Johanna Deinum

Effect of S-100 proteins and calmodulin on Ca2+-induced disassembly of brain microtubule proteins in vitro

The brain-specific S-100 protein, discovered in 1965 [l] is a mixture of two very similar proteins, the S-100a and S-100b protein. These proteins are dimers of highly homologous subunits: S-1OOa (a/I) and S-100b (/3/3) [2,3]. Both proteins are small (M, 20 000) very acidic and water soluble. It is assumed, that they are mainly located in the cytosol of glial cells [4] but they have also been found bound to membranes [5,6]. The biological activity of the S-100 proteins remains unknown. However, these proteins share two typical amino acid sequences in their primary structure, associated with the calcium-binding domain [7] which indicates that they also belong to the calcium-binding protein family, such as among others, calmodulin, troponin C and parvalbumin. Therefore, it has been proposed that a calciumsensitizing factor or factors should regulate the microtubule disassembly in vivo [ 10,111. Calmodulin has often been suggested [9,12,13] to have this role as it potentiates the disassembly effect of Ca2+ [ 121 and is found to be localized at the ends of the mitotic spindle [ 131. We now report the effect of S100 proteins on the Ca2+-induced disassembly of microtubule proteins in comparison with the effect of calmodulin. We found that S-100 protein induced disassembly of microtubules with a higher efficiency than calmodulin at mM Ca2 + levels.

Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide

BACKGROUND Plasminogen activator inhibitor type 1 (PAI-1) is an important endogenous regulator of the fibrinolytic system. Reduction of PAI-1 activity has been shown to enhance dissolution of blood clots. Like other serpins, PAI-1 binds covalently to a target serine protease, thereby irreversibly inactivating the enzyme. During this process the exposed reactive-centre loop of PAI-1 is believed to undergo a conformational change becoming inserted into beta sheet A of the serpin. Incubation with peptides from the reactive-centre loop transform serpins into a substrate for their target protease. It has been hypothesised that these peptides bind to beta sheet A, thereby hindering the conformational rearrangement leading to loop insertion and formation of the stable serpin-protease complex. RESULTS We report here the 1.95 A X-ray crystal structure of a complex of a glycosylated mutant of PAI-1, PAI-1-ala335Glu, with two molecules of the inhibitory reactive-centre loop peptide N-Ac-TVASS-NH2. Both bound peptide molecules are located between beta strands 3A and 5A of the serpin. The binding kinetics of the peptide inhibitor to immobilised PAI-1-Ala335Glu, as monitored by surface plasmon resonance, is consistent with there being two different binding sites. CONCLUSIONS This is the first reported crystal structure of a complex formed between a serpin and a serpin inhibitor. The localisation of the inhibitory peptide in the complex strongly supports the theory that molecules binding in the space between beta strands 3A and 5A of a serpin are able to prevent insertion of the reactive-centre loop into beta sheet A, thereby abolishing the ability of the serpin to irreversibly inactivate its target enzyme. The characterisation of the two binding sites for the peptide inhibitor provides a solid foundation for computer-aided design of novel, low molecular weight PAI-1 inhibitors.

Diethylstilbestrol induces metaphase arrest and inhibits microtubule assembly

Diethylstilbestrol produced a dose-dependent increase in the mitotic index of the human prostatic tumour cell line DU 145. This is the result of metaphase arrest which may be induced by the action of diethylstilbestrol on spindle microtubules. Evidence is presented to show that diethylstilbestrol affects microtubules. Diethylstilbestrol completely inhibited the assembly of isolated brain microtubules although only partial disassembly could be induced. In contrast to other microtubule poisons, the inhibitory effect of diethylstilbestrol on taxol-induced self-assembly could be reversed by the addition of GTP.

Interaction of estramustine phosphate with microtubule-associated proteins

We have reported [(1984) Cancer Res., in press] that estramustine phosphate inhibits microtubule assembly and disassembled preformed microtubules. We now present evidence that estramustine phosphate inhibits microtubule assembly by binding to the microtubule‐associated proteins. We have found that: (1) additional microtubule‐associated proteins relieved the inhibition of assembly by estramustine phosphate; (2) 3H‐labelled estramustine phosphate bound predominantly to the microtubule‐associated proteins; and (3) the content of the microtubule‐associated proteins was reduced in taxol reversed estramustine phosphate‐inhibited microtubules.

Clavatadine A, A Natural Product with Selective Recognition and Irreversible Inhibition of Factor XIa †

Bioassay-guided fractionation of a CH2Cl2/MeOH extract of the sponge Suberea clavata using the serine protease factor XIa to detect antithrombotic activity led to the isolation of the new marine natural products, clavatadines A and B. Clavatadines A and B inhibited factor XIa with IC50s of 1.3 and 27 microM, respectively. A crystal structure of protein-inhibitor (clavatadine A) complex was obtained and revealed interesting selective binding and irreversible inhibition of factor XIa. The cocrystal structure provides guidance for the design and synthesis of future factor XIa inhibitors as antithrombotic agents.

AN EPR STUDY OF NEUROSPORA TYROSINASE

It has been shown recently, that mushroom tyrosinase (EC 1.14.18.1) contains a pair of copper ions at the active site [1 ]. In contrast, Neurospora tyrosinase has been reported to contain only one copper per functional unit [2,3]. However, based on a recent determination of the molecular weight [4] this enzyme contains close to two copper ions per polypeptide chain. In analogy to the mushroom tyrosinase the copper in Neurospora tyrosinase might also be present as a copper pair. To get more information on the state of copper in Neurospora tyrosinase an EPR study was undertaken. A careful search at various temperatures revealed no EPR-detectable copper, in agreement with the results of others [2,3] and consistent with the existence of coupled dimeric copper. The reaction of NO with the native and the reduced enzyme did not give rise to any EPR signals typical for the NO-complex of hemocyanin and mushroom tyrosinase [5]. On the other hand, the reaction of the native enzyme with/~-mercaptoethanol leads to a green, enzymatically completely inactive complex [6]. This complex displays an EPR signal with rather unusual characteristics probably arising from paramagnetic copper. The green compound may represent a complex between a half reduced copper pair and /3-mercaptoethanol.

Spatial separation of the two essential thiol groups and the binding site of the exchangeable GTP in brain tubulin: A spin label study

The assembly of microtubules from tubulin prepared without glycerol was inhibited by blocking the two most reactive sulfhydryl groups of the eight free sulfhydryl groups present per tubulin dimer. The assembly was also inhibited by Cu2+ ions in a redox-reaction with the two most reactive sulfhydryl groups. These two sulfhydryl groups had almost the same reactivity towards N-ethylmaleimide and p-chloromercuribenzoate, in spite of the fact that they are located on different subunits of tubulin. It was not possible to label just one single sulfhydryl group at a time by N-ethylmaleimide, and it was not possible to decide whether one or two free sulfhydryl group(s) are needed for assembly. The EPR technique based on the interaction of spin labels with transition metals was used for the study of the distance between the two most reactive sulfhydryl groups and the sites of exchangeable GTP and Mg2+, respectively. The sulfhydryl groups were spin labelled with a nitroxide derivative of N-ethylmaleimide. Cr(III)GTP was used as a paramagnetic substitute for GTP, and Mn2+ for Mg2+. It was found that: a. The exchange of GTP and the total content of GTP were not affected by modification of the sulfhydryl groups. b. The binding sites of the exchangeable GTP and Mg2+ are located 10 A, at least, from the two most reactive sulfhydryl groups. c. The distance between the spin labels introduced on the two most reactive sulfhydryl groups was larger than 17 A. The findings indicate that there is no direct interaction between exchangeable GTP and the two most reactive sulfhydryl groups.

The Mechanism of Binding of Low-Molecular-Weight Active Site Inhibitors to Human α-Thrombin

AbstractThe thrombin inhibitors argatroban, efegatran, NAPAP, CH 1091, CH 248, inogatran and melagatran have been characterised with respect to their mechanism of binding to human α-thrombin. Stopped-flow spectrophotometry was used to follow thrombin-catalysed hydrolysis of the chromogenic substrate S-2238 in the presence of inhibitors. The rate of onset or decay of inhibition was evaluated using progress curve analysis. It was possible to obtain apparent association and dissociation rate constants from the dependence of the rates on the inhibitor concentrations. Inhibition constants calculated from the association and dissociation rate constants were in good agreement with those calculated from steady-state rates. The binding of 6 inhibitors was also monitored directly using stopped-flow spectrofluorimetry when two kinetic components were found with all inhibitors. The faster component accounted for the largest part of the change in the intrinsic fluorescence of thrombin induced by inhibitor binding and ...

Biochemical and pharmacological effects of the direct thrombin inhibitor AR-H067637

AZD0837 is in development as a new oral anticoagulant for use in thromboembolic disorders. In vivo, AZD0837 is converted to AR-H067637, a selective and reversible direct thrombin inhibitor. Established biochemical methods were used to assess and measure the biochemical and pharmacological properties of AR-H067637. Both direct Biacore binding studies of AR-H067637 with immobilised alpha-thrombin and inhibition studies using pre-steady state kinetics with thrombin in the fluid phase confirmed that AR-H067637 is a rapid-binding, reversible and potent (inhibition constant K(i) = 2-4 nM), competitive inhibitor of thrombin, as well as of thrombin bound to fibrin (clot-bound thrombin) or to thrombomodulin. The total amount of free thrombin generated in platelet-poor clotting plasma was inhibited concentration-dependently by AR-H067637, with a concentration giving half maximal inhibition (IC(50)) of 0.6 microM. Moreover, AR-H067637 is, with the exception of trypsin, a selective inhibitor for thrombin without inhibiting other serine proteases involved in haemostasis. Furthermore, no anticoagulant effect of the prodrug was found. AR-H067637 prolonged the clotting time concentration-dependently in a range of plasma coagulation assays including activated partial thromboplastin time, prothrombin time, prothrombinase-induced clotting time, thrombin time and ecarin clotting time. The two latter assays were found to be most sensitive for assessing the anticoagulant effect of AR-H067637 (plasma IC(50) 93 and 220 nM, respectively). AR-H067637 also inhibited thrombin-induced platelet activation (by glycoprotein IIb/IIIa exposure, IC(50) 8.4 nM) and aggregation (IC(50) 0.9 nM). In conclusion, AR-H067637 is a selective, reversible, competitive inhibitor of alpha-thrombin, with a predictable anticoagulant effect demonstrated in plasma coagulation assays.

Epitopes on plasminogen activator inhibitor type-1 important for binding to tissue plasminogen activator

The molecular details of the rapid complex formation between tissue plasminogen activator (tPA, E.C. 3.4.21.68) and plasminogen activator inhibitor type-1 (PAI-1) are still not fully elucidated. We have used surface plasmon resonance (SPR), the BIAcore, to characterize the binding of a large panel of monoclonal antibodies to four forms of recombinant human PAI-1, including active and latent PAI-1 as well as the complex between PAI-1 and recombinant human tc tPA or the protease part of tPA, the B-chain. Antibodies that discriminate between these different forms of PAI-1 have been identified, which is reflected by differences in k(a), k(d) as well as in Kd. In addition, in a chromogenic assay with PAI-1 and tPA we determined the IC50-values for these antibodies, i.e., studied their ability to inhibit the decrease in tPA-activity caused by PAI-1. In a competition assay using SPR, we have also been able to study whether concurrent binding of these antibodies to PAI-1 was possible. We could thereby assign the antibodies to five groups according to their binding areas. Furthermore, by using this technique, we have for the first time been able to identify three distinct epitopes on PAI-1, which are all of importance for the interaction and complex-formation with tPA. Since the antibodies that bind to one of these areas all have very poor affinity for the complex between PAI-1 and tPA, we suggest that this not previously described epitope must be located near the final binding site for tPA in this complex. Altogether, this also supports the theory of a multistep reaction between PAI-1 and tPA, in which tPA interacts with different parts of the PAI-1-molecule.