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1.
Scand J Immunol ; 66(2-3): 228-37, 2007.
Article in English | MEDLINE | ID: mdl-17635800

ABSTRACT

T lymphocytes are activated by the interaction between the T-cell antigen receptor (TCR) and peptides presented by major histocompatibility complex (MHC) molecules. The avidity of this TCR-pMHC interaction is very low. Therefore, several hypotheses have been put forward to explain how T cells become specifically activated despite this handicap: conformational change model, aggregation model, kinetic segregation model, sequential interaction model and permissive geometry model. In the present paper, we conducted experiments to distinguish between the TCR-aggregation model and the TCR-conformational change model. The results obtained using a TCR capture ELISA with Brij 98-solubilized TCR molecules from normal or activated T cells showed that the ligand-TCR interaction causes structural changes in the CD3 epsilon cytoplasmic tail as well as in the extracellular TCR beta FG loop region. Size-fractionation experiments with Brij 98-solubilized TCR/CD3/co-receptor complexes from naïve or activated CD4(+) or CD8(+) T cells demonstrated that such complexes are found as either dimers or tetramers. No monomers or multimers were detected. We propose that: (1) ligand-TCR interaction results in conformational changes in the CD3 epsilon cytoplasmic tail leading to T-cell activation; (2) CD3 epsilon cytoplasmic tail interaction with intracellular proteins may dissociate pMHC and co-receptors (CD4 or CD8) from TCR/CD3 complexes, thus leading to the arrest of T-cell activation; and (3) T-cell activation appears to occur among dimers or tetramers of TCR/CD3/co-receptor complexes interacting with self and non-self (foreign) peptide-MHC complexes.


Subject(s)
CD3 Complex/metabolism , Extracellular Fluid/metabolism , Intracellular Fluid/metabolism , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Allosteric Regulation/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/metabolism , CD3 Complex/chemistry , CD3 Complex/genetics , Cell Line, Tumor , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Ligands , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Molecular Sequence Data , Receptors, Antigen, T-Cell, alpha-beta/chemistry , Receptors, Antigen, T-Cell, alpha-beta/immunology
2.
Mol Pharmacol ; 67(5): 1485-92, 2005 May.
Article in English | MEDLINE | ID: mdl-15703384

ABSTRACT

DNA polymerase beta (Pol beta) is an error-prone enzyme whose up-regulation has been shown to be a genetic instability enhancer as well as a contributor to cisplatin resistance in tumor cells. In this work, we describe the isolation of new Pol beta inhibitors after high throughput screening of 8448 semipurified natural extracts. In vitro, the selected molecules affect specifically Pol beta-mediated DNA synthesis compared with replicative extracts from cell nuclei. One of them, masticadienonic acid (MA), is particularly attractive because it perturbs neither the activity of the purified replicative Pol delta nor that of nuclear HeLa cell extracts. With an IC50 value of 8 microM, MA is the most potent of the Pol beta inhibitors found so far. Docking simulation revealed that this molecule could substitute for single-strand DNA in the binding site of Pol beta by binding Lys35, Lys68, and Lys60, which are the main residues involved in the interaction Pol beta/single-strand DNA. Selected inhibitors also affect the Pol beta-mediated translesion synthesis (TLS) across cisplatin adducts; MA was still the most efficient. Therefore, masticadienonic acid sensitized the cisplatin-resistant 2008C13*5.25 human tumor cells. Our data suggest that molecules such as masticadienonic acid could be suitable in conjunction with cisplatin to enhance anticancer treatments.


Subject(s)
Cisplatin/pharmacology , DNA Polymerase beta/antagonists & inhibitors , DNA Polymerase beta/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Animals , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/isolation & purification , HeLa Cells , Humans , Juniperus , Pistacia , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Plant Leaves , Plant Stems , Rats
3.
Biochem J ; 340 ( Pt 3): 775-82, 1999 Jun 15.
Article in English | MEDLINE | ID: mdl-10359664

ABSTRACT

The myristoylated alanine-rich C-kinase substrate (MARCKS) purified from brain was recently characterized as a proline-directed kinase(s) substrate in vivo [Taniguchi, Manenti, Suzuki and Titani (1994) J. Biol. Chem. 269, 18299-18302]. Here we have investigated the phosphorylation of MARCKS by various cyclin-dependent kinases (Cdks) in vitro. We established that Cdk2, Cdk4 and, to a smaller extent, Cdk1 that have been immunoprecipitated from cellular extracts phosphorylate MARCKS. Comparison of MARCKS phosphorylation by protein kinase C (PKC) and by the purified cyclin E-Cdk2 complex suggested that two residues were phosphorylated by Cdk2 under these conditions. To identify these sites, Cdk2-phosphorylated MARCKS was digested with lysyl endoprotease and analysed by electrospray MS. Comparison with the digests obtained from the unphosphorylated protein demonstrated that two peptides, Gly12-Lys30 and Ala138-Lys152, were phosphorylated by cyclin E-Cdk2. The identity of these peptides was confirmed by automatic Edman degradation. On the basis of the consensus phosphorylation sequence described for Cdk2, and on MS/MS analysis of the Ala138-Lys152 peptide, we concluded that Ser27, one of the phosphorylation sites identified in vivo, and Thr150 were the Cdk2 targets in vitro. None of the other sites described in vivo were phosphorylated in these conditions. Interestingly, a preliminary phosphorylation of MARCKS by PKC improved the initial rate of phosphorylation by Cdk2 without modifying the number of sites concerned. In contrast, phosphorylation of MARCKS by Cdk2 did not significantly affect further phosphorylation by PKC.


Subject(s)
CDC2-CDC28 Kinases , Cyclin E/metabolism , Cyclin-Dependent Kinases/metabolism , Intracellular Signaling Peptides and Proteins , Membrane Proteins , Protein Serine-Threonine Kinases/metabolism , Proteins/metabolism , Proto-Oncogene Proteins , Base Sequence , CDC2 Protein Kinase/metabolism , Consensus Sequence/genetics , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase 4 , Histones/metabolism , Humans , Kinetics , Mass Spectrometry , Molecular Sequence Data , Myristoylated Alanine-Rich C Kinase Substrate , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Phosphorylation , Phosphothreonine/metabolism , Precipitin Tests , Protein Kinase C/metabolism , Proteins/chemistry , Retinoblastoma Protein/metabolism , Serine/metabolism , Serine Endopeptidases/metabolism , Substrate Specificity , Tumor Cells, Cultured
4.
Nature ; 396(6707): 184-6, 1998 Nov 12.
Article in English | MEDLINE | ID: mdl-9823900

ABSTRACT

Transforming viral proteins such as E1A force cells through the restriction point of the cell cycle into S phase by forming complexes with two cellular proteins: the retinoblastoma protein (Rb), a transcriptional co-repressor, and CBP/p300, a transcriptional co-activator. These two proteins locally influence chromatin structure: Rb recruits a histone deacetylase, whereas CBP is a histone acetyltransferase. Progression through the restriction point is triggered by phosphorylation of Rb, leading to disruption of Rb-associated repressive complexes and allowing the activation of S-phase genes. Here we show that CBP, like Rb, is controlled by phosphorylation at the G1/S boundary, increasing its histone acetyltransferase activity. This enzymatic activation is mimicked by E1A.


Subject(s)
Acetyltransferases/metabolism , Adenovirus E1A Proteins/metabolism , CDC2-CDC28 Kinases , Cell Cycle , Cyclin E/metabolism , Cyclin-Dependent Kinases/metabolism , Nuclear Proteins/metabolism , Oncogene Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins , Trans-Activators/metabolism , 3T3 Cells , Animals , CREB-Binding Protein , Cyclin-Dependent Kinase 2 , Enzyme Activation , G1 Phase , Histone Acetyltransferases , Mice , Phosphorylation , S Phase , Transcriptional Activation , Transfection
5.
Oncogene ; 16(3): 311-20, 1998 Jan 22.
Article in English | MEDLINE | ID: mdl-9467956

ABSTRACT

A unique feature of p21 that distinguishes it from the other cyclin-dependent kinase (CDK) inhibitors is its ability to associate with the proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerases delta and epsilon. While it is now well established that inhibition of cyclin/CDK complexes by p21 can result in G1 cell cycle arrest, the consequences of p21/PCNA interaction on cell cycle progression have not yet been determined. Here, we show, using a tetracycline-regulated system, that expression of wild-type p21 in p53-deficient DLD1 human colon cancer cells inhibits DNA synthesis and causes G1 and G2 cell cycle arrest. Similar effects are observed in cells expressing p21CDK-, a mutant impaired in the interaction with CDKs, but not in cells expressing p21PCNA-, a mutant deficient for the interaction with PCNA. Analysis of cells treated with a p21-derived PCNA-binding peptide provides additional evidence that the growth inhibitory effects of p21 and p21CDK result from their ability to bind to PCNA. Our results suggest that p21 might inhibit cell cycle progression by two independent mechanisms, inhibition of cyclin/CDK complexes, and inhibition of PCNA function resulting in both G1 and G2 arrest.


Subject(s)
Cyclins/metabolism , G1 Phase , G2 Phase , Proliferating Cell Nuclear Antigen/metabolism , Tumor Suppressor Protein p53/metabolism , Amino Acid Sequence , Colonic Neoplasms , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinases/metabolism , Cyclins/biosynthesis , Cyclins/genetics , DNA/biosynthesis , Gene Deletion , Gene Expression , Humans , Molecular Sequence Data , Mutagenesis , Proliferating Cell Nuclear Antigen/genetics , Solubility , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics
6.
J Biol Chem ; 272(52): 32731-4, 1997 Dec 26.
Article in English | MEDLINE | ID: mdl-9407044

ABSTRACT

In eukaryotes the activity of CDK1 (CDC2), a cyclin-dependent kinase that initiates the structural changes that culminate in the segregation of chromosomes at mitosis, is regulated by the synergistic and opposing activities of a cascade of kinases and phosphatases. Dephosphorylation of threonine 14 and tyrosine 15 of CDK1 by the CDC25 phosphatases is a key step in the activation of the CDK1-cyclin B protein kinase. Little is currently known about the role and the regulation of CDC25B. Here we report in vitro and in vivo data that indicate that CDC25B is degraded by the proteasome. This degradation is dependent upon phosphorylation by the CDK1-cyclin A complex but not by CDK1-cyclin B. These results indicate that CDK1-cyclin A phosphorylation targets CDC25B for degradation and that this might be an important component of cell cycle regulation at the G2/M transition.


Subject(s)
CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Cyclin A/metabolism , Cysteine Endopeptidases/metabolism , Multienzyme Complexes/metabolism , Phosphoprotein Phosphatases/metabolism , cdc25 Phosphatases , Calpain/antagonists & inhibitors , Cell Cycle , HeLa Cells , Humans , Leupeptins/pharmacology , Phosphorylation , Proteasome Endopeptidase Complex , Threonine/metabolism , Tyrosine/metabolism
7.
FEBS Lett ; 391(1-2): 66-70, 1996 Aug 05.
Article in English | MEDLINE | ID: mdl-8706932

ABSTRACT

The cyclin-dependent kinase (CDK) inhibitor p21Cip1 consists of two domains that interact with CDKs and proliferating cell nuclear antigen (PCNA), respectively. We have investigated the interaction between p21Cip1 and PCNA using surface plasmon resonance (SPR) technology and compared the results with those obtained from other sources such as the yeast two-hybrid system. Whilst other methods are only semi-quantitative, the SPR technique allowed us to determine the kinetic parameters of the interaction. The apparent equilibrium constant KD calculated for these kinetic parameters was 3.2 x 10(-7) M. We further demonstrate the use of SPR to study the interaction between mutant proteins and to determine their actual KD. The interaction between p21Cip1/PCNA is shown to be dependent upon the trimeric conformation of PCNA since a point mutant that abolishes PCNA-PCNA interaction also abolishes PCNA's interaction with p21Cip1. Finally, we demonstrate that SPR can be used to characterise the interaction of p21Cip1 and PCNA in the presence of short competitive peptides.


Subject(s)
Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclins/metabolism , Enzyme Inhibitors , Proliferating Cell Nuclear Antigen/metabolism , Amino Acid Sequence , Binding Sites , Biosensing Techniques , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/chemistry , Cyclins/isolation & purification , Electrophoresis, Polyacrylamide Gel , Glutathione Transferase , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Point Mutation , Proliferating Cell Nuclear Antigen/chemistry , Proliferating Cell Nuclear Antigen/isolation & purification , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Structure-Activity Relationship
8.
Eur J Biochem ; 231(3): 544-50, 1995 Aug 01.
Article in English | MEDLINE | ID: mdl-7649153

ABSTRACT

The use of the colicin A lysis protein to direct the extracellular release of a fusion protein from Escherichia coli was investigated as an approach for the preparation of recombinant animal toxins. Apamin, a bee venom neurotoxin, was used as the model toxin. It is reticulated by two disulfide bridges and interacts with small conductance Ca(2+)-activated K+ channels. Substantial amounts of free recombinant apamin were obtained by CNBr cleavage of the fusion protein [col-(1-171)-apa] and HPLC purification. It was recognized by conformation-dependent monoclonal antibodies with a K0.5 value close to that for natural apamin, indicating that folding was correct. In toxicity and binding experiments, the recombinant apamin displayed low activity. The recombinant and natural molecules differed by the amidation of the C-terminal histidine residue. Previous structure/activity relationship studies do not implicate this C-terminal residue in activity but the role of its amidation was not investigated. An apamin analog with a non-amidated C-terminal residue was then chemically synthesized. The biological properties of both recombinant and chemical molecules were determined. Amidation of the C-terminal alpha-carboxyl of apamin appears to be essential for full expression of its biological activity.


Subject(s)
Apamin/metabolism , Protein Processing, Post-Translational , Amides/metabolism , Amino Acid Sequence , Antibodies, Monoclonal/metabolism , Apamin/analogs & derivatives , Apamin/genetics , Base Sequence , Chromatography, High Pressure Liquid , Circular Dichroism , Hydrolysis , Iodine Radioisotopes , Molecular Sequence Data , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
9.
Plasmid ; 32(1): 32-40, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7991670

ABSTRACT

Heterologous expression in Escherichia coli is often limited by yield and solubility of the foreign protein in the bacterial cytoplasm. In many cases, overexpression also results in growth inhibition. In order to produce retroviral proteins that are especially difficult to overexpress in E. coli, we designed a set of beta-galactosidase fusion cassettes. Fusions with beta-galactosidase increase significantly both yield and solubility of the foreign proteins, thus making purification much easier. These cassettes allow for N- or C-terminal fusions, and the retroviral proteins can be released from the fusion by automaturation in vivo for the HIV-1 protease or cleavage by thrombine for Tat. More generally, any synthetic sequence coding for a given cleavage site can be introduced 5' or 3' to the lacZ gene through a convenient set of unique restriction sites, making these fusion cassettes highly versatile.


Subject(s)
Escherichia coli/genetics , HIV-1/genetics , Recombinant Fusion Proteins/genetics , Retroviridae Proteins/genetics , beta-Galactosidase/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Bacterial/genetics , Gene Expression Regulation, Bacterial , Genes, tat , Molecular Sequence Data , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Retroviridae Proteins/biosynthesis , Retroviridae Proteins/isolation & purification
10.
Biochimie ; 75(6): 451-8, 1993.
Article in English | MEDLINE | ID: mdl-8364095

ABSTRACT

The colicin A lysis protein (Cal) was used to direct the extracellular release of recombinant proteins produced in Escherichia coli. The cal gene, under the control of its inducible promoter, was introduced into an expression vector encoding the human growth hormone devoid of its signal sequence (Met-hGH). Cal and Met-hGH were simultaneously expressed at two different levels of Met-hGH induction. The results indicate that Cal causes the excretion of non-aggregated Met-hGH from the cytoplasm to the culture medium and that the Met-hGH is correctly folded since the released Met-hGH is antigenically indistinguishable from the authentic mature hGH and is biologically active in binding to specific receptor sites.


Subject(s)
Bacterial Proteins/metabolism , Escherichia coli/metabolism , Growth Hormone/metabolism , Lipoproteins , Bacterial Proteins/genetics , Blotting, Western , Cytoplasm/metabolism , Gene Expression , Growth Hormone/chemistry , Growth Hormone/genetics , Humans , Immunohistochemistry , Kinetics , Microscopy, Electron , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Plasmids , Protein Precursors/genetics , Recombinant Proteins/metabolism
11.
Eur J Biochem ; 202(3): 1299-305, 1991 Dec 18.
Article in English | MEDLINE | ID: mdl-1765084

ABSTRACT

The structure of the pore-forming domain of the bacterial toxin colicin A was studied by attenuated total-reflection polarized Fourier-transform infrared spectroscopy. This channel-forming fragment interacts with dimyristoylglycerophosphoglycerol (Myr2GroPGro) vesicles and forms disk-like complexes. Analysis of the shape of the amide I' band indicates that its secondary structure is not affected by the pH 5.0-7.2. However, 5-10% of the peptide amino acids adopt an alpha-helical structure upon complex formation with Myr2GroPGro, while the random-coil and beta-sheet structure contents decrease. Interestingly, the increase in alpha-helical content is essentially due to an increase in the high-frequency component of the alpha-helical domain of amide I'. The fact that only this component was 90 degrees polarized (i.e. the helix is parallel to the acyl chain) suggests that only this particular type of helix is associated with the Myr2GroPGro bilayer.


Subject(s)
Colicins/chemistry , Colicins/metabolism , Fourier Analysis , Lipid Bilayers , Phosphatidylglycerols , Protein Binding , Protein Conformation , Spectrophotometry, Ultraviolet , Thermodynamics
12.
J Mol Biol ; 217(3): 429-39, 1991 Feb 05.
Article in English | MEDLINE | ID: mdl-1704440

ABSTRACT

Six different hybrid colicins were constructed by recombining various domains of the two pore-forming colicins A and E1. These hybrid colicins were purified and their properties were studied. All of them were active against sensitive cells, although to varying degrees. From the results, one can conclude that: (1) the binding site of OmpF is located in the N-terminal domain of colicin A; (2) the OmpF, TolB and TolR dependence for translocation is also located in this domain; (3) the TolC dependence for colicin E1 is located in the N-terminal domain of colicin E1; (4) the 183 N-terminal amino acid residues of colicin E1 are sufficient to promote E1AA uptake and thus probably colicin E1 uptake; (5) there is an interaction between the central domain and C-terminal domain of colicin A; (6) the individual functioning of different domains in various hybrids suggests that domain interactions can be reconstituted in hybrids that are fully active, whereas in others that are much less active, non-proper domain interactions may interfere with translocation; (7) there is a specific recognition of the C-terminal domains of colicin A and colicin E1 by their respective immunity proteins.


Subject(s)
Colicins/metabolism , Escherichia coli Proteins , Ion Channels/physiology , Receptors, Peptide , Bacterial Outer Membrane Proteins , Biological Transport , Colicins/chemistry , Electric Conductivity , Escherichia coli/physiology , Hydrogen-Ion Concentration , Lipid Bilayers , Membrane Potentials , Membrane Transport Proteins , Receptors, Cell Surface/physiology , Recombinant Fusion Proteins/metabolism , Structure-Activity Relationship , Water-Electrolyte Balance
13.
Biochimie ; 72(2-3): 123-30, 1990.
Article in English | MEDLINE | ID: mdl-1696132

ABSTRACT

Pore-forming colicins exert their lethal effect on E coli through formation of a voltage-dependent channel in the inner (cytoplasmic-membrane) thus destroying the energy potential of sensitive cells. Their mode of action appears to involve 3 steps: i) binding to a specific receptor located in the outer membrane; ii) translocation across this membrane; iii) insertion into the inner membrane. Colicin A has been used as a prototype of pore-forming colicins. In this review, the 3 functional domains of colicin A respectively involved in receptor binding, translocation and pore formation, are defined. The components of sensitive cells implicated in colicin uptake and their interactions with the various colicin A domains are described. The 3-dimensional structure of the pore-forming domain of colicin A has been determined recently. This structure suggests a model of insertion into the cytoplasmic membrane which is supported by model membrane studies. The role of the membrane potential in channel functioning is also discussed.


Subject(s)
Bacterial Proteins/metabolism , Cell Membrane/metabolism , Colicins/metabolism , Escherichia coli Proteins , Escherichia coli/metabolism , Ion Channels/metabolism , Receptors, Cell Surface , Amino Acid Sequence , Biological Transport , Membrane Potentials , Models, Biological , Molecular Sequence Data , Peptide Hydrolases/metabolism , Protein Conformation , Receptors, Immunologic/metabolism
14.
Gene ; 80(1): 129-36, 1989 Aug 01.
Article in English | MEDLINE | ID: mdl-2507398

ABSTRACT

DNA constructs coding for human growth hormone (hGH)-releasing factor (hGRF) preceded by the specific recognition sequence for the activated blood coagulation factor X (FXa), fused in frame to the N-terminal 172-amino acid residues of colicin A, have been expressed in Escherichia coli. The construct was placed under the control of the inducible caa promoter in an operon containing a downstream gene coding for the cell lysis protein, Cal. Induction resulted in excretion of only the processed colicin A fragment. Replacement of Cal by the terminator from phage fd resulted in high expression of the hybrid protein, which was recovered as cytoplasmic aggregates. Enzymatic cleavage of the purified and renatured hybrid protein using FXa allowed the recovery of authentic hGRF.


Subject(s)
Colicins/metabolism , Growth Hormone-Releasing Hormone/genetics , Recombinant Fusion Proteins/genetics , Base Sequence , Cloning, Molecular , DNA/genetics , Escherichia coli/genetics , Factor Xa/genetics , Genes, Synthetic , Growth Hormone-Releasing Hormone/metabolism , Humans , Molecular Sequence Data , Mutation , Plasmids , Recombinant Fusion Proteins/metabolism
15.
FEMS Microbiol Lett ; 51(1): 239-43, 1989 Jul 15.
Article in English | MEDLINE | ID: mdl-2476363

ABSTRACT

The immunity protein to colicin A protects producing cells from the action of this pore-forming toxin. It is located into the cytoplasmic membrane. This protein has been 'tagged' with an epitope from the colicin A protein for which a monoclonal antibody is available. The fusion protein (named VL1) has been purified after extraction from the membrane in two steps using a chromatofocusing and an immunoadsorbant chromatography. The purified protein has then been reconstituted into lipid vesicles.


Subject(s)
Bacterial Proteins/isolation & purification , Colicins , Liposomes/metabolism , Membrane Proteins/isolation & purification , Bacterial Proteins/metabolism , Chromatography, Affinity/methods , Electrophoresis, Polyacrylamide Gel , Epitopes , Immunoblotting , Membrane Proteins/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification
16.
Eur J Biochem ; 181(1): 109-13, 1989 Apr 15.
Article in English | MEDLINE | ID: mdl-2714272

ABSTRACT

A plasmid was constructed which allowed easy and efficient production and purification of the NH2-terminal domain of colicin A. In only three steps, an homogenous 18-kDa polypeptide was obtained. The NH2- and COOH-terminal sequences of the protein were determined and showed that it corresponded to the NH2-terminal 171 amino acid residues of the 63-kDa colicin A. Although colicin A is a highly asymmetric protein, hydrodynamic studies indicated that the NH2-terminal domain (designated AT) has a globular structure. This fragment is not the receptor-binding domain of colicin A but is required for the transfer of colicin A across the outer membrane of sensitive cells. However, it has a low affinity for phospholipid films and this affinity is not pH-dependent, in contrast to that of colicin A.


Subject(s)
Colicins , Colicins/isolation & purification , Peptide Fragments/isolation & purification , Amino Acid Sequence , Base Sequence , Colicins/genetics , Electrophoresis, Polyacrylamide Gel , Immunoblotting , Liposomes , Molecular Sequence Data , Plasmids , Pressure , Surface Properties
17.
Biochemistry ; 28(6): 2509-14, 1989 Mar 21.
Article in English | MEDLINE | ID: mdl-2730878

ABSTRACT

The colicin A polypeptide chain (592 amino acid residues) contains three domains which are linearly organized and participate in the sequential steps involved in colicin action. We have compared the penetrating ability in phospholipid monolayers and the ability to promote vesicle fusion at acidic pH of colicin A and of protein derivatives containing various combinations of its domains. The NH2-terminal domain (171 amino acid residues), required for translocation across the outer membrane, has little affinity for dilauroylphosphatidylglycerol (DLPG) monolayers at all pHs tested. The central domain has a pH-dependent affinity, although lower than that of the entire colicin A. The COOH-terminal domain contains a high-affinity lipid binding site, but in addition an electrostatic interaction is required as a first step in the process of penetration into negatively charged DLPG films. In contrast to the constructs containing the ionophoric domain, the NH2-terminal domain alone has no fusogenic activity for liposomes. These results are discussed with regard to the mechanism of entry and action of colicin A in sensitive cells. Our results suggest the existence of a pH-dependent interaction between the receptor binding domain (amino acid residues 172-388) and the pore-forming domain of colicin A (amino acid residues 389-592).


Subject(s)
Colicins , Liposomes , Phosphatidylcholines , Phosphatidylethanolamines , Phosphatidylglycerols , Binding Sites , Colicins/toxicity , Energy Transfer , Hydrogen-Ion Concentration , Kinetics , Models, Theoretical , Pressure , Protein Binding , Protein Conformation , Structure-Activity Relationship , Surface Properties
18.
Biochim Biophys Acta ; 947(3): 445-64, 1988 Oct 11.
Article in English | MEDLINE | ID: mdl-3139035

ABSTRACT

The study of colicin release from producing cells has revealed a novel mechanism of secretion. Instead of a built-in 'tag', such as a signal peptide containing information for secretion, the mechanism employs coordinate expression of a small protein which causes an increase in the envelope permeability, resulting in the release of the colicin as well as other proteins. On the other hand, the mechanism of entry of colicins into sensitive cells involves the same three stages of protein translocation that have been demonstrated for various cellular organelles. They first interact with receptors located at the surface of the outer membrane and are then transferred across the cell envelope in a process that requires energy and depends upon accessory proteins (TolA, TolB, TolC, TolQ, TolR) which might play a role similar to that of the secretory apparatus of eukaryotic and prokaryotic cells. At this point, the type of colicin described in this review interacts specifically with the inner membrane to form an ion channel. The pore-forming colicins are isolated as soluble proteins and yet insert spontaneously into lipid bilayers. The three-dimensional structures of some of these colicins should soon become available and site-directed mutagenesis studies have now provided a large number of modified polypeptides. Their use in model systems, particularly those in which the role of transmembrane potential can be tested for polypeptide insertion and ionic channel gating, constitutes a powerful handle with which to improve our understanding of the dynamics of protein insertion into and across membranes and the molecular basis of membrane excitability. In addition, their immunity proteins, which exist only in one state (membrane-inserted) will also contribute to such an understanding.


Subject(s)
Colicins , Escherichia coli Proteins , Receptors, Cell Surface , Amino Acid Sequence , Base Sequence , Colicins/biosynthesis , Colicins/genetics , Colicins/immunology , Colicins/metabolism , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Mitomycin , Mitomycins/pharmacology , Models, Molecular , Molecular Sequence Data , Plasmids , Protein Biosynthesis , RNA, Messenger/metabolism , Receptors, Immunologic/metabolism , Structure-Activity Relationship
19.
FEBS Lett ; 216(2): 183-9, 1987 Jun 01.
Article in English | MEDLINE | ID: mdl-3582670

ABSTRACT

Cytoplasmic colicin A has the ability to bind to membranes and to form stable dimers. This form remains stable even in the presence of 1% SDS at 25 degrees C. Both of these properties were not observed for extracellular colicin A suggesting a possible difference in the conformation between cytoplasmic and extracellular colicin A.


Subject(s)
Colicins , Cell Membrane/metabolism , Colicins/metabolism , Cytoplasm , Extracellular Space , Macromolecular Substances , Molecular Weight , Protein Binding , Protein Conformation , Protein Processing, Post-Translational , Structure-Activity Relationship
20.
Proc Natl Acad Sci U S A ; 84(5): 1152-6, 1987 Mar.
Article in English | MEDLINE | ID: mdl-2434951

ABSTRACT

A large number of mutants introducing point mutations and deletions into the COOH-terminal domain of colicin A have been constructed by using site-directed mutagenesis. The COOH-terminal domain carries the channel activity. The effects of the alterations in the polypeptide chain on the secretion of colicin A by colicinogenic cells have been investigated. All deletions and some mutations were found to lead to protein aggregation in the cytoplasm, thereby preventing release into the medium. The mutated colicin A proteins have been purified, and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) has been assayed. Deletions in the region containing putative helices 4, 5, and 6 (predicted to be involved in pore formation) and the transitions (Ala----Asp-492, Phe----Pro-493) in helix 4 abolished the activity. No correlation was observed between mutations leading to protein aggregation and those leading to loss of channel activity. Some mutations were found to alter characteristic properties of the single channels, such as stability, current-relaxation kinetics, voltage dependence, and pore conductance. Site-directed mutagenesis provides a powerful tool for studying structure-function relationships of voltage-sensitive ionic channels.


Subject(s)
Colicins/genetics , Escherichia coli/genetics , Ion Channels/physiology , Mutation , Amino Acid Sequence , Base Sequence , Colicins/metabolism , Colicins/pharmacology , Escherichia coli/drug effects , Ion Channels/drug effects , Lipid Bilayers , Microscopy, Electron , Oligodeoxyribonucleotides/pharmacology
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