RESUMEN
The interplay between metal ion binding and the activity of thiol proteins, particularly within the protein disulfide isomerase family, remains an area of active investigation due to the critical role that these proteins play in many vital processes. This research investigates the interaction between recombinant human PDIA1 and zinc ions, focusing on the subsequent implications for PDIA1's conformational stability and enzymatic activity. Employing isothermal titration calorimetry and differential scanning calorimetry, we systematically compared the zinc binding capabilities of both oxidized and reduced forms of PDIA1 and assessed the structural consequences of this interaction. Our results demonstrate that PDIA1 can bind zinc both in reduced and oxidized states, but with significantly different stoichiometry and more pronounced conformational effects in the reduced form of PDIA1. Furthermore, zinc binding was observed to inhibit the catalytic activity of reduced-PDIA1, likely due to induced alterations in its conformation. These findings unveil a potential regulatory mechanism in PDIA1, wherein metal ion binding under reductive conditions modulates its activity. Our study highlights the potential role of zinc in regulating the catalytic function of PDIA1 through conformational modulation, suggesting a nuanced interplay between metal binding and protein stability in the broader context of cellular redox regulation.
Asunto(s)
Procolágeno-Prolina Dioxigenasa , Proteína Disulfuro Isomerasas , Humanos , Oxidación-Reducción , Procolágeno-Prolina Dioxigenasa/metabolismo , Unión Proteica , Proteína Disulfuro Isomerasas/metabolismo , Zinc/química , Zinc/metabolismoRESUMEN
The aims of the present study were to evaluate the expression of prolyl 4-hydroxylase subunit alpha 3 (P4HA3) in adipocytes and adipose tissue and to explore its effect on obesity and type 2 diabetes mellitus (T2DM). We initially demonstrated that P4HA3 was significantly upregulated in the subcutaneous adipose tissue of obesity and T2DM patients, and its functional roles in adipocyte differentiation and insulin resistance were investigated using in vitro and in vivo models. The knockdown of P4HA3 inhibited adipocyte differentiation and improved insulin resistance in 3T3-L1 cells. In C57BL/6J db/db mice fed with a high fat diet (HFD), silencing P4HA3 significantly decreased fasting blood glucose and triglycerides (TG) levels, with concomitant decrease of body weight and adipose tissue weight. Further analysis showed that P4HA3 knockdown was correlated with the augmented IRS-1/PI3K/Akt/FoxO1 signaling pathway in the adipose and hepatic tissues of obese mice, which could improve hepatic glucose homeostasis and steatosis of mice. Together, our study suggested that the dysregulation of P4HA3 may contribute to the development of obesity and T2DM.
Asunto(s)
Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Procolágeno-Prolina Dioxigenasa , Animales , Ratones , Dieta Alta en Grasa , Ratones Endogámicos C57BL , Obesidad/metabolismo , Fosfatidilinositol 3-Quinasas , Procolágeno-Prolina Dioxigenasa/metabolismoRESUMEN
BACKGROUND: Protein Disulfide Isomerases are thiol oxidoreductase chaperones from thioredoxin superfamily with crucial roles in endoplasmic reticulum proteostasis, implicated in many diseases. The family prototype PDIA1 is also involved in vascular redox cell signaling. PDIA1 is coded by the P4HB gene. While forced changes in P4HB gene expression promote physiological effects, little is known about endogenous P4HB gene regulation and, in particular, gene modulation by alternative splicing. This study addressed the P4HB splice variant landscape. RESULTS: Ten protein coding sequences (Ensembl) of the P4HB gene originating from alternative splicing were characterized. Structural features suggest that except for P4HB-021, other splice variants are unlikely to exert thiol isomerase activity at the endoplasmic reticulum. Extensive analyses using FANTOM5, ENCODE Consortium and GTEx project databases as RNA-seq data sources were performed. These indicated widespread expression but significant variability in the degree of isoform expression among distinct tissues and even among distinct locations of the same cell, e.g., vascular smooth muscle cells from different origins. P4HB-02, P4HB-027 and P4HB-021 were relatively more expressed across each database, the latter particularly in vascular smooth muscle. Expression of such variants was validated by qRT-PCR in some cell types. The most consistently expressed splice variant was P4HB-021 in human mammary artery vascular smooth muscle which, together with canonical P4HB gene, had its expression enhanced by serum starvation. CONCLUSIONS: Our study details the splice variant landscape of the P4HB gene, indicating their potential role to diversify the functional reach of this crucial gene. P4HB-021 splice variant deserves further investigation in vascular smooth muscle cells.
Asunto(s)
Procolágeno-Prolina Dioxigenasa , Proteína Disulfuro Isomerasas , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Humanos , Mutación , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Disulfuro Isomerasas/genética , Transducción de SeñalRESUMEN
BACKGROUND: Extracellular surface protein disulfide isomerase-A1 (PDI) is involved in platelet aggregation, thrombus formation and vascular remodeling. PDI performs redox exchange with client proteins and, hence, its oxidation by extracellular molecules might alter protein function and cell response. In this study, we investigated PDI oxidation by urate hydroperoxide, a newly-described oxidant that is generated through uric acid oxidation by peroxidases, with a putative role in vascular inflammation. METHODS: Amino acids specificity and kinetics of PDI oxidation by urate hydroperoxide was evaluated by LC-MS/MS and by stopped-flow. Oxidation of cell surface PDI and other thiol-proteins from HUVECs was identified using impermeable alkylating reagents. Oxidation of intracellular GSH and GSSG was evaluated with specific LC-MS/MS techniques. Cell adherence, detachment and viability were assessed using crystal violet staining, cellular microscopy and LDH activity, respectively. RESULTS: Urate hydroperoxide specifically oxidized cysteine residues from catalytic sites of recombinant PDI with a rate constant of 6 × 103 M-1 s-1. Incubation of HUVECs with urate hydroperoxide led to oxidation of cell surface PDI and other unidentified cell surface thiol-proteins. Cell adherence to fibronectin coated plates was impaired by urate hydroperoxide, as well as by other oxidants, thiol alkylating agents and PDI inhibitors. Urate hydroperoxide did not affect cell viability but significantly decreased GSH/GSSG ratio. CONCLUSIONS: Our results demonstrated that urate hydroperoxide affects thiol-oxidation of PDI and other cell surface proteins, impairing cellular adherence. GENERAL SIGNIFICANCE: These findings could contribute to a better understanding of the mechanism by which uric acid affects endothelial cell function and vascular homeostasis.
Asunto(s)
Peróxidos/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Ácido Úrico/análogos & derivados , Dominio Catalítico , Adhesión Celular/fisiología , Membrana Celular/metabolismo , Supervivencia Celular/fisiología , Cromatografía Liquida/métodos , Cisteína/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Cinética , Oxidación-Reducción , Peroxidasas/metabolismo , Agregación Plaquetaria , Procolágeno-Prolina Dioxigenasa/fisiología , Proteína Disulfuro Isomerasas/fisiología , Compuestos de Sulfhidrilo/metabolismo , Espectrometría de Masas en Tándem/métodos , Trombosis/metabolismo , Ácido Úrico/metabolismoRESUMEN
Protein disulfide isomerases including PDIA1 are implicated in cancer progression, but underlying mechanisms are unclear. PDIA1 is known to support vascular Nox1 NADPH oxidase expression/activation. Since deregulated reactive oxygen species (ROS) production underlies tumor growth, we proposed that PDIA1 is an upstream regulator of tumor-associated ROS. We focused on colorectal cancer (CRC) with distinct KRas activation levels. Analysis of RNAseq databanks and direct validation indicated enhanced PDIA1 expression in CRC with constitutive high (HCT116) vs. moderate (HKE3) and basal (Caco2) Ras activity. PDIA1 supported Nox1-dependent superoxide production in CRC; however, we first reported a dual effect correlated with Ras-level activity: in Caco2 and HKE3 cells, loss-of-function experiments indicate that PDIA1 sustains Nox1-dependent superoxide production, while in HCT116 cells PDIA1 restricted superoxide production, a behavior associated with increased Rac1 expression/activity. Transfection of Rac1G12V active mutant into HKE3 cells induced PDIA1 to become restrictive of Nox1-dependent superoxide, while in HCT116 cells treated with Rac1 inhibitor, PDIA1 became supportive of superoxide. PDIA1 silencing promoted diminished cell proliferation and migration in HKE3, not detectable in HCT116 cells. Screening of cell signaling routes affected by PDIA1 silencing highlighted GSK3ß and Stat3. Also, E-cadherin expression after PDIA1 silencing was decreased in HCT116, consistent with PDIA1 support of epithelial-mesenchymal transition. Thus, Ras overactivation switches the pattern of PDIA1-dependent Rac1/Nox1 regulation, so that Ras-induced PDIA1 bypass can directly activate Rac1. PDIA1 may be a crucial regulator of redox-dependent adaptive processes related to cancer progression.
Asunto(s)
Neoplasias del Colon/metabolismo , NADPH Oxidasa 1/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Células CACO-2 , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HCT116 , Humanos , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Transfección , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Protein disulfide isomerases are thiol oxidoreductase chaperones from thioredoxin superfamily. As redox folding catalysts from the endoplasmic reticulum (ER), their roles in ER-related redox homeostasis and signaling are well-studied. PDIA1 exerts thiol oxidation/reduction and isomerization, plus chaperone effects. Also, substantial evidence indicates that PDIs regulate thiol-disulfide switches in other cell locations such as cell surface and possibly cytosol. Subcellular PDI translocation routes remain unclear and seem Golgi-independent. The list of signaling and structural proteins reportedly regulated by PDIs keeps growing, via thiol switches involving oxidation, reduction and isomerization, S-(de)nytrosylation, (de)glutathyonylation and protein oligomerization. PDIA1 is required for agonist-triggered Nox NADPH oxidase activation and cell migration in vascular cells and macrophages, while PDIA1-dependent cytoskeletal regulation appears a converging pathway. Extracellularly, PDIs crucially regulate thiol redox signaling of thrombosis/platelet activation, e.g., integrins, and PDIA1 supports expansive caliber remodeling during injury repair via matrix/cytoskeletal organization. Some proteins display regulatory PDI-like motifs. PDI effects are orchestrated by expression levels or post-translational modifications. PDI is redox-sensitive, although probably not a mass-effect redox sensor due to kinetic constraints. Rather, the "all-in-one" organization of its peculiar redox/chaperone properties likely provide PDIs with precision and versatility in redox signaling, making them promising therapeutic targets.
Asunto(s)
Retículo Endoplásmico/metabolismo , Oxidación-Reducción , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Animales , Citoesqueleto/metabolismo , Citosol/metabolismo , Disulfuros/química , Homeostasis , Humanos , Cinética , Ratones , Chaperonas Moleculares/metabolismo , NADPH Oxidasas/metabolismo , Peróxidos/química , Procesamiento Proteico-Postraduccional , Transducción de SeñalRESUMEN
Extracellular protein disulfide isomerase (PDIA1) pool mediates thrombosis and vascular remodeling, however its externalization mechanisms remain unclear. We performed systematic pharmacological screening of secretory pathways affecting extracellular PDIA1 in endothelial cells (EC). We identified cell-surface (csPDIA1) and secreted non-particulated PDIA1 pools in EC. Such Golgi bypass also occurred for secreted PDIA1 in EC at baseline or after PMA, thrombin or ATP stimulation. Inhibitors of Type I, II and III unconventional routes, secretory lysosomes and recycling endosomes, including syntaxin-12 deletion, did not impair EC PDIA1 externalization. This suggests predominantly Golgi-independent unconventional secretory route(s), which were GRASP55-independent. Also, these data reinforce a vesicular-type traffic for PDIA1. We further showed that PDIA1 traffic is ATP-independent, while actin or tubulin cytoskeletal disruption markedly increased EC PDIA1 secretion. Clathrin inhibition enhanced extracellular soluble PDIA1, suggesting dynamic cycling. Externalized PDIA1 represents <2% of intracellular PDIA1. PDIA1 was robustly secreted by physiological levels of arterial laminar shear in EC and supported alpha 5 integrin thiol oxidation. Such results help clarify signaling and homeostatic mechanisms involved in multiple (patho)physiological extracellular PDIA1 functions.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/enzimología , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Fenómenos Biomecánicos , Células Cultivadas , Aparato de Golgi/enzimología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Integrina alfa5/metabolismo , Mecanotransducción Celular , Oxidación-Reducción , Transporte de ProteínasRESUMEN
This study analyzed the time dependence decay of the mRNA of selected genes important for the hypoxia response. The genes chosen were the two isoforms of hypoxia-inducible factors, the three isoforms of the prolyl hydroxylase domain protein, the vascular endothelial growth factor and endothelial nitric oxide synthase. mRNA and proteins were extracted from lungs obtained from control, hypoxic and 15 minutes normoxic recovered rats and analyzed by Real-time RT-PCR or by the Western Blot technique. Results indicated that in normoxia isoform 2á was the more represented hypoxia-inducible factor mRNA, and among the prolyl hydroxylase domain transcripts, isoform 3 was the least abundant. Moreover, in chronic hypoxia only hypoxia-inducible factor 1α and prolyl hydroxylase domain protein 3 increased significantly, while after 15 minutes of recovery all the mRNAs tested were decreased except endothelial nitric oxide synthase mRNA. In terms of proteins, hypoxia-inducible 1α was the isoform more significant in the nucleus, while 2á predominated in the cytosol. While the former was steady even after a brief recovery from hypoxia, the latter underwent a strong degradation. In conclusion we showed the relevance of the decay in the mRNA and protein levels upon re-oxygenation in normoxia. We believe that this has to be considered in research studies dealing with recovery from hypoxia.
Asunto(s)
Hipoxia/genética , Pulmón/metabolismo , ARN Mensajero/metabolismo , Transcripción Genética/genética , Animales , Western Blotting , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
This study analyzed the time dependence decay of the mRNA of selected genes important for the hypoxia response. The genes chosen were the two isoforms of hypoxia-inducible factors, the three isoforms of the prolyl hydroxylase domain protein, the vascular endothelial growth factor and endothelial nitric oxide synthase. mRNA and proteins were extracted from lungs obtained from control, hypoxic and 15 minutes normoxic recovered rats and analyzed by Real-time RT-PCR or by the Western Blot technique. Results indicated that in normoxia isoform 2á was the more represented hypoxia-inducible factor mRNA, and among the prolyl hydroxylase domain transcripts, isoform 3 was the least abundant. Moreover, in chronic hypoxia only hypoxia-inducible factor 1α and prolyl hydroxylase domain protein 3 increased significantly, while after 15 minutes of recovery all the mRNAs tested were decreased except endothelial nitric oxide synthase mRNA. In terms of proteins, hypoxia-inducible 1α was the isoform more significant in the nucleus, while 2á predominated in the cytosol. While the former was steady even after a brief recovery from hypoxia, the latter underwent a strong degradation. In conclusion we showed the relevance of the decay in the mRNA and protein levels upon re-oxygenation in normoxia. We believe that this has to be considered in research studies dealing with recovery from hypoxia.
Asunto(s)
Animales , Masculino , Hipoxia/genética , Pulmón/metabolismo , ARN Mensajero/metabolismo , Transcripción Genética/genética , Western Blotting , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Root hairs are single cells specialized in the absorption of water and nutrients from the soil. Growing root hairs require intensive cell-wall changes to accommodate cell expansion at the apical end by a process known as tip or polarized growth. We have recently shown that cell wall glycoproteins such as extensions (EXTs) are essential components of the cell wall during polarized growth. Proline hydroxylation, an early posttranslational modification of cell wall EXTs that is catalyzed by prolyl 4-hydroxylases (P4Hs), defines the subsequent O-glycosylation sites in EXTs. Biochemical inhibition or genetic disruption of specific P4Hs resulted in the blockage of polarized growth in root hairs. Our results demonstrate that correct hydroxylation and also further O-glycosylation on EXTs are essential for cell-wall self-assembly and, hence, root hair elongation. The changes that O-glycosylated cell-wall proteins like EXTs undergo during cell growth represent a starting point to unravel the entire biochemical pathway involved in plant development.
Asunto(s)
Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Aumento de la Célula , Raíces de Plantas/citología , Raíces de Plantas/crecimiento & desarrollo , Pared Celular/metabolismo , Glicoproteínas/metabolismo , Glicosilación , Hidroxilación , Fenotipo , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismoRESUMEN
Root hairs are single cells that develop by tip growth and are specialized in the absorption of nutrients. Their cell walls are composed of polysaccharides and hydroxyproline-rich glycoproteins (HRGPs) that include extensins (EXTs) and arabinogalactan-proteins (AGPs). Proline hydroxylation, an early posttranslational modification of HRGPs that is catalyzed by prolyl 4-hydroxylases (P4Hs), defines the subsequent O-glycosylation sites in EXTs (which are mainly arabinosylated) and AGPs (which are mainly arabinogalactosylated). We explored the biological function of P4Hs, arabinosyltransferases, and EXTs in root hair cell growth. Biochemical inhibition or genetic disruption resulted in the blockage of polarized growth in root hairs and reduced arabinosylation of EXTs. Our results demonstrate that correct O-glycosylation on EXTs is essential for cell-wall self-assembly and, hence, root hair elongation in Arabidopsis thaliana.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Pared Celular/metabolismo , Glicoproteínas/metabolismo , Hidroxiprolina/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Procolágeno-Prolina Dioxigenasa/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arabinosa/metabolismo , Conformación de Carbohidratos , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Glicoproteínas/química , Glicosilación , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Hidroxilación , Modelos Biológicos , Mutación , Pentosiltransferasa/química , Pentosiltransferasa/metabolismo , Fenotipo , Proteínas de Plantas/química , Raíces de Plantas/citología , Raíces de Plantas/metabolismo , Polisacáridos/química , Procolágeno-Prolina Dioxigenasa/genética , Prolina/metabolismo , Conformación Proteica , Procesamiento Proteico-Postraduccional , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: The Hypoxia Inducible Factor (HIF) mediates cellular adaptations to low oxygen. Prolyl-4-hydroxylases are oxygen sensors that hydroxylate the HIF alpha-subunit, promoting its proteasomal degradation in normoxia. Three HIF-prolyl hydroxylases, encoded by independent genes, PHD1, PHD2, and PHD3, occur in mammals. PHD2, the longest PHD isoform includes a MYND domain, whose biochemical function is unclear. PHD2 and PHD3 genes are induced in hypoxia to shut down HIF dependent transcription upon reoxygenation, while expression of PHD1 is oxygen-independent. The physiologic significance of the diversity of the PHD oxygen sensors is intriguing. METHODOLOGY AND PRINCIPAL FINDINGS: We have analyzed the Drosophila PHD locus, fatiga, which encodes 3 isoforms, FgaA, FgaB and FgaC that are originated through a combination of alternative initiation of transcription and alternative splicing. FgaA includes a MYND domain and is homologous to PHD2, while FgaB and FgaC are shorter isoforms most similar to PHD3. Through a combination of genetic experiments in vivo and molecular analyses in cell culture, we show that fgaB but not fgaA is induced in hypoxia, in a Sima-dependent manner, through a HIF-Responsive Element localized in the first intron of fgaA. The regulatory capacity of FgaB is stronger than that of FgaA, as complete reversion of fga loss-of-function phenotypes is observed upon transgenic expression of the former, and only partial rescue occurs after expression of the latter. CONCLUSIONS AND SIGNIFICANCE: Diversity of PHD isoforms is a conserved feature in evolution. As in mammals, there are hypoxia-inducible and non-inducible Drosophila PHDs, and a fly isoform including a MYND domain co-exists with isoforms lacking this domain. Our results suggest that the isoform devoid of a MYND domain has stronger regulatory capacity than that including this domain.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Oxígeno/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Empalme Alternativo , Animales , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Regulación Enzimológica de la Expresión Génica , Sitios Genéticos/genética , Humanos , Hipoxia/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Estadios del Ciclo de Vida/genética , Procolágeno-Prolina Dioxigenasa/química , Procolágeno-Prolina Dioxigenasa/genética , Estructura Terciaria de Proteína , ARN Mensajero/genética , Elementos de Respuesta/genética , Regulación hacia ArribaRESUMEN
The Drosophila HIFalpha homologue, Sima, is localized mainly in the cytoplasm in normoxia and accumulates in the nucleus upon hypoxic exposure. We have characterized the mechanism governing Sima oxygen-dependent subcellular localization and found that Sima shuttles continuously between the nucleus and the cytoplasm. We have previously shown that nuclear import depends on an atypical bipartite nuclear localization signal mapping next to the C-terminus of the protein. We show here that nuclear export is mediated in part by a CRM1-dependent nuclear export signal localized in the oxygen-dependent degradation domain (ODDD). CRM1-dependent nuclear export requires both oxygen-dependent hydroxylation of a specific prolyl residue (Pro850) in the ODDD, and the activity of the von Hippel Lindau tumor suppressor factor. At high oxygen tension rapid nuclear export of Sima occurs, whereas in hypoxia, Sima nuclear export is largely inhibited. HIFalpha/Sima nucleo-cytoplasmic localization is the result of a dynamic equilibrium between nuclear import and nuclear export, and nuclear export is modulated by oxygen tension.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Oxígeno/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Datos de Secuencia Molecular , Señales de Exportación Nuclear , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transgenes , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Drosophila tracheal terminal branches are plastic and have the capacity to sprout out projections toward oxygen-starved areas, in a process analogous to mammalian angiogenesis. This response involves the upregulation of FGF/Branchless in hypoxic tissues, which binds its receptor Breathless on tracheal cells. Here, we show that extra sprouting depends on the Hypoxia-Inducible Factor (HIF)-alpha homolog Sima and on the HIF-prolyl hydroxylase Fatiga that operates as an oxygen sensor. In mild hypoxia, Sima accumulates in tracheal cells, where it induces breathless, and this induction is sufficient to provoke tracheal extra sprouting. In nontracheal cells, Sima contributes to branchless induction, whereas overexpression of Sima fails to attract terminal branch outgrowth, suggesting that HIF-independent components are also required for full induction of the ligand. We propose that the autonomous response to hypoxia that occurs in tracheal cells enhances tracheal sensitivity to increasing Branchless levels, and that this mechanism is a cardinal step in hypoxia-dependent tracheal sprouting.