RESUMEN
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
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 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 ProteínaAsunto(s)
Adaptación Fisiológica/genética , Altitud , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Etnicidad/genética , Selección Genética , Mal de Altura/etnología , Mal de Altura/genética , Pueblo Asiatico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , China , Eritropoyesis/genética , Eritropoyesis/fisiología , Eritropoyetina/metabolismo , Evolución Molecular , Regulación de la Expresión Génica , Estudios de Asociación Genética , Haplotipos , Humanos , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/fisiología , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Inmunidad Innata , PPAR alfa/genética , PPAR alfa/fisiología , Polimorfismo de Nucleótido Simple , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/fisiología , América del Sur , TibetRESUMEN
High-altitude hypoxia (reduced inspired oxygen tension due to decreased barometric pressure) exerts severe physiological stress on the human body. Two high-altitude regions where humans have lived for millennia are the Andean Altiplano and the Tibetan Plateau. Populations living in these regions exhibit unique circulatory, respiratory, and hematological adaptations to life at high altitude. Although these responses have been well characterized physiologically, their underlying genetic basis remains unknown. We performed a genome scan to identify genes showing evidence of adaptation to hypoxia. We looked across each chromosome to identify genomic regions with previously unknown function with respect to altitude phenotypes. In addition, groups of genes functioning in oxygen metabolism and sensing were examined to test the hypothesis that particular pathways have been involved in genetic adaptation to altitude. Applying four population genetic statistics commonly used for detecting signatures of natural selection, we identified selection-nominated candidate genes and gene regions in these two populations (Andeans and Tibetans) separately. The Tibetan and Andean patterns of genetic adaptation are largely distinct from one another, with both populations showing evidence of positive natural selection in different genes or gene regions. Interestingly, one gene previously known to be important in cellular oxygen sensing, EGLN1 (also known as PHD2), shows evidence of positive selection in both Tibetans and Andeans. However, the pattern of variation for this gene differs between the two populations. Our results indicate that several key HIF-regulatory and targeted genes are responsible for adaptation to high altitude in Andeans and Tibetans, and several different chromosomal regions are implicated in the putative response to selection. These data suggest a genetic role in high-altitude adaption and provide a basis for future genotype/phenotype association studies necessary to confirm the role of selection-nominated candidate genes and gene regions in adaptation to altitude.
Asunto(s)
Altitud , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Genética de Población , Genoma Humano/genética , Selección Genética , Adaptación Fisiológica/genética , Variaciones en el Número de Copia de ADN/genética , Geografía , Globinas/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Filogenia , Polimorfismo de Nucleótido Simple/genética , Procolágeno-Prolina Dioxigenasa/genética , Sistema Renina-Angiotensina/genética , América del Sur , TibetRESUMEN
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.
Asunto(s)
Tipificación del Cuerpo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Larva/anatomía & histología , Oxígeno/metabolismo , Fenotipo , Procolágeno-Prolina Dioxigenasa/genética , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismoRESUMEN
Hypoxia-Inducible Factor (HIF) prolyl hydroxylase domains (PHDs) have been proposed to act as sensors that have an important role in oxygen homeostasis. In the presence of oxygen, they hydroxylate two specific prolyl residues in HIF-alpha polypeptides, thereby promoting their proteasomal degradation. So far, however, the developmental consequences of the inactivation of PHDs in higher metazoans have not been reported. Here, we describe novel loss-of-function mutants of fatiga, the gene encoding the Drosophila PHD oxygen sensor, which manifest growth defects and lethality. We also report a null mutation in dHIF-alpha/sima, which is unable to adapt to hypoxia but is fully viable in normoxic conditions. Strikingly, loss-of-function mutations of sima rescued the developmental defects observed in fatiga mutants and enabled survival to adulthood. These results indicate that the main functions of Fatiga in development, including control of cell size, involve the regulation of dHIF/Sima.