RESUMO
Poly-ADP-ribose polymerases (PARPs) are important regulators of the immune system, including TCDD-inducible poly-ADP-ribose polymerase (TIPARP), also known as poly-ADP-ribose polymerase 7 (PARP7). PARP7 negatively regulates aryl hydrocarbon receptor (AHR) and type I interferon (IFN-I) signaling, both of which have been implicated in intestinal homeostasis and immunity. Since the loss of PARP7 expression increases AHR and IFN-I signaling, we used a murine dextran sulfate sodium (DSS)-induced colitis model to investigate the effect of PARP7 loss on DSS-induced intestinal inflammation. DSS-exposed Parp7-/- mice had less body weight loss, lower disease index scores, and reduced expression of several inflammation genes, including interleukin IL-6, C-x-c motif chemokine ligand 1 (Cxcl1), and lipocalin-2, when compared with wild-type mice. However, no significant difference was observed between genotypes in the colonic expression of the AHR target gene cytochrome P450 1A1 (Cyp1a1). Moreover, no significant differences in microbial composition were observed between the genotypes. Our findings demonstrate that the absence of PARP7 protein results in an impaired immune response to colonic inflammation and suggests that PARP7 may participate in the recruitment of immune cells to the inflammation site, which may be due to its role in IFN-I signaling rather than AHR signaling.
Assuntos
ADP Ribose Transferases/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Colite/genética , Sulfato de Dextrana/efeitos adversos , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Quimiocina CXCL1/genética , Colite/induzido quimicamente , Colite/patologia , Citocromo P-450 CYP1A1/genética , Modelos Animais de Doenças , Fibroblastos/química , Fibroblastos/citologia , Técnicas de Inativação de Genes , Interferon Tipo I/metabolismo , Interleucina-6/genética , Lipocalina-2/genética , Masculino , Camundongos , Transdução de Sinais , Regulação para CimaRESUMO
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-adenosine diphosphate (ADP)-ribose polymerase (TIPARP/PARP7), an aryl hydrocarbon receptor (AHR) target gene and mono-ADP-ribosyltransferase, acts as part of a negative feedback loop to repress AHR signaling. This process is prevented by a single H532A mutation in TIPARP that destroys its catalytic activity. We hypothesized that the loss of TIPARP catalytic activity would increase sensitivity to TCDD-induced toxicity in vivo. To test this, we created a catalytically deficient mouse line (TiparpH532A) by introducing a single H532A mutation in TIPARP. Treatment of mouse embryonic fibroblasts or hepatocytes isolated from TiparpH532A mice confirmed the increased TCDD-induced expression of the AHR target genes Cyp1a1, Cyp1b1, and Tiparp. TiparpH532A mice given a single injection of 10 µg/kg TCDD, a nonlethal dose in Tiparp+/+ mice, did not survive beyond day 10. All Tiparp+/+ mice survived the 30-day treatment. TCDD-treated TiparpH532A mice displayed increased expression of AHR target genes, increased steatohepatitis and hepatotoxicity. Hepatic RNA-sequencing revealed 7-fold more differentially expressed genes in TiparpH532A mice than in Tiparp+/+ mice (4542 vs 647 genes) 6 days after TCDD treatment. Differentially expressed genes included genes involved in xenobiotic metabolism, lipid homeostasis and inflammation. Taken together, these data further support TIPARP as a critical negative regulator of AHR activity and show that loss of its catalytic activity is sufficient to increase sensitivity to TCDD-induced steatohepatitis and lethality. Since TIPARP inhibition has recently emerged as a potential anticancer therapy, the impact on AHR signaling, TCDD and polycyclic aromatic hydrocarbon toxicity will need to be carefully considered under conditions of therapeutic TIPARP inhibition.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Dibenzodioxinas Policloradas , Adenosina Difosfato Ribose , Animais , Doença Hepática Induzida por Substâncias e Drogas/genética , Fibroblastos , Camundongos , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
ADP-ribosylation is a post-translational protein modification catalyzed by a family of proteins known as poly-ADP-ribose polymerases. PARP7 (TIPARP; ARTD14) is a mono-ADP-ribosyltransferase involved in several cellular processes, including responses to hypoxia, innate immunity and regulation of nuclear receptors. Since previous studies suggested that PARP7 was regulated by 17ß-estradiol, we investigated whether PARP7 regulates estrogen receptor α signaling. We confirmed the 17ß-estradiol-dependent increases of PARP7 mRNA and protein levels in MCF-7 cells, and observed recruitment of estrogen receptor α to the promoter of PARP7. Overexpression of PARP7 decreased ligand-dependent estrogen receptor α signaling, while treatment of PARP7 knockout MCF-7 cells with 17ß-estradiol resulted in increased expression of and recruitment to estrogen receptor α target genes, in addition to increased proliferation. Co-immunoprecipitation assays revealed that PARP7 mono-ADP-ribosylated estrogen receptor α, and mass spectrometry mapped the modified peptides to the receptor's ligand-independent transactivation domain. Co-immunoprecipitation with truncated estrogen receptor α variants identified that the hinge region of the receptor is required for PARP7-dependent mono-ADP-ribosylation. These results imply that PARP7-mediated mono-ADP-ribosylation may play an important role in estrogen receptor positive breast cancer.
Assuntos
ADP-Ribosilação , Neoplasias da Mama/enzimologia , Proliferação de Células , Receptor alfa de Estrogênio/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , ADP-Ribosilação/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/genética , Estrogênios/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Transporte de Nucleosídeos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Transdução de SinaisRESUMO
Poly(ADP-ribose) polymerase 7 (PARP-7) has emerged as a critically important member of a large enzyme family that catalyzes ADP-ribosylation in mammalian cells. PARP-7 is a critical regulator of the innate immune response. What remains unclear is the mechanism by which PARP-7 regulates this process, namely because the protein targets of PARP-7 mono-ADP-ribosylation (MARylation) are largely unknown. Here, we combine chemical genetics, proximity labeling, and proteome-wide amino acid ADP-ribosylation site profiling for identifying the direct targets and sites of PARP-7-mediated MARylation in a cellular context. We found that the inactive PARP family member, PARP-13-a critical regulator of the antiviral innate immune response-is a major target of PARP-7. PARP-13 is preferentially MARylated on cysteine residues in its RNA binding zinc finger domain. Proteome-wide ADP-ribosylation analysis reveals cysteine as a major MARylation acceptor of PARP-7. This study provides insight into PARP-7 targeting and MARylation site preference.
Assuntos
ADP-Ribosilação , Cisteína/metabolismo , Proteínas de Transporte de Nucleosídeos/genética , Proteoma/genética , Proteínas de Ligação a RNA/genética , Mapeamento Cromossômico , Humanos , Proteínas de Transporte de Nucleosídeos/química , Proteoma/química , Proteínas de Ligação a RNA/químicaRESUMO
Here, we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin poly-ADP-ribose polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400-657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cells was dependent on both its catalytic activity and zinc finger domain. The catalytic activity of TIPARP was resistant to meta-iodobenzylguanidine but sensitive to iodoacetamide and hydroxylamine, implicating cysteines and acidic side chains as ADP-ribosylated target residues. Mass spectrometry identified multiple ADP-ribosylated peptides in TIPARP and AHR. Electron transfer dissociation analysis of the TIPARP peptide 33ITPLKTCFK41 revealed cysteine 39 as a site for mono-ADP-ribosylation. Mutation of cysteine 39 to alanine resulted in a small, but significant, reduction in TIPARP autoribosylation activity, suggesting that additional amino acid residues are modified, but loss of cysteine 39 did not prevent its ability to repress AHR. Our findings characterize the subcellular localization and mono-ADP-ribosyltransferase activity of TIPARP, identify cysteine as a mono-ADP-ribosylated residue targeted by this enzyme, and confirm the TIPARP-dependent mono-ADP-ribosylation of other protein targets, such as AHR.
Assuntos
ADP Ribose Transferases/genética , Cisteína/genética , Mutação de Sentido Incorreto , Poli(ADP-Ribose) Polimerases/genética , ADP Ribose Transferases/metabolismo , ADP-Ribosilação/efeitos dos fármacos , Animais , Biocatálise/efeitos dos fármacos , Células COS , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Chlorocebus aethiops , Cisteína/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Células MCF-7 , Proteínas de Transporte de Nucleosídeos , Poli(ADP-Ribose) Polimerases/metabolismo , Dibenzodioxinas Policloradas/farmacologia , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Dedos de Zinco/genéticaRESUMO
The aryl hydrocarbon receptor (AHR) mediates the toxic effects of dioxin (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin; TCDD), which includes thymic atrophy, steatohepatitis, and a lethal wasting syndrome in laboratory rodents. Although the mechanisms of dioxin toxicity remain unknown, AHR signaling in hepatocytes is necessary for dioxin-induced liver toxicity. We previously reported that loss of TCDD-inducible poly(adenosine diphosphate [ADP]-ribose) polymerase (TIPARP/PARP7/ARTD14), an AHR target gene and mono-ADP-ribosyltransferase, increases the sensitivity of mice to dioxin-induced toxicities. To test the hypothesis that TIPARP is a negative regulator of AHR signaling in hepatocytes, we generated Tiparpfl/fl mice in which exon 3 of Tiparp is flanked by loxP sites, followed by Cre-lox technology to create hepatocyte-specific (Tiparpfl/flCreAlb) and whole-body (Tiparpfl/flCreCMV; TiparpEx3-/-) Tiparp null mice. Tiparpfl/flCreAlb and TiparpEx3-/- mice given a single injection of 10 µg/kg dioxin did not survive beyond days 7 and 9, respectively, while all Tiparp+/+ mice survived the 30-day treatment. Dioxin-exposed Tiparpfl/flCreAlb and TiparpEx3-/- mice had increased steatohepatitis and hepatotoxicity as indicated by greater staining of neutral lipids and serum alanine aminotransferase activity than similarly treated wild-type mice. Tiparpfl/flCreAlb and TiparpEx3-/- mice exhibited augmented AHR signaling, denoted by increased dioxin-induced gene expression. Metabolomic studies revealed alterations in lipid and amino acid metabolism in liver extracts from Tiparpfl/flCreAlb mice compared with wild-type mice. Taken together, these data illustrate that TIPARP is an important negative regulator of AHR activity, and that its specific loss in hepatocytes is sufficient to increase sensitivity to dioxin-induced steatohepatitis and lethality.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fígado Gorduroso/induzido quimicamente , Hepatócitos/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/genética , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/metabolismo , Síndrome de Emaciação/induzido quimicamente , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Fígado Gorduroso/enzimologia , Fígado Gorduroso/genética , Expressão Gênica/efeitos dos fármacos , Hepatócitos/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos , Camundongos Knockout , Cultura Primária de Células , Deleção de Sequência , Transdução de Sinais , Síndrome de Emaciação/enzimologia , Síndrome de Emaciação/genéticaRESUMO
SUMMARY Annexins are calcium-binding proteins that have been associated in plants with different biological processes such as responses to abiotic stress and early nodulation stages. Until now, the implication of annexins during plant-pathogen interactions has not been reported. Here, a novel plant annexin gene induced in tobacco BY-2 cell suspension cultures infected with the phytopathogenic bacterium Rhodococcus fascians (strain D188) has been identified. Expression of this gene, called Ntann12, is also induced, but to a lower extent, by a strain (D188-5) that is unable to induce leafy gall formation. This gene was also induced in BY-2 cells infected with Pseudomonas syringae but not in cells infected with Agrobacterium tumefaciens or Escherichia coli. Ntann12 expression was also found to be stimulated by abiotic stress, including NaCl and abscissic acid, confirming a putative role in stress signal transduction pathways. In addition, promoter-GUS analyses using homozygous transgenic tobacco seedlings showed that the developmentally controlled expression of Ntann12 is altered upon R. fascians infection. Finally, up-regulation of Ntann12 during leafy gall ontogenesis was confirmed by RT-qPCR. Discussion is focused on the potential role of Ntann12 in biotic and abiotic stress responses and in plant development, both processes that may involve Ca(2+)-dependent signalling.