RESUMO
The primary structure of saporin-S9 and MAP-S, two type-1 ribosome-inactivating proteins isolated from the seeds of Saponaria officinalis L. and Mirabilis jalapa, respectively, was determined using a combined approach based on Edman degradation and electrospray ionization mass spectrometry (ESMS). Saporin-S9 has 253 amino acids with a calculated molecular mass of 28,492.99, which is in good agreement with that determined by ESMS (28 495 +/- 2 Da). Unlike other saporins with known primary structure, saporin-S9 contains four histidinyl residues (positions 111, 121, 216 and 248). By comparing the amino acid sequence of saporin-S9 with that of saporin-S6, we found 22 amino acid substitutions (8.7%), 13 of which are conservative and nine non-conservative. The residues known to be involved in the definition of the active site and with RNA base recognition are conserved. The four histidinyl residues and especially Lys for Gln203 contribute to the higher calculated pI value (10.17) of saporin-S9 compared with saporin-S6 (9.98). MAP-S contains 250 amino acid residues with a calculated molecular mass of 27,789.49, in good agreement with that determined by ESMS (27,789 +/- 2). Cys36 and Cys220 form a disulphide bridge and only four amino acid residues are different from the amino acid sequence of MAP, isolated from the roots of the same plant, i.e. Leu34 (Glu), Ile161 (Leu), Asp185 (Glu) and Asp191 (Glu) (in parentheses, the residues present in MAP). The reported approach can provide rapid and reliable sequence screening in the analysis of homologous proteins, including the presence of disulphide bridges.
Assuntos
Imunotoxinas , N-Glicosil Hidrolases , Proteínas de Plantas/química , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização por Electrospray , Sequência de Aminoácidos , Sequência Conservada , Brometo de Cianogênio , Endopeptidases , Metaloendopeptidases , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Proteínas Inativadoras de Ribossomos Tipo 1 , Saporinas , Homologia de Sequência , TripsinaRESUMO
Serine phosphorylation of human pro-urokinase (pro-uPA) by A431 human carcinoma cells results in a catalytically active molecule with reduced sensitivity to plasminogen activator inhibitor type 1. We mapped the phosphorylated seryl residues by analyzing the in vivo phosphorylation state of engineered pro-uPA variants carrying a COOH-terminal poly-histidine tag. Stably transfected A431 cells do not incorporate radioactive phosphate into tagged pro-uPA in which the serines 138 and 303 have been replaced with glutamic residues, although endogenous nontagged pro-uPA is 32P-labeled on A and B chains. Moreover, the catalytic-independent ability of the mono- and di-substituted "phosphorylation-like" variants to bind to the GPI-anchored urokinase receptor (uPAR) and promote adherence of differentiating U937, HL-60, and THP-1 myelomonocytic cells was examined. We found that glutamic residues as well as the naturally occurring phosphoserines at positions 138 and 303 abolish proadhesive ability, although they do not interfere with receptor binding. In addition, pro-uPA carrying Glu138/303 lacks the capability to induce a chemotactic response of THP-1 cells. The exclusive presence of Glu138 reduces pro-uPA proadhesive and chemotactic ability by 70-80%, indicating that a phosphoserine residue at the same position plays a major inhibitory role of myeloid cell response to pro-urokinase. The di-substitution does not affect pro-uPA ability to interact with vitronectin or to enhance binding of urea-denatured vitronectin to uPAR. However, unlike wild-type tagged pro-uPA, the di-substituted variant does not induce receptor polarization in pre-adherent U937 cells. Taken together, the data support the possibility that pro-uPA phosphorylation on Ser138/303 can modulate uPAR transducing ability.
Assuntos
Monócitos/citologia , Fosfosserina/metabolismo , Receptores de Superfície Celular/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Adesão Celular , Diferenciação Celular , Movimento Celular , Quimiotaxia de Leucócito , Precursores Enzimáticos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Genes , Glutamatos , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Vitronectina/metabolismoRESUMO
Bovine seminal ribonuclease (RNase) is a cytotoxin with a selective action toward tumor cells. We report here the results of an investigation that elucidate key extracellular and intracellular steps of the mechanism of its antitumor action. Seminal RNase is found to bind specifically to a large number of binding sites on the extracellular matrix of target cells, whereas other homologous RNases, including a monomeric derivative of the protein, do not bind. The key role of the pericellular matrix is confirmed by the finding that malignant cells grown in suspension bind negligible amounts of protein, and are resistant to its toxic effects, whereas the same cells, grown in monolayers, bind high amounts of seminal RNase and are killed by the protein. Seminal RNase is internalized by malignant cells, where it degrades rRNA and inhibits protein synthesis. These effects are not detectable when catalytically inactivated enzyme, or a catalytically active, monomeric derivative of the enzymes, are employed. The enzyme is bound and internalized also by the corresponding non-malignant cells, but no effects are detectable on RNA stability and on protein synthesis in these cells. This might be attributed to a different intracellular management in normal cells of the cytotoxic protein.
Assuntos
Antineoplásicos/farmacologia , Endorribonucleases/farmacologia , Animais , Sítios de Ligação , Células CHO , Linhagem Celular Transformada , Cricetinae , Endorribonucleases/metabolismo , Matriz Extracelular/metabolismo , CamundongosRESUMO
It has been previously shown that the antitumor action of bovine seminal ribonuclease (BS-RNase) is dependent on its dimeric structure. However, two distinct quaternary structures, each in equilibrium with the other, have been described for the enzyme: one in which the two subunits exchange their N-terminal ends, the other with no exchange. Antitumor activity assays, carried out on homogeneous quaternary forms of the enzyme, as well as on dimeric mutants of bovine pancreatic RNase A, reveal that another structural determinant of the antitumor activity of BS-RNase is the exchange of N-terminal ends between subunits.
Assuntos
Antineoplásicos/farmacologia , Ribonucleases/química , Ribonucleases/farmacologia , Sêmen/enzimologia , Células 3T3 , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Substâncias Macromoleculares , Masculino , Camundongos , Mutagênese Sítio-Dirigida , Pâncreas/enzimologia , Conformação Proteica , Ribonucleases/genética , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
We investigated the antitumoral effect of bovine seminal RNase (BS-RNase) in vivo and in vitro on a model system of epithelial tumor- and metastasis-derived cells as well as on epithelial tumors derived from the same system. We found that while BS-RNase significantly inhibited the growth in vitro of the epithelial tumor-derived cells, its inhibitory effect was even more dramatic on the growth of metastasis-derived cells. BS-RNase exerted no appreciable growth inhibition on normal thyroid epithelial cells. When administered in vivo to rats bearing solid carcinomas, having the same thyroid origin, BS-RNase induced a drastic reduction in the tumor weight, with no detectable toxic effects on the treated animals. These data show, for the first time on a system of neoplastically transformed epithelial cells, that BS-RNase has a potent specific antitumoral activity.
Assuntos
Antineoplásicos , Divisão Celular/efeitos dos fármacos , Neoplasias Experimentais/patologia , Ribonucleases/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Animais , Bovinos , Diferenciação Celular , Transformação Celular Viral , Relação Dose-Resposta a Droga , Epitélio/patologia , Metástase Neoplásica , Ratos , Sêmen/enzimologia , Glândula Tireoide/citologia , Neoplasias da Glândula Tireoide/patologiaRESUMO
Urokinase synthesized by human A431 epidermoid carcinoma cells is phosphorylated on serine (Mastronicola, M. R., Stoppelli, M. P., Migliaccio, A., Auricchio, F., and Blasi, F. (1990) FEBS Lett. 266, 109-114). To test the possibility that phosphorylation may have specific effects on urokinase function, the phosphorylated and nonphosphorylated forms of urokinase were separated by Fe(3+)-Sepharose chromatography. Both forms exhibit indistinguishable Km and kcat for plasminogen activation. On the other hand, their sensitivity toward the specific plasminogen activator inhibitor type 1 is different as assessed by measuring both the stability of the covalent complex and the residual enzymatic activity. Phosphorylated urokinase was 50% inhibited at a concentration of plasminogen activator inhibitor type 1 4-fold higher than nonphosphorylated urokinase (0.7 versus 0.15 nM). Furthermore about 10% of phosphorylated urokinase was resistant to plasminogen activator inhibitor type 1 at a concentration as high as 20 nM. Thus, phosphorylation affects urokinase sensitivity to plasminogen activator inhibitor type 1, therefore resulting in a net, although indirect, increase of urokinase activity. These results suggest the existence of a novel cellular regulatory mechanism of extracellular proteolysis.
Assuntos
Ativador de Plasminogênio Tipo Uroquinase/isolamento & purificação , Catálise , Humanos , Técnicas In Vitro , Cinética , Fosforilação , Fosfosserina/metabolismo , Inativadores de Plasminogênio/farmacologia , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Phosphorylation is a potent mechanism regulating the activity of many intracellular enzymes. We have discovered that the product of the human urokinase plasminogen activator gene, pro-uPA, is phosphorylated in serine in at least two human cell lines. Phosphorylation occurs within the cell during biosynthesis, and phosphorylated intracellular pro-uPA is secreted into the medium. Of the secreted pro-uPA molecules, 20-50% are phosphorylated in serine, thus representing a meaningful fraction of the total biosynthetic pro-uPA. Although the sites of phosphorylation have not yet been determined, at least two such sites must exist; in fact plasmin cleavage of phosphorylated single chain pro-uPA yields a two chain uPA in which both chains are phosphorylated. A specific function for pro-uPA phosphorylation has not yet been identified; however, it is tempting to speculate that, as in many other cases, phosphorylation may affect the activity of the enzyme, its response to inhibitors or the conversion of pro-uPA zymogen to active two-chain uPA. This would represent an additional way of regulating extracellular proteolysis, an important pathway involved in both intra- and extravascular phenomena like fibrinolysis, cell migration and invasiveness.
Assuntos
Precursores Enzimáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Serina/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Humanos , Peso Molecular , Fosfosserina/metabolismo , Testes de Precipitina , Ativador de Plasminogênio Tipo Uroquinase/biossínteseRESUMO
A specific surface receptor for urokinase plasminogen activator (uPA) recognizes the amino-terminal growth factor-like sequence of uPA, a region independent from and not required for the catalytic activity of this enzyme. The properties of the uPA receptor (uPAR) and the localization and distribution of uPA in tumor cells and tissues suggest that the uPA/uPAR interaction may be important in regulating extracellular proteolysis-dependent processes (e.g., invasion, tissue destruction). Phorbol myristate acetate (PMA), an inducer of U937 cell differentiation to macrophage-like cells, elicits a time- and concentration-dependent increase in the number of uPAR molecules as shown by binding, cross-linking, and immunoprecipitation studies. The effect of PMA is blocked by cycloheximide. Overall, the data indicate that PMA increases the synthesis of uPA. PMA treatment also causes a decrease in the affinity of the uPAR for uPA, thus uncovering another way of regulating the interaction between uPA and uPAR. In addition, the PMA treatment causes a modification of migration of the cross-linked receptor in mono- and bidimensional gel electrophoresis.