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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.
Nucleic Acids Res ; 48(13): e73, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32469060

RESUMO

Digital PCR provides high sensitivity and unprecedented accuracy in DNA quantification, but current approaches require dedicated instrumentation and have limited opportunities for multiplexing. Here, we present an isothermal platform for digital enumeration of DNA reaction products in multiplex via standard fluorescence microscopy. Circular DNA strands, which may result from a wide range of molecular detection reactions, are captured on streptavidin-coated surfaces via hybridized biotinylated primers, followed by rolling circle amplification (RCA). The addition of 15% polyethylene glycol 4000 during RCA resulted in uniform, easily recorded reaction products. Immobilized DNA circles were visualized as RCA products with 100% efficiency, as determined by droplet digital PCR. We confirmed previous reports about the influence on RCA by sequence composition and size of RCA templates, and we developed an efficient one-step restaining procedure for sequential multiplexing using toehold-triggered DNA strand displacement. Finally, we exemplify applications of this digital readout platform by demonstrating more than three orders of magnitude improved sensitivity by digital measurement of prostate specific antigen (PSA) (detection threshold ∼100 pg/l), compared to a commercial enzyme-linked immunosorbent assay (ELISA) with analogue readout (detection threshold ∼500 ng/l), using the same antibody pair.


Assuntos
DNA Circular/análise , Ensaio de Imunoadsorção Enzimática/métodos , Calicreínas/sangue , Técnicas de Amplificação de Ácido Nucleico/métodos , Antígeno Prostático Específico/sangue , Feminino , Humanos , Masculino , Hibridização de Ácido Nucleico/métodos , Estreptavidina/química
3.
Trends Biochem Sci ; 42(7): 504-515, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28566215

RESUMO

The well-oiled machinery of the cellular proteome operates via variable expression, modifications, and interactions of proteins, relaying genomic and transcriptomic information to coordinate cellular functions. In recent years, a number of techniques have emerged that serve to identify sets of proteins acting in close proximity in the course of orchestrating cellular activities. These proximity-dependent assays, including BiFC, BioID, APEX, FRET, and isPLA, have opened up new avenues to examine protein interactions in live or fixed cells. We review herein the current status of proximity-dependent in situ techniques. We compare the advantages and limitations of the methods, underlining recent progress and the growing importance of these techniques in basic research, and we discuss their potential as tools for drug development and diagnostics.


Assuntos
Proteínas/análise , Proteínas/metabolismo , Fixação de Tecidos , Animais , Técnicas Biossensoriais , Sobrevivência Celular , Transferência Ressonante de Energia de Fluorescência , Humanos , Técnicas de Amplificação de Ácido Nucleico , Análise de Componente Principal
4.
Mol Cell ; 40(4): 521-32, 2010 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-21095583

RESUMO

The versatile cytokine transforming growth factor ß (TGF-ß) regulates cellular growth, differentiation, and migration during embryonic development and adult tissue homeostasis. Activation of TGF-ß receptors leads to phosphorylation of Smad2 and Smad3, which oligomerize with Smad4 and accumulate in the nucleus where they recognize gene regulatory regions and orchestrate transcription. Termination of Smad-activated transcription involves Smad dephosphorylation, nuclear export, or ubiquitin-mediated degradation. In an unbiased proteomic screen, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a Smad-interacting partner. PARP-1 dissociates Smad complexes from DNA by ADP-ribosylating Smad3 and Smad4, which attenuates Smad-specific gene responses and TGF-ß-induced epithelial-mesenchymal transition. Thus, our results identify ADP-ribosylation of Smad proteins by PARP-1 as a key step in controlling the strength and duration of Smad-mediated transcription.


Assuntos
Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Smad/metabolismo , Transcrição Gênica , Fatores de Ribosilação do ADP/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , DNA/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Complexos Multiproteicos/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia
5.
Nucleic Acids Res ; 43(7): e45, 2015 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-25586224

RESUMO

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms.


Assuntos
Alelos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Dependovirus/genética , Interferência de RNA , Sequência de Bases , Proteína Quinase CDC2 , Linhagem Celular , Quinase 2 Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , Primers do DNA , Técnicas de Silenciamento de Genes , Humanos , Mutação , Reação em Cadeia da Polimerase , Recombinação Genética
6.
J Biol Chem ; 287(16): 12867-78, 2012 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-22378783

RESUMO

Transforming growth factor ß (TGFß) regulates many physiological processes and requires control mechanisms to safeguard proper and timely action. We have previously described how negative regulation of TGFß signaling is controlled by the serine/threonine kinase salt-inducible kinase 1 (SIK1). SIK1 forms complexes with the TGFß type I receptor and with the inhibitory Smad7 and down-regulates the type I receptor. We now demonstrate that TGFß induces SIK1 levels via a direct transcriptional mechanism that implicates the Smad proteins, and we have mapped a putative enhancer element on the SIK1 gene. We provide evidence that the ubiquitin ligase Smurf2 forms complexes and functionally cooperates with SIK1. Both the kinase activity of SIK1 and the ubiquitin ligase activity of Smurf2 are important for proper type I receptor turnover. We also show that knockdown of endogenous SIK1 and Smurf2 enhances physiological signaling by TGFß that leads to epithelial growth arrest. In conclusion, TGFß induces expression of Smad7, Smurf2, and SIK1, the products of which physically and functionally interlink to control the activity of this pathway.


Assuntos
Proteínas Serina-Treonina Quinases/genética , Ativação Transcricional/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Neoplasias da Mama , Células COS , Linhagem Celular Transformada , Chlorocebus aethiops , Regulação para Baixo/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Queratinócitos/citologia , Vison , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Mucosa Respiratória/citologia , Proteína Smad7/genética , Proteína Smad7/metabolismo
7.
Commun Biol ; 4(1): 1284, 2021 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-34773084

RESUMO

Protein interactions and posttranslational modifications orchestrate cellular responses to e.g. cytokines and drugs, but it has been difficult to monitor these dynamic events in high-throughput. Here, we describe a semi-automated system for large-scale in situ proximity ligation assays (isPLA), combining isPLA in microtiter wells with automated microscopy and computer-based image analysis. Phosphorylations and interactions are digitally recorded along with subcellular morphological features. We investigated TGF-ß-responsive Smad2 linker phosphorylations and complex formations over time and across millions of individual cells, and we relate these events to cell cycle progression and local cell crowding via measurements of DNA content and nuclear size of individual cells, and of their relative positions. We illustrate the suitability of this protocol to screen for drug effects using phosphatase inhibitors. Our approach expands the scope for image-based single cell analyses by combining observations of protein interactions and modifications with morphological details of individual cells at high throughput.


Assuntos
Processamento de Imagem Assistida por Computador , Mapeamento de Interação de Proteínas , Proteína Smad4/genética , Fator de Crescimento Transformador beta1/genética , Células HaCaT , Humanos , Fosforilação , Análise de Célula Única , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
9.
N Biotechnol ; 45: 51-59, 2018 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-29101055

RESUMO

Post-translational modifications (PTMs) regulate protein activities to help orchestrate and fine-tune cellular processes. Dysregulation of PTMs is often related with disorders and malignancies, and may serve as a precise biomarker of disease. Developing sensitive tools to measure and monitor low-abundant PTMs in tissue lysates or serum will be instrumental for opening up new PTM-based diagnostic avenues. Here, we investigate the use of solid-phase proximity ligation assay (SP-PLA) for detection of different PTMs. The assay depends on the recognition of the target protein molecule and its modification by three affinity binders. Using antibodies and lectins, we applied the method for detection of glycosylated CD44 and E-Cadherin, and phosphorylated p53 and EGFR. The assay was found to have superior dynamic range and limit of detection compared to standard ELISAs. In summary, we have established the use of SP-PLA as an appropriate method for sensitive detection of PTMs in lysates and sera, which may provide a basis for future PTM-based diagnostic and prognostic biomarkers.


Assuntos
Receptores ErbB/metabolismo , Imunoensaio , Neoplasias Gástricas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Idoso , Linhagem Celular , Receptores ErbB/análise , Feminino , Humanos , Receptores de Hialuronatos/análise , Receptores de Hialuronatos/metabolismo , Masculino , Processamento de Proteína Pós-Traducional , Neoplasias Gástricas/diagnóstico , Proteína Supressora de Tumor p53/análise
10.
Sci Rep ; 6: 32301, 2016 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-27604151

RESUMO

Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells.


Assuntos
Produtos Biológicos/metabolismo , Peptídeos Penetradores de Células/metabolismo , Endossomos/metabolismo , Substâncias Macromoleculares/metabolismo , Sítios de Ligação/genética , Produtos Biológicos/administração & dosagem , Linhagem Celular , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/genética , Sistemas de Liberação de Medicamentos/métodos , Endocitose , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células MCF-7 , Substâncias Macromoleculares/administração & dosagem , Microscopia de Fluorescência , Peptídeos/administração & dosagem , Peptídeos/genética , Peptídeos/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Imagem com Lapso de Tempo/métodos
11.
Expert Opin Drug Deliv ; 12(10): 1627-36, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25994800

RESUMO

INTRODUCTION: Macromolecular therapeutics, including enzymes, transcription factors, siRNAs, peptides and large synthetic molecules, can potentially be used to treat human diseases by targeting intracellular molecular pathways and modulating biological responses. However, large macromolecules have no ability to enter cells and require delivery vehicles. Protein transduction domains (PTDs), also known as cell-penetrating peptides (CPPs), are a diverse class of peptides that can deliver macromolecules into cells. AREAS COVERED: In this review, we cover the uptake and usage of arginine-rich PTDs/CPPs (TAT-PTD, Penetratin/Antp and 8R). We review the endocytosis-mediated uptake of these peptides and highlight three important steps: i) cell association; ii) internalization and iii) endosomal escape. We also discuss the array of different cargos that have been delivered by cationic PTDs/CPPs as well as cellular processes and biological responses that have been modulated. EXPERT OPINION: PTDs/CPPs have shown great potential to deliver otherwise undeliverable macromolecular therapeutics into cells for experimentation in cell culture and in animal disease models in vivo. Moreover, over 25 clinical trials have been performed predominantly using the TAT-PTD. However, more work is still needed. Endosomal escape and target-cell specificity remain two of the major future challenges.


Assuntos
Peptídeos Penetradores de Células/química , Sistemas de Liberação de Medicamentos , Substâncias Macromoleculares/administração & dosagem , Animais , Proteínas de Transporte , Cátions , Endocitose/fisiologia , Endossomos/metabolismo , Humanos , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo
12.
Chem Commun (Camb) ; 51(9): 1662-5, 2015 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-25500944

RESUMO

Singly and multiply modified synthetic siRNA oligonucleotides, containing isomorphic surrogate nucleobases, show high interference potency in cell culture, suggesting the highly isomorphic RNA alphabet, based on a thieno[3,4-d]-pyrimidine core, is tolerated well by the cellular silencing machinery.


Assuntos
Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Estrutura Molecular , RNA Interferente Pequeno/genética
13.
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
14.
J Exp Med ; 212(6): 833-43, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25987724

RESUMO

NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis.


Assuntos
Regulação Leucêmica da Expressão Gênica , Quinase I-kappa B/fisiologia , Leucemia Linfocítica Crônica de Células B/metabolismo , NF-kappa B/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular , Aberrações Cromossômicas , Estudos de Coortes , Citoplasma/metabolismo , Análise Mutacional de DNA , Mutação da Fase de Leitura , Deleção de Genes , Perfilação da Expressão Gênica , Humanos , Quinase I-kappa B/genética , Leucemia Linfocítica Crônica de Células B/genética , Linfoma de Células B/metabolismo , Linfoma de Zona Marginal Tipo Células B/metabolismo , Linfoma de Célula do Manto/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Resultado do Tratamento
15.
PLoS One ; 9(8): e103651, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25133494

RESUMO

BACKGROUND: Initiation, amplitude, duration and termination of transforming growth factor ß (TGFß) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFß signaling. METHODS: A robust cell model of TGFß signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFß stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference. RESULTS: TGFß signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFß target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFß-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1. CONCLUSION: Nuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGFß signaling.


Assuntos
Glicosídeo Hidrolases/fisiologia , Poli(ADP-Ribose) Polimerases/fisiologia , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Poli(ADP-Ribose) Polimerase-1 , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad7/genética , Proteína Smad7/metabolismo , Transcrição Gênica
16.
Nat Biotechnol ; 32(12): 1256-61, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25402614

RESUMO

RNA interference (RNAi) has great potential to treat human disease. However, in vivo delivery of short interfering RNAs (siRNAs), which are negatively charged double-stranded RNA macromolecules, remains a major hurdle. Current siRNA delivery has begun to move away from large lipid and synthetic nanoparticles to more defined molecular conjugates. Here we address this issue by synthesis of short interfering ribonucleic neutrals (siRNNs) whose phosphate backbone contains neutral phosphotriester groups, allowing for delivery into cells. Once inside cells, siRNNs are converted by cytoplasmic thioesterases into native, charged phosphodiester-backbone siRNAs, which induce robust RNAi responses. siRNNs have favorable drug-like properties, including high synthetic yields, serum stability and absence of innate immune responses. Unlike siRNAs, siRNNs avidly bind serum albumin to positively influence pharmacokinetic properties. Systemic delivery of siRNNs conjugated to a hepatocyte-specific targeting domain induced extended dose-dependent in vivo RNAi responses in mice. We believe that siRNNs represent a technology that will open new avenues for development of RNAi therapeutics.


Assuntos
Sistemas de Liberação de Medicamentos , Pró-Fármacos/uso terapêutico , RNA Interferente Pequeno/uso terapêutico , Animais , Humanos , Camundongos , Nanopartículas/química , Nanopartículas/uso terapêutico , Pró-Fármacos/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Albumina Sérica/química
18.
Cell Res ; 19(1): 21-35, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19030025

RESUMO

Transforming growth factor beta (TGFbeta) controls cellular behavior in embryonic and adult tissues. TGFbeta binding to serine/threonine kinase receptors on the plasma membrane activates Smad molecules and additional signaling proteins that together regulate gene expression. In this review, mechanisms and models that aim at explaining the coordination between several components of the signaling network downstream of TGFbeta are presented. We discuss how the activity and duration of TGFbeta receptor/Smad signaling can be regulated by post-translational modifications that affect the stability of key proteins in the pathway. We highlight links between these mechanisms and human diseases, such as tissue fibrosis and cancer.


Assuntos
Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Humanos , Estabilidade Proteica , Transdução de Sinais , Proteínas Smad Inibidoras/metabolismo , Proteína Smad4/metabolismo
19.
J Cell Biol ; 182(4): 655-62, 2008 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-18725536

RESUMO

Signal transduction by transforming growth factor beta (TGFbeta) coordinates physiological responses in diverse cell types. TGFbeta signals via type I and type II receptor serine/threonine kinases and intracellular Smad proteins that regulate transcription. Strength and duration of TGFbeta signaling is largely dependent on a negative-feedback program initiated during signal progression. We have identified an inducible gene target of TGFbeta/Smad signaling, the salt-inducible kinase (SIK), which negatively regulates signaling together with Smad7. SIK and Smad7 form a complex and cooperate to down-regulate the activated type I receptor ALK5. We further show that both the kinase and ubiquitin-associated domain of SIK are required for proper ALK5 degradation, with ubiquitin functioning to enhance SIK-mediated receptor degradation. Loss of endogenous SIK results in enhanced gene responses of the fibrotic and cytostatic programs of TGFbeta. We thus identify in SIK a negative regulator that controls TGFbeta receptor turnover and physiological signaling.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Animais , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Humanos , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Receptor do Fator de Crescimento Transformador beta Tipo I , Proteína Smad7/metabolismo , Ubiquitina/metabolismo
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