RESUMO
Pregnancy success requires a proper fetal maternal interaction at the establishment of implantation. Leptin has been described as a multitasking cytokine in pregnancy, particularly in the placenta, where it acts as an autocrine hormone. The expression of leptin in normal trophoblastic cells is regulated by different endogenous signals. We have previously reported that 17ß-estradiol upregulates placental leptin expression through genomic and non-genomic mechanisms. To improve the knowledge of estrogen receptor mechanisms in regulating leptin gene expression, we examined transcription nuclear factor kappa B (NFκB) effect on estradiol leptin induction in human BeWo cell line and human term placental explants. We demonstrated that estradiol induction effect on leptin expression is blocked by the inhibition of NFκB signaling. We also found that the overexpression of p65 subunit, the active form of NFκB, induces leptin expression. Moreover, downregulation of estrogen receptor alpha (ERα), through a specific siRNA, abolished NFκB effect on leptin expression. We also demonstrated that ERα enhanced NFκB signaling pathway activation in trophoblastic cells. Estradiol treatment significantly increased p65 expression and phosphorylation of the inhibitory protein κB alpha (IκBα). A reporter plasmid containing NFκB elements was also induced in response to estradiol stimulation. Localization experiments revealed that estradiol treatment induced nuclear localization of overexpressed p65. Moreover, the overexpression of ERα produced a complete displacement of p65 protein to the nucleus. Finally, immunoprecipitation experiments showed the presence of a complex containing ERα and NFκB. All these evidences suggest a cooperative behavior between ERα and NFκB transcription factors to induce leptin transcription.
Assuntos
Coriocarcinoma/patologia , Estrogênios/farmacologia , Leptina/metabolismo , NF-kappa B/metabolismo , Placenta/metabolismo , Neoplasias Uterinas/patologia , Núcleo Celular , Coriocarcinoma/genética , Coriocarcinoma/metabolismo , Feminino , Humanos , Leptina/genética , NF-kappa B/genética , Fosforilação , Placenta/efeitos dos fármacos , Gravidez , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismoRESUMO
Immunophilins are a family of intracellular receptors for immunosuppressive drugs. Those immunophilins that are related to immunosuppression are the smallest proteins of the family, i.e., FKBP12 and CyPA, whereas the other members of the family have higher molecular weight because the show additional domains to the drug-binding site. Among these extra domains, the TPR-domain is perhaps the most relevant because it permits the interaction of high molecular weight immunophilins with the 90-kDa heat-shock protein, Hsp90. This essential molecular chaperone regulates the biological function of several protein-kinases, oncogenes, protein phosphatases, transcription factors and cofactors . Hsp90-binding immunophilins where first characterized due to their association with steroid receptors. They regulate the cytoplasmic transport and the subcellular localization of these and other Hsp90 client proteins, as well as transcriptional activity, cell proliferation, cell differentiation and apoptosis. Hsp90-binding immunophilins are frequently overexpressed in several types of cancers and play a key role in cell survival. In this article we analyze the most important biological actions of the best characterized Hsp90-binding immunophilins in both steroid receptor function and cancer development and discuss the potential use of these immunophilins for therapeutic purposes as potential targets of specific small molecules.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Imunofilinas/metabolismo , Neoplasias/metabolismo , Animais , HumanosRESUMO
Hsp90 binding immunophilins FKBP51 and FKBP52 modulate steroid receptor trafficking and hormone-dependent biological responses. With the purpose to expand this model to other nuclear factors that are also subject to nuclear-cytoplasmic shuttling, we analyzed whether these immunophilins modulate NF-κB signaling. It is demonstrated that FKBP51 impairs both the nuclear translocation rate of NF-κB and its transcriptional activity. The inhibitory action of FKBP51 requires neither the peptidylprolyl-isomerase activity of the immunophilin nor its association with Hsp90. The TPR domain of FKBP51 is essential. On the other hand, FKBP52 favors the nuclear retention time of RelA, its association to a DNA consensus binding sequence, and NF-κB transcriptional activity, the latter effect being strongly dependent on the peptidylprolyl-isomerase activity and also on the TPR domain of FKBP52, but its interaction with Hsp90 is not required. In unstimulated cells, FKBP51 forms endogenous complexes with cytoplasmic RelA. Upon cell stimulation with phorbol ester, the NF-κB soluble complex exchanges FKBP51 for FKBP52, and the NF-κB biological effect is triggered. Importantly, FKBP52 is functionally recruited to the promoter region of NF-κB target genes, whereas FKBP51 is released. Competition assays demonstrated that both immunophilins antagonize one another, and binding assays with purified proteins suggest that the association of RelA and immunophilins could be direct. These observations suggest that the biological action of NF-κB in different cell types could be positively regulated by a high FKBP52/FKBP51 expression ratio by favoring NF-κB nuclear retention, recruitment to the promoter regions of target genes, and transcriptional activity.
Assuntos
Proteínas de Ligação a Tacrolimo/fisiologia , Fator de Transcrição RelA/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos , Receptores de Glucocorticoides/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
The term molecular chaperone was first used to describe the ability of nucleoplasmin to prevent the aggregation of histones with DNA during the assembly of nucleosomes. Subsequently, the name was extended to proteins that mediate the post-translational assembly of oligomeric complexes protecting them from denaturation and/or aggregation. Hsp90 is a 90-kDa molecular chaperone that represents the major soluble protein of the cell. In contrast to most conventional chaperones, Hsp90 functions as a refined sensor of protein function and its principal role in the cell is to facilitate biological activity to properly folded client proteins that already have a preserved tertiary structure. Consequently, Hsp90 is related to basic cell functions such as cytoplasmic transport of soluble proteins, translocation of client proteins to organelles, and regulation of the biological activity of key signaling factors such as protein kinases, ubiquitin ligases, steroid receptors, cell cycle regulators, and transcription factors. A growing amount of evidence links the protective action of this molecular chaperone to mechanisms related to posttranslational modifications of soluble nuclear factors as well as histones. In this article, we discuss some aspects of the regulatory action of Hsp90 on transcriptional regulation and how this effect could have impacted genetic assimilation mechanism in some organisms.
Assuntos
Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Choque Térmico HSP90/genética , Humanos , Modelos Genéticos , Chaperonas Moleculares/genética , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
The classic model of action of the glucocorticoid receptor (GR) sustains that its associated heat-shock protein of 90-kDa (HSP90) favours the cytoplasmic retention of the unliganded GR, whereas the binding of steroid triggers the dissociation of HSP90 allowing the passive nuclear accumulation of GR. In recent years, it was described a molecular machinery called transportosome that is responsible for the active retrograde transport of GR. The transportosome heterocomplex includes a dimer of HSP90, the stabilizer co-chaperone p23, and FKBP52 (FK506-binding protein of 52-kDa), an immunophilin that binds dynein/dynactin motor proteins. The model shows that upon steroid binding, FKBP52 is recruited to the GR allowing its active retrograde transport on cytoskeletal tracks. Then, the entire GR heterocomplex translocates through the nuclear pore complex. The HSP90-based heterocomplex is released in the nucleoplasm followed by receptor dimerization. Subsequent findings demonstrated that the transportosome is also responsible for the retrotransport of other soluble proteins. Importantly, the disruption of this molecular oligomer leads to several diseases. In this article, we discuss the relevance of this transport machinery in health and disease.
RESUMO
Immunophilin is the collective name given to a family of proteins that bind immunosuppressive drugs: Some immunophilins are Hsp90-binding cochaperones that affect steroid receptor function. Mood and anxiety disorders are stress-related diseases characterized by an impaired function of the mineralocorticoid and glucocorticoid receptors, two of the major regulatory elements of the hypothalamus-pituitary-adrenocortical axis. Genetic variations of the FK506-binding protein of 51-kDa, FKBP51, one of the immunophilins bound to those steroid receptor complexes, were associated with the effectiveness of treatments against depression and with a major risk-factor for the development of post-traumatic stress disorders. Interestingly, immunophilins show polymorphisms and some polymorphic isoforms of FKBP51 correlate with a greater impairment of steroid receptor functions. In this review, we discuss different aspects of the role of FKBP51 in such steroid receptor function and the impact of genetic variants of the immunophilin on the dysregulation of the stress response.
Assuntos
Transtornos de Ansiedade/metabolismo , Glucocorticoides/metabolismo , Transtornos do Humor/metabolismo , Receptores de Esteroides/metabolismo , Transtornos de Estresse Pós-Traumáticos/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Transtornos de Ansiedade/genética , Humanos , Transtornos do Humor/genética , Peptidilprolil Isomerase/metabolismo , Transtornos de Estresse Pós-Traumáticos/genéticaRESUMO
A dimer of the heat-shock protein of 90-kDa (Hsp90) represents the critical core of the chaperone complex associated to the glucocorticoid receptor (GR) oligomer. The C-terminal end of the Hsp90 dimer shapes a functional acceptor site for co-chaperones carrying tetratricopeptide repeat (TPR) domains, where they bind in a mutually exclusive and competitive manner. They impact on the biological properties of the GRâ¢Hsp90 complex and are major players of the GR transport machinery. Recently, we showed that the overexpression of a chimeric TPR peptide influences the subcellular distribution of GR. In this study, the functional role of endogenous proteins carrying TPR or TPR-like sequences on GR subcellular distribution was characterized. It is demonstrated that, contrarily to the positive influence of FKBP52 on GR nuclear accumulation, FKBP51 and 14-3-3 impaired this property. While SGT1α showed no significant effect, the overexpression of the Ser/Thr phosphatase PP5 resulted in a nearly equal nuclear-cytoplasmic redistribution of GR rather than its typical cytoplasmic localization in the absence of steroid. This observation led to analyse the influence of the phosphorylation status of GR, which resulted not linked to its nucleo-cytoplasmic shuttling mechanism. Nonetheless, it was evidenced that both PP5 and FKBP52 are related to the anchorage of the GR to nucleoskeleton structures. The influence of these TPR domain proteins on the steroid-dependent transcriptional activity of GR was also characterized. It is postulated that the pleiotropic actions of the GR in different cell types may be the consequence of the relative abundance of different TPR-domain interacting co-chaperones.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Receptores de Glucocorticoides/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Receptores de Glucocorticoides/genética , Repetições de TetratricopeptídeosRESUMO
Immunophilins are a family of proteins whose signature domain is the peptidylprolyl-isomerase domain. High molecular weight immunophilins are characterized by the additional presence of tetratricopeptide-repeats (TPR) through which they bind to the 90-kDa heat-shock protein (Hsp90), and via this chaperone, immunophilins contribute to the regulation of the biological functions of several client-proteins. Among these Hsp90-binding immunophilins, there are two highly homologous members named FKBP51 and FKBP52 (FK506-binding protein of 51-kDa and 52-kDa, respectively) that were first characterized as components of the Hsp90-based heterocomplex associated to steroid receptors. Afterwards, they emerged as likely contributors to a variety of other hormone-dependent diseases, stress-related pathologies, psychiatric disorders, cancer, and other syndromes characterized by misfolded proteins. The differential biological actions of these immunophilins have been assigned to the structurally similar, but functionally divergent enzymatic domain. Nonetheless, they also require the complementary input of the TPR domain, most likely due to their dependence with the association to Hsp90 as a functional unit. FKBP51 and FKBP52 regulate a variety of biological processes such as steroid receptor action, transcriptional activity, protein conformation, protein trafficking, cell differentiation, apoptosis, cancer progression, telomerase activity, cytoskeleton architecture, etc. In this article we discuss the biology of these events and some mechanistic aspects.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Imunofilinas/metabolismo , Animais , Proteínas de Choque Térmico HSP90/química , Humanos , Imunofilinas/química , Modelos Moleculares , Estrutura Molecular , Ligação ProteicaRESUMO
Hexameric procyanidins inhibit TNFalpha-induced NF-kappaB activation in Caco-2 cells. Most of the physiological actions of high molecular weight procyanidins could be limited to the gut lumen. Transcription factor NF-kappaB plays a central role in inflammation including human intestinal bowel disease. We investigated the capacity of a hexameric procyanidin fraction (Hex) to prevent tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation as related to oxidation and membrane interactions. In Caco-2 cells, Hex (2.5-20 microM) inhibited TNFalpha-induced NF-kappaB activation (IkappaB phosphorylation and degradation, p50 and RelA nuclear translocation, and NF-kappaB-DNA binding), inducible nitric oxide synthase expression, and cell oxidant increase. The effects on NF-kappaB activation persist beyond the period of direct exposition of cells to Hex. N-Acetylcysteine and alpha-lipoic acid inhibited TNFalpha-induced oxidant increase but did not affect NF-kappaB activation. In summary, Hex can inhibit NF-kappaB activation by interacting with the plasma membrane of intestinal cells, and through these interactions preferentially inhibits the binding of TNFalpha to its receptor and the subsequent NF-kappaB activation.
Assuntos
NF-kappa B/antagonistas & inibidores , Oxidantes/antagonistas & inibidores , Proantocianidinas/química , Proantocianidinas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Células CACO-2 , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Quinase I-kappa B/antagonistas & inibidores , Mucosa Intestinal/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Oxirredução , Fosforilação/efeitos dos fármacos , Proantocianidinas/metabolismoRESUMO
Procyanidins can exert cytoprotective, anti-inflammatory, and anticarcinogenic actions in the gastrointestinal tract. Previous evidence has shown that procyanidins can interact with synthetic membranes and protect them from oxidation and disruption. Thus, in this study we investigated the capacity of a hexameric procyanidin fraction (Hex) isolated from cocoa to protect Caco-2 cells from deoxycholic (DOC)-induced cytotoxicity, cell oxidant increase, and loss of monolayer integrity. Hex interacted with the cell membranes without affecting their integrity, as evidenced by a Hex-mediated increase in the transepithelial electrical resistance, and inhibition of DOC-induced cytotoxicity. DOC induced an increase in cell oxidants, alterations in the paracellular transport, and redistribution of the protein ZO-1 from cell-cell contacts into the cytoplasm. Hex partially inhibited all these events at concentrations ranging from 2.5 to 20 microM. Similarly, Hex (5-10 microM) inhibited the increase in cell oxidants, and the loss of integrity of polarized Caco-2 cell monolayers induced by a lipophilic oxidant (2,2'-azobis (2,4-dimethylvaleronitrile). Results show that the assayed procyanidin fraction can interact with cell membranes and protect Caco-2 cells from DOC-induced cytotoxicity, oxidant generation, and loss of monolayer integrity. At the gastrointestinal tract, large procyanidins may exert beneficial effects in pathologies such us inflammatory diseases, alterations in intestinal barrier permeability, and cancer.
Assuntos
Ácidos e Sais Biliares/toxicidade , Oxidantes/toxicidade , Proantocianidinas/farmacologia , Compostos Azo/toxicidade , Ácidos e Sais Biliares/metabolismo , Células CACO-2 , Catequina/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ácido Desoxicólico/toxicidade , Humanos , Nitrilas/toxicidade , Oxidantes/metabolismo , Proantocianidinas/químicaRESUMO
This work investigated the capacity of alpha-lipoic acid (LA) and N-acetyl-L-cysteine (NAC) to reduce zinc deficiency-induced oxidative stress, and prevent the activation of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), and the cross-talk between both activated cascades through beta-Transducin Repeat-containing Protein (beta-TrCP). IMR-32 cells were incubated in control media or media containing variable concentrations of zinc, without or with 0.5 mM LA or 1 mM NAC. Relative to control and zinc supplemented (15 microM Zn) groups, Hydrogen peroxide (H(2)O(2)) and total oxidant cell concentrations were higher, and total glutathione concentrations were lower in the zinc deficient groups (1.5 and 5 microM Zn). Both, LA and NAC, markedly reduced the increase in cell oxidants and the reduction in glutathione concentrations in the zinc deficient cells. Consistent with this, LA and NAC prevented zinc deficiency-induced activation of the early steps of NF- kappaB (IkappaBalpha phosphorylation) and AP-1 [c-Jun-N-terminal kinase (JNK) and p38 phophorylation] cascades, and the high NF-kappaB- and AP-1-DNA binding activities in total cell extracts. Thus, LA and NAC can reduce the oxidative stress associated with zinc deficiency and the subsequent triggering of NF-kappaB- and AP-1-activation in neuronal cells.
Assuntos
Acetilcisteína/farmacologia , NF-kappa B/metabolismo , Ácido Tióctico/farmacologia , Fator de Transcrição AP-1/metabolismo , Zinco/deficiência , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neuroblastoma/metabolismo , Oxidantes/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fosforilação/efeitos dos fármacos , Fator de Transcrição AP-1/antagonistas & inibidores , Zinco/farmacologiaRESUMO
The fine regulation of signalling cascades is a key event required to maintain the appropriate functional properties of a cell when a given stimulus triggers specific biological responses. In this sense, cumulative experimental evidence during the last years has shown that high molecular weight immunophilins possess a fundamental importance in the regulation of many of these processes. It was first discovered that TPR-domain immunophilins such as FKBP51 and FKBP52 play a cardinal role, usually in an antagonistic fashion, in the regulation of several members of the steroid receptor family via its interaction with the heat-shock protein of 90-kDa, Hsp90. These Hsp90-associated cochaperones form a functional unit with the molecular chaperone influencing ligand binding capacity, receptor trafficking, and hormone-dependent transcriptional activity. Recently, it was demonstrated that the same immunophilins are also able to regulate the NF-kB signalling cascade in an Hsp90 independent manner. In this article we analize these properties and discuss the relevance of this novel regulatory pathway in the context of the pleiotropic actions managed by NF-kB in several cell types and tissues.
Assuntos
NF-kappa B/metabolismo , Transdução de Sinais , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , NF-kappa B/química , Conformação Proteica , Proteínas de Ligação a Tacrolimo/químicaRESUMO
Immunophilins comprise a family of intracellular proteins with peptidyl-prolyl-(cis/trans)-isomerase activity. These foldases are abundant, ubiquitous, and able to bind immunosuppressant drugs, from which the term immunophilin derives. Family members are found in abundance in virtually all organisms and subcellular compartments, and their amino acid sequences are conserved phylogenetically. Immunophilins possess the ability to function as molecular chaperones favoring the proper folding and biological regulation of their biological actions. Their ability to interact via their TPR domains with the 90-kDa heat-shock protein, and through this chaperone, with several signalling cascade factors is of particular importance. Among the family members, the highly homologous proteins FKBP51 and FKBP52 were first characterized due to their ability to interact with steroid hormone receptors. Since then, much progress has been made in understanding the mechanisms by which they regulate receptor signaling and the resulting roles they play not only in endocrine processes, but also in cell architecture, neurodifferentiation, and tumor progression. In this article we review the most relevant features of these two immunophilins and their potential as pharmacologic targets.
Assuntos
Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/metabolismo , Humanos , Dobramento de Proteína , Estrutura Terciária de Proteína , Receptores de Esteroides/metabolismoRESUMO
Immunophilins are proteins that contain a PPIase domain as a family signature. Low-molecular-weight immunophilins were first described associated to immunosuppressive action and protein folding. Recent studies of other members of the family have led to the identification of their participation in basic processes such as protein-protein interactions, signal transduction cascades, cell differentiation, cell cycle progression, metabolic activity, apoptosis mechanisms, microorganisms infection, cancer, neurotrophism and neuroprotection, among several other physiological and pathophysiological processes. Due to all these emerging features, the development of specific ligands for immunophilins appears to have promising perspectives, in particular in the fields of cancer biology and neuroregeneration fields. We review the emerging role of immunophilins in protein transport, transcription regulation, malignancies development and neurotrophic action, in addition to a number of biological properties that transform these proteins in potential targets for novel therapeutic strategies.
Assuntos
Descoberta de Drogas , Proteínas de Choque Térmico HSP90/metabolismo , Imunofilinas/metabolismo , Animais , Descoberta de Drogas/métodos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neurogênese , Ligação Proteica , Transporte Proteico , Ativação TranscricionalRESUMO
The most relevant biological action of aldosterone in epithelial tissues is the regulation of sodium reabsorption through binding to the mineralocorticoid receptor (MR). Glucocorticoids also bind with high affinity to MR, which is usually protected by the enzyme 11ß-hydroxysteroid dehydrogenase. This activity prevents MR activation by cortisol despite the large prevalence of this steroid in plasma. Nonetheless, there are some aspects of the mechanism of action of MR that are not entirely explained by this competitive metabolic mechanism of protection. The picture is even more complicated in those tissues such as the nervous system where the enzyme is expressed at very low levels or is directly absent in various areas of the brain. Therefore, other cellular and molecular mechanisms must also intervene to allow specific aldosterone biological effects in the presence of overwhelming concentrations of glucocorticoids. In this article, we discuss some possible mechanisms that permit the specificity of action for each type of steroid, including those related to the recently discovered novel molecular mechanism of activation of corticosteroid receptors and the structural requirements of a given ligand to favor the mineralocorticoid action via MR. The relative contribution of these mechanisms may vary in different target cells allowing the fine tuning of cellular functions depending on the degree of cooperation between steroids, receptors, chaperones associated to receptors, and other factors. All these regulatory interactions can be altered in some pathophysiological situations, most of them related to stressing situations.
Assuntos
Sistema Nervoso/química , Receptores de Mineralocorticoides/fisiologia , Estresse Psicológico/metabolismo , Aldosterona/farmacologia , Aldosterona/fisiologia , Animais , Glucocorticoides/farmacologia , Glucocorticoides/fisiologia , Humanos , Sistema Nervoso/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores de Mineralocorticoides/agonistas , Transdução de Sinais/fisiologia , Estresse Psicológico/patologiaRESUMO
Procyanidins are oligomers of flavanol subunits present in large amounts in fruits and vegetables. Their consumption is associated with health benefits against colonic inflammation and colorectal cancer (CRC). Large procyanidins (with more than three subunits) are not absorbed by intestinal epithelial cells but could exert biological actions through their interactions with the cell membrane. This study investigated the capacity of hexameric procyanidins (Hex) to prevent oncogenic events initiated by deoxycholic acid (DCA), a secondary bile acid linked to the promotion of CRC. Hex interacted with Caco-2 cell membranes preferentially at the water-lipid interface. Hex (2.5-20 µM) inhibited DCA-triggered increase in cellular calcium, NADPH oxidase activation, and oxidant production. DCA promoted the activation of protein kinase B (Akt), of the mitogen-activated protein kinases ERK1/2 and p38, and of the downstream transcription factor AP-1. This activation was not triggered by calcium or oxidant increases. Hex caused a dose-dependent inhibition of DCA-mediated activation of all these signals. DCA also triggered alterations in the cell monolayer morphology and apoptotic cell death, events that were delayed by Hex. The capacity of large procyanidins to interact with the cell membrane and prevent those cell membrane-associated events can in part explain the beneficial effects of procyanidins on CRC.
Assuntos
Antioxidantes/farmacologia , Membrana Celular/efeitos dos fármacos , Neoplasias Colorretais/prevenção & controle , Ácido Desoxicólico/efeitos adversos , Proantocianidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Bile/química , Células CACO-2 , Cálcio/metabolismo , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Humanos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Oxidantes/efeitos adversos , Polimerização , Proantocianidinas/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Cytoskeletal structure is continually remodeled to accommodate normal cell growth and to respond to pathophysiological cues. As a consequence, several cytoskeleton-interacting proteins become involved in a variety of cellular processes such as cell growth and division, cell movement, vesicle transportation, cellular organelle location and function, localization and distribution of membrane receptors, and cell-cell communication. Molecular chaperones and immunophilins are counted among the most important proteins that interact closely with the cytoskeleton network, in particular with microtubules and microtubule-associated factors. In several situations, heat-shock proteins and immunophilins work together as a functionally active heterocomplex, although both types of proteins also show independent actions. In circumstances where homeostasis is affected by environmental stresses or due to genetic alterations, chaperone proteins help to stabilize the system. Molecular chaperones facilitate the assembly, disassembly and/or folding/refolding of cytoskeletal proteins, so they prevent aberrant protein aggregation. Nonetheless, the roles of heat-shock proteins and immunophilins are not only limited to solve abnormal situations, but they also have an active participation during the normal differentiation process of the cell and are key factors for many structural and functional rearrangements during this course of action. Cytoskeleton modifications leading to altered localization of nuclear factors may result in loss- or gain-of-function of such factors, which affects the cell cycle and cell development. Therefore, cytoskeletal components are attractive therapeutic targets, particularly microtubules, to prevent pathological situations such as rapidly dividing tumor cells or to favor the process of cell differentiation in other cases. In this review we will address some classical and novel aspects of key regulatory functions of heat-shock proteins and immunophilins as housekeeping factors of the cytoskeletal network.
Assuntos
Citoesqueleto/metabolismo , Imunofilinas/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Diferenciação Celular , Glicoproteínas/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Complexos Multiproteicos/metabolismo , Neurônios/fisiologia , Ligação Proteica , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas tau/metabolismoRESUMO
In the absence of hormone, corticosteroid receptors such as GR (glucocorticoid receptor) and (mineralocorticoid receptor) are primarily located in the cytoplasm. Upon steroid-binding, they rapidly accumulate in the nucleus. Regardless of their primary location, these receptors and many other nuclear factors undergo a constant and dynamic nucleocytoplasmic shuttling. All members of the steroid receptor family are known to form large oligomeric structures with the heat-shock proteins of 90-kDa (hsp90) and 70-kDa (hsp70), the small acidic protein p23, and a tetratricopeptide repeat (TPR) -domain protein such as FK506-binding proteins (FKBPs), cyclophilins (CyPs) or the serine/threonine protein phosphatase 5 (PP5). It has always been stated that the dissociation of the chaperone heterocomplex (a process normally referred to as receptor "transformation") is the first step that permits the nuclear import of steroid receptors. However the experimental evidence is consistent with a model where the chaperone machinery is required for the retrotransport of the receptor through the cytoplasm and also facilitates the passage through the nuclear pore. Recent evidence indicates that the hsp90-based chaperone system also interacts with structures of the nuclear pore such as importin ß and the integral nuclear pore glycoprotein Nup62 facilitating the passage of the untransformed receptor through the nuclear pore.
Assuntos
Núcleo Celular/metabolismo , Imunofilinas/metabolismo , Chaperonas Moleculares/metabolismo , Poro Nuclear/metabolismo , Receptores de Esteroides/metabolismo , Transporte Ativo do Núcleo Celular , Ciclofilinas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Modelos Moleculares , Chaperonas Moleculares/química , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Receptores de Esteroides/química , Proteínas de Ligação a Tacrolimo/metabolismo , beta Carioferinas/metabolismoRESUMO
In this study, we demonstrate that the subcellular localization of the mineralocorticoid receptor (MR) is regulated by tetratricopeptide domain (TPR) proteins. The high-molecular-weight immunophilin (IMM) FKBP52 links the MR-hsp90 complex to dynein/dynactin motors favoring the cytoplasmic transport of MR to the nucleus. Replacement of this hsp90-binding IMM by FKBP51 or the TPR peptide favored the cytoplasmic localization of MR. The complete movement machinery, including dynein and tubulin, could be recovered from paclitaxel/GTP-stabilized cytosol and was fully reassembled on stripped MR immune pellets. The whole MR-hsp90-based heterocomplex was transiently recovered in the soluble fraction of the nucleus after 10 min of incubation with aldosterone. Moreover, cross-linked MR-hsp90 heterocomplexes accumulated in the nucleus in a hormone-dependent manner, demonstrating that the heterocomplex can pass undissociated through the nuclear pore. On the other hand, a peptide that comprises the DNA-binding domain of MR impaired the nuclear export of MR, suggesting the involvement of this domain in the process. This study represents the first report describing the entire molecular system that commands MR nucleocytoplasmic trafficking and proposes that the MR-hsp90-TPR protein heterocomplex is dissociated in the nucleus rather than in the cytoplasm.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Motores Moleculares/metabolismo , Receptores de Mineralocorticoides/metabolismo , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Dineínas/química , Dineínas/metabolismo , Humanos , Imunofilinas/química , Imunofilinas/metabolismo , Camundongos , Microtúbulos/metabolismo , Proteínas Motores Moleculares/química , Complexos Multiproteicos , Células NIH 3T3 , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Ratos , Receptores de Mineralocorticoides/química , Receptores de Mineralocorticoides/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Ligação a Tacrolimo/deficiência , Proteínas de Ligação a Tacrolimo/genéticaRESUMO
Glucocorticoid receptor (GR) is cytoplasmic in the absence of ligand and localizes to the nucleus after steroid binding. Previous evidence demonstrated that the hsp90-based heterocomplex bound to GR is required for the efficient retrotransport of the receptor to the nuclear compartment. We examined the putative association of GR and its associated chaperone heterocomplex with structures of the nuclear pore. We found that importin beta and the integral nuclear pore glycoprotein Nup62 interact with hsp90, hsp70, p23, and the TPR domain proteins FKBP52 and PP5. Nup62 and GR were able to interact in a more efficient manner when chaperoned by the hsp90-based heterocomplex. Interestingly, the binding of hsp70 and p23 to Nup62 does not require the presence of hsp90, whereas the association of FKBP52 and PP5 is hsp90 dependent, as indicated by the results of experiments where the hsp90 function was disrupted with radicicol. The ability of both FKBP52 and PP5 to interact with Nup62 was abrogated in cells overexpressing the TPR peptide. Importantly, GR cross-linked to the hsp90 heterocomplex was able to translocate to the nucleus in digitonin-permeabilized cells treated with steroid, suggesting that GR could pass through the pore in its untransformed state.