RESUMO
ß-Thalassaemia major (ß-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of ß-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing ß-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human ß-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of ß-TM erythroblasts, which may provide a rationale for new targeted therapies of ß-TM.
Assuntos
Eritroblastos/metabolismo , Eritropoese , Proteínas de Choque Térmico HSP70/metabolismo , alfa-Globinas/metabolismo , Talassemia beta/sangue , Talassemia beta/metabolismo , Apoptose , Medula Óssea/metabolismo , Caspase 3/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular/genética , Células Cultivadas , Citoplasma/metabolismo , Ativação Enzimática , Eritroblastos/citologia , Eritroblastos/patologia , Eritropoese/genética , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Humanos , Cinética , Terapia de Alvo Molecular , Ligação Proteica , Redobramento de Proteína , Talassemia beta/patologiaRESUMO
The heat-shock protein 27 (HSP27) is up-regulated in tumor cells and released in their microenvironment. Here, we show that extracellular HSP27 has a proangiogenic effect evidenced on chick chorioallantoic membrane. To explore this effect, we test the recombinant human protein (rhHSP27) at physiopathological doses (0.1-10 µg/ml) onto human microvascular endothelial cells (HMECs) grown as monolayers or spheroids. When added onto HMECs, rhHSP27 dose-dependently accelerates cell migration (with a peak at 5 µg/ml) and favors spheroid sprouting within 12-24 h. rhHSP27 increases VEGF gene transcription and promotes secretion of VEGF-activating VEGF receptor type 2. Increased VEGF transcription is related to NF-κB activation in 30 min. All of these effects are initiated by rhHSP27 interaction with Toll-like receptor 3 (TLR3). Such an interaction can be detected by immunoprecipitation but does not seem to be direct, as we failed to detect an interaction between rhHSP27 and monomeric TLR3 by SPR analysis. rhHSP27 is rapidly internalized with a pool of TLR3 to the endosomal compartment (within 15-30 min), which is required for NF-κB activation in a cytosolic Ca(2+)-dependent manner. The HSP27/TLR3 interaction induces NF-κB activation, leading to VEGF-mediated cell migration and angiogenesis. Such a pathway provides alternative targets for antiangiogenic cancer therapy.
Assuntos
Células Endoteliais/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Neovascularização Fisiológica/fisiologia , Receptor 3 Toll-Like/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP27/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Receptor 3 Toll-Like/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by myofibroblast proliferation. Transition of epithelial/mesothelial cells into myofibroblasts [epithelial-to-mesenchymal transition (EMT)] occurs under the influence of transforming growth factor (TGF)-ß1, with Snail being a major transcription factor. We study here the role of the heat-shock protein HSP27 in fibrogenesis and EMT. In vitro, we have up- and down-modulated HSP27 expression in mesothelial and epithelial cell lines and studied the expression of different EMT markers induced by TGF-ß1. In vivo, we inhibited HSP27 with the antisense oligonucleotide OGX-427 (in phase II clinical trials as anticancer agent) in our rat subpleural/pulmonary fibrosis models. We demonstrate that HSP27 is strongly expressed during the fibrotic process in patients with IPF and in different in vivo models. We showed that HSP27 binds to and stabilizes Snail and consequently induces EMT. Conversely, HSP27 knockdown leads to Snail proteasomal degradation, thus inhibiting TGF-ß1-induced EMT. Inhibition of HSP27 with OGX-427 efficiently blocks EMT and fibrosis development. Controls in vivo were an empty adenovirus that did not induce fibrosis and a control antisense oligonucleotide. The present work opens the possibility of a new therapeutic use for HSP27 inhibitors against IPF, for which there is no conclusively effective treatment.
Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/antagonistas & inibidores , Caramujos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Caderinas/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Fibrose/metabolismo , Humanos , Oligonucleotídeos Antissenso/farmacologia , Ratos , Ratos Sprague-Dawley , Tionucleotídeos/farmacologia , Fatores de Transcrição/metabolismoRESUMO
Heat shock protein 27 (HSP27) is a chaperone whose cellular expression increases in response to various stresses and protects the cell either by inhibiting apoptotic cell death or by promoting the ubiquitination and proteasomal degradation of specific proteins. Here, we show that globin transcription factor 1 (GATA-1) is a client protein of HSP27. In 2 models of erythroid differentiation; that is, in the human erythroleukemia cell line, K562 induced to differentiate into erythroid cells on hemin exposure and CD34(+) human cells ex vivo driven to erythroid differentiation in liquid culture, depletion of HSP27 provokes an accumulation of GATA-1 and impairs terminal maturation. More specifically, we demonstrate that, in the late stages of the erythroid differentiation program, HSP27 is phosphorylated in a p38-dependent manner, enters the nucleus, binds to GATA-1, and induces its ubiquitination and proteasomal degradation, provided that the transcription factor is acetylated. We conclude that HSP27 plays a role in the fine-tuning of terminal erythroid differentiation through regulation of GATA-1 content and activity.
Assuntos
Diferenciação Celular , Células Eritroides/metabolismo , Fator de Transcrição GATA1/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Animais , Antígenos CD34/sangue , Células COS , Núcleo Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Células Eritroides/citologia , Células Eritroides/efeitos dos fármacos , Fator de Transcrição GATA1/genética , Proteínas de Choque Térmico HSP27/genética , Células HeLa , Proteínas de Choque Térmico , Humanos , Imidazóis/farmacologia , Immunoblotting , Interleucina-6/farmacologia , Células K562 , Leupeptinas/farmacologia , Chaperonas Moleculares , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Ligação Proteica , Piridinas/farmacologia , Interferência de RNA , Fator de Crescimento Transformador beta/farmacologia , Ubiquitinação/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Better identification of severe acute graft-versus-host disease (GvHD) may improve the outcome of this life-threatening complication of allogeneic hematopoietic stem cell transplantation. GvHD induces tissue damage and the release of damage-associated molecular pattern (DAMP) molecules. Here, we analyzed GvHD patients (n = 39) to show that serum heat shock protein glycoprotein 96 (Gp96) could be such a DAMP molecule. We demonstrate that serum Gp96 increases in gastrointestinal GvHD patients and its level correlates with disease severity. An increase in Gp96 serum level was also observed in a mouse model of acute GvHD. This model was used to identify complement C3 as a main partner of Gp96 in the serum. Our biolayer interferometry, yeast two-hybrid and in silico modeling data allowed us to determine that Gp96 binds to a complement C3 fragment encompassing amino acids 749-954, a functional complement C3 hot spot important for binding of different regulators. Accordingly, in vitro experiments with purified proteins demonstrate that Gp96 downregulates several complement C3 functions. Finally, experimental induction of GvHD in complement C3-deficient mice confirms the link between Gp96 and complement C3 in the serum and with the severity of the disease.
Assuntos
Complemento C3/metabolismo , Doença Enxerto-Hospedeiro/sangue , Glicoproteínas de Membrana/sangue , Chaperonas Moleculares/sangue , Adolescente , Adulto , Animais , Ativação do Complemento , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Heat shock proteins (HSPs) HSP27, HSP70 and HSP90 are powerful chaperones. Their expression is induced in response to a wide variety of physiological and environmental insults including anti-cancer chemotherapy, thus allowing the cell to survive to lethal conditions. Different functions of HSPs have been described to account for their cytoprotective function, including their role as molecular chaperones as they play a central role in the correct folding of misfolded proteins, but also their anti-apoptotic properties. HSPs are often overexpressed in cancer cells and this constitutive expression is necessary for cancer cells' survival. HSPs may have oncogene-like functions and likewise mediate "non-oncogene addiction" of stressed tumor cells that must adapt to a hostile microenvironment, thereby becoming dependent for their survival on HSPs. HSP-targeting drugs have therefore emerged as potential anti-cancer agents. This review describes the different molecules and approaches being used or proposed in cancer therapy based on the in inhibition of HSP90, HSP70 and HSP27.
Assuntos
Proteínas de Choque Térmico/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Antineoplásicos/farmacologia , Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proliferação de Células , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/terapia , Conformação Proteica , Dobramento de ProteínaRESUMO
RNA polymerase II is an essential nuclear multi subunit enzyme that transcribes nearly the whole genome. Its inhibition by the alpha-amanitin toxin leads to cell death. The enzyme of Plasmodium falciparum remains poorly characterized. Using a complementation assay in yeast as a genetic test, we demonstrate that five Plasmodium putative RNA polymerase subunits are indeed functional in vivo. The active site of this enzyme is built from the two largest subunits. Using site directed mutagenesis we were able to modify the active site of the yeast RNA polymerase II so as to introduce Plasmodium or human structural motifs. The resulting strains allow the screening of chemical libraries for potential specific inhibitors.