuolation, mitochondrial membrane damage, and macroautophagy. HO-1 promoted trapping of redox-active iron and sulfur within many cytopathological profiles without impacting ferroportin, transferrin receptor, ferritin, and IRP2 protein levels or IRP1 activity. Thus, HO-1 activity promotes mitochondrial macroautophagy and sequestration of redox-active iron in astroglia independently of classical iron mobilization pathways. Glial HO-1 may be a rational therapeutic target in AD, PD, and other human CNS conditions characterized by the unregulated deposition of brain iron.

HO-1-mediated macroautophagy: a mechanism for unregulated iron deposition in aging and degenerating neural tissues.

Hemizygous TNF(DeltaARE/+) mice are a murine model for chronic inflammation. We utilized these animals to study iron-kinetics and corresponding protein expression in an iron-deficient and iron-adequate setting. (59)Fe-absorption was determined in ligated duodenal loops in vivo. Whole body distribution of i.v. injected (59)Fe was analysed, and the organ specific expression of ferroportin, transferrin receptor-1, hepcidin and duodenal DMT-1 was quantified by real-time PCR and Western blotting. Duodenal (59)Fe-lumen-to-body transport was not affected by the genotype. Duodenal (59)Fe-retention was increased in TNF(DeltaARE/+) mice, suggesting higher (59)Fe-losses with defoliated enterocytes. Iron-deficiency increased duodenal (59)Fe-lumen-to-body transport, and higher duodenal (59)Fe-tissue retention went along with higher duodenal DMT-1, ferroportin, and liver hepcidin expression. TNF(DeltaARE/+) mice significantly increase their (59)Fe-content in inflamed joints and ilea, and correspondingly reduce splenic (59)Fe-content. Leukocyte infiltrations in the joints suggest a substantial shift of iron-loaded RES cells to inflamed tissues as the underlying mechanism. This finding was paralleled by increased non-haem iron content in joints and reduced haemoglobin and haematocrit concentrations in TNF(DeltaARE/+) mice. In conclusion, erythropoiesis in inflamed TNF(DeltaARE/+) mice could be iron-limited due to losses with exfoliated iron-loaded enterocytes and/or to increased iron-retention in RES cells that shift from the spleen to inflamed tissues.

Iron absorption and distribution in TNF(DeltaARE/+) mice, a model of chronic inflammation.

Chronic infection with hepatitis C virus (HCV) is often associated with elevated hepatic iron levels. Excess iron is known to promote oxidative stress and exacerbate liver disease. Nevertheless, biochemical studies in subgenomic HCV replicon systems showed that iron can also suppress the expression of viral RNA and proteins by inhibiting the enzymatic activity of the RNA polymerase NS5B. To explore the physiological relevance of this response, we evaluated the effects of iron during infection of permissive Huh7.5.1 hepatoma cells with HCV.

Iron inhibits replication of infectious hepatitis C virus in permissive Huh7.5.1 cells.

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The mechanisms responsible for pathological iron deposition in the aging and degenerating mammalian CNS remain poorly understood. The stress protein, HO-1 mediates the degradation of cellular heme to biliverdin/bilirubin, free iron, and CO and is up-regulated in the brains of persons with Alzheimer's disease and Parkinson's disease. HO-1 induction in primary astroglial cultures promotes deposition of non-transferrin iron, mitochondrial damage and macroautophagy, and predisposes cocultured neuronal elements to oxidative injury. To gain a better appreciation of the role of glial HO-1 in vivo, we probed for aberrant brain iron deposition using Perls' method and dynamic secondary ion mass spectrometry in novel, conditional GFAP.HMOX1 transgenic mice that selectively over-express human HO-1 in the astrocytic compartment. At 48 weeks, the GFAP.HMOX1 mice exhibited increased deposits of glial iron in hippocampus and other subcortical regions without overt changes in iron-regulatory and iron-binding proteins relative to age-matched wild-type animals. Dynamic secondary ion mass spectrometry revealed abundant FeOâ» signals in the transgenic, but not wild-type, mouse brain that colocalized to degenerate mitochondria and osmiophilic cytoplasmic inclusions (macroautophagy) documented by TEM. Sustained up-regulation of HO-1 in astrocytes promotes pathological brain iron deposition and oxidative mitochondrial damage characteristic of Alzheimer's disease-affected neural tissues. Curtailment of glial HO-1 hyperactivity may limit iron-mediated cytotoxicity in aging and degenerating neural tissues.

Unregulated brain iron deposition in transgenic mice over-expressing HMOX1 in the astrocytic compartment.

Les m&eacute;canismes responsables des d&eacute;p&ocirc;ts de fer pathologique dans le vieillissement et la d&eacute;g&eacute;n&eacute;rescence de mammif&egrave;res SNC restent mal compris. Les prot&eacute;ines de stress, HO-1 intervient dans la d&eacute;gradation de l&#39;h&egrave;me cellulaire &agrave; la biliverdine/bilirubine, le fer libre et CO et est r&eacute;gul&eacute;e dans le cerveau des personnes atteintes de la maladie d&#39;Alzheimer et de Parkinson. Induction de HO-1 dans des cultures primaires astroglials favorise les d&eacute;p&ocirc;ts de fer non-transferrine, l&eacute;sions mitochondriales et macroautophagy et pr&eacute;dispose les &eacute;l&eacute;ments neurones cocultured au dommage oxydatif. Afin de mieux appr&eacute;cier le r&ocirc;le de glial HO-1 in vivo, nous avons sond&eacute; pour les d&eacute;p&ocirc;ts de fer aberrant de cerveau &agrave; l&#39;aide de la m&eacute;thode des Perls et spectrom&eacute;trie de masse des ions secondaires dynamique dans le roman, la GFAP conditionnelle.HMOX1 souris transg&eacute;niques qui surexpriment s&eacute;lectivement humaine HO-1 dans le compartiment des astrocytes. Apr&egrave;s 48 semaines, la GFAP.HMOX1 souris pr&eacute;sentaient des d&eacute;p&ocirc;ts accrus de fer glial dans l&#39;hippocampe et autres r&eacute;gions sous-corticales sans changements manifestes dans des prot&eacute;ines fer-r&eacute;gulatrices et de fixation du fer par rapport au m&ecirc;me groupe d&#39;&acirc;ge des animaux sauvage. Spectrom&eacute;trie de masse des ions secondaires dynamique a r&eacute;v&eacute;l&eacute; des signaux FeO&#x207B; abondantes dans le transg&eacute;nique, mais le cerveau de souris non sauvage, qui &eacute;pididymaire mitochondries d&eacute;g&eacute;n&eacute;r&eacute;es et osmiophiles inclusions cytoplasmiques (macroautophagy) accompagn&eacute;es de TEM. Soutenue vers le haut-r&egrave;glement de HO-1 dans les astrocytes favorise les d&eacute;p&ocirc;ts de fer de cerveau pathologique et oxydatif mitochondrial qui caract&eacute;risent les tissus nerveux touch&eacute;es par la maladie d&#39;Alzheimer. Raccourcissement de gliale HO-1/hyperactivit&eacute; peut limiter la cytotoxicit&eacute; m&eacute;di&eacute;e par fer dans le vieillissement et la d&eacute;g&eacute;n&eacute;rescence des tissus nerveux.

D&eacute;p&ocirc;ts de fer non r&eacute;glement&eacute;e de cerveau chez des souris transg&eacute;niques surexprimant HMOX1 dans le compartiment des astrocytes.

Die Mechanismen, die f&uuml;r pathologische Eisen Abscheidung im S&auml;ugetier-Alterung und degenerative ZNS bleiben weitgehend unverstanden. Das Stress-Protein, HO-1 vermittelt die Degradierung der zellul&auml;ren H&auml;m Biliverdin/Bilirubin, freiem Eisen und CO und in den Gehirnen der Menschen mit Alzheimer und Morbus Parkinson ist oben-geregelt. HO-1-Induktion in prim&auml;ren Astroglial Kulturen f&ouml;rdert die Ablagerung von nicht-Transferrin-Eisen, mitochondriale Sch&auml;den und Macroautophagy und cocultured neuronalen Elemente zu oxidativen Sch&auml;digung pr&auml;disponiert. Um eine bessere Anerkennung der Rolle der Gliazellen HO-1 in-vivo zu gewinnen, &uuml;berpr&uuml;ften wir aberrante Gehirn Eisen Beschichtung mit Perls Methode und dynamische Sekund&auml;rionen-Massenspektrometrie in Roman, bedingte zuzuf&uuml;hren.HMOX1 transgenen M&auml;usen, die selektiv menschlichen HO-1 im astrozytische Fach over-express. Nach 48 Wochen der zuzuf&uuml;hren.HMOX1 M&auml;usen zeigte erh&ouml;hte Ablagerungen von Gliazellen Eisen im Hippocampus und anderen subkortikalen Regionen ohne offenkundige &Auml;nderungen in Eisen-regulatorischen und Eisen bindende Proteine relativ altersbezogenen Wildtyp Tiere. Dynamische Sekund&auml;rionen-Massenspektrometrie zeigte reichlich FeO&#x207B;-Signale in der transgenen, aber nicht Wildtyp-Maus-Gehirn, das entartete Mitochondrien und Osmiophilic zytoplasmatische Einschl&uuml;sse (Macroautophagy) dokumentiert durch TEM colocalized. Nachhaltige bis-Regelung der HO-1 in Astrozyten f&ouml;rdert pathologischen Gehirn Eisen Abscheidung und oxidative mitochondriale Sch&auml;den Merkmal der Alzheimer-Krankheit betroffenen Nervengewebe. Beschneidung von Gliazellen HO-1 Hyperaktivit&auml;t kann Eisen-vermittelte Zytotoxizit&auml;t in Alterungs- und Nervengewebe ausarten beschr&auml;nken.

Unregulierte Gehirn Eisen Ablagerung in transgenen M&auml;usen, die over-expressing HMOX1 im astrozytische Fach.

I meccanismi responsabili per la deposizione di ferro patologico nel SNC dei mammiferi invecchiamento e degenerazione rimangono scarsamente comprensibili. La proteina dello stress, HO 1 media la degradazione dell&#39;eme cellulare a Biliverdina/bilirubina, ferro libero e CO ed &egrave; up-regolato nel cervello delle persone con malattia di Alzheimer e di Parkinson. HO-1 induzione in primary astroglial culture favorisce la deposizione di ferro non-Transferrina, danni mitocondriali e macroautophagy e predispone gli elementi neuronali cocultured al danno ossidativo. Per ottenere un migliore apprezzamento del ruolo del glial HO-1 in vivo, abbiamo sondato per la deposizione di ferro aberrante cervello utilizzando il metodo dei Perls e spettrometria di massa di ioni secondari dinamico nel romanzo, GFAP condizionale.HMOX1 topi transgenici che selettivamente intra umana HO-1 nel vano astrocitario. A 48 settimane, i GFAP.HMOX1 topi esposti aumentati depositi di ferro gliale nell&#39;ippocampo e nelle altre regioni sottocorticali senza palesi cambiamenti nella regolamentazione-ferro e ferro-Associazione proteine rispetto alla corrispondenza et&agrave; selvaggio-tipo animali. Spettrometria di massa di ioni secondari dinamico ha rivelato abbondanti segnali FeO&#x207B; in transgenici, ma cervello del mouse non wild-type, che colocalized dei mitocondri degenerati e inclusioni citoplasmatiche osmiofilici (macroautophagy) documentati da TEM. Sostenuta up-regulation di HO-1 negli astrociti favorisce la deposizione di ferro patologica del cervello e danno ossidativo mitocondriale caratteristica dei tessuti neurali colpite dalla malattia di Alzheimer. Decurtazione di glial HO-1 iperattivit&agrave; pu&ograve; limitare la citotossicit&agrave; mediata dal ferro nell&#39;invecchiamento e degenerazione dei tessuti neurali.

Deposizione di ferro cervello non regolamentata nei topi transgenici momento HMOX1 nel vano astrocitario.

&#x8001;&#x5316;&#x3068;&#x5909;&#x6027;&#x54FA;&#x4E73;&#x985E;&#x4E2D;&#x67A2;&#x795E;&#x7D4C;&#x7CFB;&#x306E;&#x75C5;&#x7406;&#x5B66;&#x7684;&#x9244;&#x6C88;&#x7740;&#x306B;&#x8CAC;&#x4EFB;&#x304C;&#x3042;&#x308B;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306F;&#x3088;&#x304F;&#x7406;&#x89E3;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x307E;&#x307E;&#x3002;HO 1 &#x30B9;&#x30C8;&#x30EC;&#x30B9;&#x86CB;&#x767D;&#x8CEA;&#x306F;&#x7D30;&#x80DE;&#x306E;&#x30D8;&#x30E0; &#x30D3;&#x30EA;&#x30D9;&#x30EB;&#x30B8;&#x30F3;/&#x30D3;&#x30EA;&#x30EB;&#x30D3;&#x30F3;&#x3001;&#x904A;&#x96E2;&#x9244;&#x3001;CO &#x306E;&#x5206;&#x89E3;&#x3092;&#x4EF2;&#x4ECB;&#x3057;&#x3001;&#x8133;&#x306E;&#x30A2;&#x30EB;&#x30C4;&#x30CF;&#x30A4;&#x30DE;&#x30FC;&#x75C5;&#x3068;&#x30D1;&#x30FC;&#x30AD;&#x30F3;&#x30BD;&#x30F3;&#x75C5;&#x3092;&#x6301;&#x3064;&#x4EBA;&#x304C;&#x8ABF;&#x6574;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;HO 1 &#x8A98;&#x5C0E;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA; &#x30A2;&#x30B9;&#x30C8;&#x30ED;&#x30B0;&#x30EA;&#x30A2;&#x306E;&#x6587;&#x5316;&#x3067;&#x306F;&#x3001;&#x975E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30D5;&#x30A7;&#x30EA;&#x30F3;&#x9244;, &#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x640D;&#x50B7;&#x306E;&#x6C88;&#x7740;&#x3092;&#x4FC3;&#x9032;&#x3057;&#x3066;&#x57F9;&#x990A;&#x795E;&#x7D4C;&#x8981;&#x7D20;&#x3078;&#x306E;&#x9178;&#x5316;&#x7684;&#x969C;&#x5BB3;&#x306E;&#x7D20;&#x56E0;&#x3068;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;&#x30B0;&#x30EA;&#x30A2; HO 1 in vivo &#x3067;&#x306E;&#x5F79;&#x5272;&#x306E;&#x3088;&#x308A;&#x826F;&#x3044;&#x7406;&#x89E3;&#x3092;&#x5F97;&#x308B;&#x305F;&#x3081;&#x306B;&#x306F;&#x3001;&#x52D5;&#x7684;&#x4E8C;&#x6B21;&#x30A4;&#x30AA;&#x30F3;&#x8CEA;&#x91CF;&#x5206;&#x6790;&#x6CD5;&#x3068;&#x30D1;&#x30FC;&#x30EB;&#x30BA;&#x6CD5;&#x5C0F;&#x8AAC;&#x3067;&#x306F;&#x3001;&#x6761;&#x4EF6;&#x4ED8;&#x304D; GFAP &#x3092;&#x7528;&#x3044;&#x305F;&#x7570;&#x5E38;&#x8133;&#x9244;&#x6C88;&#x7740;&#x306E;&#x30D7;&#x30ED;&#x30FC;&#x30D6;&#x3002;&#x4EBA;&#x9593;&#x306E; HO 1 &#x30A2;&#x30B9;&#x30C8;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x306E;&#x30B3;&#x30F3;&#x30D1;&#x30FC;&#x30C8;&#x30E1;&#x30F3;&#x30C8;&#x3092;&#x9078;&#x629E;&#x7684;&#x306B;&#x3055;&#x305B;&#x305F;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF; &#x30DE;&#x30A6;&#x30B9;&#x3092; HMOX1&#x3002;48 &#x9031;&#x3067;&#x3001;GFAP&#x3002;HMOX1 &#x30DE;&#x30A6;&#x30B9;&#x306F;&#x6D77;&#x99AC;&#x3068;&#x4ED6;&#x306E;&#x76AE;&#x8CEA;&#x4E0B;&#x306E;&#x5730;&#x57DF;&#x898F;&#x5236;&#x9244;&#x304A;&#x3088;&#x3073;&#x9244;&#x7D50;&#x5408;&#x86CB;&#x767D;&#x8CEA;&#x76F8;&#x5BFE;&#x7684;&#x5E74;&#x9F62;&#x3092;&#x30DE;&#x30C3;&#x30C1;&#x3055;&#x305B;&#x305F;&#x91CE;&#x751F;&#x578B;&#x52D5;&#x7269;&#x306E;&#x3042;&#x304B;&#x3089;&#x3055;&#x307E;&#x306A;&#x5909;&#x5316;&#x306A;&#x3057;&#x306E;&#x30B0;&#x30EA;&#x30A2;&#x306E;&#x9244;&#x306E;&#x5897;&#x52A0;&#x306E;&#x9810;&#x91D1;&#x3092;&#x51FA;&#x5C55;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x52D5;&#x7684;&#x4E8C;&#x6B21;&#x30A4;&#x30AA;&#x30F3;&#x8CEA;&#x91CF;&#x5206;&#x6790;&#x6CD5;&#x306B;&#x3088;&#x308B;&#x8C4A;&#x5BCC;&#x306A; FeO&#x207B; &#x4FE1;&#x53F7;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF;&#x7E2E;&#x9000;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x304A;&#x3088;&#x3073;&#x30AA;&#x30B9;&#x30DF;&#x30A6;&#x30E0;&#x7D30;&#x80DE;&#x8CEA;&#x5185;&#x5C01;&#x5165;&#x306E; TEM &#x306B;&#x3088;&#x308A;&#x6587;&#x66F8;&#x5316; &#xFF08;&#x306B;&#xFF09; &#x7D50;&#x679C;&#x306A;&#x3044;&#x91CE;&#x751F;&#x578B;&#x30DE;&#x30A6;&#x30B9;&#x8133;&#x304C;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30A2;&#x30B9;&#x30C8;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x306B;&#x304A;&#x3051;&#x308B;&#x6301;&#x7D9A;&#x7684;&#x306A;&#x30A2;&#x30C3;&#x30D7;&#x898F;&#x5236;&#x306E; ho-1 &#x306E;&#x30A2;&#x30EB;&#x30C4;&#x30CF;&#x30A4;&#x30DE;&#x30FC;&#x75C5;&#x7F79;&#x60A3;&#x795E;&#x7D4C;&#x7D44;&#x7E54;&#x306E;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x9178;&#x5316;&#x7684;&#x640D;&#x50B7;&#x7279;&#x6027;&#x3068;&#x75C5;&#x614B;&#x8133;&#x9244;&#x6C88;&#x7740;&#x3092;&#x4FC3;&#x9032;&#x3057;&#x307E;&#x3059;&#x3002;&#x30B0;&#x30EA;&#x30A2; HO 1 &#x591A;&#x52D5;&#x306E;&#x524A;&#x6E1B;&#x306F;&#x3001;&#x8001;&#x5316;&#x3068;&#x795E;&#x7D4C;&#x7D44;&#x7E54;&#x306E;&#x9000;&#x5316;&#x306B;&#x9244;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x7D30;&#x80DE;&#x6BD2;&#x6027;&#x3092;&#x5236;&#x9650;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#x30A2;&#x30B9;&#x30C8;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x306E;&#x30B3;&#x30F3;&#x30D1;&#x30FC;&#x30C8;&#x30E1;&#x30F3;&#x30C8;&#x3067; HMOX1 &#x3092;&#x8D8A;&#x3048;&#x305F;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF; &#x30DE;&#x30A6;&#x30B9;&#x3067;&#x306E;&#x7121;&#x79E9;&#x5E8F;&#x306A;&#x8133;&#x9244;&#x306E;&#x6C88;&#x7740;&#x3002;

&#xC5D0;&#xC774;&#xC9D5; &#xBC0F; &#xC545;&#xD654; &#xD3EC;&#xC720;&#xB958; CNS&#xC5D0; &#xBCD1; &#xC801;&#xC778; &#xCCA0; &#xBD84; &#xC99D; &#xCC29;&#xC5D0; &#xB300; &#xD55C; &#xCC45;&#xC784; &#xBA54;&#xCEE4;&#xB2C8;&#xC998; &#xB0A8;&#xC544; &#xC81C;&#xB300;&#xB85C; &#xC774;&#xD574;. &#xC2A4;&#xD2B8;&#xB808;&#xC2A4; &#xB2E8;&#xBC31;&#xC9C8;, &#xD638; 1 biliverdin/&#xBE4C;&#xB9AC; &#xB8E8;&#xBE48;, &#xBB34;&#xB8CC; &#xCCA0; &#xBC0F; CO &#xC140;&#xB8F0;&#xB7EC; heme&#xC758; &#xC800;&#xD558;&#xB97C; &#xC911;&#xC7AC; &#xD558; &#xACE0; &#xC54C; &#xCE20; &#xD558;&#xC774; &#xBA38; &#xC9C8;&#xD658;&#xACFC; &#xD30C; &#xD0A8; &#xC2A8; &#xBCD1; &#xC788;&#xB294; &#xC0AC;&#xB78C;&#xC758; &#xBA38;&#xB9AC; &#xC704;&#xB85C;&#xC785;&#xB2C8;&#xB2E4;. &#xAE30;&#xBCF8; astroglial &#xBB38;&#xD654;&#xC5D0; &#xD638;-1 &#xC720;&#xB3C4; &#xBE44; transferrin &#xCCA0;, &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC; &#xB9AC;&#xC544; &#xC190;&#xC0C1;&#xACFC; macroautophagy, &#xC99D; &#xCC29;&#xC744; &#xCD09;&#xC9C4; &#xD558; &#xACE0; &#xC0B0;&#xD654; &#xBD80;&#xC0C1; cocultured &#xC2E0;&#xACBD; &#xC694;&#xC18C; &#xACBD;&#xD5A5;. &#xAD50; &#xD638; 1 in vivo&#xC758; &#xC5ED;&#xD560;&#xC758; &#xB354; &#xB098;&#xC740; &#xAC10;&#xC0AC;&#xB97C; &#xC5BB;&#xAE30; &#xC704;&#xD574; &#xC6B0;&#xB9AC;&#xB294; &#xC18C;&#xC124;, &#xC870;&#xAC74;&#xBD80; GFAP Perls&#xC758; &#xBA54;&#xC11C;&#xB4DC;&#xC640; &#xB3D9;&#xC801; 2 &#xCC28; &#xC774;&#xC628; &#xC9C8;&#xB7C9; &#xBD84;&#xC11D;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xD0C8; &#xC120; &#xB1CC; &#xCCA0; &#xBD84; &#xC99D; &#xCC29;&#xC5D0; &#xB300; &#xD55C; &#xC870;&#xC0AC;.&#xC120;&#xD0DD;&#xC801;&#xC73C;&#xB85C; astrocytic &#xAD6C;&#xD68D;&#xC5D0;&#xC11C; &#xC778;&#xAC04;&#xC758; &#xD638;-1 over-express HMOX1 &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xC950;. 48 &#xC8FC; GFAP.HMOX1 &#xC950;&#xC758; &#xD574; &#xB9C8;&#xC640; &#xC57C;&#xC0DD; &#xD615; &#xB3D9;&#xBB3C; &#xC77C;&#xCE58; &#xD558;&#xB294; &#xB098;&#xC774; &#xAE30;&#xC900;&#xC73C;&#xB85C; &#xADDC;&#xC81C; &#xCCA0; &#xBC0F; &#xCCA0; &#xBC14;&#xC778;&#xB529; &#xB2E8;&#xBC31;&#xC9C8;&#xC5D0; &#xBA85;&#xBC31;&#xD55C; &#xBCC0;&#xACBD; &#xC5C6;&#xC774; &#xB2E4;&#xB978; subcortical &#xC9C0;&#xC5ED; &#xAD50; &#xCCA0; &#xBD84; &#xC99D;&#xAC00; &#xC608;&#xAE08; &#xC804;&#xC2DC;. &#xB3D9;&#xC801; 2 &#xCC28; &#xC774;&#xC628; &#xC9C8;&#xB7C9; &#xBD84;&#xC11D; &#xD558;&#xC9C0;&#xB9CC;, &#xD0C0;&#xB77D; &#xD55C; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; &#xBC0F; osmiophilic &#xC138;&#xD3EC;&#xC9C8; &#xD3EC;&#xD568; (macroautophagy) &#xD3B8;&#xC73C;&#xB85C; &#xBB38;&#xC11C;&#xD654;&#xB97C; colocalized &#xD558;&#xC9C0; &#xC57C;&#xC0DD;-&#xD0C0;&#xC785;, &#xC950;&#xC758; &#xB1CC; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615;&#xC5D0; &#xD48D;&#xBD80;&#xD55C; FeO&#x207B; &#xC2E0;&#xD638;&#xB97C; &#xBC1D;&#xD614;&#xB2E4;. &#xC9C0;&#xC18D;&#xC801;&#xC778;&#xB41C; &#xC5C5; &#xB808; &#xADE4; &#xB808;&#xC774; &#xC158; &#xD638; 1&#xC758; &#xC774;&#xB2E4;&#xC5D0;&#xC11C; &#xBCD1; &#xC801;&#xC778; &#xB1CC; &#xCCA0; &#xBD84; &#xC99D; &#xCC29; &#xBC0F; &#xC54C; &#xCE20; &#xD558;&#xC774; &#xBA38; &#xBCD1;&#xC758; &#xC601;&#xD5A5;&#xC744; &#xBC1B;&#xB294; &#xC2E0;&#xACBD; &#xC870;&#xC9C1;&#xC758; &#xC0B0;&#xD654; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC; &#xB9AC;&#xC544; &#xC190;&#xC0C1; &#xD2B9;&#xC131;&#xC744; &#xCD09;&#xC9C4;&#xD569;&#xB2C8;&#xB2E4;. &#xAD50; &#xD638; 1 &#xACFC;&#xB2E4;&#xC758; &#xB2E8;&#xCD95; &#xC5D0;&#xC774;&#xC9D5; &#xBC0F; degenerating &#xC2E0;&#xACBD; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; &#xC138;&#xD3EC; &#xB3C5;&#xC131; &#xCCA0; &#xC911;&#xC7AC;&#xB97C; &#xC81C;&#xD55C;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

Over-expressing HMOX1 astrocytic &#xAD6C;&#xD68D;&#xC5D0; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xC950;&#xC5D0; &#xAD00;&#xACC4;&#xAC00; &#xC5C6;&#xB294; &#xB1CC; &#xCCA0; &#xBD84; &#xC99D; &#xCC29;.

Los mecanismos responsables de la deposici&oacute;n del hierro patol&oacute;gica en el SNC mam&iacute;fero de envejecimiento y degeneraci&oacute;n siguen siendo mal entendidos. La prote&iacute;na de estr&eacute;s, HO-1 interviene en la degradaci&oacute;n del Hem celular a biliverdina bilirrubina, hierro libre y CO y est&aacute; regulado, hasta en los cerebros de las personas con enfermedad de Alzheimer y de Parkinson. HO-1 inducci&oacute;n en culturas astroglial primaria promueve la deposici&oacute;n de hierro no-transferrina, da&ntilde;o mitocondrial y macroautophagy y predispone morfolog&iacute;a elementos neuronales a la lesi&oacute;n oxidativa. Para obtener una mejor apreciaci&oacute;n del papel de glial HO-1 in vivo, hemos sondeado para deposici&oacute;n del hierro del cerebro aberrante usando el m&eacute;todo &#39;Perls y espectrometr&iacute;a de masas de iones secundarios din&aacute;mico en novela, GFAP condicional.HMOX1 ratones transg&eacute;nicos que sobreexpresan selectivamente humano HO-1 en el compartimiento astroc&iacute;tico. A las 48 semanas, la GFAP.HMOX1 ratones exhibieron mayores dep&oacute;sitos de hierro glial en el hipocampo y otras regiones subcorticales sin cambios manifiestos en prote&iacute;nas regulatorias hierro y hierro en relaci&oacute;n con los animales de tipo salvaje de edad comparable. Espectrometr&iacute;a de masas de iones secundarios din&aacute;mica revel&oacute; abundantes se&ntilde;ales de FeO&#x207B; en los transg&eacute;nicos, pero el cerebro de rat&oacute;n no salvaje-tipo, que colocalized degeneradas mitocondrias y las inclusiones citopl&aacute;smicas osmiof&iacute;lico (macroautophagy) documentadas por TEM. Sostenido para arriba-regulaci&oacute;n de HO-1 en los astrocitos promueve la deposici&oacute;n del hierro patol&oacute;gica del cerebro y da&ntilde;o oxidativo mitocondrial caracter&iacute;stica de los tejidos neuronales afectadas por la enfermedad de Alzheimer. Reducci&oacute;n de glial hiperactividad de HO-1 puede limitar la citotoxicidad mediada por hierro en envejecimiento y degeneraci&oacute;n de los tejidos neuronales.

Deposici&oacute;n del hierro del cerebro no reglamentada en ratones transg&eacute;nicos sobreexpresi&oacute;n sistema est&aacute; da&ntilde;ado HMOX1 en el compartimiento astroc&iacute;tico.

Patients with chronic hepatitis C virus (HCV) infection frequently develop systemic iron overload, which exacerbates morbidity. Nevertheless, iron inhibits HCV replication in cell culture models and thereby exerts antiviral activity. We hypothesized that the cellular iron status is crucial for the establishment of HCV infection. We show that HCV infection of permissive Huh7.5.1 hepatoma cells promotes an iron deficient phenotype. Thus, HCV leads to increased iron regulatory protein (IRP) activity, accumulation of IRP2 and suppression of transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) in the host. These data suggest that HCV regulates cellular iron levels to bypass iron-mediated inhibition in viral replication.

Hepatitis C virus infection causes iron deficiency in Huh7.5.1 cells.

Hemojuvelin (Hjv) is a bone morphogenetic protein (BMP) co-receptor involved in the control of systemic iron homeostasis. Functional inactivation of Hjv leads to severe iron overload in humans and mice due to marked suppression of the iron-regulatory hormone hepcidin. To investigate the role of Hjv in body iron sensing, Hjv-/- mice and isogenic wild type controls were placed on a moderately low, a standard or a high iron diet for four weeks. Hjv-/- mice developed systemic iron overload under all regimens. Transferrin (Tf) was highly saturated regardless of the dietary iron content, while liver iron deposition was proportional to it. Hepcidin mRNA expression responded to fluctuations in dietary iron intake, despite the absence of Hjv. Nevertheless, iron-dependent upregulation of hepcidin was more than an order of magnitude lower compared to that seen in wild type controls. Likewise, iron signaling via the BMP/Smad pathway was preserved but substantially attenuated. These findings suggest that Hjv is not required for sensing of body iron levels and merely functions as an enhancer for iron signaling to hepcidin.

Carine Fillebeen

Lady Davis Institute for Medical Research

Jewish General Hospital

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

Carine Fillebeen

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Lady Davis Institute for Medical Research

Jewish General Hospital

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

Lady Davis Institute for Medical Research