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Introduction: Improved therapies for glioblastoma (GBM) are desperately needed and require preclinical evaluation in models that capture tumor heterogeneity and intrinsic resistance seen in patients. Epigenetic alterations have been well documented in GBM and lysine-specific demethylase 1 (LSD1/KDM1A) is amongst the chromatin modifiers implicated in stem cell maintenance, growth and differentiation. Pharmacological inhibition of LSD1 is clinically relevant, with numerous compounds in various phases of preclinical and clinical development, but an evaluation and comparison of LSD1 inhibitors in patient-derived GBM models is lacking. Methods: To assess concordance between knockdown of LSD1 and inhibition of LSD1 using a prototype inhibitor in GBM, we performed RNA-seq to identify genes and biological processes associated with inhibition. Efficacy of various LSD1 inhibitors was assessed in nine patient-derived glioblastoma stem cell (GSC) lines and an orthotopic xenograft mouse model. Results: LSD1 inhibitors had cytotoxic and selective effects regardless of GSC radiosensitivity or molecular subtype. In vivo, LSD1 inhibition via GSK-LSD1 led to a delayed reduction in tumor burden; however, tumor regrowth occurred. Comparison of GBM lines by RNA-seq was used to identify genes that may predict resistance to LSD1 inhibitors. We identified five genes that correlate with resistance to LSD1 inhibition in treatment resistant GSCs, in GSK-LSD1 treated mice, and in GBM patients with low LSD1 expression. Conclusion: Collectively, the growth inhibitory effects of LSD1 inhibition across a panel of GSC models and identification of genes that may predict resistance has potential to guide future combination therapies.
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BACKGROUND: The prostate tumor microenvironment (TME) is immunosuppressive, with few effector T cells and enrichment of inhibitory immune populations, leading to limited responses to treatments such as immune checkpoint therapies (ICTs). The immune composition of the prostate TME differs across soft tissue and bone, the most common site of treatment-refractory metastasis. Understanding immunosuppressive mechanisms specific to prostate TMEs will enable rational immunotherapy strategies to generate effective antitumor immune responses. Daratumumab (anti-CD38 antibody) and edicotinib (colony-stimulating factor-1 receptor (CSF-1R) inhibitor) may alter the balance within the prostate TME to promote antitumor immune responses. HYPOTHESIS: Daratumumab or edicotinib will be safe and will alter the immune TME, leading to antitumor responses in localized prostate cancer. PATIENTS AND METHODS: In this presurgical study, patients with localized prostate cancer received 4 weekly doses of daratumumab or 4 weeks of daily edicotinib prior to radical prostatectomy (RP). Treated and untreated control (Gleason score ≥8 in prostate biopsy) prostatectomy specimens and patient-matched pre- and post-treatment peripheral blood mononuclear cells (PBMCs) and bone marrow samples were evaluated. The primary endpoint was incidence of adverse events (AEs). The secondary endpoint was pathologic complete remission (pCR) rate. RESULTS: Twenty-five patients were treated (daratumumab, n=15; edicotinib, n=10). All patients underwent RP without delays. Grade 3 treatment-related AEs with daratumumab occurred in 3 patients (12%), and no ≥grade 3 treatment-related AEs occurred with edicotinib. No changes in serum prostate-specific antigen (PSA) levels or pCRs were observed. Daratumumab led to a decreased frequency of CD38+ T cells, natural killer cells, and myeloid cells in prostate tumors, bone marrow, and PBMCs. There were no consistent changes in CSF-1R+ immune cells in prostate, bone marrow, or PBMCs with edicotinib. Neither treatment induced T cell infiltration into the prostate TME. CONCLUSIONS: Daratumumab and edicotinib treatment was safe and well-tolerated in patients with localized prostate cancer but did not induce pCRs. Decreases in CD38+ immune cells were observed in prostate tumors, bone marrow, and PBMCs with daratumumab, but changes in CSF-1R+ immune cells were not consistently observed with edicotinib. Neither myeloid-targeted agent alone was sufficient to generate antitumor responses in prostate cancer; thus, combinations with agents to induce T cell infiltration (eg, ICTs) will be needed to overcome the immunosuppressive prostate TME.
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Antineoplásicos , Neoplasias da Próstata , Masculino , Humanos , Leucócitos Mononucleares/patologia , Antineoplásicos/uso terapêutico , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Imunossupressores , Microambiente TumoralRESUMO
Glioblastoma (GBM) is a primary brain cancer with an abysmal prognosis and few effective therapies. The ability to investigate the tumor microenvironment before and during treatment would greatly enhance both understanding of disease response and progression, as well as the delivery and impact of therapeutics. Stereotactic biopsies are a routine surgical procedure performed primarily for diagnostic histopathologic purposes. The role of investigative biopsies - tissue sampling for the purpose of understanding tumor microenvironmental responses to treatment using integrated multi-modal molecular analyses ('Multi-omics") has yet to be defined. Secondly, it is unknown whether comparatively small tissue samples from brain biopsies can yield sufficient information with such methods. Here we adapt stereotactic needle core biopsy tissue in two separate patients. In the first patient with recurrent GBM we performed highly resolved multi-omics analysis methods including single cell RNA sequencing, spatial-transcriptomics, metabolomics, proteomics, phosphoproteomics, T-cell clonotype analysis, and MHC Class I immunopeptidomics from biopsy tissue that was obtained from a single procedure. In a second patient we analyzed multi-regional core biopsies to decipher spatial and genomic variance. We also investigated the utility of stereotactic biopsies as a method for generating patient derived xenograft models in a separate patient cohort. Dataset integration across modalities showed good correspondence between spatial modalities, highlighted immune cell associated metabolic pathways and revealed poor correlation between RNA expression and the tumor MHC Class I immunopeptidome. In conclusion, stereotactic needle biopsy cores are of sufficient quality to generate multi-omics data, provide data rich insight into a patient's disease process and tumor immune microenvironment and can be of value in evaluating treatment responses. One sentence summary: Integrative multi-omics analysis of stereotactic needle core biopsies in glioblastoma.
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Cryoablation in combination with immune checkpoint therapy was previously reported to improve anti-tumor immune responses in pre-clinical studies. Here we report a pilot study of anti-CTLA-4 (tremelimumab) with (n = 15) or without (n = 14) cryoablation in patients with metastatic renal cell carcinoma (NCT02626130), 18 patients with clear cell and 11 patients with non-clear cell histologies. The primary endpoint is safety, secondary endpoints include objective response rate, progression-free survival, and immune monitoring studies. Safety data indicate ≥ grade 3 treatment-related adverse events in 16 of 29 patients (55%) including 6 diarrhea/colitis, 3 hepatitis, 1 pneumonitis, and 1 glomerulonephritis. Toxicity leading to treatment discontinuation occurs in 5 patients in each arm. 3 patients with clear cell histology experience durable responses. One patient in the tremelimumab arm experiences an objective response, the median progression-free survival for all patients is 3.3 months (95% CI: 2.0, 5.3 months). Exploratory immune monitoring analysis of baseline and post-treatment tumor tissue samples shows that treatment increases immune cell infiltration and tertiary lymphoid structures in clear cell but not in non-clear cell. In clear cell, cryoablation plus tremelimumab leads to a significant increase in immune cell infiltration. These data highlight that treatment with tremelimumab plus cryotherapy is feasible and modulates the immune microenvironment in patients with metastatic clear cell histology.
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Anticorpos Monoclonais Humanizados/administração & dosagem , Antígeno CTLA-4/antagonistas & inibidores , Carcinoma de Células Renais/tratamento farmacológico , Criocirurgia/métodos , Neoplasias Renais/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/administração & dosagem , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/cirurgia , Terapia Combinada , Feminino , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Neoplasias Renais/cirurgia , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Segurança do Paciente , Projetos Piloto , Taxa de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
Connexin36 (Cx36) is the most abundant connexin in central nervous system neurons. It forms gap junction channels that act as electrical synapses. Similar to chemical synapses, Cx36-containing gap junctions undergo activity-dependent plasticity and complex regulation. Cx36 gap junctions represent multimolecular complexes and contain cytoskeletal, regulatory and scaffolding proteins, which regulate channel conductance, assembly and turnover. The amino acid sequence of mammalian Cx36 harbors a phosphorylation site for the Ca2+/calmodulin-dependent kinase II at serine 315. This regulatory site is homologous to the serine 298 in perch Cx35 and in close vicinity to a PDZ binding domain at the very C-terminal end of the protein. We hypothesized that this phosphorylation site may serve as a molecular switch, influencing the affinity of the PDZ binding domain for its binding partners. Protein microarray and pulldown experiments revealed that this is indeed the case: phosphorylation of serine 298 decreased the binding affinity for MUPP1, a known scaffolding partner of connexin36, and increased the binding affinity for two different 14-3-3 proteins. Although we did not find the same effect in cell culture experiments, our data suggest that phosphorylation of serine 315/298 may serve to recruit different proteins to connexin36/35-containing gap junctions in an activity-dependent manner.
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Proteínas 14-3-3/metabolismo , Conexinas/metabolismo , Domínios PDZ , Animais , Conexinas/química , Sinapses Elétricas/metabolismo , Junções Comunicantes/metabolismo , Células HeLa , Humanos , Fosforilação , Ligação Proteica , Proteína delta-2 de Junções ComunicantesRESUMO
Protein S-palmitoylation is a reversible post-translational modification mediated by palmitoyl acyltransferase enzymes, a group of Zn2+-finger DHHC-domain-containing proteins (ZDHHC). Here, for the first time, we show that Zdhhc13 plays a key role in anxiety-related behaviors and motor function, as well as brain bioenergetics, in a mouse model (luc) carrying a spontaneous Zdhhc13 recessive mutation. At 3 m of age, mutant mice displayed increased sensorimotor gating, anxiety, hypoactivity, and decreased motor coordination, compared to littermate controls. Loss of Zdhhc13 in cortex and cerebellum from 3- and 24 m old hetero- and homozygous male mutant mice resulted in lower levels of Drp1 S-palmitoylation accompanied by altered mitochondrial dynamics, increased glycolysis, glutaminolysis and lactic acidosis, and neurotransmitter imbalances. Employing in vivo and in vitro models, we identified that Zdhhc13-dependent Drp1 S-palmitoylation, which acting alone or in concert, enables the normal occurrence of the fission-fusion process. In vitro and in vivo direct Zdhhc13-Drp1 protein interaction was observed, confirming Drp1 as a substrate of Zdhhc13. Abnormal fission-fusion processes result in disrupted mitochondria morphology and distribution affecting not only mitochondrial ATP output but neurotransmission and integrity of synaptic structures in the brain, setting the basis for the behavioral abnormalities described in the Zdhhc13-deficient mice.
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Aciltransferases/metabolismo , Ansiedade/fisiopatologia , Encéfalo/metabolismo , Dinaminas/metabolismo , Metabolismo Energético , Marcha , Lipoilação , Destreza Motora , Aciltransferases/deficiência , Aerobiose , Animais , Ansiedade/metabolismo , Comportamento Animal , Regulação da Expressão Gênica , Glicólise , Locomoção , Metabolômica , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Modelos Biológicos , Fenótipo , Ligação Proteica , Pele/patologiaRESUMO
PRMT5 is the primary enzyme responsible for the deposition of the symmetric dimethylarginine in mammalian cells. In an effort to understand how PRMT5 is regulated, we identified a threonine phosphorylation site within a C-terminal tail motif, which is targeted by the Akt/serum- and glucocorticoid-inducible kinases. While investigating the function of this posttranslational modification, we serendipitously discovered that its free C-terminal tail binds PDZ domains (when unphosphorylated) and 14-3-3 proteins (when phosphorylated). In essence, a phosphorylation event within the last few residues of the C-terminal tail generates a posttranslational modification-dependent PDZ/14-3-3 interaction "switch." The C-terminal motif of PRMT5 is required for plasma membrane association, and loss of this switching capacity is not compatible with life. This signaling phenomenon was recently reported for the HPV E6 oncoprotein but has not yet been observed for mammalian proteins. To investigate the prevalence of PDZ/14-3-3 switching in signal transduction, we built a protein domain microarray that harbors PDZ domains and 14-3-3 proteins. We have used this microarray to interrogate the C-terminal tails of a small group of candidate proteins and identified ERBB4, PGHS2, and IRK1 (as well as E6 and PRMT5) as conforming to this signaling mode, suggesting that PDZ/14-3-3 switching may be a broad biological paradigm.
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Proteínas 14-3-3/metabolismo , Domínios PDZ , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas 14-3-3/química , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Camundongos , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/químicaRESUMO
Prostate cancer evolution is driven by a combination of epigenetic and genetic alterations such as coordinated chromosomal rearrangements, termed chromoplexy. TMPRSS2-ERG gene fusions found in human prostate tumors are a hallmark of chromoplexy. TMPRSS2-ERG fusions have been linked to androgen signaling and depend on androgen receptor (AR)-coupled gene transcription. Here, we show that dimethylation of KDM1A at K114 (to form K114me2) by the histone methyltransferase EHMT2 is a key event controlling androgen-dependent gene transcription and TMPRSS2-ERG fusion. We identified CHD1 as a KDM1A K114me2 reader and characterized the KDM1A K114me2-CHD1 recognition mode by solving the cocrystal structure. Genome-wide analyses revealed chromatin colocalization of KDM1A K114me2, CHD1 and AR in prostate tumor cells. Together, our data link the assembly of methylated KDM1A and CHD1 with AR-dependent transcription and genomic translocations, thereby providing mechanistic insight into the formation of TMPRSS2-ERG gene fusions during prostate-tumor evolution.
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DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona Desmetilases/metabolismo , Proteínas de Fusão Oncogênica/genética , Neoplasias da Próstata/genética , Receptores Androgênicos/metabolismo , Translocação Genética , Linhagem Celular , Cristalografia por Raios X , DNA Helicases/análise , Proteínas de Ligação a DNA/análise , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade/metabolismo , Histona Desmetilases/análise , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Masculino , Metilação , Modelos Moleculares , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/análise , Transcrição GênicaRESUMO
Here we describe a spontaneous mutation in the Zdhhc13 (zinc finger, DHHC domain containing 13) gene (also called Hip14l), one of 24 genes encoding palmitoyl acyltransferase (PAT) enzymes in the mouse. This mutation (Zdhhc13luc) was identified as a nonsense base substitution, which results in a premature stop codon that generates a truncated form of the ZDHHC13 protein, representing a potential loss-of-function allele. Homozygous Zdhhc13luc/Zdhhc13luc mice developed generalized hypotrichosis, associated with abnormal hair cycle, epidermal and sebaceous gland hyperplasia, hyperkeratosis, and increased epidermal thickness. Increased keratinocyte proliferation and accelerated transit from basal to more differentiated layers were observed in mutant compared with wild-type (WT) epidermis in untreated skin and after short-term 12-O-tetradecanoyl-phorbol-13-acetate treatment and acute UVB exposure. Interestingly, this epidermal phenotype was associated with constitutive activation of NF-κB (RelA) and increased neutrophil recruitment and elastase activity. Furthermore, tumor multiplicity and malignant progression of papillomas after chemical skin carcinogenesis were significantly higher in mutant mice than WT littermates. To our knowledge, this is the first report of a protective role for PAT in skin carcinogenesis.
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Aciltransferases/genética , Predisposição Genética para Doença , Mutação , Neoplasias Cutâneas/genética , Animais , Bromodesoxiuridina/metabolismo , Códon de Terminação , Células Epidérmicas , Queratinócitos/fisiologia , Elastase de Leucócito/metabolismo , Camundongos , NF-kappa B/fisiologia , Células NIH 3T3 , Infiltração de Neutrófilos , Fenótipo , Neoplasias Cutâneas/etiologiaRESUMO
ATP-dependent chromatin remodeling complexes such as INO80 have been implicated in checkpoint regulation in response to DNA damage. However, how chromatin remodeling complexes regulate DNA damage checkpoints remain unclear. Here, we identified a mechanism of regulating checkpoint effector kinase Rad53 through a direct interaction with the INO80 chromatin remodeling complex. Rad53 is a key checkpoint kinase downstream of Mec1. Mec1/Tel1 phosphorylates the Ies4 subunit of the INO80 complex in response to DNA damage. We find that the phosphorylated Ies4 binds to the N-terminal FHA domain of Rad53. In vitro, INO80 can activate Rad53 kinase activity in an Ies4-phosphorylation-dependent manner in the absence of known activators such as Rad9. In vivo, Ies4 and Rad9 function synergistically to activate Rad53. These findings establish a direct connection between ATP-dependent chromatin remodeling complexes and checkpoint regulation.
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Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Ativação Enzimática , Dados de Sequência Molecular , Fosforilação , ProteóliseRESUMO
Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides.
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Proteínas F-Box/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Arginina/genética , Arginina/metabolismo , Biocatálise , Cristalografia por Raios X , Proteínas F-Box/química , Proteínas F-Box/genética , Humanos , Metilação , Camundongos , Dados de Sequência Molecular , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Splicing de RNA , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Especificidade por SubstratoRESUMO
Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.
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Proteínas Cromossômicas não Histona/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Lisina/análogos & derivados , Linhagem Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , Humanos , Mapas de Interação de Proteínas , Estrutura Terciária de Proteína , Proteólise , Interferência de RNA , Regulação para CimaRESUMO
The α-subunit of the cardiac voltage-gated sodium channel (NaV1.5) plays a central role in cardiomyocyte excitability. We have recently reported that NaV1.5 is post-translationally modified by arginine methylation. Here, we aimed to identify the enzymes that methylate NaV1.5, and to describe the role of arginine methylation on NaV1.5 function. Our results show that protein arginine methyl transferase (PRMT)-3 and -5 methylate NaV1.5 in vitro, interact with NaV1.5 in human embryonic kidney (HEK) cells, and increase NaV1.5 current density by enhancing NaV1.5 cell surface expression. Our observations are the first evidence of regulation of a voltage-gated ion channel, including calcium, potassium, sodium and TRP channels, by arginine methylation.
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Miocárdio/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Transferência Ressonante de Energia de Fluorescência , Humanos , Técnicas de Patch-ClampRESUMO
The FoxO family of transcription factors plays an important role in longevity and tumor suppression by regulating the expression of a wide range of target genes. FoxO3 has recently been found to be associated with extreme longevity in humans and to regulate the homeostasis of adult stem cell pools in mammals, which may contribute to longevity. The activity of FoxO3 is controlled by a variety of post-translational modifications that have been proposed to form a 'code' affecting FoxO3 subcellular localization, DNA binding ability, protein-protein interactions and protein stability. Lysine methylation is a crucial post-translational modification on histones that regulates chromatin accessibility and is a key part of the 'histone code'. However, whether lysine methylation plays a role in modulating FoxO3 activity has never been examined. Here we show that the methyltransferase Set9 directly methylates FoxO3 in vitro and in cells. Using a combination of tandem mass spectrometry and methyl-specific antibodies, we find that Set9 methylates FoxO3 at a single residue, lysine 271, a site previously known to be deacetylated by Sirt1. Methylation of FoxO3 by Set9 decreases FoxO3 protein stability, while moderately increasing FoxO3 transcriptional activity. The modulation of FoxO3 stability and activity by methylation may be critical for fine-tuning cellular responses to stress stimuli, which may in turn affect FoxO3's ability to promote tumor suppression and longevity.
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Fatores de Transcrição Forkhead/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatina , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Humanos , Metilação , Dados de Sequência Molecular , Transcrição GênicaRESUMO
Cell proliferation in primary and metastatic tumors is a fundamental characteristic of advanced breast cancer. Further understanding of the mechanism underlying enhanced cell growth will be important in identifying novel prognostic markers and therapeutic targets. Here we demonstrated that tyrosine phosphorylation of the proliferating cell nuclear antigen (PCNA) is a critical event in growth regulation of breast cancer cells. We found that phosphorylation of PCNA at tyrosine 211 (Y211) enhanced its association with the non-receptor tyrosine kinase c-Abl. We further demonstrated that c-Abl facilitates chromatin association of PCNA and is required for nuclear foci formation of PCNA in cells stressed by DNA damage as well as in unperturbed cells. Targeting Y211 phosphorylation of PCNA with a cell-permeable peptide inhibited the phosphorylation and reduced the PCNA-Abl interaction. These results show that PCNA signal transduction has an important impact on the growth regulation of breast cancer cells.
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Neoplasias da Mama/patologia , Carcinoma/patologia , Proliferação de Células , Dano ao DNA/fisiologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatina/metabolismo , Feminino , Células HEK293 , Humanos , Fragmentos de Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica/efeitos dos fármacosRESUMO
DNA CpG methylation and histone H3 lysine 9 (H3K9) methylation are two major repressive epigenetic modifications, and these methylations are positively correlated with one another in chromatin. Here we show that G9a or G9a-like protein (GLP) dimethylate the amino-terminal lysine 44 (K44) of mouse Dnmt3a (equivalent to K47 of human DNMT3A) in vitro and in cells overexpressing G9a or GLP. The chromodomain of MPP8 recognizes the dimethylated Dnmt3aK44me2. MPP8 also interacts with self-methylated GLP in a methylation-dependent manner. The MPP8 chromodomain forms a dimer in solution and in crystals, suggesting that a dimeric MPP8 molecule could bridge the methylated Dnmt3a and GLP, resulting in a silencing complex of Dnmt3a-MPP8-GLP/G9a on chromatin templates. Together, these findings provide a molecular explanation, at least in part, for the co-occurrence of DNA methylation and H3K9 methylation in chromatin.
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DNA (Citosina-5-)-Metiltransferases/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Fosfoproteínas/metabolismo , Calorimetria , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Imunoprecipitação , Fosfoproteínas/genética , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.
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Artrite Reumatoide/imunologia , NF-kappa B/metabolismo , Proteínas Metiltransferases/metabolismo , Fator de Transcrição RelA/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Montagem e Desmontagem da Cromatina/genética , Metilação de DNA , Células HEK293 , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Inflamação , Lisina/metabolismo , NF-kappa B/genética , NF-kappa B/imunologia , Ligação Proteica/genética , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/imunologia , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologiaRESUMO
Specific sites of histone tail methylation are associated with transcriptional activity at gene loci. These methyl marks are interpreted by effector molecules, which harbor protein domains that bind the methylated motifs and facilitate either active or inactive states of transcription. CARM1 and PRMT1 are transcriptional coactivators that deposit H3R17me2a and H4R3me2a marks, respectively. We used a protein domain microarray approach to identify the Tudor domain-containing protein TDRD3 as a "reader" of these marks. Importantly, TDRD3 itself is a transcriptional coactivator. This coactivator activity requires an intact Tudor domain. TDRD3 is recruited to an estrogen-responsive element in a CARM1-dependent manner. Furthermore, ChIP-seq analysis of TDRD3 reveals that it is predominantly localized to transcriptional start sites. Thus, TDRD3 is an effector molecule that promotes transcription by binding methylarginine marks on histone tails.
Assuntos
Arginina/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Guanilato Ciclase/metabolismo , Histonas/química , Histonas/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Guanilato Ciclase/genética , Humanos , Metilação , Análise Serial de Proteínas , Proteína-Arginina N-Metiltransferases/genética , Proteínas/genética , Proteínas Repressoras/genética , Transcrição Gênica/genéticaRESUMO
The p53 tumor suppressor gene contains a common single nucleotide polymorphism (SNP) that results in either an arginine or proline at position 72 of the p53 protein. This polymorphism affects the apoptotic activity of p53 but the mechanistic basis and physiologic relevance of this phenotypic difference remain unclear. Here, we describe the development of mouse models for the p53 R72P SNP using two different approaches. In both sets of models, the human or humanized p53 proteins are functional as evidenced by the transcriptional induction of p53 target genes in response to DNA damage and the suppression of early lymphomagenesis. Consistent with in vitro studies, mice expressing the 72R variant protein (p53R) have a greater apoptotic response to several stimuli compared with mice expressing the p53P variant. Molecular studies suggest that both transcriptional and nontranscriptional mechanisms may contribute to the differential abilities of the p53 variants to induce apoptosis. Despite a difference in the acute response to UV radiation, no difference in the tumorigenic response to chronic UV exposure was observed between the polymorphic mouse models. These findings suggest that under at least some conditions, the modulation of apoptosis by the R72P polymorphism does not affect the process of carcinogenesis.
Assuntos
Proteína Supressora de Tumor p53/genética , Animais , Apoptose/genética , Apoptose/efeitos da radiação , Cromossomos Artificiais Bacterianos , Códon , Éxons , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Modelos Animais , Proteínas de Fusão Oncogênica/genética , Fenótipo , Polimorfismo de Nucleotídeo Único , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/biossíntese , Raios UltravioletaRESUMO
Methylation of lysine residues on the tails of histone proteins is a major determinant of the transcription state of associated DNA coding regions. The interplay among methylation states and other histone modifications to direct transcriptional outcome is referred to as the histone code. In addition to histone methyltransferases and demethylases which function to modify the methylation state of lysine sidechains, other proteins recognize specific histone methylation marks essentially serving as code readers. While these interactions are highly specific with respect to site and methylation state of particular lysine residues, they are generally weak and therefore difficult to monitor by traditional assay techniques. Herein, we present the design and implementation of a homogeneous, miniaturizable, and sensitive assay for histone methylation-dependent interactions. We use AlphaScreen, a chemiluminescence-based technique, to monitor the interactions of chromodomains (MPP8, HP1beta and CHD1), tudor domains (JMJD2A) and plant homeodomains (RAG2) with their cognate trimethyllysine histone partners. The utility of the method was demonstrated by profiling the binding specificities of chromo- and tudor domains toward several histone marks. The simplicity of design and the sensitive and robust nature of this assay should make it applicable to a range of epigenetic studies, including the search for novel inhibitors of methylation-dependent interactions.