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1.
Protein Expr Purif ; 13(2): 243-50, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9675069

ABSTRACT

Stromelysin 1 (ST1) is a member of the matrix metalloproteinase (MMP) family probably involved in extracellular matrix degradation. Stromelysin 3 (ST3), considered by sequence homology to be a member of the MMP family of proteases, is specifically expressed in the stroma adjacent to the invasive tumoral cells, but its role in cancer progression remains to be elucidated. Genes encoding ST1 and ST3 were expressed in lepidopteran insect cells using the baculovirus expression vector system. Recombinant baculoviruses were obtained after cloning the full-length cDNA of ST1 and ST3 in plasmids pBacPAK1 and pBacPAK9, respectively. Sf9 insect cells infected with the recombinant baculovirus overexpressed the zymogen proST1 (60 kDa) in an insoluble form, a peak of expression being reached from 24 h postinfection. After solubilization in 8 M urea, and further refolding, activation, and purification, 0.3 mg of mature ST1 (30 kDa), purified to 90% homogeneity, was obtained per 5 x 10(8) infected cells. Recombinant ST1 exhibited proteolytic activity on alpha2-macroglobulin, casein, fibronectin, alpha1-antitrypsin, and laminin. The recombinant zymogen proST3 (55 kDa) was expressed as a soluble form in insect cells, maximal expression occurring at 72 h postinfection. After purification to 95% homogeneity, 2.5 mg of proST3 was obtained per 5 x 10(8) infected cells. A number of proteases including plasmin, urokinase, and ST1 were shown to be able to cleave proST3 giving rise to defined bands of 50-30 kDa. The ST3 mature form of 45 kDa (mST3) was also expressed in the baculovirus system and the obtained protein, 2. 5 mg per 5 x 10(8) infected cells purified to 80% homogeneity, was shown to be active on both casein degradation and alpha2-macroglobulin entrapment assays. Our results suggest that the baculovirus system offers a convenient and efficient means to produce ST1 and ST3 in order to carry out further biochemical studies.


Subject(s)
Matrix Metalloproteinase 3/biosynthesis , Metalloendopeptidases/biosynthesis , Recombinant Proteins/biosynthesis , Animals , Baculoviridae/genetics , Caseins/metabolism , Cloning, Molecular , DNA, Complementary/genetics , Gene Expression , Humans , Matrix Metalloproteinase 11 , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 3/metabolism , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Protein Precursors/biosynthesis , Protein Precursors/genetics , Spodoptera/cytology , Spodoptera/virology , alpha-Macroglobulins/metabolism
2.
J Biol Chem ; 272(41): 25706-12, 1997 Oct 10.
Article in English | MEDLINE | ID: mdl-9325295

ABSTRACT

To elucidate the physiological role of human stromelysin-3 (hST-3) in tumor progression and/or wound healing, insulin-like growth factor-binding protein-1 (IGFBP-1) was analyzed as a potential physiological substrate. hST-3 proteolysis generates two fragments of 16 and 9 kDa that react with IGFBP-1 monoclonal antibody, although they do not bind insulin-like growth factor-I (IGF-I) in ligand blot. N-terminal sequencing shows that hST-3 cleaves IGFBP-1 at the His140-Val141 bond located in the IGFBP-1 midregion. We show that IGFBP-1 inhibits IGF-I-induced survival and proliferation of BAF/3 cells, as well as IGF-I-mediated activation of phosphatidylinositol 3-kinase (PI 3-K). Co-incubation of the IGF-I. IGFBP-1 complex with hST-3 restores IGF-I-induced proliferation and PI 3-K kinase activity in these cells. BAF/3 proliferation is significantly increased with the hST-3-treated IGF-I.IGFBP-1 complex compared with that obtained using IGF-I alone. To produce this enhanced proliferation, IGF-I must bind to IGFBP-1 before hST-3 proteolysis, demonstrated using an IGF-I variant that does not bind IGFBP. IGFBP-1 also inhibits IGF-I-induced proliferation of the MCF-7 breast adenocarcinoma, and this inhibition was not seen in hST-3-transfected MCF-7 cells. Such proteolysis may thus play a role in in vivo tumor progression. These results indicate that hST-3 may regulate IGF-I bioavailability by proteolyzing IGFBP, thus favoring cell survival and proliferation.


Subject(s)
Insulin-Like Growth Factor Binding Protein 1/metabolism , Metalloendopeptidases/metabolism , Humans , Hydrolysis , Insulin-Like Growth Factor I/metabolism , Matrix Metalloproteinase 11 , Metalloendopeptidases/genetics , Molecular Weight , Peptide Mapping , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Transfection , Tumor Cells, Cultured
3.
Clin Exp Metastasis ; 15(1): 26-32, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9009103

ABSTRACT

To measure matrix metalloproteinase (MMP) activity in a large number of samples it is advisable to use easily automated methods. We have evaluated and compared the activity of stromelysin-1 (MMP-3), matrilysin (MMP-7), 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) by zymogram analysis and fluorescent substrate degradation assays. FITC-casein and the fluorogenic peptide Dnp-Pro-beta-cyclo-hexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-Abz)-NH 2 were used as fluorescent substrates. FITC-casein was more efficiently degraded than the fluorogenic peptide by all MMPs tested except MMP-9. MMP-2 was not significantly able to degrade the fluorogenic peptide. Gelatin zymography was the most sensitive method to detect the activity of both gelatinases but quantitation problems compromise its use. The degradation of fluorogenic substrates by MMPs could be inhibited by the chelating agent EDTA and by the tissue inhibitor of metalloproteinases 2 (TIMP-2), an MMP-specific inhibitor. Fluorometric methods represent a good alternative for MMP activity measurement, especially when a large number of samples must be processed.


Subject(s)
Collagenases/analysis , Electrophoresis, Polyacrylamide Gel , Fluorometry , Gelatinases/analysis , Matrix Metalloproteinase 3/analysis , Metalloendopeptidases/analysis , Recombinant Fusion Proteins/analysis , Calcium/physiology , Caseins/metabolism , Chelating Agents/pharmacology , Collagenases/metabolism , Edetic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Gelatinases/antagonists & inhibitors , Gelatinases/metabolism , Humans , Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase 7 , Matrix Metalloproteinase 9 , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/metabolism , Proteins/pharmacology , Recombinant Fusion Proteins/antagonists & inhibitors , Tissue Inhibitor of Metalloproteinase-2
5.
J Med Chem ; 28(1): 40-6, 1985 Jan.
Article in English | MEDLINE | ID: mdl-2981323

ABSTRACT

A series of analogues of uridine 5'-diphosphate glucose and uridine 5'-diphosphate glucosamine have been synthesized by reaction of 2,3,4,6-tetra-O-benzyl-, 2,3,4,6-tetra-O-benzoyl-, 2,3,4,6-tetra-O-acetyl-, and 2,3,4,6-tetra-O-palmitoyl-alpha-D-glucopyranose and 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranose with chlorosulfonyl isocyanate and 2',3'-O-isopropylideneuridine. Isopropylidene and acetyl groups of the resulting 5'-O-[[[[(alpha-D-glucopyranosyl)oxy]carbonyl]amino]sulfonyl] -2',3'-O-isopropylideneuridine derivatives were removed by reaction with a TFA/water (5:1) mixture and methanolic ammonia, respectively. The 5'-O-[[[[(2",3",4",6"-tetra-O-benzyl-and 2",3",4",6"-tetra-O-benzoyl-alpha-D-glucopyranosyl)oxy]carbonyl] amino]sulfonyl]-2',3'-O-isopropylideneuridine (13 and 19) and the corresponding deisopropylidenated derivatives showed antiviral activity as determined by the inhibition of the cytopathic effect induced by HSV-1 replication and by the plaque assay method. Compound 13 inhibited glycosylation of proteins in HSV-1 infected HeLa cells.


Subject(s)
Antiviral Agents/pharmacology , Carbohydrate Metabolism , Proteins/metabolism , Uridine Diphosphate Glucose/pharmacology , Uridine Diphosphate N-Acetylglucosamine/pharmacology , Uridine Diphosphate Sugars/pharmacology , HeLa Cells , Humans , Magnetic Resonance Spectroscopy , Simplexvirus/drug effects , Uridine Diphosphate Glucose/analogs & derivatives , Uridine Diphosphate N-Acetylglucosamine/analogs & derivatives
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