RESUMO
Self-DNA is present in the cytosol of many cancer cells and can promote effective immune rejection of tumor cells, but the mechanisms leading to the presence of cytosolic DNA are unknown. Here, we report that the cleavage of genomic DNA by DNA structure-specific endonuclease MUS81 and PARP-dependent DNA repair pathways leads to the accumulation of cytosolic DNA in prostate cancer cells. The number of nuclear MUS81 foci and the amount of cytosolic dsDNA increased in tandem from hyperplasia to clinical stage II prostate cancers and decreased at stage III. Cytosolic DNA generated by MUS81 stimulated DNA sensor STING-dependent type I interferon (IFN) expression and promoted phagocytic and T cell responses, resulting in type I and II IFN-mediated rejection of prostate tumor cells via mechanisms that partly depended on macrophages. Our results demonstrate that the tumor suppressor MUS81 alerts the immune system to the presence of transformed host cells.
Assuntos
Autoantígenos/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Endonucleases/metabolismo , Neoplasias da Próstata/imunologia , Linfócitos T/imunologia , Animais , Autoantígenos/imunologia , Linhagem Celular Tumoral , DNA/imunologia , Humanos , Interferon Tipo I/metabolismo , Ativação Linfocitária , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estadiamento de Neoplasias , Neoplasias Experimentais , Fagocitose , Neoplasias da Próstata/patologiaRESUMO
The measurements of plasma natriuretic peptides (NT-proBNP, proBNP and BNP) are used to diagnose heart failure but these are expensive to produce. We describe a rapid, cheap and facile production of proteins for immunoassays of heart failure. DNA encoding N-terminally His-tagged NT-proBNP and proBNP were cloned into the pJexpress404 vector. ProBNP and NT-proBNP peptides were expressed in Escherichia coli, purified and refolded in vitro. The analytical performance of these peptides were comparable with commercial analytes (NT-proBNP EC50 for the recombinant is 2.6 ng/ml and for the commercial material is 5.3 ng/ml) and the EC50 for recombinant and commercial proBNP, are 3.6 and 5.7 ng/ml respectively). Total yield of purified refolded NT-proBNP peptide was 1.75 mg/l and proBNP was 0.088 mg/l. This approach may also be useful in expressing other protein analytes for immunoassay applications. PURPOSE OF WORK: To develop a cost effective protein expression method in E. coli to obtain high yields of NT-proBNP (1.75 mg/l) and proBNP (0.088 mg/l) peptides for immunoassay use.
Assuntos
Ensaio de Imunoadsorção Enzimática/instrumentação , Escherichia coli/genética , Peptídeo Natriurético Encefálico/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Biomarcadores , Insuficiência Cardíaca , Humanos , Dados de Sequência Molecular , Peptídeo Natriurético Encefálico/química , Peptídeo Natriurético Encefálico/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Reprodutibilidade dos TestesRESUMO
The presence of damaged and microbial DNA can pose a threat to the survival of organisms. Cells express various sensors that recognize specific aspects of such potentially dangerous DNA. Recognition of damaged or microbial DNA by sensors induces cellular processes that are important for DNA repair and inflammation. Here, we review recent evidence that the cellular response to DNA damage and microbial DNA are tightly intertwined. We also discuss insights into the parameters that enable DNA sensors to distinguish damaged and microbial DNA from DNA present in healthy cells.
Assuntos
Bactérias/imunologia , Reparo do DNA/imunologia , DNA Bacteriano/imunologia , Animais , Bactérias/genética , Reparo do DNA/genética , DNA Bacteriano/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologiaRESUMO
Apoptosis is a controlled means of eliminating damaged cells without causing an inflammatory response or tissue damage. The mechanisms that contribute to the suppression of an inflammatory response upon apoptosis of cells are poorly understood. Here, we report that apoptotic cells release the interleukin-1 receptor antagonist (IL1RA). The release of IL1RA depended on the DNA damage response, caspase 9, and caspase 3.De novotranslation, classical secretion pathways, or N-glycosylation was not required for the release of IL1RA. The amounts of IL1RA released by apoptotic cells impaired IL1-induced expression of IL6 In summary, we demonstrate that the release of IL1RA in response to genotoxic stress contributes to the immunosuppressive effects of apoptotic cells.