Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 12(1): 5015, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408139

RESUMO

Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins or post-translational modifications of interest using bait-specific antibodies. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysates and identified by mass spectrometry. We demonstrate this workflow by targeting Emerin, H3K9me3 and BRG1. Amongst the main findings, our experiments reveal that the essential protein FLYWCH1 interacts with a subset of H3K9me3-marked (peri)centromeres in human cells. The ProtA-Turbo enzyme represents an off-the-shelf proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Biotina/metabolismo , Biotinilação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Espectrometria de Massas , Ligação Proteica , Proteínas/química , Proteômica
2.
Cell Death Dis ; 12(5): 469, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976119

RESUMO

HDAC1 is the prototypical human histone deacetylase (HDAC) enzyme responsible for catalyzing the removal of acetyl group from lysine residues on many substrate proteins. By deacetylating histones and non-histone proteins, HDAC1 has a profound effect on the regulation of gene transcription and many processes related to cell growth and cell death, including cell cycle progression, DNA repair, and apoptosis. Early studies reveal that, like most eukaryotic proteins, the functions and activities of HDAC1 are regulated by post-translational modifications. For example, serine phosphorylation of HDAC1 by protein kinase CK2 promotes HDAC1 deacetylase enzymatic activity and alters its interactions with proteins in corepressor complexes. Here, we describe an alternative signaling pathway by which HDAC1 activities are regulated. Specifically, we discover that EGFR activity promotes the tyrosine phosphorylation of HDAC1, which is necessary for its protein stability. A key EGFR phosphorylation site on HDAC1, Tyr72, mediates HDAC1's anti-apoptotic function. Given that HDAC1 overexpression and EGFR activity are strongly related with tumor progression and cancer cell survival, HDAC1 tyrosine phosphorylation may present a possible target to manipulate HDAC1 protein levels in future potential cancer treatment strategies.


Assuntos
Apoptose/genética , Histona Desacetilase 1/metabolismo , Receptores ErbB/metabolismo , Humanos , Fosforilação , Transfecção
3.
Stem Cells ; 39(7): 866-881, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33621399

RESUMO

A key challenge for clinical application of induced pluripotent stem cells (iPSC) to accurately model and treat human pathologies depends on developing a method to generate genetically stable cells to reduce long-term risks of cell transplant therapy. Here, we hypothesized that CYCLIN D1 repairs DNA by highly efficient homologous recombination (HR) during reprogramming to iPSC that reduces genetic instability and threat of neoplastic growth. We adopted a synthetic mRNA transfection method using clinically compatible conditions with CYCLIN D1 plus base factors (OCT3/4, SOX2, KLF4, LIN28) and compared with methods that use C-MYC. We demonstrate that CYCLIN D1 made iPSC have (a) lower multitelomeric signal, (b) reduced double-strand DNA breaks, (c) correct nuclear localization of RAD51 protein expression, and (d) reduced single-nucleotide polymorphism (SNP) changes per chromosome, compared with the classical reprogramming method using C-MYC. CYCLIN D1 iPSC have reduced teratoma Ki67 cell growth kinetics and derived neural stem cells successfully engraft in a hostile spinal cord injury (SCI) microenvironment with efficient survival, differentiation. We demonstrate that CYCLIN D1 promotes double-stranded DNA damage repair predominantly through HR during cell reprogramming to efficiently produce iPSC. CYCLIN D1 reduces general cell stress associated with significantly lower SIRT1 gene expression and can rescue Sirt1 null mouse cell reprogramming. In conclusion, we show synthetic mRNA transfection of CYCLIN D1 repairs DNA during reprogramming resulting in significantly improved genetically stable footprint in human iPSC, enabling a new cell reprogramming method for more accurate and reliable generation of human iPSC for disease modeling and future clinical applications.

4.
Genes Cancer ; 10(5-6): 119-133, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31798765

RESUMO

Temozolomide (TMZ) is an alkylating agent chemotherapy drug used as a first-line treatment for glioblastoma multiforme (GBM). O6-methyl-guanine DNA methyltransferase (MGMT) repairs DNA damage induced by TMZ; hence, elevated MGMT levels usually correlate with TMZ resistance. MGMT promoter methylation is a key regulatory mechanism for MGMT expression and is important in overcoming TMZ therapy resistance. To date, little is known about how MGMT expression is regulated beyond promoter methylation. In this work, we show an alternative mechanism by which MGMT levels are regulated independent of its promoter methylation status. We found that inhibition of the histone deacetylase HDAC8 by either HDAC8-specific inhibitor PCI34051 or HDAC8 shRNA decreases MGMT levels in GBM cell lines. Furthermore, the proteasome receptor ADRM1 participates in this MGMT regulation by interacting with HDAC8. Interestingly, this interaction is disrupted by TMZ exclusively in TMZ sensitive cells, suggesting that this MGMT regulatory pathway might be inactivated in TMZ resistant cells. Consequently, HDAC8 inhibition in GBM cell lines increases DNA damage and cell cycle arrest and, eventually, decreases cell viability, likely due to the decrease in MGMT protein levels.

5.
Mol Cell Oncol ; 5(4): e1445942, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30250909

RESUMO

The NF-κB pathway regulates cell physiology under stress conditions. We have recently described a novel NF-κB regulatory mechanism, by which SIRT6 induces cysteine monoubiquitination of the methyltransferase SUV39H1. This causes SUV39H1 dissociation from the gene encoding the NF-κB inhibitor IκBα,  increasing its expression and leading to NF-κB pathway inactivation.

6.
Nat Commun ; 9(1): 101, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29317652

RESUMO

Sirtuins are NAD+-dependent deacetylases that facilitate cellular stress response. They include SirT6, which protects genome stability and regulates metabolic homeostasis through gene silencing, and whose loss induces an accelerated aging phenotype directly linked to hyperactivation of the NF-κB pathway. Here we show that SirT6 binds to the H3K9me3-specific histone methyltransferase Suv39h1 and induces monoubiquitination of conserved cysteines in the PRE-SET domain of Suv39h1. Following activation of NF-κB signaling Suv39h1 is released from the IκBα locus, subsequently repressing the NF-κB pathway. We propose that SirT6 attenuates the NF-κB pathway through IκBα upregulation via cysteine monoubiquitination and chromatin eviction of Suv39h1. We suggest a mechanism based on SirT6-mediated enhancement of a negative feedback loop that restricts the NF-κB pathway.


Assuntos
Cisteína/metabolismo , Metiltransferases/metabolismo , NF-kappa B/metabolismo , Domínios PR-SET , Proteínas Repressoras/metabolismo , Sirtuínas/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Cromatina/metabolismo , Cisteína/genética , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Metiltransferases/genética , Camundongos , Inibidor de NF-kappaB alfa/metabolismo , Células NIH 3T3 , Ligação Proteica , Proteínas Repressoras/genética , Transdução de Sinais , Sirtuínas/genética , Ubiquitinação , Regulação para Cima
7.
Genes Chromosomes Cancer ; 52(4): 423-30, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23340989

RESUMO

Suv39h1 mediates heterochromatin formation in pericentric and telomeric regions by trimethylation of lysine 9 of histone 3 (H3K9me3). Yet, its role in the induction of chromosomal instability is poorly understood. We established a leukemia model by retrovirally expressing Myc in wild-type and histone methyltransferase Suv39h1-deficient hematopoietic cells and characterized the resulting leukemias for chromosomal instability. All mice that received cells overexpressing Myc developed myeloid leukemia with a median survival of 44 days posttransplantation. Myc-overexpressing wild-type leukemias demonstrated clones with numerical chromosomal aberrations (5/16). In secondary transplantations of these leukemic cells, structural changes, mostly end-to-end fusions of chromosomes, appeared (10/12). In contrast, leukemic cells overexpressing Myc with reduced or no Suv39h1 expression had a normal karyotype in primary, secondary, and tertiary transplantations (16/16). Myc-transduced Suv39h1-deficient cells showed less critically short telomeres (P < 0.05) compared with Myc-transduced wild-type bone marrow cells. Gene expression analysis showed upregulation of genes involved in the alternative lengthening of telomeres (ALT) mechanism. Thus, we hypothesize that loss of Suv39h1 implies activation of the ALT mechanism, in turn ensuring telomere length and stability. Our data show for the first time that Suv39h1 deficiency may prevent chromosomal instability by more efficient telomere stabilization in hematopoietic bone marrow cells overexpressing Myc.


Assuntos
Instabilidade Cromossômica , Leucemia Mieloide/genética , Metiltransferases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Repressoras/genética , Animais , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Feminino , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hibridização in Situ Fluorescente , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Masculino , Metiltransferases/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/deficiência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cariotipagem Espectral , Telômero/genética , Homeostase do Telômero/genética , Encurtamento do Telômero/genética
8.
Cancer Cell ; 21(6): 719-21, 2012 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-22698398

RESUMO

Recently reporting in Nature, Barber et al. demonstrated that SIRT7 maintains critical features that define cancer cells by removing the acetylation mark on lysine 18 of histone H3. Interestingly, hypoacetylation of H3K18 has been described as a general marker of tumor prognosis and oncoviral transformation.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...