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1.
Nat Commun ; 11(1): 605, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001718

RESUMO

Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets.


Assuntos
Optogenética , Proteínas/metabolismo , Actinas/metabolismo , Movimento Celular/efeitos da radiação , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Luz , Engenharia de Proteínas
2.
iScience ; 9: 63-70, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30384134

RESUMO

Actin has been linked to processes spanning the whole gene expression cascade, from regulating specific transcription factors, such as myocardin-related transcription factor, to chromatin remodeling and RNA polymerase function. However, whether actin controls the transcription of only specific genes or has a global role in gene expression has remained elusive. Our genome-wide analysis reveals, for the first time, that actin interacts with essentially all transcribed genes in Drosophila ovaries. Actin co-occupies the majority of gene promoters together with Pol II, and on highly expressed genes, these two proteins also associate with gene bodies. Mechanistically, actin is required for Pol II recruitment to gene bodies, and manipulation of nuclear transport factors for actin leads to the decreased expression of eggshell genes. Collectively, these results uncover a global role for actin in transcription and demonstrate the in vivo importance of balanced nucleocytoplasmic shuttling of actin in the transcriptional control of a developmental process.

3.
Nat Cell Biol ; 19(12): 1386-1388, 2017 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-29184179

RESUMO

After mitosis, the nucleus must be rebuilt and chromatin decondensed to permit interphase genomic functions, but decondensation mechanisms are poorly understood. Now, the traditional cytoskeletal protein actin is shown to form transient nuclear filaments that are required for chromatin decondensation and nuclear expansion at mitotic exit.


Assuntos
Actinas/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Interfase/fisiologia , Mitose/fisiologia , Modelos Biológicos
4.
Mol Cancer Ther ; 13(11): 2537-46, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25277384

RESUMO

Activation of the p53 pathway has been considered a therapeutic strategy to target cancers. We have previously identified several p53-activating small molecules in a cell-based screen. Two of the compounds activated p53 by causing DNA damage, but this modality was absent in the other four. We recently showed that one of these, BMH-21, inhibits RNA polymerase I (Pol I) transcription, causes the degradation of Pol I catalytic subunit RPA194, and has potent anticancer activity. We show here that three remaining compounds in this screen, BMH-9, BMH-22, and BMH-23, cause reorganization of nucleolar marker proteins consistent with segregation of the nucleolus, a hallmark of Pol I transcription stress. Further, the compounds destabilize RPA194 in a proteasome-dependent manner and inhibit nascent rRNA synthesis and expression of the 45S rRNA precursor. BMH-9- and BMH-22-mediated nucleolar stress was detected in ex vivo-cultured human prostate tissues indicating good tissue bioactivity. Testing of closely related analogues showed that their activities were chemically constrained. Viability screen for BMH-9, BMH-22, and BMH-23 in the NCI60 cancer cell lines showed potent anticancer activity across many tumor types. Finally, we show that the Pol I transcription stress by BMH-9, BMH-22, and BMH-23 is independent of p53 function. These results highlight the dominant impact of Pol I transcription stress on p53 pathway activation and bring forward chemically novel lead molecules for Pol I inhibition, and, potentially, cancer targeting.


Assuntos
Nucléolo Celular/efeitos dos fármacos , RNA Polimerase I/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Células HCT116 , Humanos , Masculino , Melanoma/tratamento farmacológico , Osteossarcoma/tratamento farmacológico , Próstata/efeitos dos fármacos
5.
Cancer Cell ; 25(1): 77-90, 2014 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-24434211

RESUMO

We define the activity and mechanisms of action of a small molecule lead compound for cancer targeting. We show that the compound, BMH-21, has wide and potent antitumorigenic activity across NCI60 cancer cell lines and represses tumor growth in vivo. BMH-21 binds GC-rich sequences, which are present at a high frequency in ribosomal DNA genes, and potently and rapidly represses RNA polymerase I (Pol I) transcription. Strikingly, we find that BMH-21 causes proteasome-dependent destruction of RPA194, the large catalytic subunit protein of Pol I holocomplex, and this correlates with cancer cell killing. Our results show that Pol I activity is under proteasome-mediated control, which reveals an unexpected therapeutic opportunity.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , RNA Polimerase I/efeitos dos fármacos , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Nucleus ; 4(4): 315-25, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23782956

RESUMO

CRM1 is an export factor that together with its adaptor NMD3 transports numerous cargo molecules from the nucleus to cytoplasm through the nuclear pore. Previous studies have suggested that CRM1 and NMD3 are detected in the nucleolus. However, their localization with subnucleolar domains or participation in the activities of the nucleolus are unclear. We demonstrate here biochemically and using imaging analyses that CRM1 and NMD3 co-localize with nucleolar marker proteins in the nucleolus. In particular, their nucleolar localization is markedly increased by inhibition of RNA polymerase I (Pol I) transcription by actinomycin D or by silencing Pol I catalytic subunit, RPA194. We show that CRM1 nucleolar localization is dependent on its activity and the expression of NMD3, whereas NMD3 nucleolar localization is independent of CRM1. This suggests that NMD3 provides nucleolar tethering of CRM1. While inhibition of CRM1 by leptomycin B inhibited processing of 28S ribosomal (r) RNA, depletion of NMD3 did not, suggesting that their effects on 28S rRNA processing are distinct. Markedly, depletion of NMD3 and inhibition of CRM1 reduced the rate of pre-47S rRNA synthesis. However, their inactivation did not lead to nucleolar disintegration, a hallmark of Pol I transcription stress, suggesting that they do not directly regulate transcription. These results indicate that CRM1 and NMD3 have complex functions in pathways that couple rRNA synthetic and processing engines and that the rRNA synthesis rate may be adjusted according to proficiency in rRNA processing and export.


Assuntos
Nucléolo Celular/metabolismo , Carioferinas/metabolismo , RNA Ribossômico/biossíntese , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Células HeLa , Humanos , Células Tumorais Cultivadas
7.
PLoS One ; 8(3): e59096, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23554979

RESUMO

UV damage activates cellular stress signaling pathways, causes DNA helix distortions and inhibits transcription by RNA polymerases I and II. In particular, the nucleolus, which is the site of RNA polymerase I transcription and ribosome biogenesis, disintegrates following UV damage. The disintegration is characterized by reorganization of the subnucleolar structures and change of localization of many nucleolar proteins. Here we have queried the basis of localization change of nucleophosmin (NPM), a nucleolar granular component protein, which is increasingly detected in the nucleoplasm following UV radiation. Using photobleaching experiments of NPM-fluorescent fusion protein in live human cells we show that NPM mobility increases after UV damage. However, we show that the increase in NPM nucleoplasmic abundance after UV is independent of UV-activated cellular stress and DNA damage signaling pathways. Unexpectedly, we find that proteasome activity affects NPM redistribution. NPM nucleolar expression was maintained when the UV-treated cells were exposed to proteasome inhibitors or when the expression of proteasome subunits was inhibited using RNAi. However, there was no evidence of increased NPM turnover in the UV damaged cells, or that ubiquitin or ubiquitin recycling affected NPM localization. These findings suggest that proteasome activity couples to nucleolar protein localizations in UV damage stress.


Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular , Nucléolo Celular/metabolismo , Humanos , Inibidores de Proteassoma/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/efeitos da radiação , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos da radiação , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Estresse Fisiológico , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/efeitos da radiação , Ubiquitina/metabolismo , Raios Ultravioleta/efeitos adversos
8.
J Cell Physiol ; 227(6): 2605-12, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21882188

RESUMO

Transforming growth factor-ß (TGF-ß) is a diverse cytokine regulating growth, apoptosis, differentiation, adhesion, invasion, and extracellular matrix production. Dysregulation of TGF-ß is associated with fibrotic disorders and epithelial-mesenchymal transition, and has been linked with idiopathic pulmonary fibrosis (IPF). Cysteine-rich protein 1 (CRP1) is a small LIM-domain containing protein involved in smooth muscle differentiation. Here, we show that TGF-ß1 increases the expression of CRP1 protein and that CRP1 levels increase in a biphasic fashion. A rapid transient (15-45 min) increase in CRP1 is followed by a subsequent, sustained increase in CRP1 a few hours afterwards that lasts several days. We find that TGF-ß1 regulates the expression of CRP1 through Smad and non-conventional p38 MAPK signaling pathways in a transcription-independent manner and that the induction occurs concomitant with an increase in myofibroblast differentiation. Using CRP1 silencing by shRNA, we identify CRP1 as a novel factor mediating cell contractility. Furthermore, we localize CRP1 to fibroblastic foci in IPF lungs and find that CRP1 is significantly more expressed in IPF as compared to control lung tissue. The results show that CRP1 is a novel TGF-ß1 regulated protein that is expressed in fibrotic lesions and may be relevant in the IPF disease.


Assuntos
Proteínas de Transporte/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Proteínas com Domínio LIM/metabolismo , Pulmão/metabolismo , Miofibroblastos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Proteínas de Transporte/genética , Estudos de Casos e Controles , Diferenciação Celular , Linhagem Celular Tumoral , Forma Celular , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Proteínas com Domínio LIM/genética , Pulmão/patologia , Camundongos , Miofibroblastos/patologia , Células NIH 3T3 , Interferência de RNA , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad2/metabolismo , Fatores de Tempo , Transfecção , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
Mol Cell Proteomics ; 10(10): M111.009241, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21778410

RESUMO

The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner.


Assuntos
Nucléolo Celular/metabolismo , Dano ao DNA , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Proteoma/análise , Antígenos Nucleares/análise , Antígenos Nucleares/metabolismo , Nucléolo Celular/efeitos da radiação , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Exossomos/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Marcação por Isótopo , Autoantígeno Ku , Microscopia Eletrônica de Transmissão , Proteínas Nucleares/genética , Proteoma/genética , Proteoma/metabolismo , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/metabolismo , Radiação Ionizante , Estresse Fisiológico , Transcrição Genética , Raios Ultravioleta
10.
PLoS One ; 5(9): e12996, 2010 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-20885994

RESUMO

Manipulation of the activity of the p53 tumor suppressor pathway has demonstrated potential benefit in preclinical mouse tumor models and has entered human clinical trials. We describe here an improved, extensive small-molecule chemical compound library screen for p53 pathway activation in a human cancer cell line devised to identify hits with potent antitumor activity. We uncover six novel small-molecule lead compounds, which activate p53 and repress the growth of human cancer cells. Two tested compounds suppress in vivo tumor growth in an orthotopic mouse model of human B-cell lymphoma. All compounds interact with DNA, and two activate p53 pathway in a DNA damage signaling-dependent manner. A further screen of a drug library of approved drugs for medicinal uses and analysis of gene-expression signatures of the novel compounds revealed similarities to known DNA intercalating and topoisomerase interfering agents and unexpected connectivities to known drugs without previously demonstrated anticancer activities. These included several neuroleptics, glycosides, antihistamines and adrenoreceptor antagonists. This unbiased screen pinpoints interference with the DNA topology as the predominant mean of pharmacological activation of the p53 pathway and identifies potential novel antitumor agents.


Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/fisiopatologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Proteína Supressora de Tumor p53/genética
11.
Photodermatol Photoimmunol Photomed ; 26(2): 70-7, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20415737

RESUMO

BACKGROUND: Cysteine-rich protein 1 (CRP1) is a growth-inhibitory cytoskeletal protein that is induced by ultraviolet (UV) C radiation radiation in fibroblasts. Our aim was to investigate the effects of UV radiation on CRP1 in keratinocytes, the main cell type subjected to UV radiation in the human body. METHODS: The effects of physiologically relevant doses of UVB radiation on CRP1 protein levels were studied in cultured primary keratinocytes and transformed cell lines (HaCaT, A-431) by immunoblotting. UVB-induced keratinocyte apoptosis was assessed by flow cytometry and monitoring caspase activity. Expression of CRP1 in human skin in vivo was studied by immunohistochemistry in samples of normal skin, actinic keratosis (AK) representing UV-damaged skin and squamous cell carcinoma (SCC), a UV-induced skin cancer. RESULTS: CRP1 expression increased by UVB radiation in primary but not in immortalized keratinocytes. Upon high, apoptosis-inducing doses of UV radiation, CRP1 was cleaved in a caspase-dependent manner. In normal skin, CRP1 was expressed in smooth muscle cells, vasculature, sweat glands, sebaceous glands and hair root sheath, but very little CRP1 was present in keratinocytes. CRP1 expression was elevated in basal cells in AK but not in SCC. CONCLUSION: CRP1 expression is regulated by UVB in human keratinocytes, suggesting a role for CRP1 in the phototoxic responses of human skin.


Assuntos
Apoptose/efeitos da radiação , Queratinócitos/metabolismo , Proteínas Nucleares/biossíntese , Pele/metabolismo , Raios Ultravioleta/efeitos adversos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Caspases/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Feminino , Humanos , Queratinócitos/patologia , Ceratose Actínica/metabolismo , Ceratose Actínica/patologia , Masculino , Pessoa de Meia-Idade , Pele/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
12.
PLoS Pathog ; 6(3): e1000818, 2010 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-20333249

RESUMO

Nucleophosmin (NPM) is a multifunctional nuclear phosphoprotein and a histone chaperone implicated in chromatin organization and transcription control. Oncogenic Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). In the infected host cell KSHV displays two modes of infection, the latency and productive viral replication phases, involving extensive viral DNA replication and gene expression. A sustained balance between latency and reactivation to the productive infection state is essential for viral persistence and KSHV pathogenesis. Our study demonstrates that the KSHV v-cyclin and cellular CDK6 kinase phosphorylate NPM on threonine 199 (Thr199) in de novo and naturally KSHV-infected cells and that NPM is phosphorylated to the same site in primary KS tumors. Furthermore, v-cyclin-mediated phosphorylation of NPM engages the interaction between NPM and the latency-associated nuclear antigen LANA, a KSHV-encoded repressor of viral lytic replication. Strikingly, depletion of NPM in PEL cells leads to viral reactivation, and production of new infectious virus particles. Moreover, the phosphorylation of NPM negatively correlates with the level of spontaneous viral reactivation in PEL cells. This work demonstrates that NPM is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA.


Assuntos
Quinase 6 Dependente de Ciclina/metabolismo , Herpesvirus Humano 8/crescimento & desenvolvimento , Proteínas Nucleares/metabolismo , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/virologia , Latência Viral/fisiologia , Acetilação , Antígenos Virais/genética , Antígenos Virais/metabolismo , Linhagem Celular Tumoral , Herpesvirus Humano 8/genética , Humanos , Proteínas Nucleares/genética , Fosforilação/fisiologia , RNA Interferente Pequeno , Treonina/metabolismo , Replicação Viral/fisiologia
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