RESUMO
Post-translational modifications provide sensitive and flexible mechanisms to dynamically modulate protein function in response to specific signalling inputs. In the case of transcription factors, changes in phosphorylation state can influence protein stability, conformation, subcellular localization, cofactor interactions, transactivation potential and transcriptional output. Here we show that the evolutionarily conserved transcription factor Eyes absent (Eya) belongs to the phosphatase subgroup of the haloacid dehalogenase (HAD) superfamily, and propose a function for it as a non-thiol-based protein tyrosine phosphatase. Experiments performed in cultured Drosophila cells and in vitro indicate that Eyes absent has intrinsic protein tyrosine phosphatase activity and can autocatalytically dephosphorylate itself. Confirming the biological significance of this function, mutations that disrupt the phosphatase active site severely compromise the ability of Eyes absent to promote eye specification and development in Drosophila. Given the functional importance of phosphorylation-dependent modulation of transcription factor activity, this evidence for a nuclear transcriptional coactivator with intrinsic phosphatase activity suggests an unanticipated method of fine-tuning transcriptional regulation.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Proteínas do Olho/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anticorpos Fosfo-Específicos/imunologia , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Indução Embrionária , Olho/embriologia , Olho/enzimologia , Olho/metabolismo , Proteínas do Olho/química , Proteínas do Olho/genética , Regulação da Expressão Gênica , Cinética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Conformação Proteica , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Especificidade por Substrato , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Background: Immunotherapies have demonstrated efficacy across a diverse set of tumors supporting further evaluation in glioblastoma. The objective of this study was to evaluate the safety/tolerability and describe immune-mediated effects of nivolumab ± ipilimumab in patients with recurrent glioblastoma. Exploratory efficacy outcomes are also reported. Methods: Patients were randomized to receive nivolumab 3 mg/kg every 2 weeks (Q2W; NIVO3) or nivolumab 1 mg/kg + ipilimumab 3 mg/kg every 3 weeks (Q3W) for 4 doses, then nivolumab 3 mg/kg Q2W (NIVO1+IPI3). An alternative regimen of nivolumab 3 mg/kg + ipilimumab 1 mg/kg Q3W for 4 doses, then nivolumab 3 mg/kg Q2W (NIVO3+IPI1) was investigated in a nonrandomized arm. Results: Forty patients were enrolled (NIVO3, n = 10; NIVO1+IPI3, n = 10; NIVO3+IPI1, n = 20). The most common treatment-related adverse events (AEs) were fatigue (NIVO3, 30%; NIVO1+IPI3, 80%; NIVO3+IPI1, 55%) and diarrhea (10%, 70%, 30%, respectively). AEs leading to discontinuation occurred in 10% (NIVO3), 30% (NIVO1+IPI3), and 20% (NIVO3+IPI1) of patients. Three patients achieved a partial response (NIVO3, n = 1; NIVO3+IPI1, n = 2) and 8 had stable disease for ≥12 weeks (NIVO3, n = 2; NIVO1+IPI3, n = 2; NIVO3+IPI1, n = 4 [Response Assessment in Neuro-Oncology criteria]). Most patients (68%) had tumor-cell programmed death ligand-1 expression ≥1%. Immune-mediated effects mimicking radiographic progression occurred in 2 patients. Conclusions: Nivolumab monotherapy was better tolerated than nivolumab + ipilimumab; the tolerability of the combination was influenced by ipilimumab dose. These safety and exploratory findings merit further investigation of immunotherapies in glioblastoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Adulto , Idoso , Neoplasias Encefálicas/patologia , Estudos de Coortes , Feminino , Seguimentos , Glioblastoma/patologia , Humanos , Ipilimumab/administração & dosagem , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Nivolumabe/administração & dosagem , PrognósticoRESUMO
OBJECTIVE: To develop an objective, readily measurable pharmacodynamic biomarker of glucocorticoid (GC) activity. METHODS: Genes modulated by prednisolone were identified from in vitro studies using peripheral blood mononuclear cells from normal healthy volunteers. Using the criteria of a >2-fold change relative to vehicle controls and an adjusted P value cutoff of less than 0.05, 64 up-regulated and 18 down-regulated genes were identified. A composite score of the up-regulated genes was generated using a single-sample gene set enrichment analysis algorithm. RESULTS: GC gene signature expression was significantly elevated in peripheral blood leukocytes from normal healthy volunteers following oral administration of prednisolone. Expression of the signature increased in a dose-dependent manner, peaked at 4 hours postadministration, and returned to baseline levels by 48 hours after dosing. Lower expression was detected in normal healthy volunteers who received a partial GC receptor agonist, which is consistent with the reduced transactivation potential of this compound. In cohorts of patients with systemic lupus erythematosus and patients with rheumatoid arthritis, expression of the GC signature was negatively correlated with the percentages of peripheral blood lymphocytes and positively correlated with peripheral blood neutrophil counts, which is consistent with the known biology of the GC receptor. Expression of the signature largely agreed with reported GC use in these populations, although there was significant interpatient variability within the dose cohorts. CONCLUSION: The GC gene signature identified in this study represents a pharmacodynamic marker of GC exposure.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/administração & dosagem , Leucócitos Mononucleares/efeitos dos fármacos , Prednisolona/administração & dosagem , Administração Oral , Artrite Reumatoide/sangue , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Biomarcadores/sangue , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Feminino , Voluntários Saudáveis , Humanos , Contagem de Leucócitos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/genética , Masculino , Testes Farmacogenômicos , Regulação para Cima/efeitos dos fármacosRESUMO
The mammalian TOR (mTOR) pathway integrates nutrient- and growth factor-derived signals to regulate growth, the process whereby cells accumulate mass and increase in size. mTOR is a large protein kinase and the target of rapamycin, an immunosuppressant that also blocks vessel restenosis and has potential anticancer applications. mTOR interacts with the raptor and GbetaL proteins to form a complex that is the target of rapamycin. Here, we demonstrate that mTOR is also part of a distinct complex defined by the novel protein rictor (rapamycin-insensitive companion of mTOR). Rictor shares homology with the previously described pianissimo from D. discoidieum, STE20p from S. pombe, and AVO3p from S. cerevisiae. Interestingly, AVO3p is part of a rapamycin-insensitive TOR complex that does not contain the yeast homolog of raptor and signals to the actin cytoskeleton through PKC1. Consistent with this finding, the rictor-containing mTOR complex contains GbetaL but not raptor and it neither regulates the mTOR effector S6K1 nor is it bound by FKBP12-rapamycin. We find that the rictor-mTOR complex modulates the phosphorylation of Protein Kinase C alpha (PKCalpha) and the actin cytoskeleton, suggesting that this aspect of TOR signaling is conserved between yeast and mammals.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citoesqueleto/metabolismo , Proteínas Quinases/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sequência Conservada , Primers do DNA , Drosophila , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Componentes do Gene , Células HeLa , Humanos , Immunoblotting , Dados de Sequência Molecular , Fosforilação , Testes de Precipitina , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína Companheira de mTOR Insensível à Rapamicina , Proteína Regulatória Associada a mTOR , Alinhamento de Sequência , Análise de Sequência de DNA , Sirolimo/metabolismo , Serina-Treonina Quinases TOR , TransfecçãoRESUMO
The Whitehead siRNA (short interfering RNA) Selection Web Server (http://jura.wi.mit.edu/bioc/siRNA) automates the design of short oligonucleotides that can specifically 'knock down' expression of target genes. These short sequences are about 21 nt in length, and when synthesized as double stranded RNA and introduced into cell culture, can reduce or eliminate the function of the target gene. Depending on the length of a gene, there are potentially numerous combinations of possible 21mers. Some experimental evidence has already shown that not all 21mers in a gene have the same effectiveness at silencing gene function. Our tool incorporates published design rules and presents the scientist with information about uniqueness of the 21mers within the genome, thermodynamic stability of the double stranded RNA duplex, GC content, presence of SNPs and other features that may contribute to the effectiveness of a siRNA.
Assuntos
RNA Interferente Pequeno/química , Software , Algoritmos , Animais , Humanos , Internet , Camundongos , Oligonucleotídeos/química , Interferência de RNA , Interface Usuário-ComputadorRESUMO
Smad proteins are a class of tumor suppressors that play critical roles in inhibiting the proliferation of a variety of cell types by modulating the transcriptions of target genes. Despite recent advances, the mechanism of their nuclear import is not completely understood. Smad proteins contain a conserved basic motif in their N-terminal MH1 domains that resembles a nuclear localization signal (NLS). Previous studies indicate that in receptor-regulated Smads such as Smad1 and Smad3 this motif determines their interactions with nuclear import receptors and mediates their ligand-induced nuclear translocation. Common-Smads such as Smad4 display constant nucleocytoplasmic shuttling and are capable of autonomous nuclear import and export. Mutations of the basic motif in Smad4 disrupted its nuclear accumulation. However, this motif is not sufficient to confer nuclear translocation to a fused heterologous protein, suggesting that it is only part of the bona fide Smad4 NLS. We mapped the Smad4 NLS by fusing various segments of Smad4 sequence covering the basic motif to GFP and tested the localization of the fusion proteins. We identified an extended NLS, starting from the basic motif and extending into the DNA-binding region (AA 45-110), that is sufficient to confer nuclear localization to GFP. Among the 14 basic residues in the NLS, only four (K45, K46, K48 and R81) are critical for import. This NLS is critical not only for autonomous nuclear import of Smad4, but also for its nuclear translocation in the presence of activated R-Smads, further confirming the functional relevance of the Smad4 NLS in TGF-beta signal transduction. Structural modeling demonstrated that the four critical basic residues are all solvent exposed and map to a single localized segment on one surface of the Smad4 MH1 domain. Their distribution and spacing resemble a classical bipartite NLS. Smad4 displays specific binding to importin alpha through its MH1 domain, which was abrogated by loss-of-function mutations in Smad4 NLS. Finally, the Smad4 NLS is essential for its transcriptional activity since loss-of-function NLS mutants are also transcriptionally inactive.
Assuntos
Proteínas de Ligação a DNA/química , Sinais Direcionadores de Proteínas/fisiologia , Transativadores/química , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Rim , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteína Smad3 , Proteína Smad4 , Transativadores/metabolismo , Transcrição Gênica , Transfecção , Fator de Crescimento Transformador beta/fisiologiaRESUMO
BACKGROUND: IL-23 is a cytokine produced by dendritic cells, T-cells and macrophages that plays a critical regulatory role in the inflammatory and autoimmune responses. We describe the development and preclinical validation of a highly sensitive Luminex(®) assay specific to IL-23 that is suitable for its measurement in support of early-phase clinical trials. RESULTS: Intra-assay precision for the BioSource™ ELISA was under 12.3%, and under 5.2% for the eBioscience(®) ELISA. In comparison, the Luminex assays provided an intra-assay precision under 6.2%. The measured inter-assay precision was less than 15.6% for the BioSource ELISA, under 33% for the eBioscience and less than 10% for the Luminex assays. CONCLUSIONS: The Luminex method described provides a way to measure IL-23 in clinical samples either as a single biomarker or as a panel of biomarkers. The assay should prove useful to scientists and clinicians investigating the biology of IL-23 and to those needing to monitor changes in IL-23 as part of a clinical study.
Assuntos
Imunoensaio/métodos , Interleucina-23/sangue , Animais , Humanos , Interleucina-23/metabolismo , Limite de Detecção , Microesferas , Ratos , Reprodutibilidade dos Testes , Transdução de SinaisRESUMO
BACKGROUND: The selective inhibitor of T-cell costimulation, belatacept, blocks CD28-mediated T-cell activation by binding CD80 and CD86 on antigen-presenting cells. Understanding the extent to which belatacept binds to its targets in patients may enable correlation of belatacept exposure to receptor saturation as a pharmacodynamic measure of costimulation blockade. METHODS: Flow cytometry-based receptor competition assays were developed to monitor concentration-dependent occupancy of CD80 and CD86 receptors in whole blood and dendritic cell cultures in vitro. Receptor occupancy was correlated with inhibition of mixed leukocyte reactions and clinical validation was obtained by comparing receptor saturation in whole blood from healthy volunteers and in de novo renal transplant recipients participating in studies comparing cyclosporine and belatacept-based immunosuppression. RESULTS: Belatacept saturated CD80 and CD86 receptors in whole blood and dendritic cell cultures, although the belatacept concentrations required for CD86-receptor saturation were approximately 10-fold higher than those required for CD80 saturation (IC50=0.102 microg/mL vs. 0.009 microg/mL). Primary alloresponses were inhibited at the belatacept concentration required for CD86-receptor saturation, but not at the lower concentration needed to saturate CD80. Whole blood from belatacept-treated patients had significantly lower levels of free CD86 receptors versus pretransplant levels, healthy volunteers, or cyclosporine-treated patients. CD86-receptor saturation correlated with belatacept dose/dose frequency and remained consistently more than 80%. CONCLUSIONS: These results suggest that belatacept-mediated inhibition of alloresponses involved in transplant rejection correlates with CD86 saturation, indicating that CD86-receptor occupancy may be a valid pharmacodynamic measure of costimulation blockade and provide the first direct clinical evidence that belatacept binds to one of its targets.
Assuntos
Antígeno B7-1/imunologia , Antígeno B7-2/imunologia , Imunoconjugados/uso terapêutico , Imunossupressores/uso terapêutico , Transplante de Rim/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Abatacepte , Animais , Antígenos CD/imunologia , Ciclosporina/uso terapêutico , Células Dendríticas/imunologia , Citometria de Fluxo , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/imunologia , Antígenos HLA/imunologia , Humanos , Imunoconjugados/farmacologia , Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Monócitos/imunologia , Distribuição Aleatória , Receptores de Antígenos de Linfócitos T/efeitos dos fármacos , OvinosRESUMO
The Bcr-Abl fusion protein kinase causes chronic myeloid leukemia and is targeted by the signal transduction inhibitor STI-571/Gleevec/imatinib (STI-571). Sequencing of the BCR-ABL gene in patients who have relapsed after STI-571 chemotherapy has revealed a limited set of kinase domain mutations that mediate drug resistance. To obtain a more comprehensive survey of the amino acid substitutions that confer STI-571 resistance, we performed an in vitro screen of randomly mutagenized BCR-ABL and recovered all of the major mutations previously identified in patients and numerous others that illuminate novel mechanisms of acquired drug resistance. Structural modeling implies that a novel class of variants acts allosterically to destabilize the autoinhibited conformation of the ABL kinase to which STI-571 preferentially binds. This screening strategy is a paradigm applicable to a growing list of target-directed anti-cancer agents and provides a means of anticipating the drug-resistant amino acid substitutions that are likely to be clinically problematic.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores Enzimáticos/farmacologia , Mutagênese Sítio-Dirigida , Piperazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Benzamidas , Variação Genética , Humanos , Mesilato de Imatinib , Concentração Inibidora 50 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/genéticaRESUMO
mTOR/RAFT1/FRAP is the target of the immunosuppressive drug rapamycin and the central component of a nutrient- and hormone-sensitive signaling pathway that regulates cell growth. We report that mTOR forms a stoichiometric complex with raptor, an evolutionarily conserved protein with at least two roles in the mTOR pathway. Raptor has a positive role in nutrient-stimulated signaling to the downstream effector S6K1, maintenance of cell size, and mTOR protein expression. The association of raptor with mTOR also negatively regulates the mTOR kinase activity. Conditions that repress the pathway, such as nutrient deprivation and mitochondrial uncoupling, stabilize the mTOR-raptor association and inhibit mTOR kinase activity. We propose that raptor is a missing component of the mTOR pathway that through its association with mTOR regulates cell size in response to nutrient levels.
Assuntos
Proteínas Quinases/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Divisão Celular , Linhagem Celular Transformada , Tamanho Celular , Sequência Conservada , Meios de Cultura , Evolução Molecular , Humanos , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas/genética , Proteína Regulatória Associada a mTOR , Sequências Repetitivas de Aminoácidos , Proteínas Quinases S6 Ribossômicas/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TORRESUMO
mTOR and raptor are components of a signaling pathway that regulates mammalian cell growth in response to nutrients and growth factors. Here, we identify a member of this pathway, a protein named GbetaL that binds to the kinase domain of mTOR and stabilizes the interaction of raptor with mTOR. Like mTOR and raptor, GbetaL participates in nutrient- and growth factor-mediated signaling to S6K1, a downstream effector of mTOR, and in the control of cell size. The binding of GbetaL to mTOR strongly stimulates the kinase activity of mTOR toward S6K1 and 4E-BP1, an effect reversed by the stable interaction of raptor with mTOR. Interestingly, nutrients and rapamycin regulate the association between mTOR and raptor only in complexes that also contain GbetaL. Thus, we propose that the opposing effects on mTOR activity of the GbetaL- and raptor-mediated interactions regulate the mTOR pathway.