RESUMO
For a long time, proteins with enzymatic activity have not been usually considered to carry out other functions different from catalyzing chemical reactions within or outside the cell. Nevertheless, in the last few years several reports have uncovered the participation of numerous enzymes in other processes, placing them in the category of moonlighting proteins. Some moonlighting enzymes have been shown to participate in complex processes such as cell adhesion. Cell adhesion plays a physiological role in multiple processes: it enables cells to establish close contact with one another, allowing communication; it is a key step during cell migration; it is also involved in tightly binding neighboring cells in tissues, etc. Importantly, cell adhesion is also of great importance in pathophysiological scenarios like migration and metastasis establishment of cancer cells. Cell adhesion is strictly regulated through numerous switches: proteins, glycoproteins and other components of the cell membrane. Recently, several cell membrane enzymes have been reported to participate in distinct steps of the cell adhesion process. Here, we review a variety of examples of membrane bound enzymes participating in adhesion of immune cells.
Assuntos
Adesão Celular/fisiologia , Leucócitos/enzimologia , 5'-Nucleotidase/imunologia , 5'-Nucleotidase/fisiologia , Proteínas ADAM/imunologia , Proteínas ADAM/fisiologia , ADP-Ribosil Ciclase/imunologia , ADP-Ribosil Ciclase/fisiologia , ADP-Ribosil Ciclase 1/imunologia , ADP-Ribosil Ciclase 1/fisiologia , Antígenos CD/imunologia , Antígenos CD/fisiologia , Antígenos CD13/imunologia , Antígenos CD13/fisiologia , Adesão Celular/imunologia , Membrana Celular/enzimologia , Membrana Celular/imunologia , Dipeptidil Peptidase 4/imunologia , Dipeptidil Peptidase 4/fisiologia , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/fisiologia , Humanos , Leucócitos/imunologia , Leucócitos/fisiologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/fisiologia , Modelos BiológicosRESUMO
As vascular disease is complex and the various manifestations are influenced by differences in vascular bed architecture, exposure to shear and mechanical forces, cell types involved, and inflammatory responses, in vivo models are necessary to recapitulate the complex physiology and dynamic cellular interactions during pathogenesis. Murine knockout models are commonly used tools for investigators to study the role of a specific gene or pathway in multifaceted disease traits. Although valuable, these models are not perfect, and this is particularly true in regard to CD73 (cluster of differentiation 73), the extracellular enzyme that generates adenosine from AMP. At baseline, CD73-deficient mice do not present with an overt phenotype, whereas CD73-deficient humans present with the complex phenotype of vascular calcification, arteriomegaly and tortuosity, and calcification in small joints. In this review, we highlight the differences between the mouse and human systems and discuss the potential to leverage findings in mice to inform us on the human conditions.
Assuntos
5'-Nucleotidase/fisiologia , Doenças Vasculares/genética , 5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/deficiência , 5'-Nucleotidase/genética , 5'-Nucleotidase/imunologia , Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/fisiologia , Humanos , Inflamação , Artropatias/genética , Artropatias/patologia , Camundongos Knockout , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Fenótipo , Especificidade da Espécie , Trombofilia/genética , Túnica Média/patologia , Calcificação Vascular/genética , Remodelação VascularRESUMO
PURPOSE: Trimodal therapy is a reasonable bladder-preserving option to radical cystectomy. However, many tumors are radioresistive. In this sense, the identification of new prognostic and predictive biomarkers that allow the selection of patients with better responses to radiation therapy would improve outcomes. With the aim of using ecto-5'-nucleotidase/CD73 as a predictive biomarker, the role of this enzyme in the context of radiotherapy in T24 human bladder cancer cell line was investigated. METHODS: T24 cell line was exposure to a single dose of radiation (4 Gray) and trypan blue assay (pharmacological assays of viability/cumulative population doubling), flow cytometry (cell cycle/cell death/active caspase-3/ecto-5'-nucleotidase/CD73 protein staining), DAPI staining (nuclear morphometric assay), RT-PCR and real-time PCR, malachite green method (ectonucleotidase enzymatic assay), and HPLC (analysis of AMP metabolism) were carried out. T24 cell line in which ecto-5'-nucleotidase/CD73 has been completely silenced (5'KO) was also used. RESULTS: The exposure of T24 cell line to a single dose (4 Gray) of radiation-induced cell death and triggered a transitory increase in ecto-5'-nucleotidase/CD73 expression, increased ectonucleotidase activity, and led to adenosine and inosine accumulation in the extracellular medium. Pharmacological inhibition or knocking out ecto-5'-nucleotidase/CD73 rescued cells' proliferative capacity, reducing their sensitivity to radiation. CONCLUSION: Our findings show that the induction of ecto-5'-nucleotidase/CD73 by radiation contributes to the radiosensitivity of T24 cell line.
Assuntos
5'-Nucleotidase/fisiologia , Tolerância a Radiação/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/radioterapia , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Proteínas Ligadas por GPI/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Técnicas de Silenciamento de Genes , Humanos , Doses de Radiação , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismoRESUMO
PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.
Assuntos
Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Mucosa Olfatória/citologia , 5'-Nucleotidase/fisiologia , Animais , Antígenos CD34/fisiologia , Diferenciação Celular/fisiologia , Plasticidade Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Criopreservação , Osso Etmoide/citologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Mucosa Olfatória/crescimento & desenvolvimento , Coelhos , Antígenos Thy-1/fisiologiaRESUMO
Glioblastoma multiform is the most common and aggressive type of brain tumor. The overexpression of ecto-5'-nucleotidase/CD73 (ecto-5'-NT/CD73), an adhesion molecule and the main enzymatic source of extracellular adenosine, has been reported in tumor cells, and it is emerging as a component of glioma progression. Here, we evaluated the involvement of ecto-5'-NT/CD73 in cell adhesion through its interaction with different components of the extracellular matrix in the human U138MG glioma cell line. The results indicated that adenosine induced an increase in glioma cell adhesion. The treatment of glioma cells with adenosine receptor antagonists, APCP (α,ß-methylene ADP) and dipyridamole prevented the adenosine effect, indicating the participation of extracellular and intracellular signaling pathways in cell adhesion mediated by adenosine. The ECM protein laminin (lam) and chondroitin sulfate (ChS) modulated the ecto-5'-NT/CD73 activity and glioma adhesion in a parallel manner, suggesting the involvement of purinergic signaling in the effects mediated by the extracellular matrix. Taken together, these results suggest that ecto-5'-NT/CD73, an important producer of extracellular adenosine, may modulate glioma cell adhesion and tumor cell-extracellular matrix interactions.
Assuntos
5'-Nucleotidase/fisiologia , Glioma/patologia , Adenosina/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Proteínas Ligadas por GPI/fisiologia , Glioma/enzimologia , Humanos , Antagonistas de Receptores Purinérgicos P1 , PurinasRESUMO
NTPDase (EC 3.6.1.5) is an enzyme that hydrolyzes extracellular nucleoside tri-and/ or diphosphates to form ATP, which can serve as a substrate for ecto-5'- nucleotidase (EC 3.1.3.5), releasing adenosine, an inhibitor of platelet aggregation and an immunosuppressant agent. In this study, the activity of enzymes that hydrolyze adenine nucleotides was investigated in lymphocytes and platelets of immunosuppressed rats. NTPDase and ecto-5'-nucleotidase activities were determined by colorimetric assay with quantification of the inorganic phosphate released. A significant increase in NTPDase activity was observed in lymphocytes (about 30% in ATP hydrolysis and 80% in ADP hydrolysis, at p<0.05 and p<0.01, respectively). In platelets, there was a significant increase in 5'-nucleotidase activity in immunosuppressed rats (p<0.01) when compared with controls. These results suggest that the hydrolysis of adenine nucleotides is modified in the immunosuppressed state, possibly to compensate for alterations that occur and to avoid the adverse effects of therapy.
Assuntos
5'-Nucleotidase/fisiologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Antígenos CD/fisiologia , Apirase/fisiologia , Plaquetas/enzimologia , Tolerância Imunológica , Linfócitos/enzimologia , Animais , Humanos , Hidrólise , Masculino , Ratos , Ratos WistarRESUMO
Extracellular nucleotides and nucleosides act as signaling molecules involved in a wide spectrum of biological effects. Their levels are controlled by a complex cell surface-located group of enzymes called ectonucleotidases. There are four major families of ectonucleotidases, nucleoside triphosphate diphosphohydrolases (NTPDases/CD39), ectonucleotide pyrophosphatase/phosphodiesterases (E-NPPs), alkaline phosphatases and ecto-5'-nucleotidase. In the last few years, substantial progress has been made toward the molecular identification of members of the ectonucleotidase families and their enzyme structures and functions. In this review, there is an emphasis on the involvement of NTPDase and 5'-nucleotidase activities in disease processes in several tissues and cell types. Brief background information is given about the general characteristics of these enzymes, followed by a discussion of their roles in thromboregulatory events in diabetes, hypertension, hypercholesterolemia and cancer, as well as in pathological conditions where platelets are less responsive, such as in chronic renal failure. In addition, immunomodulation and cell-cell interactions involving these enzymes are considered, as well as ATP and ADP hydrolysis under different clinical conditions related with alterations in the immune system, such as acute lymphoblastic leukemia (ALL), B-chronic lymphocytic leukemia (B-CLL) and infections associated with human immunodeficiency virus (HIV). Finally, changes in ATP, ADP and AMP hydrolysis induced by inborn errors of metabolism, seizures and epilepsy are discussed in order to highlight the importance of these enzymes in the control of neuronal activity in pathological conditions. Despite advances made toward understanding the molecular structure of ectonucleotidases, much more investigation will be necessary to entirely grasp their role in physiological and pathological conditions.
Assuntos
5'-Nucleotidase/fisiologia , Antígenos CD/fisiologia , Apirase/fisiologia , Coagulação Sanguínea/fisiologia , Adenosina/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD/farmacologia , Apirase/farmacologia , Aterosclerose/sangue , Aterosclerose/enzimologia , Plaquetas/fisiologia , Comunicação Celular/fisiologia , Doenças Desmielinizantes/enzimologia , Epilepsia/enzimologia , Humanos , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/fisiopatologia , Infarto do Miocárdio/sangue , Infarto do Miocárdio/enzimologia , Neoplasias/dietoterapia , Neoplasias/enzimologia , Ativação Plaquetária/fisiologia , Agregação Plaquetária/efeitos dos fármacos , Convulsões/enzimologia , Transdução de Sinais , Tamoxifeno/uso terapêutico , Trombose/fisiopatologiaRESUMO
Hepatic 5'-nucleotidases of vertebrates were investigated for localization in the lysosomes and the plasma membrane, microheterogeneity of the glycosylphosphatidylinositol (GPI)-anchor moiety and minimal requirement of the C-terminal signal peptide for GPI attachment. Using PIPLC of Bacillus thuringiensis and subcellular fractionation by Percoll gradient centrifugation, we found that chicken liver 5'-nucleotidase can be transferred from plasma membrane to lysosomes in the GPI-anchored or soluble form. Bovine liver ecto 5'-nucleotidase was solubilized by PIPLC, purified to a homogeneous state, and analyzed for the structures of GPI-anchor isoforms by HPLC and ESI-MS in combination with glycosidase treatments, after peptide-bond cleavage by CNBr or trypsin. Several isomers of the GPI anchor were thus characterized; major components contained two phosphorylethanolamine residues, whereas the component containing three phosphorylethanolamine residues was present only as a small percentage of the total. The cleavage/attachment site of the GPI anchor in the C-terminal of 5'-nucleotidase was shown to be Ser523. The peptide region cleaved off at the posttranslational processing has a length of 25 amino acid residues which contains a hydrophobic stretch of 17 amino acids. By site-directed mutagenesis, we determined the minimal length of the hydrophobic peptide to be 13 amino acids for expression of 5'-nucleotidase as a GPI-anchored form on the COS cell surface. When peptide length was shortened to less than 13 amino acids, the expressed enzyme was not sorted to the cell surface but present within, or secreted out of the cells.