RESUMEN

ELYS is a nucleoporin that localizes to the nuclear side of the nuclear pore complex (NPC) in interphase cells. In mitosis, it serves as an assembly platform that interacts with chromatin and then with nucleoporin subcomplexes to initiate post-mitotic NPC assembly. Here we identify ELYS as a major binding partner of the membrane protein VAPB during mitosis. In mitosis, ELYS becomes phosphorylated at many sites, including a predicted FFAT (two phenylalanines in an acidic tract) motif, which mediates interaction with the MSP (major sperm protein)-domain of VAPB. Binding assays using recombinant proteins or cell lysates and co-immunoprecipitation experiments show that VAPB binds the FFAT motif of ELYS in a phosphorylation-dependent manner. In anaphase, the two proteins co-localize to the non-core region of the newly forming nuclear envelope. Depletion of VAPB results in prolonged mitosis, slow progression from meta- to anaphase and in chromosome segregation defects. Together, our results suggest a role of VAPB in mitosis upon recruitment to or release from ELYS at the non-core region of the chromatin in a phosphorylation-dependent manner.

Asunto(s)

Proteínas de Unión al ADN , Mitosis , Unión Proteica , Factores de Transcripción , Proteínas de Transporte Vesicular , Humanos , Anafase , Cromatina/metabolismo , Segregación Cromosómica , Células HeLa , Proteínas de la Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Fosforilación , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Transporte Vesicular/metabolismo

RESUMEN

The aminopeptidase DPP9 removes dipeptides from N-termini of substrates having a proline or alanine in second position. Although linked to several pathways including cell survival and metabolism, the molecular mechanisms underlying these outcomes are poorly understood. We identified a novel interaction of DPP9 with Filamin A, which recruits DPP9 to Syk, a central kinase in B-cell signalling. Syk signalling can be terminated by degradation, requiring the ubiquitin E3 ligase Cbl. We show that DPP9 cleaves Syk to produce a neo N-terminus with serine in position 1. Pulse-chases combined with mutagenesis studies reveal that Ser1 strongly influences Syk stability. Furthermore, DPP9 silencing reduces Cbl interaction with Syk, suggesting that DPP9 processing is a prerequisite for Syk ubiquitination. Consistently, DPP9 inhibition stabilizes Syk, thereby modulating Syk signalling. Taken together, we demonstrate DPP9 as a negative regulator of Syk and conclude that DPP9 is a novel integral aminopeptidase of the N-end rule pathway.

RESUMEN

The intracellular prolyl peptidase DPP9 is implied to be involved in various cellular pathways including amino acid recycling, antigen maturation, cellular homeostasis, and viability. Interestingly, the major RNA transcript of DPP9 contains two possible translation initiation sites, which could potentially generate a longer (892 aa) and a shorter version (863 aa) of DPP9. Although the endogenous expression of the shorter DPP9 form has been previously verified, it is unknown whether the longer version is expressed, and what is its biological significance. By developing specific antibodies against the amino-terminal extension of the putative DPP9-long form, we demonstrate for the first time the endogenous expression of this longer isoform within cells. Furthermore, we show that DPP9-long represents a significant fraction of total DPP9 in cells, under steady-state conditions. Using biochemical cell fractionation assays in combination with immunofluorescence studies, we find the two isoforms localize to separate subcellular compartments. Whereas DPP9-short is present in the cytosol, DPP9-long localizes preferentially to the nucleus. This differential localization is attributed to a classical monopartite nuclear localization signal (K(K/R)X(K/R)) in the N-terminal extension of DPP9-long. Furthermore, we detect prolyl peptidase activity in nuclear fractions, which can be inhibited by specific DPP8/9 inhibitors. In conclusion, a considerable fraction of DPP9, which was previously considered as a purely cytosolic peptidase, localizes to the nucleus and is active there, raising the intriguing possibility that the longer DPP9 isoform may regulate the activity or stability of nuclear proteins, such as transcription factors.

Asunto(s)

Núcleo Celular/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Señales de Localización Nuclear , Secuencia de Aminoácidos , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/análisis , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/fisiología , Células HeLa , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Isoenzimas/fisiología , Datos de Secuencia Molecular , Transporte de Proteínas

RESUMEN

Sumoylation affects many cellular processes by regulating the interactions of modified targets with downstream effectors. Here we identified the cytosolic dipeptidyl peptidase 9 (DPP9) as a SUMO1 interacting protein. Surprisingly, DPP9 binds to SUMO1 independent of the well known SUMO interacting motif, but instead interacts with a loop involving Glu(67) of SUMO1. Intriguingly, DPP9 selectively associates with SUMO1 and not SUMO2, due to a more positive charge in the SUMO1-loop. We mapped the SUMO-binding site of DPP9 to an extended arm structure, predicted to directly flank the substrate entry site. Importantly, whereas mutants in the SUMO1-binding arm are less active compared with wild-type DPP9, SUMO1 stimulates DPP9 activity. Consistent with this, silencing of SUMO1 leads to a reduced cytosolic prolyl-peptidase activity. Taken together, these results suggest that SUMO1, or more likely, a sumoylated protein, acts as an allosteric regulator of DPP9.

Asunto(s)

Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Proteína SUMO-1/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Regulación Enzimológica de la Expresión Génica , Células HeLa , Humanos , Cinética , Datos de Secuencia Molecular , Unión Proteica , Proteína SUMO-1/química , Proteína SUMO-1/genética , Sumoilación

RESUMEN

Protein degradation is an essential process that continuously takes place in all living cells. Regulated degradation of most cellular proteins is initiated by proteasomes, which produce peptides of varying length. These peptides are rapidly cleaved to single amino acids by cytoplasmic peptidases. Proline-containing peptides pose a specific problem due to structural constrains imposed by the pyrrolidine ring that prevents most peptidases from cleavage. Here we show that DPP9, a poorly characterized cytoplasmic prolyl-peptidase, is rate-limiting for destruction of proline-containing substrates both in cell extracts and in intact cells. We identified the first natural substrate for DPP9, the RU1(34-42) antigenic peptide (VPYGSFKHV). RU1(34-42) is degraded in vitro by DPP9, and down-regulation of DPP9 in intact cells results in increased presentation of this antigen. Together our findings demonstrate an important role for DPP9 in peptide turnover and antigen presentation.

Asunto(s)

Citoplasma/enzimología , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Prolina/metabolismo , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Secuencia de Bases , Línea Celular , Citosol/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Regulación Enzimológica de la Expresión Génica , Antígenos HLA-A/inmunología , Antígenos HLA-A/metabolismo , Humanos , Cinética , Ratones , Especificidad por Sustrato

RESUMEN

Sumoylation of proteins in vitro has evolved as an indispensable tool for the functional analysis of this post-translational modification. In this article we present detailed protocols for bacterial production of mammalian proteins necessary to perform in vitro sumoylation reactions, namely the E1 activating enzyme Aos1/Uba2 (SAE1/SAE2), the E2 conjugating enzyme Ubc9, SUMO-1 (identical protocols can be used for SUMO-2/3), and the catalytic domain of the E3 ligase RanBP2/Nup358. Two alternative procedures are described for the E1 enzyme, one depending on co-expression of His-Aos1 and untagged Uba2, and a second protocol for separate expression of His-Aos1 and Uba2-His and subsequent reconstitution of the active dimer. Two example conditions for in vitro sumoylation of RanGAP1 and Sp100 in the absence or presence of the SUMO E3 ligase RanBP2, respectively, are provided. Both protocols can be adapted easily to test in vitro conjugation of other target proteins and/or E3 ligases.

Asunto(s)

Técnicas de Laboratorio Clínico , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Animales , Catálisis , Humanos , Chaperonas Moleculares/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Enzimas Ubiquitina-Conjugadoras/aislamiento & purificación

RESUMEN

To determine mechanisms of neonatal parasite antigen (Ag)-specific immune suppression associated with placental Plasmodium falciparum infection, we isolated cord blood mononuclear cells (CBMCs) from Gabonese neonates born to mothers with differing histories of P. falciparum infection and performed ex vivo and in vitro studies to evaluate immune regulatory activity. We found increased ex vivo percentages of CD4(+)CD25(hi) and CD4(+)CD25(+)CTLA-4(+) cells and increased interleukin (IL)-10 responses to parasite Ag in vitro in CBMCs from neonates born to mothers with placental P. falciparum infection at delivery. Depleting CBMCs of CD4(+)CD25(+) cells before cell culture led to the abrogation of parasite Ag-specific IL-10 responses, to enhanced interferon- gamma responses, and to enhanced expression of CD25 on CD8(+) T cells and of major histocompatibility complex class I and II on monocytes. These data demonstrate that parasite Ag-specific CD4(+) regulatory cells are generated in utero as a consequence of placental P. falciparum infection.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Sangre Fetal/inmunología , Malaria Falciparum/inmunología , Placenta/parasitología , Complicaciones Parasitarias del Embarazo/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Animales , Femenino , Sangre Fetal/citología , Humanos , Recién Nacido , Malaria Falciparum/parasitología , Placenta/inmunología , Enfermedades Placentarias/inmunología , Enfermedades Placentarias/parasitología , Plasmodium falciparum/inmunología , Embarazo , Complicaciones Parasitarias del Embarazo/parasitología

RESUMEN

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates the adherence of P. falciparum-infected erythrocytes to placental syncytiotrophoblasts via interactions with chondroitin sulfate A (CSA), a characteristic of pregnancy-associated malaria. Pregnancy-associated malaria predicts increased susceptibility of newborns to malaria, and it is postulated that transplacental passage of parasite antigen induces immune regulatory activity in the neonate. We wished to examine the immune responsiveness to a CSA-binding domain of PfEMP1, the DBL-gamma3 domain, in cord and maternal venous blood obtained from pregnancies with various histories of P. falciparum infection. We assessed in vitro T-cell cytokine and plasma immunoglobulin G (IgG) and IgM responses to four peptides corresponding to highly conserved regions of a DBL-gamma3 domain common to central African parasite isolates. The presence of placental P. falciparum infection at delivery was associated with elevated frequencies of DBL-gamma3 peptide-specific CD3+ interleukin-10-positive T cells in cord blood, while treatment and clearance of infection prior to delivery was associated with elevated frequencies of CD3+ gamma interferon-positive T cells. DBL-gamma3 peptide-specific IgM antibodies were detected in 12 of 60 (20%) cord plasma samples from those born to mothers with P. falciparum infection during pregnancy. Consistent with polyclonal anti-PfEMP1 antibody responses that are associated with protection against pregnancy-associated malaria, the presence of maternal IgG antibodies with specificity for one of the DBL-gamma3 peptides showed a parity-dependent profile. These data demonstrate that peptides corresponding to conserved regions of the DBL-gamma3 domain of PfEMP1 are immunogenic in P. falciparum-infected mothers and their offspring.

Asunto(s)

Sulfatos de Condroitina/metabolismo , Sangre Fetal/inmunología , Epítopos Inmunodominantes/inmunología , Malaria Falciparum/inmunología , Complicaciones Parasitarias del Embarazo/inmunología , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Sangre/inmunología , Citocinas/sangre , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Recién Nacido , Datos de Secuencia Molecular , Péptidos/inmunología , Placenta/inmunología , Embarazo , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Linfocitos T/inmunología

RESUMEN

Insulin-like growth factor 1 (IGF-1) and plasminogen activator inhibitor-1 (PAI-1) appear to play a crucial role in a number of processes associated with growth and tissue remodelling. IGF-1 was shown to enhance PAI-1 expression in primary hepatocytes and HepG2 hepatoma cells, but the molecular mechanisms underlying this effect have not been fully elucidated. In this study, we investigated the transcriptional mechanism and the signaling pathway by which IGF-1 mediates induction of PAI-1 expression in HepG2 cells. By using human PAI-1 promoter reporter gene assays we found that mutation of the hypoxia responsive element (HRE), which could bind hypoxia-inducible factor-1 (HIF-1), nearly abolished the induction by IGF-1. We found that IGF-1-induced up-regulation of PAI-1 expression was associated with activation of HIF-1 alpha. Furthermore,IGF-1 enhanced HIF-1alpha protein levels and HIF-1 DNA-binding to each HRE,E4 and E5 as shown by EMSA. Mutation of the E-boxes, E4 and E5, did not affect the IGF-1-dependent induction of PAI-1 promoter constructs under normoxia but abolished the effect of IGF-1 under hypoxia. Inhibition of either the PI3K by LY294002 or ERK1/2 by U0126 reduced HIF-1alpha protein levels while both inhibitors together completely abolished the IGF-1 effect on HIF-1alpha. Remarkably, transfection of HepG2 cells with vectors expressing a dominant-negative PDK1 or the PKB inhibitor, TRB3, did not influence while dominant-negative Raf inhibited the IGF-1 effect on HIF-1alpha. Thus, IGF-1 activates human PAI-1 gene expression through activation of the PI3-kinase and ERK1/2 via HIF-1alpha.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas Nucleares/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , ADN/genética , ADN/metabolismo , Genes Reporteros/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neoplasias/genética , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Transcripción Genética/efectos de los fármacos

RESUMEN

BACKGROUND: Women with semi-immunity to malaria who live in regions where the disease is endemic are at increased risk for more frequent and severe episodes of malaria during pregnancy. Recent findings indicate that this increased risk might persist beyond delivery, but the underlying mechanisms for this change in risk are poorly understood. METHODS: One hundred fifty women were included in a cohort study in Lambaréné, Gabon, and were actively followed up weekly for 10 weeks after delivery, as were nonpregnant control women who had been matched to them by location and age. Parasites in samples of placenta and blood were genotyped by use of polymerase chain reaction amplification of the merozoite surface antigen 2 gene and the subtelomeric variable open reading frame gene of Plasmodium falciparum. RESULTS: Eleven puerperal women had cases of clinical malaria, compared with 1 control woman (rate ratio, 9.8; P=.006). Eighteen puerperal women had P. falciparum parasitemia, compared with 6 control women (rate ratio, 2.7; P=.03). Five of 16 puerperal women (31%) with parasitemia on follow-up had identical parasites in their placentas and blood, and 11 of these cases (69%) were the result of reinfection. Puerperal women remained at equal risk for the development of parasitemia throughout the first 10 weeks after delivery. Use of bed nets, use of chloroquine prophylaxis during pregnancy, presence of malaria episodes during pregnancy, gravidity, and age were not associated with the acquisition of parasitemia during follow-up. CONCLUSIONS: Compared with nonpregnant women, puerperal women have a considerably increased risk for the development of malaria and/or parasitemia. This increased risk is caused both by the recurrence of P. falciparum parasitemia and by the increased susceptibility to new infections, although the latter plays a more significant role.

Asunto(s)

Malaria Falciparum/epidemiología , Malaria Falciparum/fisiopatología , Plasmodium falciparum , Infección Puerperal/epidemiología , Infección Puerperal/fisiopatología , Adulto , Animales , Sangre/parasitología , Estudios de Cohortes , Femenino , Gabón/epidemiología , Genotipo , Humanos , Incidencia , Malaria Falciparum/parasitología , Parasitemia/epidemiología , Parasitemia/parasitología , Placenta/parasitología , Plasmodium falciparum/clasificación , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Periodo Posparto , Embarazo , Complicaciones Parasitarias del Embarazo/epidemiología , Estudios Prospectivos , Infección Puerperal/parasitología , Factores de Riesgo

RESUMEN

Available evidence suggests that immune cells from neonates born to mothers with placental Plasmodium falciparum (Pf) infection are sensitized to parasite Ag in utero but have reduced ability to generate protective Th1 responses. In this study, we detected Pf Ag-specific IFN-gamma(+) T cells in cord blood from human neonates whose mothers had received treatment for malaria or who had active placental Pf infection at delivery, with responses being significantly reduced in the latter group. Active placental malaria at delivery was also associated with reduced expression of monocyte MHC class I and II in vivo and following short term in vitro coculture with Pf Ag compared with levels seen in neonates whose mothers had received treatment during pregnancy. Given that APC activation and Th1 responses are driven in part by IFN-gamma and down-regulated by IL-10, we examined the role of these cytokines in modulating the Pf Ag-specific immune responses in cord blood samples. Exogenous recombinant human IFN-gamma and neutralizing anti-human IL-10 enhanced T cell IFN-gamma production, whereas recombinant human IFN-gamma also restored MHC class I and II expression on monocytes from cord blood mononuclear cells cocultured with Pf Ag. Accordingly, active placental malaria at delivery was associated with increased frequencies of Pf Ag-specific IL-10(+)CD4(+) T cells in cord blood mononuclear cell cultures from these neonates. Generation and maintenance of IL-10(+) T cells in utero may thus contribute to suppression of APC function and Pf Ag-induced Th1 responses in newborns born to mothers with placental malaria at delivery, which may increase susceptibility to infection later in life.

Asunto(s)

Sangre Fetal/inmunología , Interferón gamma/fisiología , Interleucina-10/fisiología , Malaria Falciparum/inmunología , Enfermedades Placentarias/inmunología , Plasmodium falciparum/inmunología , Complicaciones Parasitarias del Embarazo/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos de Protozoos/inmunología , Células Cultivadas , Femenino , Sangre Fetal/citología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase II/biosíntesis , Humanos , Recién Nacido , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Malaria Falciparum/parasitología , Enfermedades Placentarias/parasitología , Embarazo , Complicaciones Parasitarias del Embarazo/parasitología , Células TH1/inmunología , Células TH1/metabolismo

RESUMEN

The plasminogen activator inhibitor-1 (PAI-1) expression can be enhanced by hypoxia and other stimuli leading to the mobilization of intracellular calcium. Thus, it was the aim of the present study to investigate the role of calcium in the hypoxia-dependent PAI-1 expression. It was shown that the Ca(2+)-ionophore A23187 and the cell permeable Ca(2+)-chelator BAPTA-am (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester) induced PAI-1 mRNA and protein expression under normoxia and hypoxia in HepG2 cells. Transfection experiments with wild-type and hypoxia response element (HRE)-mutated PAI promoter constructs revealed that the HRE binding hypoxiainducible factor-1 (HIF-1) mediated the response to A23187 and BAPTA-am. Although A23187 induced a striking and stable induction of HIF-1alpha, BAPTA-am only mediated a fast and transient increase. By using actinomycin D and cycloheximide we showed that A23187 induced HIF-1alpha mRNA expression, whereas BAPTA-am acted after transcription. Although A23187 activated extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), as well as protein kinase B, it appeared that the enhancement of HIF-1alpha by A23187 was only mediated via the ERK pathway. By contrast, BAPTA-am exerted its effects via inhibition of HIF-prolyl hydroxylase activity and von Hippel-Lindau tumor repressor protein (VHL) interaction. Thus, calcium appeared to have a critical role in the regulation of the HIF system and subsequent activation of the PAI-1 gene expression.

Asunto(s)

Calcio/fisiología , Ácido Egtácico/análogos & derivados , Regulación Neoplásica de la Expresión Génica/genética , Inhibidor 1 de Activador Plasminogénico/genética , Factores de Transcripción/fisiología , Calcimicina/farmacología , Línea Celular Tumoral , Quelantes/farmacología , Cicloheximida/farmacología , Dactinomicina/farmacología , Ácido Egtácico/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Procolágeno-Prolina Dioxigenasa/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética

RESUMEN

It has been proposed that hydroxyl radicals (.OH) generated in a perinuclear iron-dependent Fenton reaction are involved in O(2)-dependent gene expression. Thus, it was the aim of this study to localize the cellular compartment in which the Fenton reaction takes place and to determine whether scavenging of.OH can modulate hypoxia-inducible factor 1 (HIF-1)-dependent gene expression. The Fenton reaction was localized by using the nonfluorescent dihydrorhodamine (DHR) 123 that is irreversibly oxidized to fluorescent rhodamine 123 while scavenging.OH together with gene constructs allowing fluorescent labeling of mitochondria, endoplasmic reticulum (ER), Golgi apparatus, peroxisomes, or lysosomes. A 3D two-photon confocal laser scanning microscopy showed.OH generation in distinct hot spots of perinuclear ER pockets. This ER-based Fenton reaction was strictly pO(2)-dependent. Further colocalization experiments showed that the O(2)-sensitive transcription factor HIF-1alpha was present at the ER under normoxia, whereas HIF-1alpha was present only in the nucleus under hypoxia. Inhibition of the Fenton reaction by the.OH scavenger DHR attenuated HIF-prolyl hydroxylase activity and interaction with von Hippel-Lindau protein, leading to enhanced HIF-1alpha levels, HIF-1alpha transactivation, and activated expression of the HIF-1 target genes plasminogen activator inhibitor 1 and heme oxygenase 1. Further,.OH scavenging appeared to enhance redox factor 1 (Ref-1) binding and, thus, recruitment of p300 to the transactivation domain C because mutation of the Ref-1 binding site cysteine 800 abolished DHR-induced transactivation. Thus, the localized Fenton reaction appears to impact the expression of hypoxia-regulated genes by means of HIF-1alpha stabilization and coactivator recruitment.

Asunto(s)

Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Hipoxia/metabolismo , Núcleo Celular/metabolismo , Genes Reporteros , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Técnicas In Vitro , Oxidación-Reducción , Rodamina 123/metabolismo , Rodaminas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo