RESUMO
Surface plasmon resonance (SPR) permits the quantitative analysis of therapeutic antibody concentrations and impurities including bacteria, Protein A, Protein G and small molecule ligands leached from chromatography media. The use of surface plasmon resonance has gained popularity within the biopharmaceutical industry due to the automated, label free, real time interaction that may be exploited when using this method. The application areas to assess protein interactions and develop analytical methods for biopharmaceutical downstream process development, quality control, and in-process monitoring are reviewed.
Assuntos
Anticorpos/análise , Técnicas Biossensoriais/métodos , Proteínas Imobilizadas/química , Técnicas de Imunoadsorção , Ressonância de Plasmônio de Superfície/métodos , Anticorpos/química , Cinética , Ligação ProteicaRESUMO
Thymus-specific serine protease (TSSP) was initially reported as a putative protease specifically expressed in the endosomal compartment of cortical thymic epithelial cells (cTEC). As such, TSSP is potentially involved in the presentation of the self-peptides that are bound to MHC class II molecules expressed at the cTEC surface and are involved in the positive selection of CD4(+) thymocytes. We tested this hypothesis by generating mutant mice deprived of Prss16, the gene encoding TSSP. TSSP-deficient mice produced normal numbers of T cells, despite a decrease in the percentage of cTEC expressing high surface levels of MHC class II. By using sensitive transgenic models expressing MHC class II-restricted TCR transgenes (Marilyn and OT-II), we showed that the absence of TSSP markedly impaired the selection of Marilyn and OT-II CD4(+) T cells. In contrast, selection of CD8(+) T cells expressing an MHC class I-restricted TCR transgene (OT-I) was unaffected. Therefore, TSSP is involved in the positive selection of some CD4(+) T lymphocytes and likely constitutes the first serine protease to play a function in the intrathymic presentation of self-peptides bound to MHC class II complexes.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Epiteliais/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Serina Endopeptidases/imunologia , Timo/imunologia , Animais , Linfócitos T CD4-Positivos/enzimologia , Células Epiteliais/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Serina Endopeptidases/genética , Timo/enzimologiaRESUMO
p53 exerts its tumor suppressor function mainly through transcriptional induction of target genes involved in several processes, including cell cycle checkpoints, apoptosis, and regulation of cell redox status. p53 antioxidant function is dependent on its transcriptional activity and proceeds by sequential induction of antioxidant and proapoptotic targets. However, none of the thus far renowned p53 targets have proved able to abolish on their own the intracellular reactive oxygen species (ROS) accumulation caused by p53 deficiency, therefore pointing to the existence of other prominent and yet unknown p53 antioxidant targets. Here, we show that TP53INP1 represents such a target. Indeed, TP53INP1 transcript induction on oxidative stress is strictly dependent on p53. Mouse embryonic fibroblasts (MEF) and splenocytes derived from TP53INP1-deficient (inp1(-/-)) mice accumulate intracellular ROS, whereas overexpression of TP53INP1 in p53-deficient MEFs rescues ROS levels to those of p53-proficient cells, indicating that TP53INP1 antioxidant function is p53 independent. Furthermore, accumulation of ROS in inp1(-/-) cells on oxidant challenge is associated with decreased expression of p53 targets p21/Cdkn1a, Sesn2, TAp73, Puma, and Bax. Mutation of p53 Ser(58) (equivalent to human p53 Ser(46)) abrogates transcription of these genes, indicating that TP53INP1-mediated p53 Ser(58) phosphorylation is implicated in this process. In addition, TP53INP1 deficiency results in an antioxidant (N-acetylcysteine)-sensitive acceleration of cell proliferation. Finally, TP53INP1 deficiency increases oxidative stress-related lymphoma incidence and decreases survival of p53(+/-) mice. In conclusion, our data show that TP53INP1 is a major actor of p53-driven oxidative stress response that possesses both a p53-independent intracellular ROS regulatory function and a p53-dependent transcription regulatory function.
Assuntos
Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Peróxido de Hidrogênio/farmacologia , Linfoma/genética , Linfoma/metabolismo , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Pancreatic cancer is a disease with an extremely poor prognosis. Tumor protein 53-induced nuclear protein 1 (TP53INP1) is a proapoptotic stress-induced p53 target gene. In this article, we show by immunohistochemical analysis that TP53INP1 expression is dramatically reduced in pancreatic ductal adenocarcinoma (PDAC) and this decrease occurs early during pancreatic cancer development. TP53INP1 reexpression in the pancreatic cancer-derived cell line MiaPaCa2 strongly reduced its capacity to form s.c., i.p., and intrapancreatic tumors in nude mice. This anti-tumoral capacity is, at least in part, due to the induction of caspase 3-mediated apoptosis. In addition, TP53INP1(-/-) mouse embryonic fibroblasts (MEFs) transformed with a retrovirus expressing E1A/ras(V12) oncoproteins developed bigger tumors than TP53INP1(+/+) transformed MEFs or TP53INP1(-/-) transformed MEFs with restored TP53INP1 expression. Finally, TP53INP1 expression is repressed by the oncogenic micro RNA miR-155, which is overexpressed in PDAC cells. TP53INP1 is a previously unknown miR-155 target presenting anti-tumoral activity.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , MicroRNAs/genética , Neoplasias Pancreáticas/prevenção & controle , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/prevenção & controle , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Camundongos Nus , Transplante de Neoplasias , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , RNA Neoplásico/genética , Transplante HeterólogoRESUMO
Tumor protein 53-induced nuclear protein 1 (TP53INP1) is an antiproliferative and proapoptotic protein involved in cell stress response. To address its physiological roles in colorectal cancer and colitis, we generated and tested the susceptibility of Trp53inp1-deficient mice to the development of colorectal tumors induced by injection of the carcinogen azoxymethane followed by dextran sulfate sodium (DSS)-induced chronic colitis. Trp53inp1-deficient mice showed an increased incidence and multiplicity of tumors compared to those of wild-type (WT) mice. Furthermore, acute colitis induced by DSS treatment was more severe in Trp53inp1-deficient mice than in WT mice. Treatment with the antioxidant N-acetylcysteine prevented colitis and colitis-associated tumorigenesis more efficiently in WT mice than in Trp53inp1-deficient mice, suggesting a higher oxidative load in the latter. Consistently, we demonstrated by electron spin resonance and spin trapping that colons derived from deficient mice produced more free radicals than those of the WT during colitis and that the basal blood level of the antioxidant ascorbate was decreased in Trp53inp1-deficient mice. Collectively, these results indicate that the oxidative load is higher in Trp53inp1-deficient mice than in WT mice, generating a more-severe DSS-induced colitis, which favors development of colorectal tumors in Trp53inp1-deficient mice. Therefore, TP53INP1 is a potential target for the prevention of colorectal cancer in patients with inflammatory bowel disease.
Assuntos
Proteínas de Transporte/metabolismo , Colite/metabolismo , Colite/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Proteínas Nucleares/metabolismo , Doença Aguda , Animais , Proteínas de Transporte/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Doença Crônica , Colite/complicações , Colite/genética , Neoplasias do Colo/complicações , Neoplasias do Colo/genética , Sulfato de Dextrana/farmacologia , Peroxidação de Lipídeos , Camundongos , Camundongos Knockout , Mutação/genética , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Estresse Oxidativo , Espécies Reativas de OxigênioRESUMO
Following injection into recombinase-activating gene-deficient (Rag1(-/-)) mice, pro-B cells lacking the Pax5 transcription factor (Pax5(-/-)) develop into most major hematopoietic lineages, with the notable exception of B cells. We assessed whether Pax5(-/-) pro-B cells that were also rendered deficient for the linker for activation of T cells (LAT), an adaptor essential for T cell receptor signaling, can be used for the rapid in vivo analysis of mutant forms of LAT. We showed that Pax5(-/-) Lat(-/-) pro-B cell lines can be infected with recombinant retroviruses expressing a LAT cDNA and sorted for the expression of LAT. When injected into Rag1(-/-) mice, they restore normal intrathymic T cell development and give rise to functional peripheral T cells. Considering that the handling of Pax5(-/-) pro-B cell lines is easier than that of bone marrow hematopoietic precursors, we used them for the rapid functional analysis of a novel Lat allelic series. When compared to knock-in and transgenic approaches, a major advantage of our Pax5(-/-) pro-B cell-based experimental approach consists in the production of mice bearing a given mutation within 2-3 months. Therefore, it constitutes a powerful first-line screen for mutations worth fastidious knock-in approaches.