RESUMO
ADP-ribosylation of proteins is emerging as an important regulatory mechanism. Depending on the family member, ADP-ribosyltransferases either conjugate a single ADP-ribose to a target or generate ADP-ribose chains. Here we characterize Parp9, a mono-ADP-ribosyltransferase reported to be enzymatically inactive. Parp9 undergoes heterodimerization with Dtx3L, a histone E3 ligase involved in DNA damage repair. We show that the Dtx3L/Parp9 heterodimer mediates NAD+-dependent mono-ADP-ribosylation of ubiquitin, exclusively in the context of ubiquitin processing by E1 and E2 enzymes. Dtx3L/Parp9 ADP-ribosylates the carboxyl group of Ub Gly76. Because Gly76 is normally used for Ub conjugation to substrates, ADP-ribosylation of the Ub carboxyl terminus precludes ubiquitylation. Parp9 ADP-ribosylation activity therefore restrains the E3 function of Dtx3L. Mutation of the NAD+ binding site in Parp9 increases the DNA repair activity of the heterodimer. Moreover, poly(ADP-ribose) binding to the Parp9 macrodomains increases E3 activity. Dtx3L heterodimerization with Parp9 enables NAD+ and poly(ADP-ribose) regulation of E3 activity.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Células HEK293 , Humanos , Mutação , NAD/metabolismo , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Fatores de Tempo , Transfecção , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR-Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.
Assuntos
Bioensaio/métodos , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Corantes Fluorescentes/química , Genes Reporter , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , DNA Ligase Dependente de ATP/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , HumanosRESUMO
Natural killer (NK) cells play a critical role in controlling malignancies. Susceptibility or resistance to lung cancer, for example, specifically depends on NK cell function. Nevertheless, intrinsic factors that control NK cell-mediated clearance of lung cancer are unknown. Here we report that NK cells exposed to exogenous major histocompatibility class I (MHCI) provide a significant immunologic barrier to the growth and progression of malignancy. Clearance of lung cancer is facilitated by up-regulation of NKG2D, NKp46, and other activating receptors upon exposure to environmental MHCI. Surface expression of the inhibitory receptor Ly49C/I, on the other hand, is down-regulated upon exposure to tumor-bearing tissue. We thus demonstrate that NK cells exhibit dynamic plasticity in surface expression of both activating and inhibitory receptors based on the environmental context. Our data suggest that altering the activation state of NK cells may contribute to immunologic control of lung and possibly other cancers.
Assuntos
Antígenos Ly/imunologia , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Receptores Imunológicos/imunologia , Receptores de Células Matadoras Naturais/metabolismo , Animais , Citotoxicidade Imunológica , Regulação para Baixo , Antígenos de Histocompatibilidade Classe I/metabolismo , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regulação para CimaRESUMO
CRISPR-Cas9-induced DNA damage may have deleterious effects at high-copy-number genomic regions. Here, we use CRISPR base editors to knock out genes by changing single nucleotides to create stop codons. We show that the CRISPR-STOP method is an efficient and less deleterious alternative to wild-type Cas9 for gene-knockout studies. Early stop codons can be introduced in â¼17,000 human genes. CRISPR-STOP-mediated targeted screening demonstrates comparable efficiency to WT Cas9, which indicates the suitability of our approach for genome-wide functional screenings.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Códon de Terminação/genética , Inativação Gênica , Códon sem Sentido , Regulação da Expressão Gênica , Marcação de Genes/métodos , Vetores Genéticos , Células HEK293 , Humanos , PlasmídeosRESUMO
OBJECTIVE: To understand role of barrier molecules in melanomas. BACKGROUND: We have reported poor patient survival and low immune infiltration of melanomas that overexpress a set of genes that include filaggrin (FLG), dystonin (DST), junction plakoglobin (JUP), and plakophilin-3 (PKP3), and are involved in cell-cell adhesions. We hypothesized that these associations are causal, either by interfering with immune cell infiltration or by enhancing melanoma cell growth. METHODS: FLG and DST were knocked out by CRISPR/Cas9 in human DM93 and murine B16-F1 melanoma cells. PKP3 and JUP were overexpressed in murine B16-AAD and human VMM39 melanoma cells by lentiviral transduction. These cell lines were evaluated in vitro for cell proliferation and in vivo for tumor burden, immune composition, cytokine expression, and vascularity. RESULTS: Immune infiltrates were not altered by these genes. FLG/DST knockout reduced proliferation of human DM93 melanoma in vitro, and decreased B16-F1 tumor burden in vivo. Overexpression of JUP, but not PKP3, in B16-AAD significantly increased tumor burden, increased VEGF-A, reduced IL-33, and enhanced vascularity. CONCLUSIONS: FLG and DST support melanoma cell growth in vitro and in vivo. Growth effects of JUP were only evident in vivo, and may be mediated, in part, by enhancing angiogenesis. In addition, growth-promoting effects of FLG and DST in vitro suggest that these genes may also support melanoma cell proliferation through angiogenesis-independent pathways. These findings identify FLG, DST, and JUP as novel therapeutic targets whose down-regulation may provide clinical benefit to patients with melanoma.
Assuntos
Biomarcadores Tumorais/metabolismo , Distonina/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Melanoma/patologia , Neovascularização Patológica/metabolismo , gama Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/metabolismo , Proteínas Filagrinas , Citometria de Fluxo , Imunofluorescência , Humanos , Melanoma/imunologia , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
In mammals, formation of new nephrons ends perinatally due to consumption of mesenchymal progenitor cells. Premature depletion of progenitors due to prematurity or postnatal loss of nephrons due to injury causes chronic kidney disease and hypertension. Intensive efforts are currently invested in designing regenerative strategies to form new nephron progenitors from pluripotent cells, which upon further differentiation provide a potential source of new nephrons. To know if reprogramed renal cells can maintain their identity and fate requires knowledge of the epigenetic states of native nephron progenitors and their progeny. In this article, we summarize current knowledge and gaps in the epigenomic landscape of the developing kidney. We now know that Pax2/PTIP/H3K4 methyltransferase activity provides the initial epigenetic specification signal to the metanephric mesenchyme. During nephrogenesis, the cap mesenchyme housing nephron progenitors is enriched in bivalent chromatin marks; as tubulogenesis proceeds, the tubular epithelium acquires H3K79me2. The latter mark is uniquely induced during epithelial differentiation. Analysis of histone landscapes in clonal metanephric mesenchyme cell lines and in Wilms tumor and normal fetal kidney has revealed that promoters of poised nephrogenesis genes carry bivalent histone signatures in progenitors. Differentiation or stimulation of Wnt signaling promotes resolution of bivalency; this does not occur in Wilms tumor cells consistent with their developmental arrest. The use of small cell number ChIP-Seq should facilitate the characterization of the chromatin landscape of the metanephric mesenchyme and various nephron compartments during nephrogenesis. Only then we will know if stem and somatic cell reprogramming into kidney progenitors recapitulates normal development.
Assuntos
Rim/citologia , Néfrons/citologia , Animais , Diferenciação Celular/fisiologia , Epigenômica , Feminino , Humanos , Rim/embriologia , Rim/metabolismo , Mesoderma/citologia , Néfrons/metabolismo , Gravidez , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
The functional, bulk, and interfacial shear rheological properties of hazelnut protein isolate were studied at different pH values between 3.0 and 8.0 and ionic strength levels between 0.0 and 1.0 M. The results showed that pH significantly affected protein solubility, emulsion properties, water and oil holding capacities, foam stability, surface hydrophobicity, and free -SH groups. The highest surface hydrophobicity, free -SH groups, and better functional properties were observed at pH 8.0. Protein solubility also increased with increasing ionic strength up to 0.6 M. The emulsion and foam stability of hazelnut protein isolate showed similar changes with protein solubility. The flow behavior of hazelnut protein suspensions was found to be shear thinning with the highest consistency index at pH 3.0 and the lowest at pH 6.0, however, the ionic strength did not significantly affect the consistency coefficient but did cause a significant change in the flow behavior index, with the lowest value observed at 0.6 M. The best gel structure in hazelnut proteins was observed at pH 3.0 and 4.0. The addition of ions at 0.4 and 0.6 M concentrations resulted in an improved viscoelastic character. The hazelnut protein isolate was also found to form solid-like viscoelastic layers at both air-water and oil-water interfaces, with the interfacial adsorption behavior affected by both pH and ionic strength. Overall, these results suggest that pH and ionic strength have significant effects on the functional and rheological properties of hazelnut protein isolate, which may have the potential as an auxiliary substance in food systems.
Assuntos
Corylus , Emulsões/química , Proteínas/química , Concentração Osmolar , Concentração de Íons de Hidrogênio , Reologia , Água/químicaRESUMO
Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia - histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism.
Assuntos
Conexinas/metabolismo , Desacetilase 6 de Histona/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Acetilação , Animais , Células Cultivadas , Conexinas/genética , Conexinas/fisiologia , Células HEK293 , Desacetilase 6 de Histona/genética , Humanos , Células Jurkat , Lisina/genética , Lisina/metabolismo , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Técnicas de Patch-Clamp , Receptores Adrenérgicos alfa 1/genética , Transdução de Sinais/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
BACKGROUND & AIMS: The inflammatory response to intestinal damage promotes healing through mechanisms that are incompletely understood. Gene expression of cluster of differentiation 74 (CD74), the receptor for cytokine macrophage migration inhibitory factor, is increased in patients with inflammatory bowel disease (IBD), however, the role of CD74 signaling in intestinal inflammation remains poorly understood. The aim of this study was to determine the functional role of CD74 signaling in intestinal inflammation. METHODS: We studied the characteristics of CD74 protein expression in human IBD and experimental colitis. The functional role of CD74 signaling in the intestine was investigated using cellular models; wild-type, CD74-/-, and bone marrow chimera mice; neutralizing anti-CD74 antibodies; flow cytometry; immunohistochemistry; immunofluorescence; immunoblotting; and clustered regularly interspaced short palindromic repeats and associated protein 9 technology. RESULTS: In IBD patients and experimental colitis, CD74-receptor protein expression was increased in inflamed intestinal tissue, prominently in the crypt epithelial cells. By using distinct but complementary chemical and non-chemically induced mouse models of colitis with genetic and antibody neutralization approaches, we found that CD74 signaling was necessary for gut repair. Mechanistically, we found that the macrophage migration inhibitory factor cytokine, which also is increased in colitis, stimulated the CD74 receptor, enhancing intestinal epithelial cell proliferation through activation of the protein kinase B and the extracellular signal-regulated kinase pathways. Our data also suggest that CD74 signaling in immune cells was not essential for mucosal healing. CONCLUSIONS: CD74 signaling is strongly activated during intestinal inflammation and protects the host by promoting epithelial cell regeneration, healing, and maintaining mucosal barrier integrity. Enhancing the CD74 pathway may represent a unique therapeutic strategy for promoting healing in IBD.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Mucosa Intestinal/patologia , Transdução de Sinais/imunologia , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Biópsia , Transplante de Medula Óssea , Linhagem Celular Tumoral , Colite Ulcerativa/genética , Colite Ulcerativa/parasitologia , Colite Ulcerativa/patologia , Doença de Crohn/patologia , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Entamoeba histolytica/patogenicidade , Células Epiteliais/imunologia , Células Epiteliais/patologia , Perfilação da Expressão Gênica , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Permeabilidade , Cultura Primária de Células , Regeneração/imunologia , Transdução de Sinais/genética , Quimeras de TransplanteRESUMO
Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation. Recently, the RNA-guidable feature of CRISPR-Cas9 has been utilized for imaging of chromatin within live cells. However, these methods are mostly applicable to highly repetitive regions, whereas imaging regions with low or no repeats remains as a challenge. To address this challenge, we design single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification. These engineered sgRNAs enable multicolour labelling of low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four unique sgRNAs. We achieve tracking of native chromatin loci throughout the cell cycle and determine differential positioning of transcriptionally active and inactive regions in the nucleus. These results demonstrate the feasibility of our approach to monitor the position and dynamics of both repetitive and non-repetitive genomic regions in live cells.
Assuntos
Ciclo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Estudos de Viabilidade , Loci Gênicos , Células HEK293 , Células HeLa , Humanos , Microscopia Intravital , Microscopia Confocal , Epitélio Pigmentado da Retina/citologiaRESUMO
Transcription factors have traditionally been viewed with skepticism as viable drug targets, but they offer the potential for completely novel mechanisms of action that could more effectively address the stem cell like properties, such as self-renewal and chemo-resistance, that lead to the failure of traditional chemotherapy approaches. Core binding factor is a heterodimeric transcription factor comprised of one of 3 RUNX proteins (RUNX1-3) and a CBFß binding partner. CBFß enhances DNA binding of RUNX subunits by relieving auto-inhibition. Both RUNX1 and CBFß are frequently mutated in human leukemia. More recently, RUNX proteins have been shown to be key players in epithelial cancers, suggesting the targeting of this pathway could have broad utility. In order to test this, we developed small molecules which bind to CBFß and inhibit its binding to RUNX. Treatment with these inhibitors reduces binding of RUNX1 to target genes, alters the expression of RUNX1 target genes, and impacts cell survival and differentiation. These inhibitors show efficacy against leukemia cells as well as basal-like (triple-negative) breast cancer cells. These inhibitors provide effective tools to probe the utility of targeting RUNX transcription factor function in other cancers.
Assuntos
Antineoplásicos/farmacologia , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Subunidade beta de Fator de Ligação ao Core/metabolismo , Neoplasias/metabolismo , Regulação Alostérica/efeitos dos fármacos , Antineoplásicos/química , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Subunidades alfa de Fatores de Ligação ao Core/química , Subunidade beta de Fator de Ligação ao Core/química , Subunidade beta de Fator de Ligação ao Core/genética , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Leucemia , Modelos Moleculares , Conformação Molecular , Mutação , Neoplasias/genética , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Dextransucrase is produced by Leuconostoc, Streptococcus and Lactobacillus Species. The enzyme synthesizes dextran and acceptor products some of which act as prebiotics that are increasingly used in such industries as food, medicine, and cosmetics. B-512F Leuconostoc mesenteroides dextransucrase (DSR-S) is the preferred enzyme in commercial production of dextran and prebiotics. In the present work, a novel dextransucrase which is efficient in prebiotics production was designed. The enzyme was produced at optimal conditions in Escherichia coli by truncation and fusion to glutathione S-transferase (GST) in the gene from Leuconostoc mesenteroides B-512 FMC. The novel enzyme (MW: 119 kDa) was active and carried out dextran biosynthesis and acceptor reactions effectively. The novel dextransucrase (fTDSR-S) was produced by truncating signal, variable, and the glucan-binding regions in the gene and fusion of gst gene at the 5' end. fTDSR-S was characterized in detail and compared to the DSR-S. Truncation and fusion resulted in an increase in fTDSR-S biosynthesis in E. coli BL21 (DE3) by 35 fold. fTDSR-S leads to production of dextran as well as increased acceptor reactions. Due to GST fusion, it was possible to immobilize fTDSR-S covalently onto Eupergit C successfully. It was also found that the size of the active site of dextransucrase is 49 amino acids shorter than that reported previously in the literature.
Assuntos
Proteínas de Bactérias/química , Enzimas Imobilizadas/química , Glucosiltransferases/química , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Biocatálise , Cloreto de Cálcio/química , Domínio Catalítico , Dextranos/biossíntese , Enzimas Imobilizadas/biossíntese , Enzimas Imobilizadas/genética , Escherichia coli , Glucosiltransferases/biossíntese , Glucosiltransferases/genética , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Concentração de Íons de Hidrogênio , Cinética , Leuconostoc/enzimologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
One application of next-generation sequencing (NGS) is the targeted resequencing of interested genes which has not been used in viral integration site analysis of gene therapy applications. Here, we combined targeted sequence capture array and next generation sequencing to address the whole genome profiling of viral integration sites. Human 293T and K562 cells were transduced with a HIV-1 derived vector. A custom made DNA probe sets targeted pLVTHM vector used to capture lentiviral vector/human genome junctions. The captured DNA was sequenced using GS FLX platform. Seven thousand four hundred and eighty four human genome sequences flanking the long terminal repeats (LTR) of pLVTHM fragment sequences matched with an identity of at least 98% and minimum 50 bp criteria in both cells. In total, 203 unique integration sites were identified. The integrations in both cell lines were totally distant from the CpG islands and from the transcription start sites and preferentially located in introns. A comparison between the two cell lines showed that the lentiviral-transduced DNA does not have the same preferred regions in the two different cell lines.