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1.
Mol Biol (Mosk) ; 36(1): 76-83, 2002.
Article in Russian | MEDLINE | ID: mdl-11862716

ABSTRACT

In all secreted proteins related to the epidermal growth factor (EGF), EGF domains that occur in a mature factor are each encoded by two exons, and those that do not, by one exon. During splicing, additional exon 3a can be inserted between exons 3 and 4, which code for the EGF domain of the mature heparin-binding EGF-like growth factor (HB-EGF). The resulting mRNA codes for the short form of HB-EGF (SF HB-EGF), which retains the signal peptide, the propeptide, and the heparin-binding domain. However, its EGF domain lacks the C-terminal subdomain essential for the interaction with the EGF receptor (EGFR). Structural analysis suggested that SF HB-EGF is a secreted polypeptide that has high affinity for heparin, but weakly, if at all, interacts with EGFR. Data obtained in three different systems indicated that SF HB-EGF possesses a mitogenic activity but utilizes a signal transduction pathway other than that of HB-EGF.


Subject(s)
Epidermal Growth Factor/genetics , Heparin/metabolism , Alternative Splicing , Amino Acid Sequence , Animals , Cells, Cultured , Cloning, Molecular , DNA/biosynthesis , DNA/drug effects , Epidermal Growth Factor/chemistry , Epidermal Growth Factor/metabolism , ErbB Receptors/metabolism , Exons , Heparin-binding EGF-like Growth Factor , Intercellular Signaling Peptides and Proteins , Mammals , Mitogens/pharmacology , Molecular Sequence Data , Protein Structure, Tertiary , RNA, Messenger , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Sequence Homology, Amino Acid , Signal Transduction
2.
Toxicon ; 39(11): 1723-8, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11595634

ABSTRACT

The plant toxin ricin consists of two disulfide-linked polypeptides with different functions. The A-chain enters the cytosol and inactivates the ribosomes enzymatically, whereas the B-chain has lectin properties and binds to carbohydrates at the cell surface. This binding is a requirement for translocation of the A-chain to the cytosol. The bound toxin is endocytosed and transported retrograde through the Golgi apparatus to the endoplasmic reticulum where it appears to be translocated to the cytosol by the sec61p complex.


Subject(s)
Ricin/toxicity , Animals , Biological Transport , Cytosol/metabolism , Humans , Models, Molecular , Ricin/chemistry , Ricin/metabolism
3.
J Cell Sci ; 114(Pt 9): 1677-89, 2001 May.
Article in English | MEDLINE | ID: mdl-11398757

ABSTRACT

Endocytic uptake and intracellular transport of acidic fibroblast growth factor (aFGF) was studied in cells transfected with FGF receptor 4 with mutations in the cytoplasmic part. Endocytic uptake in HeLa cells was reduced but not abolished when the tyrosine kinase of the receptor was inactivated by mutations or deletions. The tyrosine kinase-dependent endocytosis of aFGF was prevented by the expression of a dominant negative dynamin mutant that blocks endocytosis from coated pits and caveolae. However, more than half of the total endocytic uptake of aFGF was not affected under these conditions, indicating an endocytic uptake mechanism not involving coated pits or caveolae. Mutation or deletion of a putative caveolin-binding sequence did not prevent the localization of part of the receptors to a low density, caveolin-containing subcellular fraction. Whereas wild-type receptor transfers the growth factor from early endosomes to the recycling compartment, kinase negative, full length receptors were inefficient in this respect and the growth factor instead accumulated in lysosomes. By contrast, when most of the intracellular part of the receptor, including the kinase domain, was removed, aFGF was transported to the recycling compartment, as in cells that express wild-type receptors, suggesting the presence of a kinase-regulated targeting signal in the cytoplasmic tail.


Subject(s)
Fibroblast Growth Factor 1/metabolism , Mutation , Receptors, Cytoplasmic and Nuclear/metabolism , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Caveolin 1 , Caveolins/metabolism , DNA Primers , Endocytosis , HeLa Cells , Humans , Microscopy, Fluorescence , Molecular Sequence Data , Protein Binding , Protein Transport , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/genetics , Transfection
5.
Biochemistry ; 40(14): 4349-58, 2001 Apr 10.
Article in English | MEDLINE | ID: mdl-11284691

ABSTRACT

A number of proteins are able to enter cells from the extracellular environment, including protein toxins, growth factors, viral proteins, homeoproteins, and others. Many such translocating proteins, or parts of them, appear to be able to carry with them cargo into the cell, and a basic sequence from the HIV-1 Tat protein has been reported to provide intracellular delivery of several fused proteins. For evaluating the efficiency of translocation to the cytosol, this TAT-peptide was fused to the diphtheria toxin A-fragment (dtA), an extremely potent inhibitor of protein synthesis which normally is delivered efficiently to the cytosol by the toxin B-fragment. The fusion of the TAT-peptide to dtA converted the protein to a heparin-binding protein that bound avidly to the cell surface. However, no cytotoxicity of this protein was detected, indicating that the TAT-peptide is unable to efficiently deliver enzymatically active dtA to the cytosol. Interestingly, the fused TAT-peptide potentiated the binding and cytotoxic effect of the corresponding holotoxin. We made a fusion protein between VP22, another membrane-permeant protein, and dtA, and also in this case we detected association with cells in the absence of a cytotoxic effect. The data indicate that transport of dtA into the cell by the TAT-peptide and VP22 is inefficient.


Subject(s)
Diphtheria Toxin/metabolism , Gene Products, tat/physiology , Peptide Fragments/metabolism , Peptide Fragments/physiology , Viral Structural Proteins/physiology , Animals , Biological Transport, Active/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Carrier Proteins/toxicity , Cell Adhesion/drug effects , Cell Adhesion/genetics , Cell Membrane/genetics , Cell Membrane/metabolism , Chlorocebus aethiops , Cytosol/metabolism , Diphtheria Toxin/genetics , Diphtheria Toxin/toxicity , Drug Synergism , Gene Products, tat/genetics , Glycoproteins/genetics , Glycoproteins/metabolism , Glycoproteins/toxicity , HIV-1/physiology , Heparin/metabolism , Heparin/pharmacology , Herpesvirus 1, Human/physiology , Humans , LDL-Receptor Related Protein-Associated Protein , Peptide Fragments/genetics , Peptide Fragments/toxicity , Plasmids/chemical synthesis , Plasmids/toxicity , Protein Binding/drug effects , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/toxicity , Vero Cells , tat Gene Products, Human Immunodeficiency Virus
6.
Mol Biol (Mosk) ; 35(1): 136-45, 2001.
Article in Russian | MEDLINE | ID: mdl-11234372

ABSTRACT

A new mRNA coding for the heparin-binding EGF-like growth factor (HB-EGF) was found in Vero cells. The corresponding cDNA had C-156 in place of T, which resulted in a loss of the NheI site and a substitution of Leu-33 with Pro in the HB-EGF precursor. The known and new forms of the precursor were accordingly termed L and P. A possible conformational change in the corresponding propeptide region were assumed to affect processing of soluble secreted HB-EGF. The L and P mRNAs are differently expressed in various cell lines and have the identical 5'-untranslated sequences. Possibly, they are transcribed from one promoter and then alternatively spliced. Stimulation of resting Vero cells with tetraphorbol ester (TPA) substantially increased production of the L form, decreased production of the P form, and did not affect expression of the total HB-EGF mRNA. This was associated with an increase in binding of the diphtheria toxin, suggesting that the L HB-EGF precursor acts as its receptor.


Subject(s)
Epidermal Growth Factor/genetics , Protein Isoforms/genetics , Receptors, Cell Surface/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Blotting, Western , Cell Line , Cloning, Molecular , DNA, Complementary , Epidermal Growth Factor/chemistry , Heparin-binding EGF-like Growth Factor , Intercellular Signaling Peptides and Proteins , Molecular Sequence Data , Protein Isoforms/chemistry , RNA, Messenger/genetics , Receptors, Cell Surface/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Tetradecanoylphorbol Acetate/pharmacology
7.
J Biol Chem ; 276(10): 7202-8, 2001 Mar 09.
Article in English | MEDLINE | ID: mdl-11113144

ABSTRACT

Here we demonstrate that ricin is able to interact with the molecular chaperone calreticulin both in vitro and in vivo. The interaction occurred with ricin holotoxin, but not with free ricin A chain; and it was prevented in the presence of lactose, suggesting that it was mediated by the lectin activity of the ricin B chain. This lectin is galactose-specific, and metabolic labeling with [(3)H]galactose or treating galactose oxidase-modified calreticulin with sodium [(3)H]borohydride indicated that Vero cell calreticulin possesses a terminally galactosylated oligosaccharide. Brefeldin A treatment indicated that the intracellular interaction occurred initially in a post-Golgi stack compartment, possibly the trans-Golgi network, whereas the reductive separation of ricin subunits occurred in an earlier part of the secretory pathway, most probably the endoplasmic reticulum (ER). Intoxicating Vero cells with ricin whose A chain had been modified to include either a tyrosine sulfation site or the sulfation site plus available N-glycosylation sites, in the presence of Na(2)35SO(4), confirmed that calreticulin interacted with endocytosed ricin that had already undergone retrograde transport to both the Golgi and the ER. Although we cannot exclude the possibility that the interaction between ricin and calreticulin is an indirect one, the data presented are consistent with the idea that calreticulin may function as a recycling carrier for retrograde transport of ricin from the Golgi to the ER.


Subject(s)
Calcium-Binding Proteins/metabolism , Ribonucleoproteins/metabolism , Ricin/chemistry , Ricin/metabolism , Animals , Brefeldin A/pharmacology , Calreticulin , Chlorocebus aethiops , Endocytosis , Endoplasmic Reticulum/metabolism , Galactose/metabolism , Glycosylation , Golgi Apparatus/metabolism , Lactose/pharmacology , Lectins/metabolism , Male , Plasmids/metabolism , Precipitin Tests , Protein Binding , Protein Synthesis Inhibitors/pharmacology , Protein Transport , Rats , Rats, Sprague-Dawley , Tyrosine/metabolism , Vero Cells
8.
Biochemistry ; 39(49): 15091-100, 2000 Dec 12.
Article in English | MEDLINE | ID: mdl-11106487

ABSTRACT

Acidic fibroblast growth factor (aFGF) is transported to the cytosol and the nucleus when added to cells expressing FGF receptors, implying that aFGF must cross cellular membranes. Since protein translocation across membranes commonly requires extensive unfolding of the protein, we were interested in testing whether this is also necessary for membrane translocation of aFGF. We therefore constructed mutant growth factors with intramolecular disulfide bonds to prevent complete unfolding. Control experiments demonstrated that translocation of aFGF by the diphtheria toxin pathway, which requires extensive unfolding of the protein, was prevented by disulfide bond formation, indicating that the introduced disulfide bonds interfered with the unfolding of the growth factor. On the other hand, when the growth factor as such was added to cells expressing FGF receptors, the disulfide-bonded mutants were translocated to the cytosol and the nucleus equally well as wild-type aFGF. The possibility that the translocation of the mutants was due to reduction of the disulfide bonds prior to translocation was tested in experiments using an irreversibly cross-linked mutant. Also this mutant was transported to the cytosol and to the nucleus. The results suggest that extensive unfolding is not required for membrane translocation of aFGF.


Subject(s)
Cell Nucleus/metabolism , Cytosol/metabolism , Fibroblast Growth Factor 1/metabolism , Protein Folding , Protein Transport , 3T3 Cells , Animals , Cell Membrane/metabolism , Cross-Linking Reagents , Diphtheria Toxin/genetics , Diphtheria Toxin/metabolism , Disulfides , Fibroblast Growth Factor 1/genetics , Intracellular Membranes/metabolism , Mice , Models, Molecular , Mutation , Phosphorylation , Protein Conformation , Protein Denaturation , Receptors, Fibroblast Growth Factor/metabolism , Recombinant Fusion Proteins/metabolism
9.
Biochem J ; 352 Pt 3: 629-35, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11104667

ABSTRACT

Acidic fibroblast growth factor (aFGF) intracellular binding protein (FIBP) is a protein found mainly in the nucleus that might be involved in the intracellular function of aFGF. Here we present a comparative analysis of the deduced amino acid sequences of human, murine and Drosophila FIBP analogues and demonstrate that FIBP is an evolutionarily conserved protein. The human gene spans more than 5 kb, comprising ten exons and nine introns, and maps to chromosome 11q13.1. Two slightly different splice variants found in different tissues were isolated and characterized. Sequence analysis of the region surrounding the translation start revealed a CpG island, a classical feature of widely expressed genes. Functional studies of the promoter region with a luciferase reporter system suggested a strong transcriptional activity residing within 600 bp of the 5' flanking region.


Subject(s)
Carrier Proteins/genetics , Chromosomes, Human, Pair 11/genetics , Fibroblast Growth Factor 1/metabolism , Promoter Regions, Genetic/genetics , Alternative Splicing/genetics , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Line , Cloning, Molecular , Codon, Initiator/genetics , Conserved Sequence/genetics , CpG Islands/genetics , Drosophila melanogaster/genetics , Exons/genetics , Humans , In Situ Hybridization, Fluorescence , Intercellular Signaling Peptides and Proteins , Intracellular Signaling Peptides and Proteins , Introns/genetics , Mice , Molecular Sequence Data , Physical Chromosome Mapping , RNA, Messenger/analysis , RNA, Messenger/genetics , Sequence Alignment , Transfection
10.
J Biol Chem ; 275(16): 11972-80, 2000 Apr 21.
Article in English | MEDLINE | ID: mdl-10766827

ABSTRACT

Acidic fibroblast growth factor (aFGF) is a potent mitogen for many cells. Exogenous aFGF is able to enter the cytosol and nucleus of sensitive cells. There are indications that both activation of the receptor tyrosine kinase and translocation of aFGF to the nucleus are of importance for mitogenesis. However, the mechanism of transport of aFGF from the cell surface to the nucleus is poorly understood. In this work we demonstrate that inhibition of phosphatidylinositol (PI) 3-kinase by chemical inhibitors and by expression of a dominant negative mutant of PI 3-kinase blocks translocation of aFGF to the cytosol and nucleus. Translocation to the cytosol and nucleus was monitored by cell fractionation, by farnesylation of aFGF modified to contain a farnesylation signal, and by phosphorylation by protein kinase C of aFGF added externally to cells. If aFGF is fused to diphtheria toxin A-fragment, it can be artificially translocated from the cell surface to the cytoplasm by the diphtheria toxin pathway. Upon further incubation, the fusion protein enters the nucleus due to a nuclear localization sequence in aFGF. We demonstrate here that upon inhibition of PI 3-kinase the fusion protein remains in the cytosol. We also provide evidence that the phosphorylation status of the fusion protein does not regulate its nucleocytoplasmic distribution.


Subject(s)
Cell Nucleus/metabolism , Cytosol/metabolism , Fibroblast Growth Factor 1/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases , 3T3 Cells , Animals , Biological Transport , COS Cells , Cell Fractionation , Cells, Cultured , Chromones/pharmacology , Diphtheria Toxin/metabolism , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/pharmacology , Mice , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Recombinant Fusion Proteins/metabolism
11.
J Cell Sci ; 113 ( Pt 10): 1827-38, 2000 May.
Article in English | MEDLINE | ID: mdl-10769213

ABSTRACT

The ability of COS cells to bind and internalise acidic fibroblast growth factor (aFGF) was studied after transient transfection of the cells with wild-type and mutated fibroblast growth factor receptor 4. In one case the tyrosine kinase of the receptor was inactivated by a point mutation in the active site, whereas in other cases parts of the receptor were deleted to remove various parts of the cytoplasmic domain. In all cases the receptors were expressed at the cell surface at a high level and the cells bound labelled growth factor efficiently and internalised it by endocytosis. Translocation of externally added aFGF across cellular membranes to reach the cytosol and nucleus was measured as transport of labelled growth factor to the nuclear fraction obtained by centrifugation, by farnesylation of growth factor modified to carry a CAAX motif, and by phosphorylation of the growth factor at a site specific for protein kinase C. Whereas both full-length receptors (with and without an active kinase domain) facilitated translocation of the growth factor to the cytosol and nucleus, as assessed by these methods, the mutants of the receptor where the C terminus was deleted, were unable to do so. In contrast, a receptor containing only the 57 most C-terminal amino acids of the cytoplasmic domain in addition to the juxtamembrane, transmembrane and extracellular domains, was in fact able to mediate translocation of aFGF to the cytosol. These data indicate that information contained in the C terminus of the receptor is required for translocation.


Subject(s)
Cell Nucleus/metabolism , Cytosol/metabolism , Fibroblast Growth Factor 1/metabolism , Receptors, Fibroblast Growth Factor/genetics , Receptors, Fibroblast Growth Factor/metabolism , Animals , Binding Sites/physiology , Biological Transport/physiology , COS Cells , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Membrane/metabolism , Endocytosis/physiology , Gene Deletion , Phosphotransferases/metabolism , Point Mutation/genetics , Protein Structure, Tertiary , Transfection
13.
J Biol Chem ; 275(6): 4363-8, 2000 Feb 11.
Article in English | MEDLINE | ID: mdl-10660606

ABSTRACT

Diphtheria toxin A-fragment enters the cytosol of target cells, where it inhibits protein synthesis by catalyzing ADP-ribosylation of elongation factor 2 (EF-2). We have here analyzed toxin-induced protein synthesis inhibition in single cells by autoradiography and compared it with inhibition of protein synthesis in the whole cell culture. The data show that half-maximal protein synthesis inhibition in the whole cell population after a short incubation time is achieved by partially inhibiting protein synthesis in basically all the cells, while half-maximal protein synthesis inhibition after a long incubation time is due to a complete protein synthesis block in about half the cells in the population. We have also compared stable and unstable A-fragment mutants with respect to the kinetics of cell intoxication. While the toxicity of the stable mutants increased with time, the unstable mutants showed a similar toxicity at early and late time points. When studying the kinetics of cell intoxication by toxins with short cytosolic half-life, we could not detect any recovery of protein synthesis at late time points when all the mutant A-fragments should be degraded. This indicates that the ADP-ribosylation of EF-2 cannot be reversed by an endogenous activity in the cells. The data indicate that entry of toxin into a cell is not associated with an immediate block in protein synthesis, and that prolonged action of single A-fragment molecules in the cytosol is sufficient to obtain complete protein synthesis inhibition at low toxin concentrations.


Subject(s)
Cytosol/metabolism , Diphtheria Toxin/pharmacology , Peptide Fragments/pharmacology , Protein Biosynthesis , Protein Synthesis Inhibitors/pharmacology , Animals , Chlorocebus aethiops , Diphtheria Toxin/genetics , Hydrogen-Ion Concentration , Kinetics , Mutation , Peptide Elongation Factor 2/metabolism , Peptide Fragments/genetics , Vero Cells
14.
J Biol Chem ; 274(48): 34443-9, 1999 Nov 26.
Article in English | MEDLINE | ID: mdl-10567425

ABSTRACT

Ricin acts by translocating to the cytosol the enzymatically active toxin A-chain, which inactivates ribosomes. Retrograde intracellular transport and translocation of ricin was studied under conditions that alter the sensitivity of cells to the toxin. For this purpose tyrosine sulfation of mutant A-chain in the Golgi apparatus, glycosylation in the endoplasmic reticulum (ER) and appearance of A-chain in the cytosolic fraction was monitored. Introduction of an ER retrieval signal, a C-terminal KDEL sequence, into the A-chain increased the toxicity and resulted in more efficient glycosylation, indicating enhanced transport from Golgi to ER. Calcium depletion inhibited neither sulfation nor glycosylation but inhibited translocation and toxicity, suggesting that the toxin is translocated to the cytosol by the pathway used by misfolded proteins that are targeted to the proteasomes for degradation. Slightly acidified medium had a similar effect. The proteasome inhibitor, lactacystin, sensitized cells to ricin and increased the amount of ricin A-chain in the cytosol. Anti-Sec61alpha precipitated sulfated and glycosylated ricin A-chain, suggesting that retrograde toxin translocation involves Sec61p. The data indicate that retrograde translocation across the ER membrane is required for intoxication.


Subject(s)
Cytosol/metabolism , Endoplasmic Reticulum/metabolism , Ricin/metabolism , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Animals , Antibodies/immunology , Biological Transport/drug effects , Calcium/pharmacology , Chlorocebus aethiops , Cysteine Endopeptidases/drug effects , Cysteine Proteinase Inhibitors/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , Glycosylation , Humans , Hydrogen-Ion Concentration , Ionophores/pharmacology , Membrane Proteins/immunology , Monensin/pharmacology , Multienzyme Complexes/drug effects , Mutation , Precipitin Tests , Proteasome Endopeptidase Complex , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/toxicity , Ricin/genetics , Ricin/toxicity , SEC Translocation Channels , Sulfates/metabolism , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Vero Cells
15.
Mol Biol Cell ; 10(11): 3835-48, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10564275

ABSTRACT

Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolin-containing fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfona te, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment.


Subject(s)
Caveolins , Fibroblast Growth Factor 1/metabolism , Receptors, Fibroblast Growth Factor/genetics , Animals , Biological Transport , Caveolin 1 , Cell Line , Cross-Linking Reagents , Cytoskeleton/metabolism , Fluorescent Antibody Technique , Humans , Iodine Radioisotopes , Membrane Proteins/metabolism , Nocodazole/pharmacology , Phosphotyrosine/analysis , Protein-Tyrosine Kinases/metabolism , Receptor, Fibroblast Growth Factor, Type 4 , Succinimides/chemistry , Transfection , Transferrin/metabolism
16.
Biochem J ; 343 Pt 1: 199-207, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10493930

ABSTRACT

Diphtheria toxin enters the cytosol of mammalian cells where it inhibits cellular protein synthesis, leading to cell death. Recently we found that the addition of a signal for N-end-rule-mediated protein degradation to diphtheria toxin substantially reduced its intracellular stability and toxicity. These results prompted us to construct a toxin containing a degradation signal that is removable through the action of a viral protease. In principle, such a toxin would be preferentially stabilized, and thus activated, in cells expressing the viral protease in the cytosol, i.e. virus-infected cells, thereby providing a specific eradication of these cells. In the present work we describe the construction of toxins that contain a signal for N-end-rule-mediated degradation just upstream of a cleavage site for the protease from HIV type 1 (HIV-1 PR). We show that the toxins are cleaved by HIV-1 PR exclusively at the introduced sites, and thereby are converted from unstable to stable proteins. Furthermore, this cleavage substantially increased the ability of the toxins to inhibit cellular protein synthesis. However, the toxins were unable to selectively eradicate HIV-1-infected cells, apparently due to low cytosolic HIV-1 PR activity, since we could not detect cleavage of the toxins by HIV-1 PR in infected cells. Alternative strategies for the construction of toxins that can specifically be activated by viral proteases are discussed.


Subject(s)
Antigens, Bacterial , Bacterial Toxins/pharmacokinetics , Biotransformation , Diphtheria Toxin/pharmacokinetics , HIV Protease/metabolism , Amino Acid Sequence , Bacterial Toxins/genetics , Bacterial Toxins/pharmacology , Base Sequence , DNA Primers , Diphtheria Toxin/genetics , Diphtheria Toxin/pharmacology , HeLa Cells , Humans , Hydrolysis , Molecular Sequence Data , Mutagenesis , Protein Synthesis Inhibitors/pharmacology
17.
J Biol Chem ; 274(25): 18081-6, 1999 Jun 18.
Article in English | MEDLINE | ID: mdl-10364261

ABSTRACT

Acidic fibroblast growth factor (aFGF) contains a phosphorylation site recognized by protein kinase C. A non-mitogenic mutant growth factor is devoid of this phosphorylation site. We have changed amino acids in and close to the phosphorylation site and studied the consequences of this for binding of the growth factor to high affinity receptors as well as to heparin. We have also studied the ability of the mutants to stimulate DNA synthesis and cell proliferation as well as phosphorylation of mitogen-activated protein kinase and the ability of the growth factor mutants to be transported to the nucleus. The results indicate that while the mutations strongly affect the ability of the growth factor to bind to heparin, they do not affect much the binding to the specific FGF receptors, activation of mitogen-activated protein kinase or transport of the growth factor to the nucleus. The mutations affect to various extents the ability of the growth factor to stimulate DNA synthesis and to induce cell multiplication. We find that phosphorylation of aFGF is not required for mitogenic activity. The data suggest that altered interaction of the growth factor with a cellular component different from the receptor, possibly a component in the nucleus, is the reason for the different mitogenicity of the different growth factor mutants.


Subject(s)
Fibroblast Growth Factor 1/genetics , 3T3 Cells , Animals , Binding Sites , Biological Transport , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Division , Cell Nucleus/metabolism , DNA Replication/genetics , Fibroblast Growth Factor 1/chemistry , Heparin/metabolism , Mice , Mutation , Phosphorylation , Protein Kinase C/metabolism , Receptors, Fibroblast Growth Factor/metabolism
19.
Biochemistry ; 37(45): 15737-46, 1998 Nov 10.
Article in English | MEDLINE | ID: mdl-9843379

ABSTRACT

Solving the crystallographic structure of the ring-shaped heptamer formed by protective antigen (PA), the B moiety of anthrax toxin, has focused attention on understanding how this oligomer mediates membrane translocation of the toxin's A moieties. We have developed an assay for translocation in which radiolabeled ligands are bound to proteolytically activated PA (PA63) at the surface of CHO or L6 cells, and translocation across the plasma membrane is induced by lowering the pH. The cells are then treated with Pronase E to degrade residual surface-bound material, and protected ligands are quantified after fractionation by SDS-PAGE. Translocation was most efficient (35%-50%) with LFN, the N-terminal PA binding domain of the anthrax lethal factor (LF). Intact LF, edema factor (EF), or fusion proteins containing LFN fused to certain heterologous proteins [the diphtheria toxin A chain (DTA) or dihydrofolate reductase (DHFR)] were less efficiently translocated (15%-20%); and LFN fusions to several other proteins were not translocated at all. LFN with different N-terminal residues was found to be degraded according to the N-end rule by the proteasome, and translocation of LFN fused to a mutant form of DHFR with a low affinity for methotrexate (MTX) protected cells from the effects of MTX. Both results are consistent with a cytosolic location of protected proteins. Evidence that a protein must unfold to be translocated was obtained in experiments showing that (i) translocation of LFNDTA was blocked by introduction of an artificial disulfide into the DTA moiety, and (ii) translocation of LFNDHFR and LFNDTA was blocked by their ligands (MTX and adenine, respectively). These results demonstrate that the acid-induced translocation by anthrax toxin closely resembles that of diphtheria toxin, despite the fact that these two toxins are unrelated and form pores by different mechanisms.


Subject(s)
Antigens, Bacterial/metabolism , Bacillus anthracis/immunology , Bacterial Toxins/metabolism , Amino Acid Sequence , Animals , Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Biological Transport/genetics , Biological Transport/immunology , Biomarkers , CHO Cells , Cell Line , Cell Membrane/genetics , Cell Membrane/metabolism , Cell Membrane/microbiology , Cricetinae , Methotrexate/antagonists & inhibitors , Methotrexate/toxicity , Molecular Sequence Data , Pronase/metabolism , Protein Folding , Rats , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Tetrahydrofolate Dehydrogenase/genetics , Tetrahydrofolate Dehydrogenase/pharmacology
20.
Biochem J ; 336 ( Pt 1): 213-22, 1998 Nov 15.
Article in English | MEDLINE | ID: mdl-9806903

ABSTRACT

In addition to its extracellular action, there is evidence that acidic fibroblast growth factor (aFGF) acts inside cells. To identify intracellular proteins interacting with aFGF, we screened a HeLa cell library in the yeast two-hybrid system using pLex-aFGF as a bait. A clone binding to aFGF, but not to the non-mitogenic mutant aFGF-K132E, was isolated and characterized. The insert contained an open reading frame corresponding to a novel protein of 42 kDa. The protein, termed aFGF intracellular binding protein (FIBP), is mainly hydrophilic and does not contain an N-terminal signal sequence. In vitro-translated FIBP bound specifically to a fusion protein of maltose-binding protein and aFGF. FIBP became post-translationally associated with microsomes added to the cell-free protein synthesizing system, and the membrane-associated protein bound aFGF with high efficiency. Immunoblots and fluorescence microscopy demonstrated that the protein is present in nuclei and, to a lesser extent, associated with mitochondria and other cytoplasmic membranes. The possibility is discussed that FIBP may be involved in the mitogenic action of aFGF.


Subject(s)
Carrier Proteins/genetics , Fibroblast Growth Factor 1/metabolism , Mitogens/metabolism , Amino Acid Sequence , Animals , COS Cells , Carrier Proteins/metabolism , Chlorocebus aethiops , Cloning, Molecular , DNA Replication , DNA, Complementary , Fibroblast Growth Factor 1/genetics , Fluorescent Antibody Technique, Indirect , HeLa Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Fluorescence , Molecular Sequence Data , Protein Binding , RNA, Messenger/genetics , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism , Vero Cells
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