RESUMO
Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signaling and repair proteins to the sites of DNA lesions. Coordinated protein SUMOylation and ubiquitylation have crucial roles in regulating the dynamic assembly of protein complexes at these sites. However, how SUMOylation influences protein ubiquitylation at DSBs is poorly understood. We show herein that Rnf4, an E3 ubiquitin ligase that targets SUMO-modified proteins, accumulates in DSB repair foci and is required for both homologous recombination (HR) and non-homologous end joining repair. To establish a link between Rnf4 and the DNA damage response (DDR) in vivo, we generated an Rnf4 allelic series in mice. We show that Rnf4-deficiency causes persistent ionizing radiation-induced DNA damage and signaling, and that Rnf4-deficient cells and mice exhibit increased sensitivity to genotoxic stress. Mechanistically, we show that Rnf4 targets SUMOylated MDC1 and SUMOylated BRCA1, and is required for the loading of Rad51, an enzyme required for HR repair, onto sites of DNA damage. Similarly to inactivating mutations in other key regulators of HR repair, Rnf4 deficiency leads to age-dependent impairment in spermatogenesis. These findings identify Rnf4 as a critical component of the DDR in vivo and support the possibility that Rnf4 controls protein localization at DNA damage sites by integrating SUMOylation and ubiquitylation events.
Assuntos
Reparo do DNA , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Alelos , Animais , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Genótipo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/genética , Rad51 Recombinase/metabolismo , Radiação Ionizante , Sumoilação , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
BACKGROUND: alpha(1)-Acid glycoprotein (alpha(1)-AGP) is an acute phase protein in most mammalian species whose concentration rises 2-5-fold during an acute phase reaction. Its serum concentration has often been used as a marker of disease, including inflammatory bowel disease (IBD). High alpha(1)-AGP levels were found to have a prognostic value for an increased risk of relapse in IBD. AIMS: To investigate a possible role for increased serum levels of alpha(1)-AGP in the development of IBD. METHODS: Dextran sodium sulphate (DSS) 2% was added to the drinking water of transgenic mice, overexpressing the rat alpha(1)-AGP gene, to induce acute colitis, thus mimicking the conditions of relapse. Clinical parameters, inflammatory parameters, and histological analyses on colon sections were performed. RESULTS: Homozygous alpha(1)-AGP-transgenic mice started losing weight and showed rectal bleeding significantly earlier than heterozygous transgenic or wild-type mice. Survival time of homozygous transgenic mice was significantly shorter compared with heterozygous and wild-type mice. The higher susceptibility of homozygous alpha(1)-AGP-transgenic mice to DSS induced acute colitis was also reflected in higher local myeloperoxidase levels, higher inflammation scores of the colon, and higher systemic levels of interleukin 6 and serum amyloid P component. Local inflammatory parameters were also significantly different in heterozygous transgenic mice compared with wild-type mice, indicating a local dosage effect. In homozygous transgenic mice, significantly higher amounts of bacteria were found in organs but IgA levels were only slightly lower than those of control mice. CONCLUSION: Sufficiently high serum levels of alpha(1)-AGP result in a more aggressive development of acute colitis.
Assuntos
Colite/sangue , Orosomucoide/metabolismo , Doença Aguda , Animais , Colite/induzido quimicamente , Contagem de Colônia Microbiana , Sulfato de Dextrana , Modelos Animais de Doenças , Feminino , Hemorragia Gastrointestinal/sangue , Imunoglobulina A/sangue , Interleucina-6/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peroxidase/sangue , Componente Amiloide P Sérico/análise , Redução de Peso/efeitos dos fármacosRESUMO
Tumour necrosis factor (TNF) is a major mediator in septic shock and several inflammatory diseases such as hepatitis. Galactosamine (GalN) sensitises experimental animals for TNF and the combination TNF/GalN leads to a lethal inflammatory hepatitis. We describe that a single injection of lipopolysaccharide (LPS), interleukin-1 (IL-1) or TNF can desensitise against the lethality induced by TNF/GalN, but also against changes in metabolic parameters such as hypothermia and transaminase release, in a dose responsive way. We also describe the desensitising capacity of a component present in Mouse Liver Extract (MLE). The MLE desensitises mice against the effects of TNF/GalN in a dose responsive way. The activity of the MLE is heat labile and does not involve LPS, TNF, IL-1 or TNF soluble receptors. We describe partial and complete purification of the factor. Partially pure material protects mice against all changes induced by TNF/GalN. The protection is dose dependent and heat labile and also possible in endotoxin-hyporesponsive C3H/HeJ mice. The pure material protects against lethality, hypothermia and AST release and it appears as a heat labile protein of relative molecular weight of 70 kDa probably with a break down product of 35 kDa.
Assuntos
Galactosamina/antagonistas & inibidores , Interleucina-1/administração & dosagem , Lipopolissacarídeos/administração & dosagem , Extratos Hepáticos/isolamento & purificação , Extratos Hepáticos/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Aspartato Aminotransferases/sangue , Dessensibilização Imunológica , Relação Dose-Resposta a Droga , Feminino , Galactosamina/imunologia , Galactosamina/toxicidade , Injeções Intraperitoneais , Interleucina-1/imunologia , Lipopolissacarídeos/imunologia , Fígado/química , Extratos Hepáticos/química , Camundongos , Camundongos Endogâmicos C57BL , Peso Molecular , Proteínas/imunologia , Proteínas/isolamento & purificação , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/toxicidadeRESUMO
Tumor necrosis factor (TNF) is an essential mediator in the pathogenesis of Gram-negative septic shock. Injection of TNF into normal mice leads to systemic, lethal inflammation, which is indistinguishable from lipopolysaccharide (LPS)-induced lethal inflammation. alpha(2)-macroglobulin (A2M) is a major positive acute phase protein with broad-spectrum protease-inhibitory activity. Mouse A2M-deficient (MAM-/-) mice were significantly protected against lethal systemic inflammation induced by TNF. The protection is not due to faster clearance of the injected TNF. The induction of tolerance to TNF-induced lethality by repetitive administration of small doses of human TNF for five consecutive days was equally efficient in both mutant mice compared to wild-type mice. In D-(+)-galactosamine (GalN)-sensitized mice, TNF induces lethal inflammatory hepatitis. MAM(-/-) mice are equally sensitive to the lethal combination of TNF/GalN. Furthermore, interleukin-1-induced desensitization to TNF/GalN was not impaired in MAM(-/-) mice. We conclude that MAM plays a mediating role in TNF-induced lethal shock and that MAM deficiency does not reduce changes in efficiency of tolerance and desensitization to TNF and TNF/GalN-induced lethality, respectively.
Assuntos
Choque/induzido quimicamente , Fator de Necrose Tumoral alfa/farmacologia , alfa-Macroglobulinas/deficiência , Animais , Dessensibilização Imunológica , Galactosamina/farmacologia , Hepatite Animal/induzido quimicamente , Mediadores da Inflamação/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidores de Proteases/imunologia , alfa-Macroglobulinas/genética , alfa-Macroglobulinas/imunologiaRESUMO
The proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) induces lethal hepatitis when injected into D-(+)-galactosamine-sensitized mice on the one hand or systemic inflammatory response syndrome (SIRS) in normal mice on the other hand. We studied whether serum amyloid P component (SAP), the major acute-phase protein in mice, plays a protective role in both lethal models. For this purpose, we used SAP(0/0) mice generated by gene targeting. We studied the lethal response of SAP(0/0) or SAP(+/+) mice to both lethal triggers but found no differences in the sensitivity of both types of mice. We also investigated whether SAP is involved in establishing two types of endogenous protection: one using a single injection of interleukin-1beta (IL-1beta) for desensitization and clearly involving a liver protein, the other by tolerizing mice for 5 days using small doses of human TNF-alpha. Although after IL-1beta or after tolerization the SAP levels in the serum had risen fourfold in the control mice and not in the SAP(0/0) mice, the same extents of desensitization and tolerization were achieved. Finally, we observed that the induction of hemorrhagic necrosis in the skin of mice by two consecutive local injections with TNF-alpha was not altered in SAP(0/0) mice. We conclude that the presence or absence of SAP has no influence on the sensitivity of mice to TNF-alpha-induced hepatitis, SIRS, and hemorrhagic necrosis or on the endogenous protective mechanisms of desensitization or tolerization.
Assuntos
Hepatite Animal/etiologia , Componente Amiloide P Sérico/fisiologia , Pele/patologia , Síndrome de Resposta Inflamatória Sistêmica/etiologia , Fator de Necrose Tumoral alfa/toxicidade , Animais , Galactosamina , Interleucina-1/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , NecroseRESUMO
Resistance to gram-negative infection can be induced by pretreating animals with several agents such as turpentine and interleukin (IL)-1. Because these agents are powerful inducers of acute phase proteins, we wondered whether these proteins, more particularly alpha(1)-acid glycoprotein (alpha(1)-AGP), are involved in nonspecific resistance to infection. Turpentine and IL-1 protect completely against a lethal challenge of Klebsiella pneumoniae when given 48 and 12-48 h before the challenge, respectively. alpha(1)-AGP induction in the serum reached peak values 48 h after turpentine and 12-48 h after IL-1 injection. Administration of alpha(1)-AGP, 2 h before a challenge of K. pneumoniae, significantly increased the survival. Numbers of bacteria cultured from blood and organs were significantly lower in mice pretreated with a protective dose of turpentine, IL-1, or alpha(1)-AGP. These data suggest that alpha(1)-AGP is a possible mediator in turpentine- or IL-1-induced protection because time points of maximal induction of alpha(1)-AGP by turpentine or IL-1 and of optimal protection by alpha(1)-AGP coincide. Transgenic overexpression of rat alpha(1)-AGP protected mice from a K. pneumoniae infection. Bacterial counts in blood and organs were significantly lower in transgenic mice, and only in control mice were large necrotic areas, apoptosis, and blood clots observed in the spleen. Our data suggest that alpha(1)-AGP prevents gram-negative infections and may be an essential component in nonspecific resistance to infection.
Assuntos
Infecções por Bactérias Gram-Negativas/fisiopatologia , Infecções por Klebsiella/fisiopatologia , Klebsiella pneumoniae/isolamento & purificação , Orosomucoide/fisiologia , Animais , Bovinos , Feminino , Infecções por Bactérias Gram-Negativas/sangue , Imunidade Inata , Interleucina-1/farmacologia , Rim/microbiologia , Infecções por Klebsiella/sangue , Infecções por Klebsiella/prevenção & controle , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Orosomucoide/biossíntese , Orosomucoide/genética , Ratos , Baço/microbiologia , Taxa de Sobrevida , Terebintina/farmacologiaRESUMO
alpha 1-Acid glycoprotein (AGP) is an acute phase protein produced by hepatocytes. Although its exact biological function remains controversial, it was shown to protect galactosamine-sensitized or normal mice against hepatitis and lethal shock induced by tumor necrosis factor (TNF). Rat-AGP-transgenic mice, constitutively producing several mg AGP per ml serum were tested for their response to a combined challenge with TNF and D-(+)-galactosamine. A previously characterized, single transgenic line (9.5-5) was used. In contrast to our expectations both heterozygous or homozygous transgenic mice were not protected by the endogenously overproduced AGP. However, both transgenic and non-transgenic mice were protected by pretreatment with interleukin-1, an effect which we believe is mediated by the induction of acute phase proteins like AGP. Furthermore, both types of mice were protected by exogenous bovine AGP, suggesting that the lack of protection by endogenous AGP is not because of a repressed response to AGP. Finally, we demonstrate that purified AGP from the serum of transgenic mice is as protective as the AGP from non-transgenic mice or rats. The results suggest that AGP is protective only when its concentration is rapidly induced, perhaps because the endogenous steady state synthesis of AGP, in non-transgenic as well as transgenic mice, is coupled to the production of an AGP-binding factor. This study provides an interesting example of differences in outcome to a lethal challenge between an acute administered and a chronically produced protective protein.