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Biomedical subjects

M Zurini

Publications and source records attributed to M Zurini.

At least 19 recordsLinked to original sources
PubMed2001

Sanglifehrin A, a novel cyclophilin-binding compound showing immunosuppressive activity with a new mechanism of action.

We report here on the characterization of the novel immunosuppressant Sanglifehrin A (SFA). SFA is a representative of a class of macrolides produced by actinomycetes that bind to cyclophilin A (CypA), the binding protein of the fungal cyclic peptide cyclosporin A (CsA). SFA interacts with high affinity with the CsA binding side of CypA and inhibits its peptidyl-prolyl isomerase activity. The mode of action of SFA is different from known immunosuppressive drugs. It has no effect on the phosphatase activity of calcineurin, the target of the immunosuppressants CsA and FK506 when complexed to their binding proteins CypA and FK binding protein, respectively. Moreover, its effects are independent of binding of cyclophilin. SFA inhibits alloantigen-stimulated T cell proliferation but acts at a later stage than CsA and FK506. In contrast to these drugs, SFA does not affect IL-2 transcription or secretion. However, it blocks IL-2-dependent proliferation and cytokine production of T cells, in this respect resembling rapamycin. SFA inhibits the proliferation of mitogen-activated B cells, but, unlike rapamycin, it has no effect on CD154/IL-4-induced Ab synthesis. The activity of SFA is also different from that of other known late-acting immunosuppressants, e.g., mycophenolate mofetil or brequinar, as it does not affect de novo purine and pyrimidine biosynthesis. In summary, we have identified a novel immunosuppressant, which represents, in addition to CsA, FK506 and rapamycin, a fourth class of immunophilin-binding metabolites with a new, yet undefined mechanism of action.

Animals↗
PubMed2001

Optimization of the anti-(human CD3) immunotoxin DT389-scFv(UCHT1) N-terminal sequence to yield a homogeneous protein.

The production and regulatory approval processes for biopharmaceuticals require detailed characterization of potential products. Therapeutic proteins should preferably be homogeneous, although limited, reproducible, heterogeneity may be tolerated. A diphtheria toxin-based anti-(human CD3) immunotoxin, DT389-scFv(UCHT1), was expressed in Escherichia coli and purified following refolding [DT389 corresponds to amino acids 1-389 of diphtheria toxin, scFv is single-chain variable-region antibody fragment and UCHT1is an anti-(human CD3) monoclonal antibody]. Biochemical characterization of this molecule by MS and N-terminal sequencing by Edman degradation revealed that the protein was heterogeneous at the N-terminus, containing species both with (60%) and without (40%) the initiator methionine residue. In an attempt to generate an N-terminally homogeneous molecule, a panel of seven N-terminal variants was designed, based on the published specificity of bacterial methionine aminopeptidase. Following bacterial expression, partial purification and separation on SDS/PAGE, these proteins were subjected to N-terminal sequencing by Edman degradation. Three of the mutants yielded a 100% homogeneous amino acid sequence. By contrast, the original DT389-scFv(UCHT1) protein and four variant proteins yielded two sequences with varying ratios corresponding to species with and without methionine. The N-terminal sequences of the three homogeneous clones were MLADD and MLDD, where the methionine was completely retained, and SADD, where the methionine was completely removed. One of the homogeneous mutants (SADD) was expressed, refolded and purified and found to be equipotent with the parent immunotoxin. Thus, using a rational mutagenesis approach, three N-terminally homogeneous variants of DT389-scFv(UCHT1) have been identified, at least one of which is functionally indistinguishable from the parent immunotoxin. This approach is generally applicable to biopharmaceutical production and immunotoxin development in particular.

Antibodies, Monoclonal↗
PubMed2000

Coordinate activation of endogenous p38alpha, beta, gamma, and delta by inflammatory stimuli.

The p38 family of mitogen-activated protein kinases is believed to mediate a variety of leukocyte responses to pro-inflammatory stimuli. There are four members of the p38 family, and although activation of the different members has been studied in transiently transfected cells much less is known about activation of the endogenous p38s, particularly in myeloid lineage cells. To investigate activation of endogenous p38s, we have made monoclonal antibodies specific for each p38 and have used these antibodies to study p38 activation by pro-inflammatory stimuli in several human monocytic cell lines. Without stimulation endogenous p38alpha kinase activity was readily detectable, whereas that of p38beta, gamma, and delta was barely measurable. In response to inflammatory stimuli, we observed a time- and dose-dependent activation of all four p38s. The kinetics of activation of each of the p38s were similar for each stimulus used, suggesting a common upstream activation pathway. Simultaneous activation of the p38s suggests that all four may be important in inflammation.

Antibodies, Monoclonal↗
PubMed2000

Phosphorylation of eIF-4E on Ser 209 in response to mitogenic and inflammatory stimuli is faithfully detected by specific antibodies.

Phosphorylation of Ser 209 is thought to modulate the activity of the cap-binding factor eIF-4E which is a crucial component in the initiation complex for cap-dependent translation of mRNA. We report here the full reconstitution of the p38 Map kinase cascade leading to phosphorylation of eIF-4E in vitro and the generation of antibodies specific for phospho-serine 209 in eIF-4E. These antibodies were used to probe the phosphorylation of eIF-4E in mammalian cells stimulated with mitogens and pro-inflammatory cytokines. Treatment of human dermal fibroblasts with FCS led to a transient hyperphosphorylation, followed by hypophosphorylation and return to normal state phosphorylation at 16 h after the initial stimulation. By using a potent small molecular weight inhibitor of Mnk1, the upstream kinase for eIF-4E, we observed a rapid dephosphorylation of eIF-4E within 45 min after addition of the inhibitor, suggesting a high turnover of phosphate on eIF-4E mediated by Mnk1 and a yet unidentified phosphatase.

Antibodies↗
PubMed2000

Measurement of cdk4 kinase activity using an affinity peptide-tagging technology.

Cyclin-dependent kinases such as Cdk4 are involved in the control of cell cycle progression, and misregulation of Cdk4 has been implicated in many types of cancers. In the present study, we report the development of a novel homogeneous assay using an affinity peptide-tagging technology for rapidly discovering Cdk4 inhibitors. The DNA sequence encoding a streptavidin recognition motif, or StrepTag (AWRHPQFGG), was cloned and expressed at the C-terminus of a fusion protein of a 152-amino acid hyperphosphorylation domain (Rb152) of the retinoblastoma protein (Rb) linked to GST at the N-terminus. This affinity peptide-tagged protein (GST-Rb152-StrepTag), which contains the two known phosphorylation sites of Rb, specifically phosphorylated by Cdk4 in vivo, was used as a substrate in the current in vitro kinase assay. After phosphorylation, scintillation proximity assay (SPA) scintillant beads coated with streptavidin were added. Radiolabeled GST-Rb152-StrepTag was brought in close proximity to the SPA scintillant beads through the interaction between StrepTag and streptavidin, resulting in the emission of light from beads. By applying the affinity peptide-tagging technology, we have eliminated the separation and wash steps which are normally required in a radioactive filtration assay. Therefore, this homogeneous method is simple, robust, and highly amenable to high-throughput screening of Cdk4-specific inhibitors. Furthermore, the affinity peptide tagging technique reported here is a simple, generic method that can be applied to many recombinant proteins for the development of kinase and protein-protein interaction assays.

Affinity Labels↗
PubMed1999

Structural and conformational requirements for high-affinity binding to the SH2 domain of Grb2(1).

Following earlier work on cystine-bridged peptides, cyclic phosphopeptides containing nonreducible mimics of cystine were synthesized that show high affinity and specificity toward the Src homology (SH2) domain of the growth factor receptor-binding protein (Grb2). Replacement of the cystine in the cyclic heptapeptide cyclo(CYVNVPC) by D-alpha-acetylthialysine or D-alpha-lysine gave cyclo(YVNVP(D-alpha-acetyl-thiaK)) (22) and cyclo(YVNVP(D-alpha-acetyl-K)) (30), which showed improved binding 10-fold relative to that of the control peptide KPFYVNVEF (1). NMR spectroscopy and molecular modeling experiments indicate that a beta-turn conformation centered around YVNV is essential for high-affinity binding. X-ray structure analyses show that the linear peptide 1 and the cyclic compound 21 adopt a similar binding mode with a beta-turn conformation. Our data confirm the unique structural requirements of the ligand binding site of the SH2 domain of Grb2. Moreover, the potency of our cyclic lactams can be explained by the stabilization of the beta-turn conformation by three intramolecular hydrogen bonds (one mediated by an H2O molecule). These stable and easily accessible cyclic peptides can serve as templates for the evaluation of phosphotyrosine surrogates and further chemical elaboration.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Use of organic solvents and small molecules for locating binding sites on proteins in solutions.

Application of a modified ePHOGSY and other NMR experiments to an H2O-DMSO solution of the protein FKBP12 identified the presence of one molecule of DMSO bound in the substrate binding site. It occupies the same spatial region occupied by the pipecolidine moiety of the immunosuppressive drugs FK506 and Rapamycin complexed to the protein. The binding constant K(D) for ths DMSO molecule was only 275 mM. A substructure search of small molecules similar to DMSO resulted in the identification of molecules with improved binding affinity. This work represents a clear example of the powerful interplay of molecular modelling and NMR.

Binding Sites↗
PubMed1998

Isolation, identification and immunosuppressive activity of a new IMM-125 metabolite from human liver microsomes. Identification of its cyclophilin A-IMM-125 metabolite complex by nanospray tandem mass spectrometry.

The isolation from human liver microsomes and identification by electrospray mass spectrometry and tandem mass spectrometry of a new metabolite of IMM-125 resulting from the biotransformation of the amino acid 1 vinylic methyl group to a carboxylic acid, called the IMM-125-COOH metabolite, is described. It was found that the complex of this new metabolite with cyclophilin A is formed less easily than the corresponding cyclophilin A-IMM-125-CH2OH main metabolite and cyclophilin A-IMM-125 complexes. However, when formed, the IMM-125-COOH metabolite-cyclophilin A complex requires more collision-induced dissociation (CID) to dissociate the complex than the complexes formed with the two other ligands. The nanospray tandem mass spectrum of the IMM-125-COOH metabolite-cyclophilin A complex (m/z 1755) gives rise to cyclophilin A-ligand complexes of m/z 1751 by elimination of CO2 and of m/z 1749 by loss of CO2 and H2O or glycerol. Since immunosuppressive activity is known to be dependent on the formation of a binary complex between cyclophilin A and the drug and since the target for the binary complex was found to be the calcium- and calmodulin-dependent protein phosphatase, calcineurin, it could be interesting to measure for structurally related immunosuppressive drugs the CID energy necessary to dissociate the binary complexes in order to evaluate whether a correlation with the phosphatase activity could be derived.

Biotransformation↗
PubMed1994

Solution structure of a cysteine rich domain of rat protein kinase C.

Intracellular protein phosphorylation by protein kinase C (PKC) plays a major role in the translation of extracellular signals into cellular events. Speculations on the structural basis for PKC activation are based on sequence homology between their cysteine-rich domains (CRD) and the DNA-binding 'zinc-fingers'. We produced a fragment comprising the second CRD (CRD2) of rat PKC-alpha and determined its three-dimensional structure in solution by NMR spectroscopy. This revealed that CRD2 adopts a globular fold allowing two non-consecutive sets of zinc-binding residues to form two separate metal-binding sites. The fold is different to those previously proposed and allows insight into the molecular topology of a family of homologous proteins.

Amino Acid Sequence↗
PubMed1993

In vitro and in vivo characterization of a neutral boron-containing thrombin inhibitor.

Peptide boronic acid derivatives have proven to be very potent inhibitors of serine proteases with boroarginine derivatives being particularly potent thrombin inhibitors. The importance of the charged side chain of arginine has been investigated by synthesizing a derivative in which this side chain has been replaced by a neutral one. This boronic acid derivative, D-benzyloxycarbonyl (Z)-Phe-Pro-methoxypropylglycine-pinanediol (MpgC10H16), inhibited thrombin by a competitive mechanism with an inhibition constant (Ki) of 8.9 nM. In comparison to boroarginine derivatives, Z-D-Phe-Pro-boroMpgC10H16 displayed higher selectivity for thrombin over trypsin (Ki = 1.1 microM) and plasmin (Ki = 15.7 microM). Prolongation of thrombin time and activated partial thromboplastin time were observed with micromolar concentrations of Z-D-Phe-Pro-boroMpgC10H16. In a thrombin-dependent in vitro aggregation assay with human platelets, Z-D-Phe-Pro-boroMpgC10H16 inhibited aggregation with an IC50 of 85 nM. When tested in a thrombin-dependent platelet accumulation model in the rat, a bolus injection of (Z)-D-Phe-Pro-boroMpgC10H16 (0.3-3 mg/kg) inhibited platelet accumulation. Thus, the substitution of the charged guanidino group in the P1 side chain by the neutral methoxy group resulted in a potent and highly selective thrombin inhibitor with an interesting pharmacological profile with in vitro as well as in vivo models.

Animals↗
PubMed1990

Crystallisation and preliminary X-ray diffraction studies of cyclophilin-tetrapeptide and cyclophilin-cyclosporin complexes.

Recombinant human cyclophilin has been co-crystallised with a number of peptides to give crystals suitable for X-ray analysis. The crystal complexes for which heavy-atom derivatives have been prepared and X-ray data collected are: cyclophilin with N-acetyl-Ala-Ala-Pro-Ala-amidomethyl-coumarin (I) which crystallises in space group P2(1)2(1)2(1) with a = 108.2, b = 123.0, c = 35.8 A, and cyclophilin with cyclosporin (II) which crystallises as tetragonal plates in space group P4(1)2(1)2 or P4(3)2(1)2 with a = b = 94.98, c = 278.55 A.

Amino Acid Isomerases↗
PubMed1988

A C-terminal, calmodulin-like regulatory domain from the plasma membrane Ca2+-pumping ATPase.

A cDNA that encodes what appears to be the inhibitory domain of the plasma membrane calcium-pumping ATPase (Ca2+-ATPase) has been isolated by screening a lambda gt11 bovine brain cDNA library with antibodies prepared against the human erythrocyte membrane Ca2+-ATPase. This screening resulted in isolation of a bacteriophage containing a 1.5-kilobase cDNA insert encoding a 71-residue polypeptide, the remainder being a large 3' terminal noncoding region. A portion of this deduced peptide sequence was identical to that of a peptide isolated from a V8 protease digest of the human erythrocyte Ca2+-ATPase except for 1 residue. Antibodies purified by immunoabsorption to the fusion protein containing this cDNA-encoded polypeptide reacted only with those fragments of a limited trypsin digest of the human erythrocyte Ca2+-ATPase that contain the inhibitory domain. Moreover, these antibodies were able to partially stimulate basal enzyme activity and block further activation by calmodulin. The encoded polypeptide bears homology to the glutamic acid-rich regions N-terminal to the Ca2+-binding loops of calmodulin and to a lesser extent with the loops themselves. This encoded polypeptide also represents the C terminus of the Ca2+-ATPase. Portions of the isolated cDNA were homologous to the 3' noncoding region of the sarcoplasmic reticulum Ca2+-ATPase cDNA, indicating a possible mechanism for the evolution of these distinct membrane Ca2+ pumps.

Amino Acid Sequence↗
PubMed1986

The calcium pump of plasma membranes.

The calcium pump of plasma membranes is an ATPase of the E1E2 type; that is, it forms a phosphoenzyme during the reaction cycle and is inhibited by vanadate. It differs from the Ca2+-transporting ATPase of sarcoplasmic reticulum in molecular mass, immunological properties and Ca2+/ATP stoichiometry. Its affinity for calcium, which is low in the absence of calmodulin (Km, 10-20 microM), is increased by the latter (to a Km of about 0.5 microM). The effect of calmodulin is mimicked by acidic phospholipids (including the phosphorylated products of phosphatidylinositol), long-chain polyunsaturated fatty acids, and controlled treatment with a number of proteases. The ATPase has been purified to homogeneity from a number of plasma membranes using calmodulin affinity chromatography. The purified enzyme (a single polypeptide of molecular mass 138 kDa) pumps calcium into reconstituted liposomes in exchange for protons. Controlled trypsin proteolysis has shown that about one-third of the enzyme mass can be removed without impairing calcium transport. It has also indicated that the ability to bind calmodulin and to respond to it resides in a 9 kDa sequence of the enzyme molecule. The sequence contains a 4 kDa domain that binds calmodulin, and a 5 kDa domain which is essential for the stimulation.

Animals↗