RESUMO
Expression of hyperactive RAF kinases, such as the oncogenic B-RAF-V600E mutant, in normal human cells triggers a proliferative arrest that blocks tumor formation. We discovered that glucocorticoids delayed the entry into senescence induced by B-RAF-V600E in human fibroblasts, and allowed senescence bypass when the cells were regularly passaged, but that they did not allow proliferation of cells that were already senescent. Transcriptome and siRNA analyses revealed that the EGR1 gene is one target of glucocorticoid action. Transcription of the EGR1 gene is activated by the RAF-MEK-ERK MAPK pathway and acts as a sensor of hyper-mitogenic pathway activity. The EGR1 transcription factor regulates the expression of p15 and p21 (encoded by CDKN2B and CDKN1A, respectively) that are redundantly required for the proliferative arrest of BJ fibroblasts upon expression of B-RAF-V600E. Our results highlight the need to evaluate the action of glucocorticoid on cancer progression in melanoma, thyroid and colon carcinoma in which B-RAF-V600E is a frequent oncogene, and cancers in which evasion from senescence has been shown.
Assuntos
Senescência Celular/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fibroblastos/metabolismo , Glucocorticoides/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Substituição de Aminoácidos , Linhagem Celular , Senescência Celular/genética , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor de Quinase Dependente de Ciclina p21 , Proteína 1 de Resposta de Crescimento Precoce/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
Irreparable double-strand breaks (DSBs) in response to ionizing radiation (IR) trigger prolonged DNA damage response (DDR) and induce premature senescence. Profound chromatin reorganization with formation of senescence-associated heterochromatin foci (SAHF) is an essential epigenetic mechanism for controlling the senescence-associated secretory phenotype (SASP). To decipher molecular mechanisms provoking continuous DDR leading to premature senescence, radiation-induced DSBs (53BP1-foci) and dynamics of histone variant H2A.J incorporation were analyzed together with chromatin re-modeling in human fibroblasts after IR exposure. High-resolution imaging by transmission electron microscopy revealed that persisting 53BP1-foci developed into DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS), consistently located at the periphery of SAHFs. Quantitative immunogold-analysis by electron microscopy revealed that H2A.J, steadily co-localizing with 53BP1, is increasingly incorporated into DNA-SCARS during senescence progression. Strikingly, shRNA-mediated H2A.J depletion in fibroblasts modified senescence-associated chromatin re-structuring and abolished SASP, thereby shutting down the production of inflammatory mediators. These findings provide mechanistic insights into biological phenomena of SASP and suggest that H2A.J inhibition could ablate SASP, without affecting the senescence-associated growth arrest.
Assuntos
Senescência Celular/efeitos da radiação , Dano ao DNA , Variação Genética , Histonas/biossíntese , Histonas/genética , Radiação Ionizante , Epigênese Genética , Heterocromatina/genética , Heterocromatina/metabolismo , Heterocromatina/ultraestrutura , HumanosRESUMO
The histone chaperone Asf1 and the checkpoint kinase Rad53 are found in a complex in budding yeast cells in the absence of genotoxic stress. Our data suggest that this complex involves at least three interaction sites. One site involves the H3-binding surface of Asf11 with an as yet undefined surface of Rad53. A second site is formed by the Rad53-FHA1 domain binding to Asf1-T(270) phosphorylated by casein kinase II. The third site involves the C-terminal 21 amino acids of Rad53 bound to the conserved Asf1 N-terminal domain. The structure of this site showed that the Rad53 C-terminus binds Asf1 in a remarkably similar manner to peptides derived from the histone cochaperones HirA and CAF-I. We call this binding motif, (R/K)R(I/A/V) (L/P), the AIP box for Asf1-Interacting Protein box. Furthermore, C-terminal Rad53-F(820) binds the same pocket of Asf1 as does histone H4-F(100). Thus Rad53 competes with histones H3-H4 and cochaperones HirA/CAF-I for binding to Asf1. Rad53 is phosphorylated and activated upon genotoxic stress. The Asf1-Rad53 complex dissociated when cells were treated with hydroxyurea but not methyl-methane-sulfonate, suggesting a regulation of the complex as a function of the stress. We identified a rad53 mutation that destabilized the Asf1-Rad53 complex and increased the viability of rad9 and rad24 mutants in conditions of genotoxic stress, suggesting that complex stability impacts the DNA damage response.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperonas de Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Proteínas de Ciclo Celular/química , Quinase do Ponto de Checagem 2 , Cristalografia por Raios X , Dano ao DNA , Histonas/metabolismo , Hidroxiureia/farmacologia , Modelos Moleculares , Chaperonas Moleculares/química , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Peptídeos/química , Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/química , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
PURPOSE: Histone variant H2A.J is associated with premature senescence after ionizing radiation (IR) and modulates senescence-associated secretory phenotype (SASP). Using constitutive H2A.J knock-out mice, the role of H2A.J was investigated in radiation dermatitis. METHODS AND MATERIALS: H2A.J wild-type (WT) and knock-out (KO) mice were exposed to moderate or high IR doses (≤20 Gy, skinfold IR). Radiation-induced skin reactions were investigated up to 2 weeks post-IR at macroscopic and microscopic levels. H2A.J and other senescence markers, as well as DNA damage and proliferation markers, were studied by immunohistochemistry, immunofluorescence, and electron microscopy. After high-dose IR, protein-coding transcriptomes were analyzed by RNA sequencing, immune cell infiltration by flow cytometry, and gene expression by reverse transcription polymerase chain reaction in (non-) irradiated WT versus KO skin. RESULTS: In WT skin, epidermal keratinocytes showed time- and dose-dependent H2A.J accumulation after IR exposure. Unexpectedly, stronger inflammatory reactions with increased epidermal thickness and progressive hair follicle loss were observed in irradiated KO versus WT skin. Clearly more radiation-induced senescence was observed in keratinocyte populations of KO skin after moderate and high doses, with hair follicle stem cells being particularly badly damaged, leading to follicle atrophy. After high-dose IR, transcriptomic analysis revealed enhanced senescence-associated signatures in irradiated KO skin, with intensified release of SASP factors. Flow cytometric analysis indicated increased immune cell infiltration in both WT and KO skin; however, specific chemokine-mediated signaling in irradiated KO skin led to more neutrophil recruitment, thereby aggravating radiation toxicities. Increased skin damage in irradiated KO skin led to hyperproliferation, abnormal differentiation, and cornification of keratinocytes, accompanied by increased upregulation of transcription-factor JunB. CONCLUSIONS: Lack of radiation-induced H2A.J expression in keratinocytes is associated with increased senescence induction, modulation of SASP expression, and exacerbated inflammatory skin reactions. Hence, epigenetic H2A.J-mediated gene expression in response to IR regulates keratinocyte immune functions and plays an essential role in balancing the inflammatory response during radiation dermatitis.
Assuntos
Histonas , Radiodermite , Animais , Camundongos , Histonas/metabolismo , Pele/efeitos da radiação , Queratinócitos/fisiologia , Radiação Ionizante , Senescência Celular/efeitos da radiaçãoRESUMO
PURPOSE: Radiation-induced senescence is characterized by profound changes in chromatin organization with the formation of Senescence-Associated-Heterochromatin-Foci (SAHF) and DNA-Segments-with-Chromatin-Alterations-Reinforcing-Senescence (DNA-SCARS). Importantly, senescent cells also secrete complex combinations of pro-inflammatory factors, referred as Senescence-Associated-Secretory-Phenotype (SASP). Here, we analyzed the epigenetic mechanism of histone variant H2A.J in establishing radiation-induced senescence. EXPERIMENTAL DESIGN: Primary and genetically-modified lung fibroblasts with down- or up-regulated H2A.J expression were exposed to ionizing radiation and were analyzed for the formation of SAHF and DNA-SCARS by immunofluorescence microscopy. Dynamic changes in chromatin organization and accessibility, transcription factor recruitment, and transcriptome signatures were mapped by ATAC-seq and RNA-seq analysis. The secretion of SASP factors and potential bystander effects were analyzed by ELISA and RT-PCR. Lung tissue of mice exposed to different doses were analyzed by the digital image analysis of H2A.J-immunohistochemistry. RESULTS: Differential incorporation of H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of senescence. Integrative analyses of ATAC-seq and RNA-seq datasets indicate that H2A.J-associated changes in chromatin accessibility of regulatory regions decisively modulates transcription factor recruitment and inflammatory gene expression, resulting in an altered SASP secretome. In lung parenchyma, pneumocytes show dose-dependent H2A.J expression in response to radiation-induced DNA damage, therefore contributing to pro-inflammatory tissue reactions. CONCLUSIONS: The fine-tuned incorporation of H2A.J defines the epigenetic landscape for driving the senescence programme in response to radiation-induced DNA damage. Deregulated H2A.J deposition affects chromatin remodeling, transcription factor recruitment, and the pro-inflammatory secretome. Our findings provide new mechanistic insights into DNA-damage triggered epigenetic mechanisms governing the biological processes of radiation-induced injury.
Assuntos
Cicatriz , Histonas , Animais , Camundongos , Histonas/metabolismo , Cromatina , Heterocromatina , Fatores de Transcrição/metabolismo , Radiação IonizanteRESUMO
Senescence is a cellular stress response that involves prolonged cell survival, a quasi-irreversible proliferative arrest and a modification of the transcriptome that sometimes includes inflammatory gene expression. Senescent cells are resistant to apoptosis, and if not eliminated by the immune system they may accumulate and lead to chronic inflammation and tissue dysfunction. Senolytics are drugs that selectively induce cell death in senescent cells, but not in proliferative or quiescent cells, and they have proved a viable therapeutic approach in multiple mouse models of pathologies in which senescence is implicated. As the catalogue of senolytic compounds is expanding, novel survival strategies of senescent cells are uncovered, and variations in sensitivity to senolysis between different types of senescent cells emerge. We propose herein a mechanistic classification of senolytic drugs, based on the level at which they target senescent cells: directly disrupting BH3 protein networks that are reorganized upon senescence induction; downregulating survival-associated pathways essential to senescent cells; or modulating homeostatic processes whose regulation is challenged in senescence. With this approach, we highlight the important diversity of senescent cells in terms of physiology and pathways of apoptosis suppression, and we describe possible avenues for the development of more selective senolytics.
Assuntos
Senescência Celular , Senoterapia , Animais , Apoptose , Sobrevivência Celular , Modelos Animais de Doenças , CamundongosRESUMO
Aging-related diseases such as cancer can be traced to the accumulation of molecular disorder including increased DNA mutations and epigenetic drift. We provide a comprehensive review of recent results in mice and humans on modifications of DNA methylation and histone variants during aging and in cancer. Accumulated errors in DNA methylation maintenance lead to global decreases in DNA methylation with relaxed repression of repeated DNA and focal hypermethylation blocking the expression of tumor suppressor genes. Epigenetic clocks based on quantifying levels of DNA methylation at specific genomic sites is proving to be a valuable metric for estimating the biological age of individuals. Histone variants have specialized functions in transcriptional regulation and genome stability. Their concentration tends to increase in aged post-mitotic chromatin, but their effects in cancer are mainly determined by their specialized functions. Our increased understanding of epigenetic regulation and their modifications during aging has motivated interventions to delay or reverse epigenetic modifications using the epigenetic clocks as a rapid readout for efficacity. Similarly, the knowledge of epigenetic modifications in cancer is suggesting new approaches to target these modifications for cancer therapy.
Assuntos
Envelhecimento/genética , Metilação de DNA/genética , Histonas/genética , Mutação/genética , Neoplasias/genética , Sequência de Aminoácidos , Animais , Epigênese Genética , Histonas/química , HumanosRESUMO
Cellular senescence is a state of terminal proliferative arrest that plays key roles in aging by preventing stem cell renewal and by inducing the expression of a series of inflammatory factors including many secreted proteins with paracrine effects. The in vivo identification of senescent cells is difficult due to the absence of universal biomarkers. Chromatin modifications are key aspects of the senescence transition and may provide novel biomarkers. We used a combined protein profiling and bottom-up mass spectrometry approach to characterize the isoforms and post-translational modifications of chromatin proteins over time in post-mitotic human fibroblasts in vitro. We show that the H2B type 1-K variant is specifically enriched in deep senescent cells with persistent DNA damage. This accumulation was not observed in quiescent cells or in cells induced into senescence without DNA damage by expression of the RAF kinase. Similarly, HMGA1a di-methylated and HMGA1b tri-phosphorylated forms accumulated exclusively in the chromatin of cells in deep senescent conditions with persistent DNA damage. H2B type 1-K and modified HMGA1 may thus represent novel biomarkers of senescent cells containing persistent DNA damage.
RESUMO
Cellular senescence is an irreversible growth arrest that occurs as a result of damaging stimuli, including DNA damage and/or telomere shortening. Here, we investigate histone variant H2A.J as a new biomarker to detect senescent cells during human skin aging. Skin biopsies from healthy volunteers of different ages (18-90 years) were analyzed for H2A.J expression and other parameters involved in triggering and/or maintaining cellular senescence. In the epidermis, the proportions of H2A.J-expressing keratinocytes increased from ≈20% in young to ≈60% in aged skin. Inverse correlations between Ki67- and H2A.J staining in germinative layers may reflect that H2A.J-expressing cells having lost their capacity to divide. As cellular senescence is triggered by DNA-damage signals, persistent 53BP1-foci, telomere lengths, and telomere-associated damage foci were analyzed in epidermal keratinocytes. Only slight age-related telomere attrition and few persistent nuclear 53BP1-foci, occasionally colocalizing with telomeres, suggest that unprotected telomeres are not a significant cause of senescence during skin aging. Quantification of integrin-α6+ basal cells suggests that the number and function of stem/progenitor cells decreased during aging and their altered proliferation capacities resulted in diminished tissue renewal with epidermal thinning. Collectively, our findings suggest that H2A.J is a sensitive marker of epidermal aging in human skin.
RESUMO
The expression of BRAF-V600E triggers oncogene-induced senescence in normal cells and is implicated in the development of several cancers including melanoma. Here, we report that cardioglycosides such as ouabain are potent senolytics in BRAF senescence. Sensitization by ATP1A1 knockdown and protection by supplemental potassium showed that senolysis by ouabain was mediated by the Na,K-ATPase pump. Both ion transport inhibition and signal transduction result from cardioglycosides binding to Na,K-ATPase. An inhibitor of the pump that does not trigger signaling was not senolytic despite blocking ion transport, demonstrating that signal transduction is required for senolysis. Ouabain triggered the activation of Src, p38, Akt, and Erk in BRAF-senescent cells, and signaling inhibitors prevented cell death. The expression of BRAF-V600E increased ER stress and autophagy in BRAF-senescent cells and sensitized the cell to senolysis by ouabain. Ouabain inhibited autophagy flux, which was restored by signaling inhibitors. Consequently, we identified autophagy inhibitor chloroquine as a novel senolytic in BRAF senescence based on the mode of action of cardioglycosides. Our work underlies the interest of characterizing the mechanisms of senolytics to discover novel compounds and identifies the endoplasmic reticulum stress-autophagy tandem as a new vulnerability in BRAF senescence that can be exploited for the development of further senolytic strategies.
Assuntos
Autofagia/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Cloroquina/farmacologia , Ouabaína/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismoRESUMO
Accumulation of senescent cells is an important contributor to chronic inflammation upon aging. The inflammatory phenotype of senescent cells was previously shown to be driven by cytoplasmic DNA. Here, we propose that cytoplasmic double-stranded RNA has a similar effect. We find that several cell types driven into senescence by different routes share an accumulation of long promoter RNAs and 3' gene extensions rich in retrotransposon sequences. Accordingly, these cells display increased expression of genes involved in response to double stranded RNA of viral origin downstream of the interferon pathway. The RNA accumulation is associated with evidence of reduced RNA turnover, including in some cases, reduced expression of RNA exosome subunits. Reciprocally, depletion of RNA exosome subunit EXOSC3 accelerated expression of multiple senescence markers. A senescence-like RNA accumulation was also observed in cells exposed to oxidative stress, an important trigger of cellular senescence. Altogether, we propose that in a subset of senescent cells, repeat-containing transcripts stabilized by oxidative stress or reduced RNA exosome activity participate in driving and maintaining the permanent inflammatory state characterizing cellular senescence.
Assuntos
Senescência Celular/genética , Estabilidade de RNA/genética , RNA/metabolismo , Linhagem Celular , Dano ao DNA , Humanos , Inflamação/metabolismo , Estresse Oxidativo/genética , Fenótipo , RNA/genética , RNA de Cadeia Dupla/efeitos adversos , RNA de Cadeia Dupla/genética , Retroelementos/genéticaRESUMO
H2A.J is a poorly studied mammalian-specific variant of histone H2A. We used immunohistochemistry to study its localization in various human and mouse tissues. H2A.J showed cell-type specific expression with a striking enrichment in luminal epithelial cells of multiple glands including those of breast, prostate, pancreas, thyroid, stomach, and salivary glands. H2A.J was also highly expressed in many carcinoma cell lines and in particular, those derived from luminal breast and prostate cancer. H2A.J thus appears to be a novel marker for luminal epithelial cancers. Knocking-out the H2AFJ gene in T47D luminal breast cancer cells reduced the expression of several estrogen-responsive genes which may explain its putative tumorigenic role in luminal-B breast cancer.
Assuntos
Glândulas Endócrinas/metabolismo , Células Epiteliais/metabolismo , Histonas/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Glândulas Endócrinas/patologia , Células Epiteliais/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Variação Genética , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos/genética , Gravidez , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
Histones are subjected to extensive post-translational modifications (PTMs) that are known to play key roles in many biological processes. In this study, we report a fast, efficient, highly reproducible, and easily automated method involving ultra-high performance liquid chromatography (UHPLC) coupled to a high resolution/high mass accuracy LTQ-Orbitrap mass spectrometer to profile core histone modifications/variants from WI-38 primary human fibroblasts. The whole analysis was performed on intact unfractionated histones within 19 min, which is â¼3-fold faster than previously published procedures. High mass accuracy measurements combined with top-down tandem mass spectrometry (MS) experiments enable accurate histone identification. Experimental and biological variations were thoroughly assessed and were 8% and 16% on average, respectively. With a sample preparation reduced to the minimum, characterization of the most abundant histones can be achieved in a single experiment. Semi-quantitative information can be obtained with respect to the relative abundances of the detected isoforms through a label-free approach. Isoform identities and relative distributions were further confirmed by the LC-MS/MS analysis of tryptic digests. Overall, our UHPLC-MS approach for histone profiling offers a sensitive and reproducible tool that will be of great value for exploring PTMs and variants and can readily be applied to clinical or pharmaceutical studies.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Histonas/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Histonas/metabolismo , Humanos , Fosfoproteínas/análise , Isoformas de Proteínas/análise , Processamento de Proteína Pós-Traducional , Reprodutibilidade dos TestesRESUMO
PURPOSE: This study addresses the sensitivity of different peripheral CD4+ T-lymphocyte subsets to irradiation (IR) and identifies potential targets for the prevention or treatment of radiation-induced toxicity. METHODS: This study was performed on peripheral blood mononuclear cells or sorted peripheral memory lymphocytes of CCR6+ mucosa-homing Th17/CCR6negTh and regulatory T subtypes of healthy volunteers. Cells were irradiated with a 2 Gy with or without pharmacologic inhibitors of different signaling pathways. Senescence of irradiated cells was assessed by resistance to apoptosis and determination of various senescence-associated biomarkers (senescence associated b-galactosidase activity, p16Ink4a-, p21Cdkn1a-, gH2A.X-, H2A.J expression). Cytokine production was measured in supernatants of irradiated cells by Luminex technology. RESULTS: Not all CD4+ memory T lymphocyte subsets were equally radiosensitive. High sensitivity of CCR6+Th17 lymphocytes to IR-induced senescence was shown by expression of the histone variant H2A.J, higher SA-b-Gal activity, and upregulation of p16Ink4a and p21Cdkn1a expression. Lower Annexin V staining and cleaved caspase-3, and higher expression of antiapoptotic genes Bcl-2 and Bcl-xL LF, showed that CCR6+Th17 lymphocytes were more resistant to IR-induced apoptosis than CCR6neg memory Th and regulatory T lymphocytes. After a 2 Gy IR, both CCR6+Th17 and CCR6neg cells acquired a moderate senescence-associated secretory phenotype, but only CCR6+Th17 cells secreted interleukin 8 (IL-8) and vascular endothelial growth factor-A (VEGF-A). Pharmacologic targeting of reactive oxygen species (ROS), mitogen-activated protein kinases (MAPKs), and mammalian target of rapamycin (mTOR) signaling pathways prevented the expression of senescent markers and IL-8 and VEGF-A expression by CCR6+Th17 cells after IR. CONCLUSIONS: This study suggests that IR induces senescence of CCR6+Th17 lymphocytes associated with secretion of IL-8 and VEGF-A that may be detrimental to the irradiated tissue. ROS-MAPKs signaling pathways are candidate targets to prevent this CCR6+Th17-dependent radiation-induced potential toxicity. Finally, the ratio of circulating H2A.J+ senescent CCR6+ Th17/CD4+ T lymphocytes may be a candidate marker of individual intrinsic radiosensitivity.
Assuntos
Senescência Celular/efeitos da radiação , Lesões por Radiação/prevenção & controle , Receptores CCR6/metabolismo , Células Th17/citologia , Células Th17/efeitos da radiação , Senescência Celular/efeitos dos fármacos , Senescência Celular/imunologia , Humanos , Terapia de Alvo Molecular , Lesões por Radiação/imunologia , Segurança , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Transdução de Sinais/efeitos da radiação , Células Th17/efeitos dos fármacos , Células Th17/imunologiaRESUMO
Asf1 is a histone chaperone that favors histone H3/H4 assembly and disassembly. We solved the structure of the conserved domain of human ASF1A in complex with the C-terminal helix of histone H3 using nuclear magnetic resonance spectroscopy. This structure is fully compatible with an association of ASF1 with the heterodimeric form of histones H3/H4. In our model, ASF1 substitutes for the second H3/H4 heterodimer that is normally found in heterotetrameric H3/H4 complexes. This result constitutes an essential step in the fundamental understanding of the mechanisms of nucleosome assembly by histone chaperones. Point mutations that perturb the Asf1/histone interface were designed from the structure. The decreased binding affinity of the Asf1-H3/H4 complex correlates with decreased levels of H3-K56 acetylation and phenotypic defects in vivo.
Assuntos
Proteínas de Ciclo Celular/química , Chaperonas Moleculares/química , Acetilação , Proteínas de Ciclo Celular/genética , Células Cultivadas , Dimerização , Histonas/química , Humanos , Espectroscopia de Ressonância Magnética , Chaperonas Moleculares/genética , Mutação Puntual , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Anti-silencing function 1 (ASF1) is a conserved H3-H4 histone chaperone involved in histone dynamics during replication, transcription, and DNA repair. Overexpressed in proliferating tissues including many tumors, ASF1 has emerged as a promising therapeutic target. Here, we combine structural, computational, and biochemical approaches to design peptides that inhibit the ASF1-histone interaction. Starting from the structure of the human ASF1-histone complex, we developed a rational design strategy combining epitope tethering and optimization of interface contacts to identify a potent peptide inhibitor with a dissociation constant of 3 nM. When introduced into cultured cells, the inhibitors impair cell proliferation, perturb cell-cycle progression, and reduce cell migration and invasion in a manner commensurate with their affinity for ASF1. Finally, we find that direct injection of the most potent ASF1 peptide inhibitor in mouse allografts reduces tumor growth. Our results open new avenues to use ASF1 inhibitors as promising leads for cancer therapy.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Desenho de Fármacos , Chaperonas Moleculares/metabolismo , Peptídeos/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Epitopos/química , Epitopos/metabolismo , Feminino , Histonas/química , Histonas/metabolismo , Humanos , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Chaperonas Moleculares/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Termodinâmica , Transplante HomólogoRESUMO
The Snf1/AMP-activated kinases are involved in a wide range of stress responses in eukaryotic cells. We discovered a novel role for the Snf1 kinase in the cellular response to genotoxic stress in yeast. snf1 mutants are hypersensitive to hydroxyurea (HU), methyl-methane sulfonate, and cadmium, but they are not sensitive to several other genotoxic agents. HU inhibits ribonucleotide reductase (RNR), and deletion of SNF1 also increased the growth defects of an rnr4 ribonucleotide reductase mutant. The snf1 mutant has a functional checkpoint response to HU insofar as cells arrest division normally and derepress the transcription of RNR genes. The sensitivity of snf1 to HU or to RNR4 deletion may be due to posttranscriptional defects in RNR function or to defects in the repair of, and recovery from, stalled replication forks. The Mig3 repressor was identified as one target of Snf1 in this pathway. Genetic and biochemical analyses suggest that a weak kinase activity is sufficient to confer resistance to HU, whereas a high level of kinase activity is required for optimal growth on carbon sources other than glucose. Quantitative regulation of Snf1 kinase activity may contribute to the specificity of the effector responses that it controls.
Assuntos
Inibidores Enzimáticos/farmacologia , Hidroxiureia/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleotídeo Redutases/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Cádmio/farmacologia , Dano ao DNA , Farmacorresistência Fúngica/genética , Genes Fúngicos , Peptídeos e Proteínas de Sinalização Intracelular , Metanossulfonato de Metila/farmacologia , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleotídeo Redutases/genética , Fase S/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Yaf9 is one of three proteins in budding yeast containing a YEATS domain. We show that Yaf9 is part of a large complex and that it coprecipitates with three known subunits of the NuA4 histone acetyltransferase. Although Esa1, the catalytic subunit of NuA4, is essential for viability, we found that yaf9 Delta mutants are viable but hypersensitive to microtubule depolymerizing agents and synthetically lethal with two different mutants of the mitotic apparatus. Microtubules depolymerized more readily in the yaf9Delta mutant compared to the wild type in the presence of nocodazole, and recovery of microtubule polymerization and cell division from limiting concentrations of nocodazole was inhibited. Two other NuA4 mutants (esa1-1851 and yng2 Delta) and nonacetylatable histone H4 mutants were also sensitive to benomyl. Furthermore, wild-type budding yeast were more resistant to benomyl when grown in the presence of trichostatin A, a histone deacetylase inhibitor. These results strongly suggest that acetylation of histone H4 by NuA4 is required for the cellular resistance to spindle stress.
Assuntos
Acetiltransferases/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fuso Acromático/genética , Acetilação , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Actinas/genética , Sequência de Aminoácidos , Benomilo/farmacologia , Cromatina/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/efeitos dos fármacos , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases , Ácidos Hidroxâmicos/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Nocodazol/farmacologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Fuso Acromático/efeitos dos fármacos , Transcrição GênicaRESUMO
The senescence of mammalian cells is characterized by a proliferative arrest in response to stress and the expression of an inflammatory phenotype. Here we show that histone H2A.J, a poorly studied H2A variant found only in mammals, accumulates in human fibroblasts in senescence with persistent DNA damage. H2A.J also accumulates in mice with aging in a tissue-specific manner and in human skin. Knock-down of H2A.J inhibits the expression of inflammatory genes that contribute to the senescent-associated secretory phenotype (SASP), and over expression of H2A.J increases the expression of some of these genes in proliferating cells. H2A.J accumulation may thus promote the signalling of senescent cells to the immune system, and it may contribute to chronic inflammation and the development of aging-associated diseases.