RESUMO
A cycle of palmitoylation/depalmitoylation of H-Ras mediates bidirectional trafficking between the Golgi apparatus and the plasma membrane, but nothing is known about how this cycle is regulated. We show that the prolyl isomerase (PI) FKBP12 binds to H-Ras in a palmitoylation-dependent fashion and promotes depalmitoylation. A variety of inhibitors of the PI activity of FKBP12, including FK506, rapamycin, and cycloheximide, increase steady-state palmitoylation. FK506 inhibits retrograde trafficking of H-Ras from the plasma membrane to the Golgi in a proline 179-dependent fashion, augments early GTP loading of Ras in response to growth factors, and promotes H-Ras-dependent neurite outgrowth from PC12 cells. These data demonstrate that FKBP12 regulates H-Ras trafficking by promoting depalmitoylation through cis-trans isomerization of a peptidyl-prolyl bond in proximity to the palmitoylated cysteines.
Assuntos
Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína 1A de Ligação a Tacrolimo/fisiologia , Acilação , Animais , Lipoilação , Células PC12 , Transporte Proteico , Proteínas Proto-Oncogênicas p21(ras)/química , Ratos , Transdução de Sinais , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
Tacrolimus is a widely used immunosuppressive drug that inhibits the phosphatase calcineurin when bound to the 12 kDa FK506-binding protein (FKBP12). When this binding occurs in T cells, it leads to immunosuppression. Tacrolimus also causes side effects, however, such as hypertension and hyperkalemia. Previously, we reported that tacrolimus stimulates the renal thiazide-sensitive sodium chloride cotransporter (NCC), which is necessary for the development of hypertension. However, it was unclear if tacrolimus-induced hypertension resulted from tacrolimus effects in renal epithelial cells directly or in extrarenal tissues, and whether inhibition of calcineurin was required. To address these questions, we developed a mouse model in which FKBP12 could be deleted along the nephron. FKBP12 disruption alone did not cause phenotypic effects. When treated with tacrolimus, however, BP and the renal abundance of phosphorylated NCC were lower in mice lacking FKBP12 along the nephron than in control mice. Mice lacking FKBP12 along the nephron also maintained a normal relationship between plasma potassium levels and the abundance of phosphorylated NCC with tacrolimus treatment. In cultured cells, tacrolimus inhibited dephosphorylation of NCC. Together, these results suggest that tacrolimus causes hypertension predominantly by inhibiting calcineurin directly in cells expressing NCC, indicating thiazide diuretics may be particularly effective for lowering BP in tacrolimus-treated patients with hypertension.
Assuntos
Hipertensão/induzido quimicamente , Hipertensão/prevenção & controle , Imunossupressores/efeitos adversos , Proteína 1A de Ligação a Tacrolimo/fisiologia , Tacrolimo/efeitos adversos , Animais , Deleção de Genes , Rim , Masculino , Camundongos , Proteína 1A de Ligação a Tacrolimo/genéticaRESUMO
Genetic analysis of protein function requires a rapid means of inactivating the gene under study. Typically, this exploits temperature-sensitive mutations or promoter shut-off techniques. We report the adaptation to Schizosaccharomyces pombe of the anchor-away technique, originally designed in budding yeast by Laemmli lab. This method relies on a rapamycin-mediated interaction between the FRB- and FKBP12-binding domains to relocalize nuclear proteins of interest to the cytoplasm. We demonstrate a rapid nuclear depletion of abundant proteins as proof of principle.
Assuntos
Componente 4 do Complexo de Manutenção de Minicromossomo/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/fisiologia , Sirolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo/fisiologia , Cruzamentos Genéticos , DNA Fúngico/química , DNA Fúngico/genética , Microscopia de Fluorescência , Plasmídeos , Reação em Cadeia da Polimerase , Transformação GenéticaRESUMO
FK506 binding protein 12.6 kDa (FKBP12.6), a protein that regulates ryanodine Ca(2+) release channels, may act as an important regulator of insulin secretion. In this study, the role of FKBP12.6 in the control of insulin secretion and blood glucose is clarified using FKBP12.6(-/-) mice. FKBP12.6(-/-) mice showed significant fed hyperinsulinemia but exhibited normoglycemia, fasting normoinsulinemia, and normal body weight compared with wild-type (WT) littermate control mice. Deletion of FKBP12.6 resulted in enhanced glucose-stimulated insulin secretion (GSIS) both in vivo and in vitro, a result that is due to enhanced glucose-induced islet Ca(2+) elevation. After a high-fat dietary challenge (HF diet) for 3 mo, FKBP12.6(-/-) mice displayed higher body weight, hyperinsulinemia, and lower fed blood glucose concentrations compared with WT mice. FKBP12.6(-/-) mice displayed hyperinsulinemia, and resistance to HF diet-induced hyperglycemia, suggesting that FKBP12.6 plays an important role in insulin secretion and blood glucose control, and raising the possibility that it may be a potential therapeutic target for the treatment of type 2 diabetes.
Assuntos
Glicemia/metabolismo , Gorduras na Dieta/efeitos adversos , Hiperglicemia/metabolismo , Hiperinsulinismo/etiologia , Insulina/metabolismo , Proteína 1A de Ligação a Tacrolimo/deficiência , Animais , Hiperglicemia/prevenção & controle , Resistência à Insulina/fisiologia , Secreção de Insulina , Camundongos , Camundongos Knockout , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/fisiologiaRESUMO
The discovery of rapamycin from a soil sample on Easter Island in the mid 60's marked the beginning of an exciting field of research in cell biology and medicine. While it was first used as an antifungal and as an immunosuppressive drug, more recent studies confirmed rapamycin's antiproliferative properties over a variety of solid tumors. Research aimed at identifying its mechanism of action uncovered mTOR (mammalian target of rapamycin), a protein kinase that regulates mRNA translation and protein synthesis, an essential step in cell division and proliferation. Recent evidence suggests a more complex role for mTOR in the regulation of several growth factor-stimulated protein kinases, including the proto-oncogene Akt. This article reviews mTOR function and regulation, and briefly details the future challenges for anti-cancer therapies based on mTOR inhibition.
Assuntos
Serina-Treonina Quinases TOR/fisiologia , Animais , Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Autofagia/fisiologia , Humanos , Imunossupressores/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Estrutura Molecular , Complexos Multiproteicos/fisiologia , Fosforilação , Biossíntese de Proteínas/fisiologia , Proteínas Quinases/fisiologia , Processamento de Proteína Pós-Traducional , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-akt/fisiologia , RNA Mensageiro/genética , Ribossomos/fisiologia , Sirolimo/química , Sirolimo/isolamento & purificação , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteína 1A de Ligação a Tacrolimo/fisiologia , Fatores de Transcrição/fisiologiaRESUMO
The functional characterization of the FKBP12 encoding gene from the phytopathogenic fungus Botrytis cinerea was carried out. B. cinerea genome sequence owns a single ortholog, named BcFKBP12, encoding a FK506-binding protein of 12kDa. BcFKBP12 mediates rapamycin sensitivity both in B. cinerea and in Saccharomyces cerevisiae, a property unique to FKBP12 proteins, probably via the inhibition of the protein kinase TOR (target of rapamycin). The relative abundance of the prolyl isomerase appeared to be regulated and increased in response to the presence of extracellular nutrients. Surprisingly, the BcFKBP12 deletion did not affect the pathogenic development of the strain B05.10, while it was reported to cause a reduction of the virulence of the strain T4. We report for the first time the BcFKBP12 involvement in the sulfur repression of the synthesis of a secreted serine protease. Rapamycin treatment did not relieve the sulfur repression of the reporter system in the wild-type strain. Thus BcFKBP12 may participate in sulfur regulation and its contribution seems to be independent of TOR.
Assuntos
Botrytis/fisiologia , Botrytis/patogenicidade , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica , Enxofre/metabolismo , Proteína 1A de Ligação a Tacrolimo/fisiologia , Antifúngicos/farmacologia , DNA Fúngico/genética , Deleção de Genes , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Serina Endopeptidases/biossíntese , Sirolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo/genéticaRESUMO
Various substitution mutations in ALK2, a transmembrane serine/threonine kinase receptor for bone morphogenetic proteins (BMPs), have been identified in patients with genetic disorders such as fibrodysplasia ossificans progressiva (FOP), diffuse intrinsic pontine glioma (DIPG) and heart defects. In this study, we characterized the ALK2 mutants R258G, G328V and F246Y, which were identified in patients with severe FOP, DIPG and unusual hereditary skeletal dysplasia, respectively. Both R258G and G328V were gain-of-function mutations, but F246Y was equivalent to wild-type ALK2. We also examined the effect of the suppressor FKBP12 on the signal transduction of a further 14 ALK2 mutations associated with FOP and/or DIPG. To varying extents FKBP12 over-expression suppressed the basal signaling induced by thirteen of the ALK2 mutants, whereas PF197-8L was uniquely resistant. In the PF197-8L mutant, the modelled ALK2 residue L197 induced a steric clash with the D36 residue in FKBP12 and dissociated their interaction. The co-expression of BMP type II receptors or stimulation with ligands relieved the suppression by FKBP12 by disrupting the interaction between mutant ALK2 and FKBP12. Taken together, FKBP12 binds to and suppresses mutant ALK2 proteins associated with FOP and DIPG, except for PF197-8L.
Assuntos
Receptores de Ativinas Tipo I/genética , Doenças do Desenvolvimento Ósseo/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/fisiologia , Neoplasias do Tronco Encefálico/genética , Glioma/genética , Miosite Ossificante/genética , Proteína 1A de Ligação a Tacrolimo/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Humanos , Camundongos , Miosite Ossificante/patologia , Ossificação Heterotópica/genética , Transdução de SinaisRESUMO
The ascomycete Botrytis cinerea is a broad-spectrum plant pathogen. Here, we describe the first macroarray transcriptomic study of the fungus in real-time infection conditions. Infection of Arabidopsis thaliana leaves by B.cinerea was monitored using macroarrays, containing 3032 genes. Variance analysis revealed that 7% of B.cinerea genes are differentially expressed during infection and allowed us to identify 27 genes significantly up-regulated in planta. Among them, two genes have already been associated with fungal pathogenicity, while eight genes have unidentified functions. The 27 genes were separated into three groups according to their expression profile. The first group showed maximal expression at the early stage following fungal penetration, the second one showed maximal expression at the outset of the colonization of plant leaves and the third group showed maximal expression when the colonization of plant leaves was completed. A gene of the last group (BcPIC5), which is homologous to FKBP12 proteins, was disrupted in order to determine its role in pathogenicity. At seven days post-inoculation, the lesions caused by the DeltaBcPIC5 mutant on bean leaves were reduced by 69% and did not further expand compared to the wild-type. These results confirm that transcriptomic analysis under infection conditions can be very valuable for the identification of fungal genes related to pathogenicity.
Assuntos
Arabidopsis/microbiologia , Botrytis/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/fisiologia , Folhas de Planta/microbiologia , Proteína 1A de Ligação a Tacrolimo/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Botrytis/patogenicidade , Marcação de Genes , Dados de Sequência Molecular , Mutação , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteína 1A de Ligação a Tacrolimo/genética , Transcrição Gênica , Regulação para CimaRESUMO
Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease (PD). We have used fluorescence correlation spectroscopy (FCS) to study alpha-SYN aggregation in vitro and discovered that this process is clearly accelerated by addition of FK506 binding proteins (FKBPs). This effect was observed both with E. coli SlyD FKBP and with human FKBP12 and was counteracted by FK506, a specific inhibitor of FKBP. The alpha-SYN aggregates formed in the presence of FKBP12 showed fibrillar morphology. The rotamase activity of FKBP apparently accelerates the folding and subsequent aggregation of alpha-SYN. Since FK506 and other non-immunosuppressive FKBP inhibitors are known to display neuroregenerative and neuroprotective properties in disease models, the observed inhibition of rotamase activity and alpha-SYN aggregation, may explain their mode of action. Our results open perspectives for the treatment of PD with immunophilin ligands that inhibit a specific member of the FKBP family.
Assuntos
Proteínas de Escherichia coli/farmacologia , Peptidilprolil Isomerase/farmacologia , Proteína 1A de Ligação a Tacrolimo/farmacologia , alfa-Sinucleína/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/fisiologia , Humanos , Microscopia Eletrônica , Nefelometria e Turbidimetria , Peptidilprolil Isomerase/isolamento & purificação , Peptidilprolil Isomerase/fisiologia , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Espectrometria de Fluorescência , Tacrolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo/fisiologia , alfa-Sinucleína/efeitos dos fármacos , alfa-Sinucleína/genética , alfa-Sinucleína/ultraestruturaRESUMO
Although signaling from phosphatidylinositol 3-kinase (PI3K) and AKT to mechanistic target of rapamycin (mTOR) is prominently dysregulated in high-grade glial brain tumors, blockade of PI3K or AKT minimally affects downstream mTOR activity in glioma. Allosteric mTOR inhibitors, such as rapamycin, incompletely block mTORC1 compared with mTOR kinase inhibitors (TORKi). Here, we compared RapaLink-1, a TORKi linked to rapamycin, with earlier-generation mTOR inhibitors. Compared with rapamycin and Rapalink-1, TORKi showed poor durability. RapaLink-1 associated with FKBP12, an abundant mTOR-interacting protein, enabling accumulation of RapaLink-1. RapaLink-1 showed better efficacy than rapamycin or TORKi, potently blocking cancer-derived, activating mutants of mTOR. Our study re-establishes mTOR as a central target in glioma and traces the failure of existing drugs to incomplete/nondurable inhibition of mTORC1.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Complexos Multiproteicos/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos BALB C , Sirolimo/uso terapêutico , Proteína 1A de Ligação a Tacrolimo/fisiologiaRESUMO
Multiple intracellular receptors of the FK506 binding protein (FKBP) family of peptidylprolyl cis/trans-isomerases are potential targets for the immunosuppressive drug FK506. Inhibition of the protein phosphatase calcineurin (CaN), which has been implicated in the FK506-mediated blockade of T cell proliferation, was shown to involve a gain of function in the FKBP12/FK506 complex. We studied the potential of six human FKBPs to contribute to CaN inhibition by comparative examination of inhibition constants of the respective FK506/FKBP complexes. Interestingly, these FKBPs form tight complexes with FK506, exhibiting comparable dissociation constants, but the resulting FK506/FKBP complexes differ greatly in their affinity for CaN, with IC50 values in the range of 0.047-17 microM. The different capacities of FK506/FKBP complexes to affect CaN activity are partially caused by substitutions corresponding to the amino acid side chains K34 and I90 of FKBP12. Only the FK506 complexes of FKBP12, FKBP12.6, and FKBP51 showed high affinity to CaN; small interfering RNA against these FKBP allowed defining the contribution of individual FKBP in an NFAT reporter gene assay. Our results allow quantitative correlation between FK506-mediated CaN effects and the abundance of the different FKBPs in the cell.
Assuntos
Inibidores de Calcineurina , Inibidores Enzimáticos/metabolismo , Imunossupressores/farmacologia , Complexos Multienzimáticos/metabolismo , Proteínas de Ligação a Tacrolimo/fisiologia , Sequência de Aminoácidos , Animais , Calcineurina/metabolismo , Calmodulina/fisiologia , Bovinos , Inibidores Enzimáticos/farmacologia , Humanos , Imunossupressores/metabolismo , Células Jurkat , Dados de Sequência Molecular , Complexos Multienzimáticos/fisiologia , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Sirolimo/metabolismo , Sirolimo/farmacologia , Especificidade por Substrato , Tacrolimo/metabolismo , Tacrolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo/fisiologia , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Peptide bond isomerases are involved in important physiological processes that can be targeted in order to treat neurodegenerative disease, cancer, diseases of the immune system, allergies, and many others. The folding helper enzyme class of Peptidyl-Prolyl-cis/trans Isomerases (PPIases) contains the three enzyme families of cyclophilins (Cyps), FK506 binding proteins (FKBPs), and parvulins (Pars). Although they are structurally unrelated, all PPIases catalyze the cis/trans isomerization of the peptide bond preceding the proline in a polypeptide chain. This process not only plays an important role in de novo protein folding, but also in isomerization of native proteins. The native state isomerization plays a role in physiological processes by influencing receptor ligand recognition or isomer-specific enzyme reaction or by regulating protein function by catalyzing the switch between native isomers differing in their activity, e.g., ion channel regulation. Therefore elucidating PPIase involvement in physiological processes and development of specific inhibitors will be a suitable attempt to design therapies for fatal and deadly diseases.
Assuntos
Ciclofilinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Peptidilprolil Isomerase/antagonistas & inibidores , Dobramento de Proteína , Proteína 1A de Ligação a Tacrolimo/antagonistas & inibidores , Animais , Doenças Cardiovasculares/tratamento farmacológico , Catálise , Ciclofilinas/fisiologia , Ciclosporina/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/uso terapêutico , Humanos , Imunossupressores/farmacologia , Inflamação/tratamento farmacológico , Peptidilprolil Isomerase de Interação com NIMA , Neoplasias/tratamento farmacológico , Peptidilprolil Isomerase/fisiologia , Traumatismo por Reperfusão/tratamento farmacológico , Dermatopatias/tratamento farmacológico , Proteína 1A de Ligação a Tacrolimo/fisiologiaRESUMO
FK506 is a commonly used immunosuppressant that mediates its action by exclusively interacting with the cytosolic immunophilin, FK506 binding protein 12 (FKBP12). Although FK506-induced acute osteoporosis is now well recognised, its precise mode of action in osteoblasts remains unclear. Therefore, in the present study we characterised FKBP12 in osteoblasts and investigated the role of FK506 in modulating osteoblast-specific transcription factors, core-binding factor alpha1 (Cbfa1) and osterix gene expression in ROS 17/2.8 cells. RT-PCR, immunolocalisation and Western blotting studies were employed to identify and characterise FKBP12 in rat primary osteoblasts and osteoblast-like osteosarcoma ROS 17/2.8 cells. Western blotting extracts of these cells revealed the 12 kDa and hitherto unreported 10 kDa FKBP isoform that were immunolocalised predominantly to the cytosol. The transient exposure of ROS 17/2.8 cells to H2O2 (100 microM) was found to elevate FKBP12 mRNA after 10 min and protein expression after 24 h. Both PTH (10(-9) M) and 1,25 (OH)2D3 (Vitamin D3) (10(-7) M) suppressed FKBP12 protein expression. FK506 in the therapeutic range (25 nmol/L) suppressed expression of Cbfa1 and osterix mRNA. The inhibition of Cbfa1 isoforms II/III expression was evident at 30 min and the extent of inhibition was sustained at 6 h. Osterix inhibition was also seen after 30 min, however, it became maximal after 6 h. The dose-dependant inhibition of osterix in these cells, carried out using 1.25, 12.5 and 125 nmol/L of FK506 was maximal at 1.25 nmol/L. Cbfa1 isoforms II/III were also maximally inhibited at 1.25 nmol/L; interestingly, the inhibition became less marked at higher concentrations of FK506. Similar dose of FK506 was found to inhibit ROS 17/2.8 cell proliferation; the inhibitory effect however was greater in insulin-stimulated cells. The results of this study suggest that immunosuppressant-induced osteoporosis, which is known to involve accelerated bone resorption by increase in osteoclastogenesis, may in fact also be accentuated by the inhibition of osteoblast differentiation and function.
Assuntos
Proteínas de Neoplasias/biossíntese , Proteína 1A de Ligação a Tacrolimo/fisiologia , Fatores de Transcrição/biossíntese , Fosfatase Alcalina/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core , Fatores de Ligação ao Core , Citosol/enzimologia , Citosol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Isoformas de Proteínas/fisiologia , RNA Mensageiro/biossíntese , Ratos , Tacrolimo/metabolismo , Tacrolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo/biossíntese , Proteína 1A de Ligação a Tacrolimo/metabolismo , Fatores de Transcrição/genéticaRESUMO
FKBP12 as an immunophilin that binds to two well-known immunosuppressive macrolides, FK506 and rapamycin, has attracted immense attention and its role in mediating the immunosuppressive functions of these macrolides has been extensively studied. Since FKBP12 is a well-conserved protein among many species and is also highly expressed in almost all cells, it must play important roles in cellular function in the absence of macrolides. In one such a role, FKBP12 interacts with and regulates the functional state of the ryanodine Ca2+ channel receptor by altering protein conformation and coordinating multi-protein complex formation. This review summarizes another physiological role of FKBP12 as an interactor and a regulator of the type I serine/threonine kinase receptors of TGF-beta superfamily. Current data, derived from detailed biochemical studies as well as from functional studies in various systems, suggest that FKBP12 functions as a "guardian" for the type I receptors to prevent them from leaky signaling under sub-optimal ligand concentrations, thereby providing a molecular "gradient reader" for TGF-beta family morphogens. This aspect of FKBP12 function may be critical for cellular responsiveness to morphogenetic gradients of the TGF-beta family members during early development, serving to assure the translation of different ligand concentrations into different signaling readouts.
Assuntos
Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismo , Animais , Canais de Cálcio/química , Previsões , Humanos , Imunofilinas/metabolismo , Macrolídeos/farmacologia , Estrutura Terciária de Proteína/fisiologia , Proteína 1A de Ligação a Tacrolimo/efeitos dos fármacos , Proteína 1A de Ligação a Tacrolimo/fisiologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Rapamycin (RAPA) is a well-known immunosuppressant, the action of which is mediated by the immunophilin FKBP12. Upon RAPA binding, FKBP12 forms ternary complexes with phosphatidyl inositol related kinases known as the target of RAPA (TOR), which can lead to a mitotic block at the G1-S phase transition. Such an antiproliferative effect makes RAPA an attractive anticancer, antifungal or antiparasitic compound. In this study, we found the helminth parasite Schistosoma mansoni to be insensitive to the drug. In order to elucidate the mechanism underlying RAPA resistance, the S. mansoni drug receptor FKBP12 (SmFKBP12) was cloned for functional analysis. Western blot experiments showed that the protein is constitutively expressed in all life cycle stages and in both male and female parasites. The Escherichia coli-synthesised recombinant protein possessed enzymatic activity, which was inhibitable by RAPA. Moreover, SmFKBP12 was able to complement mutant Saccharomyces cerevisiae cells lacking FKBP12 in their RAPA sensitivity phenotype, leading us to conclude that SmFKBP12 is expressed in yeast in a functional form and capable of interacting with the drug and yeast TOR kinase. Even though the wild type SmFKBP12 appeared to restore a large part of RAPA sensitivity, a mutation of Asp(89)-Lys(90) to Pro(89)-Gly(90) in the schistosome protein was found to be more effective and restored drug sensitivity to the same level as the endogenous yeast protein. Despite ternary complex formation, our results suggest that additional unknown factors other than a functional drug receptor are implicated in drug resistance mechanisms.
Assuntos
Imunossupressores/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Esquistossomicidas/farmacologia , Sirolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo/fisiologia , Sequência de Aminoácidos , Animais , Western Blotting , Clonagem Molecular , Dados de Sequência Molecular , Peptidilprolil Isomerase/metabolismo , Proteínas Recombinantes/metabolismo , Schistosoma mansoni/metabolismo , Esquistossomicidas/metabolismo , Alinhamento de Sequência , Sirolimo/análise , Sirolimo/metabolismo , Proteína 1A de Ligação a Tacrolimo/genética , Leveduras/metabolismoRESUMO
BACKGROUND: Rapamycin is a recently introduced immunosuppressive agent. Its effect on lymphocytes has been extensively studied. Whether it can also modulate dendritic cell (DC) function is unknown. METHODS: The effect of rapamycin on differentiation, antigen uptake, and the immunostimulatory capacity of human DC was examined. DC were derived from monocytes upon culture with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor in the presence or absence of rapamycin (0.1-100 ng/mL). Surface phenotype and antigen uptake capacity of DC were assessed by flow cytometry. Immunostimulatory capacity was measured by mixed lymphocyte culture. RESULTS: Rapamycin reduced DC recovery and increased DC apoptosis. DC differentiated in the presence of rapamycin (rapa-DC) had increased expression of CD1a, CD1b, and CD1c and decreased expression of MHC I, MHC II, CD80, CD86, and CD40. Antigen uptake receptor expression (mannose receptor, CD32, CD91, CD46) was decreased, and receptor-mediated endocytosis of fluorescein isothiocyanate-dextran was markedly impaired in rapa-DC, as were fluid phase endocytosis of Lucipher Yellow and phagocytic activity of bacteria and dead or apoptotic cells. CD40 ligand-induced production of both IL-12 and IL-10 was reduced in rapa-DC, and allogeneic T lymphocyte responses were moderately impaired when rapa-DC were used as stimulator cells. Neither cyclosporine nor FK506 affected DC function. However, the effects of rapamycin on DC could be completely inhibited by a 10-fold excess of FK506 but not by up to 100-fold excess of cyclosporine. CONCLUSION: Rapamycin has a unique and profound inhibitory effect on DC function, which seems to be at least in part mediated by the FKBP immunophilins.
Assuntos
Antígenos/metabolismo , Células Dendríticas/efeitos dos fármacos , Imunossupressores/farmacologia , Sirolimo/farmacologia , Diferenciação Celular/efeitos dos fármacos , Polaridade Celular , Células Cultivadas , Citocinas/biossíntese , Células Dendríticas/imunologia , Endocitose/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-4/farmacologia , Fagocitose/efeitos dos fármacos , Proteína 1A de Ligação a Tacrolimo/fisiologiaRESUMO
Neuroimmunophilin ligands are a class of compounds that hold great promise for the treatment of nerve injuries and neurological disease. In contrast to neurotrophins (e.g., nerve growth factor), these compounds readily cross the blood-brain barrier, being orally effective in a variety of animal models of ischaemia, traumatic nerve injury and human neurodegenerative disorders. A further distinction is that neuroimmunophilin ligands act via unique receptors that are unrelated to the classical neurotrophic receptors (e.g., trk), making it unlikely that clinical trials will encounter the same difficulties found with the neurotrophins. Another advantage is that two neuroimmunophilin ligands (cyclosporin A and FK-506) have already been used in humans (as immunosuppressant drugs). Whereas both cyclosporin A and FK-506 demonstrate neuroprotective actions, only FK-506 and its derivatives have been clearly shown to exhibit significant neuroregenerative activity. Accordingly, the neuroprotective and neuroregenerative properties seem to arise via different mechanisms. Furthermore, the neuroregenerative property does not involve calcineurin inhibition (essential for immunosuppression). This is important since most of the limiting side effects produced by these drugs arise via calcineurin inhibition. A major breakthrough for the development of this class of compounds for the treatment of human neurological disorders was the ability to separate the neuroregenerative property of FK-506 from its immunosuppressant action via the development of non-immunosuppressant (non-calcineurin inhibiting) derivatives. Further studies revealed that different receptor subtypes, or FK-506-binding proteins (FKBPs), mediate immunosuppression and nerve regeneration (FKBP-12 and FKBP-52, respectively, the latter being a component of steroid receptor complexes). Thus, steroid receptor chaperone proteins represent novel targets for future drug development of novel classes of compounds for the treatment of a variety of human neurological disorders, including traumatic injury (e.g., peripheral nerve and spinal cord), chemical exposure (e.g., vinca alkaloids, Taxol) and neurodegenerative disease (e.g. , diabetic neuropathy and Parkinson's disease).
Assuntos
Ciclosporina/uso terapêutico , Imunossupressores/uso terapêutico , Regeneração Nervosa/fisiologia , Doenças do Sistema Nervoso/tratamento farmacológico , Proteína 1A de Ligação a Tacrolimo/fisiologia , Tacrolimo/uso terapêutico , Animais , Lesões Encefálicas/tratamento farmacológico , Síndrome do Túnel Carpal/tratamento farmacológico , Ciclosporina/farmacologia , Humanos , Imunofilinas/farmacologia , Imunofilinas/uso terapêutico , Imunossupressores/farmacologia , Ligantes , Regeneração Nervosa/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Tacrolimo/química , Tacrolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo/efeitos dos fármacosRESUMO
We focused on immunophilin isoforms in order to clarify the neuroimmunophilins which were identified as targets for the immunophilin ligands to elicit a neuroprotective effect. Although the expressions of five FK506-binding protein (FKBP) mRNAs were detected in both SH-SY5Y (human neuroblastoma) and U251 (human glioma) cell lysates, the FKBP12 mRNA expression was detected in only the SH-SY5Y cells, and not the U251 cells. However, we found that the SH-SY5Y and the U251 cells were equipotent in the intensity of cellular protection of FK506 (an immunosuppressive immunophilin ligand) and GPI1046 (a non-immunosuppressive FK506 analog), indicating that the protective effect and glutathione activation of FK506 and GPI1046 had little need to bind FKBP12. Therefore, we conclude that the neuroprotective and antioxidant properties of immunophilin ligands are independent on the FKBP12 pathway.
Assuntos
Peróxido de Hidrogênio/farmacologia , Imunossupressores/farmacologia , Peptidilprolil Isomerase , Proteína 1A de Ligação a Tacrolimo/fisiologia , Proteínas de Ligação a Tacrolimo/genética , Tacrolimo/farmacologia , Actinas/genética , Actinas/metabolismo , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Colorimetria , Dimetil Sulfóxido/farmacologia , Glutationa/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Fármacos Neuroprotetores/farmacologia , Pirrolidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Proteína 1A de Ligação a Tacrolimo/efeitos dos fármacos , Proteína 1A de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Células Tumorais CultivadasAssuntos
Cálcio/metabolismo , Morte Súbita/etiologia , Mutação , Miocárdio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Taquicardia Ventricular/etiologia , Animais , Cardiomiopatias/etiologia , Humanos , Camundongos , Taquicardia Ventricular/genética , Proteína 1A de Ligação a Tacrolimo/fisiologiaRESUMO
The immunophilin 12-kDa FK506 binding protein (FKBP12) stabilizes intracellular Ca(2+) release channel (CRC) activity in different tissues. In this work, the presence of FKBP12 in rat vas deferens (RVD) and its possible contribution to RVD function was investigated. Treatment under appropriate pH, temperature, and ionic conditions was used to strip FKBP12 from CRC binding sites; Western blotting revealed FKBP12 in control but not in treated homogenates. Disruption of the FKBP12-CRC complex in RVD decreased the Ca(2+) content of sarcoplasmic reticulum (SR) by increasing Ca(2+) leakage through the ryanodine receptor (RyR3 isoform) but not through 1,4,5-inositol trisphosphate receptors (IP(3)R1 and IP(3)R3 isoforms). The decrease of SR Ca(2+) content was not related to inhibition of SERCA ATPase. It seems that dissociation of FKBP12-RyR leads to conformational changes in RyR that make it difficult for ryanodine to access its binding site. Rapamycin, which is commonly used as a pharmacological tool to disrupt the FKBP12-RyR complex, decreased phenylephrine-induced contractions in RVD epididymal halves. The data suggest that FKBP12 is expressed in RVD in a labile association with RyR3. Disruption of the FKBP12-RyR3 complex may lead to modifications of RVD physiology and in consequence may compromise male fertility.