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1.
Nucleic Acids Res ; 50(22): e129, 2022 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-36189884

RESUMO

Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by the dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to fixed adherent cells agreed with expectations from expression profiles of the cells. We also introduce a proximity ligation variant of TEMA to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug-target interactions at spatial resolution in protein arrays, cells and in tissues.


Assuntos
Terapia de Alvo Molecular , Dasatinibe/farmacologia , Sondas de Oligonucleotídeos , Análise Serial de Proteínas , Proteínas , Gefitinibe/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Terapia de Alvo Molecular/métodos
2.
Mol Cell Proteomics ; 15(6): 1848-56, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26929218

RESUMO

The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF-ß signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF-ß signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF-ß signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA.Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.


Assuntos
Anticorpos de Cadeia Única/análise , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células MCF-7 , Biblioteca de Peptídeos , Fosforilação , Transdução de Sinais/efeitos dos fármacos
3.
Nat Methods ; 11(8): 841-6, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24997862

RESUMO

The spatial organization of membrane-bound ligands is thought to regulate receptor-mediated signaling. However, direct regulation of receptor function by nanoscale distribution of ligands has not yet been demonstrated, to our knowledge. We developed rationally designed DNA origami nanostructures modified with ligands at well-defined positions. Using these 'nanocalipers' to present ephrin ligands, we showed that the nanoscale spacing of ephrin-A5 directs the levels of EphA2 receptor activation in human breast cancer cells. Furthermore, we found that the nanoscale distribution of ephrin-A5 regulates the invasive properties of breast cancer cells. Our ligand nanocaliper approach has the potential to provide insight into the roles of ligand nanoscale spatial distribution in membrane receptor-mediated signaling.


Assuntos
Nanotecnologia , Receptores de Superfície Celular/metabolismo , Endocitose , Ligantes
4.
J Cell Biol ; 163(4): 723-8, 2003 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-14638857

RESUMO

The Notch and transforming growth factor-beta (TGF-beta) signaling pathways play critical roles in the control of cell fate during metazoan development. However, mechanisms of cross-talk and signal integration between the two systems are unknown. Here, we demonstrate a functional synergism between Notch and TGF-beta signaling in the regulation of Hes-1, a direct target of the Notch pathway. Activation of TGF-beta signaling up-regulated Hes-1 expression in vitro and in vivo. This effect was abrogated in myogenic cells by a dominant-negative form of CSL, an essential DNA-binding component of the Notch pathway. TGF-beta regulated transcription from the Hes-1 promoter in a Notch-dependent manner, and the intracellular domain of Notch1 (NICD) cooperated synergistically with Smad3, an intracellular transducer of TGF-beta signals, to induce the activation of synthetic promoters containing multimerized CSL- or Smad3-binding sites. NICD and Smad3 were shown to interact directly, both in vitro and in cells, in a ligand-dependent manner, and Smad3 could be recruited to CSL-binding sites on DNA in the presence of CSL and NICD. These findings indicate that Notch and TGF-beta signals are integrated by direct protein-protein interactions between the signal-transducing intracellular elements from both pathways.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Receptor Cross-Talk/fisiologia , Receptores de Superfície Celular/metabolismo , Transativadores/metabolismo , Fatores de Transcrição , Fator de Crescimento Transformador beta/metabolismo , Animais , Sítios de Ligação/fisiologia , Células Cultivadas , Embrião de Galinha , Estrutura Terciária de Proteína/fisiologia , Receptor Notch1 , Transdução de Sinais/fisiologia , Proteína Smad3 , Regulação para Cima/fisiologia
5.
Curr Biol ; 12(1): 35-45, 2002 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-11790301

RESUMO

BACKGROUND: Members of the GATA family of zinc finger transcription factors are genetically controlled "master" regulators of development in the hematopoietic and nervous systems. Whether GATA factors also serve to integrate epigenetic signals on target promoters is, however, unknown. The transforming growth factor-beta (TGF-beta) superfamily is a large group of phylogenetically conserved secreted factors controlling cell proliferation, differentiation, migration, and survival in multiple tissues. RESULTS: GATA-3, a key regulator of T helper cell development, was found to directly interact with Smad3, an intracellular signal transducer of TGF-beta. Complex formation required a central region in GATA-3 and the N-terminal domain of Smad3. GATA-3 mediated recruitment of Smad3 to GATA binding sites independently of Smad3 binding to DNA, and the two factors cooperated synergistically to regulate transcription from the IL-5 promoter in a TGF-beta-dependent manner. Treatment of T helper cells with TGF-beta promoted the formation of an endogenous Smad3/GATA-3 nuclear complex and stimulated production of the Th2 cytokine IL-10 in a Smad3- and GATA-3-dependent manner. CONCLUSIONS: Although Smad proteins are known to interact with a number of general transcription factors, these are insufficient to explain the tissue-specific biology of TGF-beta proteins. Through its interaction with Smad3, GATA-3 is able to integrate a genetic program of cell differentiation with an extracellular signal, providing a molecular framework for the effects of TGF-beta on the development and function of specific subsets of immune cells and possibly other cell types.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Transativadores/metabolismo , Transativadores/fisiologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Sítios de Ligação , Células COS , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/química , Fator de Transcrição GATA3 , Genes Reporter , Humanos , Interleucina-10/biossíntese , Interleucina-5/genética , Células Jurkat , Substâncias Macromoleculares , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteína Smad3 , Linfócitos T/imunologia , Transativadores/química , Ativação Transcricional
6.
PLoS One ; 11(4): e0154214, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27110718

RESUMO

BACKGROUND: The diagnosis of malignant melanoma currently relies on clinical inspection of the skin surface and on the histopathological status of the excised tumor. The serum marker S100B is used for prognostic estimates at later stages of the disease, but analyses are marred by false positives and inadequate sensitivity in predicting relapsing disorder. OBJECTIVES: To investigate SOX10 as a potential biomarker for melanoma and vitiligo. METHODS: In this study we have applied proximity ligation assay (PLA) to detect the transcription factor SOX10 as a possible serum marker for melanoma. We studied a cohort of 110 melanoma patients. We further investigated a second cohort of 85 patients with vitiligo, which is a disease that also affects melanocytes. RESULTS: The specificity of the SOX10 assay in serum was high, with only 1% of healthy blood donors being positive. In contrast, elevated serum SOX10 was found with high frequency among vitiligo and melanoma patients. In patients with metastases, lack of SOX10 detection was associated with treatment benefit. In two responding patients, a change from SOX10 positivity to undetectable levels was seen before the response was evident clinically. CONCLUSIONS: We show for the first time that SOX10 represents a promising new serum melanoma marker for detection of early stage disease, complementing the established S100B marker. Our findings imply that SOX10 can be used to monitor responses to treatment and to assess if the treatment is of benefit at stages earlier than what is possible radiologically.


Assuntos
Biomarcadores Tumorais/genética , Melanócitos/metabolismo , Melanoma/diagnóstico , Fatores de Transcrição SOXE/genética , Neoplasias Cutâneas/diagnóstico , Vitiligo/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Bioensaio , Biomarcadores Tumorais/sangue , Estudos de Casos e Controles , Estudos de Coortes , Diagnóstico Precoce , Feminino , Humanos , Metástase Linfática , Masculino , Melanócitos/patologia , Melanoma/sangue , Melanoma/genética , Melanoma/patologia , Pessoa de Meia-Idade , Subunidade beta da Proteína Ligante de Cálcio S100/sangue , Subunidade beta da Proteína Ligante de Cálcio S100/genética , Fatores de Transcrição SOXE/sangue , Sensibilidade e Especificidade , Neoplasias Cutâneas/sangue , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Resultado do Tratamento , Vitiligo/sangue , Vitiligo/genética , Vitiligo/patologia
7.
J Mol Biol ; 427(21): 3407-15, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25937570

RESUMO

The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Activity of the signaling pathway is associated with cell density sensing and tissue organization. Furthermore, the Hippo pathway helps to coordinate cellular processes through crosstalk with growth-factor-mediated signaling pathways such as TGFß. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGFß (Smad2/3) signaling pathway by using in situ proximity ligation assays. We investigated the formation of protein complexes between YAP/TAZ and Smad2/3 and examined how these interactions were affected by TGFß stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGFß induces formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells. Under sparse cell conditions, the complexes were detected to a higher degree and were predominantly located in the nucleus, while under dense culture conditions, the complexes were fewer and mainly located in the cytoplasm. Surprisingly, we could not detect any YAP/TAZ-Smad2/3 complexes in HT29 cells. To examine if Smad4 deficiency was responsible for the absence of interactions, we treated HaCaT cells with siRNA targeting Smad4. However, we could still observe complex formation in the siRNA-treated cells, suggesting that Smad4 is not essential for the YAP-Smad2/3 interaction. In conclusion, this study shows localized, density-dependent formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells and provides evidence supporting a crosstalk between the Hippo and the TGFß signaling pathways.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias do Colo/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Smad Reguladas por Receptor/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/análise , Linhagem Celular Tumoral , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/patologia , Via de Sinalização Hippo , Humanos , Fosfoproteínas/análise , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/análise , Proteínas Smad Reguladas por Receptor/análise , Proteína Smad2/análise , Proteína Smad2/metabolismo , Proteína Smad3/análise , Proteína Smad3/metabolismo , Fatores de Transcrição/análise , Fator de Crescimento Transformador beta/análise , Proteínas de Sinalização YAP
8.
Neuromolecular Med ; 15(3): 458-69, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23666762

RESUMO

The microtubule-affinity regulating kinase (MARK) family consists of four highly conserved members that have been implicated in phosphorylation of tau protein, causing formation of neurofibrillary tangles in Alzheimer's disease (AD). Understanding of roles by individual MARK isoform in phosphorylating tau has been limited due to lack of antibodies selective for each MARK isoform. In this study, we first applied the proximity ligation assay on cells to select antibodies specific for each MARK isoform. In cells, a CagA peptide specifically and significantly inhibited tau phosphorylation at Ser²6² mediated by MARK4 but not other MARK isoforms. We then used these antibodies to study expression levels of MARK isoforms and interactions between tau and individual MARK isoforms in postmortem human brains. We found a strong and significant elevation of MARK4 expression and MARK4-tau interactions in AD brains, correlating with the Braak stages of the disease. These results suggest the MARK4-tau interactions are of functional importance in the progression of AD and the results also identify MARK4 as a promising target for AD therapy.


Assuntos
Doença de Alzheimer/enzimologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Especificidade de Anticorpos , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Encéfalo/enzimologia , Progressão da Doença , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Células NIH 3T3 , Proteínas do Tecido Nervoso/imunologia , Emaranhados Neurofibrilares/metabolismo , Fragmentos de Peptídeos/farmacologia , Fosforilação , Fosfosserina/análise , Mapeamento de Interação de Proteínas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
9.
Development ; 130(24): 6089-99, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14597575

RESUMO

The bone morphogenetic protein (BMP) and Notch signaling pathways are crucial for cellular differentiation. In many cases, the two pathways act similarly; for example, to inhibit myogenic differentiation. It is not known whether this inhibition is caused by distinct mechanisms or by an interplay between Notch and BMP signaling. Here we demonstrate that functional Notch signaling is required for BMP4-mediated block of differentiation of muscle stem cells, i.e. satellite cells and the myogenic cell line C2C12. Addition of BMP4 during induction of differentiation dramatically reduced the number of differentiated satellite and C2C12 cells. Differentiation was substantially restored in BMP4-treated cultures by blocking Notch signaling using either the gamma-secretase inhibitor L-685,458 or by introduction of a dominant-negative version of the Notch signal mediator CSL. BMP4 addition to C2C12 cells increased transcription of two immediate Notch responsive genes, Hes1 and Hey1, an effect that was abrogated by L-685,458. A 3 kb Hey1-promoter reporter construct was synergistically activated by the Notch 1 intracellular domain (Notch 1 ICD) and BMP4. The BMP4 mediator SMAD1 mimicked BMP activation of the Hey1 promoter. A synthetic Notch-responsive promoter containing no SMAD1 binding sites responded to SMAD1, indicating that DNA-binding activity of SMAD1 is not required for activation. Accordingly, Notch 1 ICD and SMAD1 interacted in binding experiments in vitro. Thus, the data presented here provide evidence for a direct interaction between the Notch and BMP signaling pathways, and indicate that Notch has a crucial role in the execution of certain aspects of BMP-mediated differentiation control.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/fisiologia , Proteínas de Membrana/metabolismo , Músculos/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteína Morfogenética Óssea 4 , Carbamatos/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Dipeptídeos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Camundongos , Regiões Promotoras Genéticas , Receptores Notch , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Smad , Proteína Smad1 , Células-Tronco/citologia , Transativadores/metabolismo
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