RESUMO
Emerging digital assessment of biomarkers by linking health-related data obtained from wearable electronic devices and embedded health and fitness sensors in smartphones is opening up the possibility of creating a continuous remote-monitoring platform for disease management. It is considered that the built-in flashlight of smartphones may be utilized to remotely program genetically engineered designer cells for on-demand delivery of protein-based therapeutics. Here, the authors present smartphone-induced insulin release in ß-cell line (iß-cell) technology for traceless light-triggered rapid insulin secretion, employing the light-activatable receptor melanopsin to induce calcium influx and membrane depolarization upon illumination. This iß-cell-based system enables repeated, reversible secretion of insulin within 15 min in response to light stimulation, with a high induction fold both in vitro and in vivo. It is shown that programmable percutaneous remote control of implanted microencapsulated iß-cells with a smartphone's flashlight rapidly reverses hyperglycemia in a mouse model of type-1 diabetes.
Assuntos
Diabetes Mellitus , Smartphone , Animais , Glucose , Homeostase , Insulina , Secreção de Insulina , CamundongosRESUMO
Presenilin (PSEN) 1 and 2 are the catalytic components of the γ-secretase complex, which cleaves a variety of proteins, including the amyloid precursor protein (APP). Proteolysis of APP leads to the formation of the APP intracellular domain (AICD) and amyloid ß that is crucially involved in the pathogenesis of Alzheimer's disease. Prolyl-4-hydroxylase-domain (PHD) proteins regulate the hypoxia-inducible factors (HIFs), the master regulators of the hypoxic response. We previously identified the FK506 binding protein 38 (FKBP38) as a negative regulator of PHD2. Genetic ablation of PSEN1/2 has been shown to increase FKBP38 protein levels. Therefore, we investigated the role of PSEN1/2 in the oxygen sensing pathway using a variety of genetically modified cell and mouse lines. Increased FKBP38 protein levels and decreased PHD2 protein levels were found in PSEN1/2-deficient mouse embryonic fibroblasts and in the cortex of forebrain-specific PSEN1/2 conditional double knock-out mice. Hypoxic HIF-1α protein accumulation and transcriptional activity were decreased, despite reduced PHD2 protein levels. Proteolytic γ-secretase function of PSEN1/2 was needed for proper HIF activation. Intriguingly, PSEN1/2 mutations identified in Alzheimer patients differentially affected the hypoxic response, involving the generation of AICD. Together, our results suggest a direct role for PSEN in the regulation of the oxygen sensing pathway via the APP/AICD cleavage cascade.
Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Fator 1 Induzível por Hipóxia/metabolismo , Mutação , Neurônios/metabolismo , Presenilina-1/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Córtex Cerebral/metabolismo , Fibroblastos/metabolismo , Hipóxia/genética , Hipóxia/metabolismo , Fator 1 Induzível por Hipóxia/genética , Camundongos , Presenilina-1/metabolismo , Ativação TranscricionalRESUMO
The prolyl-4-hydroxylase domain 1-3 (PHD1-3) enzymes are regulating the protein stability of the α-subunit of the hypoxia-inducible factor-1 (HIF-1), which mediates oxygen-dependent gene expression. PHD2 is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. In human cancers, HIF-1α is overexpressed as a result of intratumoral hypoxia which in turn promotes tumor progression. The role of PHD2 for tumor progression is in contrast far from being thoroughly understood. Therefore, we established PHD2 knockdown clones of MDA-MB-231 breast cancer cells and analyzed their tumor-forming potential in a SCID mouse model. Tumor progression was significantly impaired in the PHD2 knockdown MDA-MB-231 cells, which could be partially rescued by re-establishing PHD2 expression. In a RNA profile screen, we identified the secreted phosphoprotein 1 (SPP1) as one target, which is differentially regulated as a consequence of the PHD2 knockdown. Knockdown of PHD2 drastically reduced the SPP1 expression in MDA-MB-231 cells. A correlation of SPP1 and PHD2 expression was additionally verified in 294 invasive breast cancer biopsies. In subsequent analyses, we identified that PHD2 alters the processing of transforming growth factor (TGF)-ß1, which is highly involved in SPP1 expression. The altered processing capacity was associated with a dislocation of the pro-protein convertase furin. Thus, our data demonstrate that in MDA-MB-231 cells PHD2 might affect tumor-relevant TGF-ß1 target gene expression by altering the TGF-ß1 processing capacity.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Fator de Crescimento Transformador beta1/metabolismo , Animais , Neoplasias da Mama/patologia , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia , Camundongos , Osteopontina/genética , Transdução de Sinais , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neoplastic expression of the onconeuronal cerebellar degeneration-related antigen Cdr2 in ovary and breast tumors is associated with paraneoplastic cerebellar degeneration (PCD). Cdr2 protein expression is normally restricted to neurons, but aberrant Cdr2 expression has mainly been described for breast and ovarian tumors. Previously, we found strong Cdr2 protein expression in the papillary subtype of renal cell carcinoma (pRCC) and showed that Cdr2 interacts with the hypoxia-inducible factor (HIF) prolyl-4-hydroxylase PHD1. High Cdr2 protein levels are associated with decreased HIF-dependent gene expression in cells as well as in clinical pRCC samples, providing a possible explanation why pRCCs are the most hypovascular renal tumors. Here, we demonstrate that strong Cdr2 protein expression in clinical samples from pRCC patients correlates with elevated PHD1 protein levels, suggesting that increased PHD1 activity attenuates HIF-dependent gene expression. Interestingly, survival analysis revealed a significant correlation between high levels of Cdr2 expression and worse patient outcome in clear cell (cc) RCC patients. These findings provide evidence that Cdr2 might represent an important tumor antigen in kidney cancer and possibly in other cancer types as well. In contrast to ovary and breast tumor patients who develop PCD, no Cdr2 auto-antibodies were detected in the serum of pRCC patients, which is in line with the fact that pRCC patients have not been reported to display paraneoplastic neurodegenerative syndromes. This suggests that, despite a shared target antigen, tumor immunity and autoimmunity only partially overlap, and also highlights to which extent immuno-surveillance against cancer can be clinically silent.
Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Autoanticorpos/sangue , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/mortalidade , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia , Neoplasias Renais/diagnóstico , Neoplasias Renais/mortalidade , Proteínas do Tecido Nervoso/imunologia , Taxa de SobrevidaRESUMO
Cells are responding to hypoxia via prolyl-4-hydroxylase domain (PHD) enzymes, which are responsible for oxygen-dependent hydroxylation of the hypoxia-inducible factor (HIF)-1α subunit. To gain further insight into PHD function, we generated knockdown cell models for the PHD2 isoform, which is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. Induction of a PHD2 knockdown in tetracycline-inducible HeLa PHD2 knockdown cells resulted in increased F-actin formation as detected by phalloidin staining. A similar effect could be observed in the stably transfected PHD2 knockdown cell clones 1B6 and 3B7. F-actin is at least in part responsible for shaping cell morphology as well as regulating cell migration. Cell migration was impaired significantly as a consequence of PHD2 knockdown in a scratch assay. Mechanistically, PHD2 knockdown resulted in activation of the RhoA (Ras homolog gene family member A)/Rho-associated kinase pathway with subsequent phosphorylation of cofilin. Because cofilin phosphorylation impairs its actin-severing function, this may explain the F-actin phenotype, thereby providing a functional link between PHD2-dependent signaling and cell motility.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Movimento Celular , Citoesqueleto/metabolismo , Células HeLa , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia , Modelos Biológicos , Fosforilação , Polímeros/química , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de ProteínaRESUMO
Wearable smart electronic devices, such as smart watches, are generally equipped with green-light-emitting diodes, which are used for photoplethysmography to monitor a panoply of physical health parameters. Here, we present a traceless, green-light-operated, smart-watch-controlled mammalian gene switch (Glow Control), composed of an engineered membrane-tethered green-light-sensitive cobalamin-binding domain of Thermus thermophilus (TtCBD) CarH protein in combination with a synthetic cytosolic TtCBD-transactivator fusion protein, which manage translocation of TtCBD-transactivator into the nucleus to trigger expression of transgenes upon illumination. We show that Apple-Watch-programmed percutaneous remote control of implanted Glow-controlled engineered human cells can effectively treat experimental type-2 diabetes by producing and releasing human glucagon-like peptide-1 on demand. Directly interfacing wearable smart electronic devices with therapeutic gene expression will advance next-generation personalized therapies by linking biopharmaceutical interventions to the internet of things.
Assuntos
Proteínas de Bactérias/efeitos da radiação , Diabetes Mellitus Tipo 2/terapia , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Optogenética/métodos , Transativadores/efeitos da radiação , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Engenharia Celular , Diabetes Mellitus Tipo 2/genética , Feminino , Engenharia Genética , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células HEK293 , Humanos , Luz , Masculino , Células-Tronco Mesenquimais , Camundongos , Camundongos Obesos , Optogenética/instrumentação , Fotopletismografia/instrumentação , Domínios Proteicos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiação , Thermus thermophilus/genética , Transativadores/genética , Transativadores/metabolismo , Transgenes , Dispositivos Eletrônicos VestíveisRESUMO
Prolyl-4-hydroxylase domain (PHD) proteins are 2-oxoglutarate and dioxygen-dependent enzymes that mediate the rapid destruction of hypoxia-inducible factor alpha subunits. Whereas PHD1 and PHD3 proteolysis has been shown to be regulated by Siah2 ubiquitin E3 ligase-mediated polyubiquitylation and proteasomal destruction, protein regulation of the main oxygen sensor responsible for hypoxia-inducible factor alpha regulation, PHD2, remained unknown. We recently reported that the FK506-binding protein (FKBP) 38 specifically interacts with PHD2 and determines PHD2 protein stability in a peptidyl-prolyl cis-trans isomerase-independent manner. Using peptide array binding assays, fluorescence spectroscopy, and fluorescence resonance energy transfer analysis, we defined a minimal linear glutamate-rich PHD2 binding domain in the N-terminal part of FKBP38 and showed that this domain forms a high affinity complex with PHD2. Vice versa, PHD2 interacted with a non-linear N-terminal motif containing the MYND (myeloid, Nervy, and DEAF-1)-type Zn(2+) finger domain with FKBP38. Biochemical fractionation and immunofluorescence analysis demonstrated that PHD2 subcellular localization overlapped with FKBP38 in the endoplasmic reticulum and mitochondria. An additional fraction of PHD2 was found in the cytoplasm. In cellulo PHD2/FKBP38 association, as well as regulation of PHD2 protein abundance by FKBP38, is dependent on membrane- anchored FKBP38 localization mediated by the C-terminal transmembrane domain. Mechanistically our data indicate that PHD2 protein stability is regulated by a ubiquitin-independent proteasomal pathway involving FKBP38 as adaptor protein that mediates proteasomal interaction. We hypothesize that FKBP38-bound PHD2 is constantly degraded whereas cytosolic PHD2 is stable and able to function as an active prolyl-4-hydroxylase.
Assuntos
Membranas Intracelulares/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia , Immunoblotting , Camundongos , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Pró-Colágeno-Prolina Dioxigenase/química , Pró-Colágeno-Prolina Dioxigenase/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
The heterodimeric hypoxia-inducible transcription factors (HIFs) are central regulators of the response to low oxygenation. HIF-alpha subunits are constitutively expressed but rapidly degraded under normoxic conditions. Oxygen-dependent hydroxylation of two conserved prolyl residues by prolyl-4-hydroxylase domain-containing enzymes (PHDs) targets HIF-alpha for proteasomal destruction. We identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as a novel interactor of PHD2. Yeast two-hybrid, glutathione S-transferase pull-down, coimmunoprecipitation, colocalization, and mammalian two-hybrid studies confirmed specific FKBP38 interaction with PHD2, but not with PHD1 or PHD3. PHD2 and FKBP38 associated with their N-terminal regions, which contain no known interaction motifs. Neither FKBP38 mRNA nor protein levels were regulated under hypoxic conditions or after PHD inhibition, suggesting that FKBP38 is not a HIF/PHD target. Stable RNA interference-mediated depletion of FKBP38 resulted in increased PHD hydroxylation activity and decreased HIF protein levels and transcriptional activity. Reconstitution of FKBP38 expression abolished these effects, which were independent of the peptidyl prolyl cis/trans isomerase activity. Downregulation of FKBP38 did not affect PHD2 mRNA levels but prolonged PHD2 protein stability, suggesting that FKBP38 is involved in PHD2 protein regulation.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Estabilidade Enzimática , Expressão Gênica , Genes Reporter , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia , Oxigênio/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
The Per-ARNT-Sim (PAS) domain serine/threonine kinase PASKIN, or PAS kinase, links energy flux and protein synthesis in yeast and regulates glycogen synthase in mammals. A recent report suggested that PASKIN mRNA, protein, and kinase activity are increased in pancreatic islet beta-cells under hyperglycemic conditions and that PASKIN is necessary for insulin gene expression. We previously generated Paskin knockout mice by targeted replacement of the kinase domain with the beta-geo fusion gene encoding beta-galactosidase reporter activity. Here we show that no 5-bromo-4-chloro-3-indolyl-ss-d-galactopyranoside (X-gal) staining was observed in islet beta-cells derived from Paskin knockout mice, irrespective of the ambient glucose concentration, whereas adenoviral expression of the lacZ gene in beta-cells showed strong X-gal staining. No induction of PASKIN mRNA could be detected in insulinoma cell lines or in islet beta-cells. Increasing glucose concentrations resulted in PASKIN-independent induction of insulin mRNA levels and insulin release. PASKIN mRNA levels were high in testes but undetectable in pancreas and in islet beta-cells. Finally, blood glucose levels and glucose tolerance after intraperitoneal glucose injection were indistinguishable between Paskin wild-type and knockout mice. These results suggest that Paskin gene expression is not induced by glucose in pancreatic beta-cells and that glucose-stimulated insulin production is independent of PASKIN.
Assuntos
Glucose/farmacologia , Insulina/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Animais , Técnicas de Cultura de Células , Regulação da Expressão Gênica , Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genéticaRESUMO
Diagnosis marks the beginning of any successful therapy. Because many medical conditions progress asymptomatically over extended periods of time, their timely diagnosis remains difficult, and this adversely affects patient prognosis. Focusing on hypercalcemia associated with cancer, we aimed to develop a synthetic biology-inspired biomedical tattoo using engineered cells that would (i) monitor long-term blood calcium concentration, (ii) detect onset of mild hypercalcemia, and (iii) respond via subcutaneous accumulation of the black pigment melanin to form a visible tattoo. For this purpose, we designed cells containing an ectopically expressed calcium-sensing receptor rewired to a synthetic signaling cascade that activates expression of transgenic tyrosinase, which produces melanin in response to persistently increased blood Ca2+ We confirmed that the melanin-generated color change produced by this biomedical tattoo could be detected with the naked eye and optically quantified. The system was validated in wild-type mice bearing subcutaneously implanted encapsulated engineered cells. All animals inoculated with hypercalcemic breast and colon adenocarcinoma cells developed tattoos, whereas no tattoos were seen in animals inoculated with normocalcemic tumor cells. All tumor-bearing animals remained asymptomatic throughout the 38-day experimental period. Although hypercalcemia is also associated with other pathologies, our findings demonstrate that it is possible to detect hypercalcemia associated with cancer in murine models using this cell-based diagnostic strategy.
Assuntos
Cálcio/sangue , Hipercalcemia/sangue , Hipercalcemia/diagnóstico , Biologia Sintética/métodos , Animais , Neoplasias da Mama/sangue , Linhagem Celular , Neoplasias do Colo/sangue , Feminino , Humanos , Hipercalcemia/etiologia , Melaninas/sangue , Camundongos , Neoplasias/sangue , Neoplasias/complicaçõesRESUMO
Cellular oxygen is sensed by prolyl-4-hydroxylase domain (PHD) proteins that hydroxylate hypoxia-inducible factor (HIF) alpha subunits. Under normoxic conditions, hydroxylated HIFalpha is bound by the von Hippel-Lindau (pVHL) tumor suppressor, leading to ubiquitinylation and proteasomal degradation. Under hypoxic conditions, hydroxylation becomes reduced, leading to HIFalpha stabilization. The authors recently showed that changes in PHD abundance and activity can regulate HIFalpha stability under normoxic as well as under hypoxic conditions. Thus, the PHD oxygen sensors themselves represent effectors of cellular signalling pathways as well as potential drug targets. Here, a cell-free in vitro microtiter plate-based peptide hydroxylation assay was used to investigate the influence of ferrous iron, Krebs cycle intermediates, transition metals, and vitamin C and other antioxidants on the activity of purified PHD1 to 3. PHD activity depends not only on oxygen availability but is also regulated by iron, vitamin C, and Krebs cycle intermediates, suggesting a physiological relevance of their cellular concentrations. Copper but not iron, cobalt, or nickel salts catalyzed vitamin C oxidation. While vitamin C is essential for PHD activity in vitro, N-acetyl-L-cysteine had no effect, and gallic acid or n-propyl gallate efficiently inhibited the activity of all three PHDs, demonstrating different functions of these antioxidants.
Assuntos
Pró-Colágeno-Prolina Dioxigenase/metabolismo , Animais , Ácido Ascórbico/metabolismo , Células CHO , Cricetinae , Cricetulus , Genes Reporter , Ferro/metabolismo , Cinética , Oxirredução , Consumo de OxigênioRESUMO
The prolyl-4-hydroxylase domain (PHD) oxygen sensor proteins hydroxylate hypoxia-inducible transcription factor (HIF)-alpha (alpha) subunits, leading to their subsequent ubiquitinylation and degradation. Since oxygen is a necessary cosubstrate, a reduction in oxygen availability (hypoxia) decreases PHD activity and, subsequently, HIF-alpha hydroxylation. Non-hydroxylated HIF-alpha cannot be bound by the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL), and HIF-alpha proteins thus become stabilized. HIF-alpha then heterodimerizes with HIF-beta (beta) to form the functionally active HIF transcription factor complex, which targets approximately 200 genes involved in adaptation to hypoxia. The three HIF-alpha PHDs are of a different nature compared with the prototype collagen prolyl-4-hydroxylase, which hydroxylates a mass protein rather than a rare transcription factor. Thus, novel assays had to be developed to express and purify functionally active PHDs and to measure PHD activity in vitro. A need also exists for such assays to functionally distinguish the three different PHDs in terms of substrate specificity and drug function. We provide a detailed description of the expression and purification of the PHDs as well as of an HIF-alpha-dependent and a HIF-alpha-independent PHD assay.
Assuntos
Pró-Colágeno-Prolina Dioxigenase/biossíntese , Pró-Colágeno-Prolina Dioxigenase/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Cromatografia em Camada Fina , Descarboxilação , Glutaratos/química , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Oxirredução , Oxigênio/química , Oxigênio/metabolismo , Peptídeos/química , Peptídeos/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/isolamento & purificação , Extratos de Tecidos/químicaRESUMO
The hypoxia-inducible factor 1 (HIF-1) was initially identified as a transcription factor that regulated erythropoietin gene expression in response to a decrease in oxygen availability in kidney tissue. Subsequently, a family of oxygen-dependent protein hydroxylases was found to regulate the abundance and activity of three oxygen-sensitive HIFalpha subunits, which, as part of the HIF heterodimer, regulated the transcription of at least 70 different effector genes. In addition to responding to a decrease in tissue oxygenation, HIF is proactively induced, even under normoxic conditions, in response to stimuli that lead to cell growth, ultimately leading to higher oxygen consumption. The growing cell thus profits from an anticipatory increase in HIF-dependent target gene expression. Growth stimuli-activated signaling pathways that influence the abundance and activity of HIFs include pathways in which kinases are activated and pathways in which reactive oxygen species are liberated. These pathways signal to the HIF protein hydroxylases, as well as to HIF itself, by means of covalent or redox modifications and protein-protein interactions. The final point of integration of all of these pathways is the hypoxia-response element (HRE) of effector genes. Here, we provide comprehensive compilations of the known growth stimuli that promote increases in HIF abundance, of protein-protein interactions involving HIF, and of the known HIF effector genes. The consensus HRE derived from a comparison of the HREs of these HIF effectors will be useful for identification of novel HIF target genes, design of oxygen-regulated gene therapy, and prediction of effects of future drugs targeting the HIF system.
Assuntos
Hipóxia Celular/fisiologia , Sequência Consenso , Regulação da Expressão Gênica/fisiologia , Fator 1 Induzível por Hipóxia/fisiologia , Oxigênio/fisiologia , Sequências Reguladoras de Ácido Nucleico , Transdução de Sinais/fisiologia , Animais , Divisão Celular , Dano ao DNA , Desenho de Fármacos , Epigênese Genética , Previsões , Terapia Genética , Humanos , Fator 1 Induzível por Hipóxia/química , Fator 1 Induzível por Hipóxia/genética , Oxigenases de Função Mista , Modelos Biológicos , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Proteínas Quinases/fisiologia , Subunidades Proteicas , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologiaRESUMO
Chronic lung hypoxia causes vascular remodeling with pulmonary artery smooth muscle cell (SMCPA) hyperplasia, resulting in pulmonary hypertension and cor pulmonale. We investigated SMCPA and pulmonary artery adventitial fibroblasts (FBPA) for their proliferative response to hypoxia. Strong SMCPA growth occurred under hypoxic conditions in SMCPA/FBPA co-cultures, but not in SMCPA monocultures. SMCPA growth was fully reproduced by transferring serum-free supernatant from hypoxic cultured FBPA to normoxic SMCPA. Hypoxia-inducible-transcription-factor subtypes (HIF-1alpha, HIF-2alpha, HIF-3alpha) and its dependent target genes, carrying the hypoxia-responsive-element as regulatory component, were strongly activated in both hypoxic FBPA and SMCPA. HIF-transcription-factor decoy technique, employed to FBPA during hypoxic culturing, blocked the mitogenic activity of FBPA conditioned medium on SMCPA. The data suggest that hypoxia-driven gene regulation in pulmonary artery fibroblasts results in a mitogenic stimulus on adjacent pulmonary artery smooth muscle cells, and HIF-transcription-decoy may offer a new therapeutic approach to suppress these events.
Assuntos
Fibroblastos/metabolismo , Músculo Liso Vascular/metabolismo , Artéria Pulmonar/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Divisão Celular , Hipóxia Celular/fisiologia , Células Cultivadas , Fibroblastos/citologia , Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Músculo Liso Vascular/citologia , Artéria Pulmonar/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genéticaRESUMO
The hypoxia-inducible transcription factor (HIF) is a key component of the cellular adaptation mechanisms to hypoxic conditions. HIFα subunits are degraded by prolyl-4-hydroxylase domain (PHD) enzyme-dependent prolyl-4-hydroxylation of LxxLAP motifs that confer oxygen-dependent proteolytic degradation. Interestingly, only three non-HIFα proteins contain two conserved LxxLAP motifs, including the putative RNA helicase with a zinc finger domain HELZ. However, HELZ proteolytic regulation was found to be oxygen-independent, supporting the notion that a LxxLAP sequence motif alone is not sufficient for oxygen-dependent protein destruction. Since biochemical pathways involving RNA often require RNA helicases to modulate RNA structure and activity, we used luciferase reporter gene constructs and metabolic labeling to demonstrate that HELZ overexpression activates global protein translation whereas RNA-interference mediated HELZ suppression had the opposite effect. Although HELZ interacted with the poly(A)-binding protein (PABP) via its PAM2 motif, PABP was dispensable for HELZ function in protein translation. Importantly, downregulation of HELZ reduced translational initiation, resulting in the disassembly of polysomes, in a reduction of cell proliferation and hypophosphorylation of ribosomal protein S6.
Assuntos
Iniciação Traducional da Cadeia Peptídica , RNA Helicases/metabolismo , Proteína S6 Ribossômica/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sequência Conservada , Genes Reporter/genética , Humanos , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Oxigênio/farmacologia , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas de Ligação a Poli(A)/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Helicases/química , Análise de Sequência de ProteínaAssuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Animais , Fracionamento Celular/métodos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Células Cultivadas , Cromatografia em Camada Fina/métodos , Humanos , Mamíferos , Camundongos , Oligodesoxirribonucleotídeos , EspectrofotometriaRESUMO
Protein stability of hypoxia-inducible factor (HIF)alpha subunits is regulated by the oxygen-sensing prolyl-4-hydroxylase domain (PHD) enzymes. Under oxygen-limited conditions, HIFalpha subunits are stabilized and form active HIF transcription factors that induce a large number of genes involved in adaptation to hypoxic conditions with physiological implications for erythropoiesis, angiogenesis, cardiovascular function and cellular metabolism. Oxygen-sensing is regulated by the co-substrate-dependent activity and hypoxia-inducible abundance of the PHD enzymes which trigger HIFalpha stability even under low oxygen conditions. Because HIFalpha itself is notoriously reluctant to the development of antagonists, an increase in PHD activity would offer an interesting alternative to the development of drugs that interfere specifically with the HIF signalling pathway. Interestingly, among the recently discovered PHD interacting proteins were not only novel downstream targets but also upstream regulators of PHDs. Their PHD isoform-specific interaction offers the possibility to target distinct PHD isoforms and their non-identical downstream signalling pathways. This review summarizes our current knowledge on PHD interacting proteins, including upstream regulators, chaperonins, scaffolding proteins, and novel downstream transcription factors.
Assuntos
Sistemas de Liberação de Medicamentos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Animais , Chaperoninas/metabolismo , Humanos , Pró-Colágeno-Prolina Dioxigenase/efeitos dos fármacos , Isoformas de Proteínas , Estabilidade Proteica , Transdução de Sinais/efeitos dos fármacosRESUMO
The activating transcription factor-4 (ATF-4) is translationally induced under anoxic conditions, mediates part of the unfolded protein response following endoplasmic reticulum (ER) stress, and is a critical regulator of cell fate. Here, we identified the zipper II domain of ATF-4 to interact with the oxygen sensor prolyl-4-hydroxylase domain 3 (PHD3). The PHD inhibitors dimethyloxalylglycine (DMOG) and hypoxia, or proteasomal inhibition, all induced ATF-4 protein levels. Hypoxic induction of ATF-4 was due to increased protein stability, but was independent of the ubiquitin ligase von Hippel-Lindau protein (pVHL). A novel oxygen-dependent degradation (ODD) domain was identified adjacent to the zipper II domain. Mutations of 5 prolyl residues within this ODD domain or siRNA-mediated down-regulation of PHD3, but not of PHD2, was sufficient to stabilize ATF-4 under normoxic conditions. These data demonstrate that PHD-dependent oxygen-sensing recruits both the hypoxia-inducible factor (HIF) and ATF-4 systems, and hence not only confers adaptive responses but also cell fate decisions.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Dioxigenases/fisiologia , Oxigênio/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Fator 4 Ativador da Transcrição/química , Sequência de Aminoácidos , Hipóxia Celular/fisiologia , Dioxigenases/química , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
PASKIN links energy flux and protein synthesis in yeast, regulates glycogen synthesis in mammals, and has been implicated in glucose-stimulated insulin production in pancreatic beta-cells. Using newly generated monoclonal antibodies, PASKIN was localized in the nuclei of human testis germ cells and in the midpiece of human sperm tails. A speckle-like nuclear pattern was observed for endogenous PASKIN in HeLa cells in addition to its cytoplasmic localization. By yeast two-hybrid screening, we identified the multifunctional eukaryotic translation elongation factor eEF1A1 as a novel interaction partner of PASKIN. This interaction was mapped to the PAS A and kinase domains of PASKIN and to the C-terminus of eEF1A1 using mammalian two-hybrid and GST pull-down assays. Kinase assays, mass spectrometry and site-directed mutagenesis revealed PASKIN auto-phosphorylation as well as eEF1A1 target phosphorylation mainly but not exclusively at Thr432. Wild-type but not kinase-inactive PASKIN increased the in vitro translation of a reporter cRNA. Whereas eEF1A1 did not localize to the nucleus, it co-localizes with PASKIN to the cytoplasm of HeLa cells. The two proteins also showed a remarkably similar localization in the midpiece of the sperm tail. These data suggest regulation of eEF1A1 by PASKIN-dependent phosphorylation in somatic as well as in sperm cells.
Assuntos
Fator 1 de Elongação de Peptídeos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espermatozoides/metabolismo , Anticorpos Monoclonais , Sequência de Bases , Núcleo Celular/metabolismo , Sistema Livre de Células , Citoplasma/metabolismo , Primers do DNA/genética , Expressão Gênica , Células HeLa , Humanos , Técnicas In Vitro , Masculino , Fator 1 de Elongação de Peptídeos/química , Fator 1 de Elongação de Peptídeos/genética , Fosforilação , Biossíntese de Proteínas , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Cauda do Espermatozoide/metabolismo , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Prolyl 4-hydroxylase domain (PHD) proteins are oxygen-dependent enzymes that hydroxylate hypoxia-inducible transcription factor (HIF) alpha-subunits, leading to their subsequent ubiquitination and degradation. Paradoxically, the expression of two family members (PHD2 and PHD3) is induced in hypoxic cell culture despite the reduced availability of the oxygen co-substrate, and it has been suggested that they become functionally relevant following re-oxygenation to rapidly terminate the HIF response. Here we show that PHDs are also induced in hypoxic mice in vivo, albeit in a tissue-specific manner. As demonstrated under chronically hypoxic conditions in vitro, PHD2 and PHD3 show a transient maximum but remain up-regulated over more than 10 days, suggesting a feedback down-regulation of HIF-1alpha which then levels off at a novel set point. Indeed, hypoxic induction of PHD2 and PHD3 is paralleled by the attenuation of endogenous HIF-1alpha. Using an engineered oxygen-sensitive reporter gene in a cellular background lacking endogenous HIF-1alpha and hence inducible PHD expression, we could show that increased exogenous PHD levels can compensate for a wide range of hypoxic conditions. Similar data were obtained in a reconstituted cell-free system in vitro. In summary, these results suggest that due to their high O2 Km values, PHDs have optimal oxygen-sensing properties under all physiologically relevant oxygen concentrations; increased PHDs play a functional role even under oxygen-deprived conditions, allowing the HIF system to adapt to a novel oxygen threshold and to respond to another hypoxic insult. Furthermore, such an autoregulatory oxygen-sensing system would explain how a single mechanism works in a wide variety of differently oxygenated tissues.