Mechanistic information on the reaction of cis- and trans-[RuCl2(DMSO)4] with d(T2GGT2) derived from MALDI-TOF and HPLC studies

Reactions of trans and cis isomers of the Ru(II) complex [RuCl(2)(DMSO)(4)] with single-stranded hexanucleotide d(T(2)GGT(2)) were studied in aqueous solutions in the absence and presence of excess chloride by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). Despite the different reactive species formed from the two isomers in aqueous solution, similar reaction products are obtained in their interaction with d(T(2)GGT(2)). Both [RuCl(2)(DMSO)(4)] isomers bind to the oligonucleotide in the bidentate mode to form thermodynamically stable bis-guanosine adducts, Ru(G-N7)(2). Significant differences were observed in the reaction rates, however the reaction with trans- [RuCl(2)(DMSO)(4)] is ca. 5-10 times faster in comparison to that observed for the cis analogue. This difference is interpreted in terms of different rate-limiting steps for the trans and cis complexes, respectively. It is suggested that the rate of the reaction with the trans isomer is controlled by dissociation of a Cl(-) ligand from the initially formed trans,cis,cis-[RuCl(2)(DMSO)(2)(H(2)O)(2)]. In the contrast, release of a dimethyl sulfoxide molecule from the reactive species cis,fac-[RuCl(2)(DMSO)(3)(H(2)O)] is likely to be rate limiting for the cis analogue. Significant influence of electrostatic interactions on the reaction rate was observed for the trans isomer. Mechanistic interpretation of the observed reactivity trends based on data obtained from UV-Vis spectroscopy, HPLC and MALDI-TOF MS studies is presented and discussed within the paper.     

Mechanistic studies of the autoactivation of PAK2: a two-step model of cis initiation followed by trans amplification

Protein kinase activation, via autophosphorylation of the activation loop, is a common regulatory mechanism in phosphorylation-dependent signaling cascades. Despite the prevalence of this reaction and its importance in biological regulation, the molecular mechanisms of autophosphorylation are poorly understood. In this study, we developed a kinetic approach to distinguish quantitatively between cis- and trans-pathways in an autocatalytic reaction. Using this method, we have undertaken a detailed kinetic analysis for the autoactivation mechanism of p21-activated protein kinase 2 (PAK2). PAK2 is regulated in vivo and in vitro by small GTP-binding proteins, Cdc42 and Rac. Full activation of PAK2 requires autophosphorylation of the conserved threonine, Thr(402), in the activation loop of its catalytic kinase domain. Analyses of the time courses of substrate reaction during PAK2 autoactivation suggest that autophosphorylation of Thr(402) in PAK2 obeys a two-step mechanism of cis initiation, followed by trans amplification. The unphosphorylated PAK2 undergoes an intramolecular (cis) autophosphorylation on Thr(402) to produce phosphorylated PAK2, and this newly formed active PAK2 then phosphorylates other PAK2 molecules at Thr(402) in an intermolecular (trans) manner. Based on the kinetic equation derived, all microscopic kinetic constants for the cis and trans autophosphorylation have been estimated quantitatively. The advantage of the new method is not only its usefulness in the study of fast activation reactions, but its convenience in the study of substrate effects on modification reaction. It would be particularly useful when the regulatory mechanism of the autophosphorylation reaction toward certain enzymes is being assessed.     

Linear systems analysis of the ciliary steering behavior associated with negative-phototaxis in Chlamydomonas reinhardtii

In response to light stimulation Chlamydomonas reinhardtii changes the beating frequency, beating pattern, and beating synchrony of the trans and cis cilia to steer the freely-swimming cell relative to light sources. To understand the cell steering behavior the impulse responses of the beating frequency and stroke velocity of each cilium have been obtained with high temporal resolution on cells held with a micropipette. Interestingly the response of each cilium is quite different. The trans cilium responds with less delay than the cis cilium for both beating frequency and stroke velocity. For light stimulation at 2 Hz, the critical cell-rotation frequency, both responses of the trans and cis cilia are about 180 degrees out of phase. The trans-cilium beating frequency response peaks at a stimulus frequency of 5-6 Hz, higher than the cis at 1-2 Hz. The stroke velocities of the trans and cis cilia have the same stimulus-frequency response (2 Hz), but the trans cilium has a shorter delay than the cis. The times to maximum response are much shorter than the time for a rotation of the cell. The use of two different approaches that enable the trans cilium to respond ahead of the cis for both the beating frequency and stroke velocity responses suggests the importance of both responses to phototaxis. Internal cell processing responsible for the time course of the responses is proposed.     

Evidence that the substrate backbone conformation is critical to phosphorylation by p42 MAP kinase

The effect of prolyl bond isomers on the substrate recognition capabilities of various endoproteases may be investigated in a reaction where both cis/trans isomers co-exist. Here we address the question of whether enzyme reactions at the side chain of an amino acid preceding proline proceed through an isomer specific pathway. The proline-directed p42 mitogen-activated protein kinase (ERK2) was used to phosphorylate the serine side chain in Pro-Arg-Ser-Pro-Phe-4-nitroanilide under conditions where different amounts of cis prolyl isomer of the substrate were present. Initial phosphorylation rates were calculated ranging between zero at 100% cis isomer and around 60 pM/min at the equilibrium content of 83.5% trans isomer. In the presence of the peptidyl-prolyl cis/trans isomerase human hFKBP12 (500 nM), cis/trans isomerization proceeds rapidly, permitting the maximal phosphorylation rate to be observed in the dead time of the experiment. Results show that correct signature sequences are not sufficient to render potential substrates reactive to proline-directed enzymatic phosphorylations, but that the conformational state of the peptide bond following serine (threonine) is a critical determinant. Therefore, catalysis by peptidyl-prolyl cis/trans isomerases may add a new level of control to intracellular protein phosphorylations.     

Analysis of heterophilic and homophilic interactions of cadherins using the c-Jun/c-Fos dimerization domains

Cadherin-mediated cell-cell adhesion is initiated by cis dimerization of cadherin ectodomains at the cell surface followed by an antiparallel trans interaction of dimers on opposing cells. To resolve open questions concerning the molecular details and specificity of cis and trans interactions, ectodomains of E- and P-cadherin were analyzed by chemical cross-linking and by electron microscopy. At the high intrinsic concentration created by artificial oligomerization the N-terminal cadherin (CAD)-domain of P-cadherin are forming ring-like cis dimers. At 2 mm Ca(2+)-associated rings involving two cis dimers indicate trans contacts in electron micrographs. cis and trans interactions were further analyzed by heterodimerization of the ectodomains of E-cadherin (ECAD) and P-cadherin (PCAD) through the leucine zipper domains of c-Jun and c-Fos. ECADJun/ECADFos dimers predominantly form ring-like cis dimers at 1 mm Ca(2+) and double-ringed trans contacts above 2 mm Ca(2+). The Ca(2+)-dependent tetrameric trans contacts of ECADJun/ECADFos dimers are also detectable after chemical cross-linking. Only cis contacts but no trans interactions are observed for heterodimers of ECADFos and the Trp-2 to Ala mutant ECADW2AJun arguing for a decisive role of Trp-2 in trans but not cis interaction. Neither cis nor trans interaction was found for heterodimers of ECADJun and PCADFos suggesting that specificity for homophilic interactions already exists at the level of cis dimerization.     

Photocyclization of trans-1-(1'-naphthyl)-2-(3-hydroxyphenyl)ethene: evidence for adiabatic 1trans*-->1cis* photoisomerization.

The photochemistry of trans- and cis-1-(1'-naphthyl)-2-(3-hydroxyphenyl)ethene in cyclohexane and acetonitrile was examined. In cyclohexane fluorescence is the main deactivation channel for the 1trans* isomer while photocyclization is the main reaction of the 1cis* isomer. The weighty formation of hydroxychrysene following one photon absorption by the trans isomer furnished evidence of an adiabatic 1trans*-->1cis* isomerization. The photoreactivity data in acetonitrile indicated the influence of solvent polarity on the shape of the excited state surface.     

Synthesis of Mitomycin C and Decarbamoylmitomycin C N2 deoxyguanosine-adducts

Mitomycin C (MC) and Decarbamoylmitomycin C (DMC) – a derivative of MC lacking the carbamate on C10 – are DNA alkylating agents. Their cytotoxicity is attributed to their ability to generate DNA monoadducts as well as intrastrand and interstrand cross-links (ICLs). The major monoadducts generated by MC and DMC in tumor cells have opposite stereochemistry at carbon one of the guanine–mitosene bond: trans (or alpha) for MC and cis (or beta) for DMC. We hypothesize that local disruptions of DNA structure from trans or cis adducts are responsible for the different biochemical responses produced by MC and DMC. Access to DNA substrates bearing cis and trans MC/DMC lesions is essential to verify this hypothesis. Synthetic oligonucleotides bearing trans lesions can be obtained by bio-mimetic methods. However, this approach does not yield cis adducts. This report presents the first chemical synthesis of a cis mitosene DNA adduct. We also examined the stereopreference exhibited by the two drugs at the mononucleotide level by analyzing the formation of cis and trans adducts in the reaction of deoxyguanosine with MC or DMC using a variety of activation conditions. In addition, we performed Density Functional Theory calculations to evaluate the energies of these reactions. Direct alkylation under autocatalytic or bifunctional conditions yielded preferentially alpha adducts with both MC and DMC. DFT calculations showed that under bifunctional activation, the thermodynamically favored adducts are alpha, trans, for MC and beta, cis, for DMC. This suggests that the duplex DNA structure may stabilize/oriente the activated pro-drugs so that, with DMC, formation of the thermodynamically favored beta products are possible in a cellular environment.     

Comparison of radiolysis products of thymine and thymidine with e.s.r. results.

Radicals determined by e.s.r. spectrometry of irradiated thymine or thymidine and radiolytic products generated under tha ction of gamma rays in aerated aqueous solutions have been compared. This comparison lies mainly in the fact that a radical R gives rapidly the corresponding peroxide ROOH. The authors have isolated and characterized twenty peroxides, i.e., the four isomers cis (-), cis (+), trans (-), trans(+) of 6-hydroperoxy-5-hydroxy-5,6-dihydrothymidine; the four isomers cis (-), cis (+), trans (-), trans (+) of 5-hydroperoxy-6-hydroxy-5,6-dihydrothymidine; 5-hydroperoxy-2-deoxyuridin;cis and trans 6-hydroperoxy-5-hydroxy-5,6-dihydrothymine; cis and trans 5-hydroperoxy-6-hydroxy-5,6-dihydrothymine; 5-hydroperoxymethyl-uracil; 5-hydroperoxy-5,6-dihydrothymine;cis and trans 6-hydroperoxy-5,6-dihydrothymine; 5-hydroperoxy-5-methyl barbituric acid; 5-hydroperoxy-5-methyl hydantoin; trans 5,6-dihydroperoxy-5,6-dihydrothymine. Most of thethymine and thymidine radicals hypothesized or described in the literature were correlated to these peroxides. However, the presence of certain peroxides could not be explained by recognized radicals. Taking advantage of this fact, the existence of new thymine or thymidine radicals so far unknown can be predicted.     

Isomer-specific effects of conjugated linoleic acid on mineralized bone nodule formation from human osteoblast-like cells

Mixed isomers of conjugated linoleic acid (CLA) have been shown to have variable effects on bone formation and resorption in animals. The variable effects of CLA on bone physiology may be due to the different isomers present in common commercial preparations of CLA, and the effects of the predominant individual isomers (9cis,11trans and 10trans,12cis CLA) are not clear. The objective of this study was to determine the effects of individual and mixed isomers of CLA on mineralized bone nodule formation and alkaline phosphatase (ALP) activity in vitro using long-term cultures of SaOS-2 cells. Mineralized bone nodules were stained using the von Kossa method, and ALP activity in cell lysates was measured as a marker of early osteoblast differentiation. The 9cis,11trans isomer increased the number (~4- to 11-fold) and size (~2- to 5-fold) of mineralized bone nodules from 25 to 100 microM, but the 10trans,12cis isomer did not. The increase in mineralized bone nodule formation by 9cis,11trans CLA was accompanied by a variable increase in ALP activity. These results show that the 9cis,11trans isomer of CLA increases the formation of mineralized bone nodules using bone cells of human origin, and provide evidence for isomer-specific effects of CLA on bone health.     

Radiosensitizing effect of conjugated linoleic acid on tumor cells MCF-7 and MDA-MB-231

Apoptotic pathways in breast cancer cells are frequently altered, reducing the efficiency of radiotherapy. Conjugated linoleic acid (CLA), known to trigger apoptosis, was tested as radiosensitizer in breast cancer cells MCF-7 and MDA-MB-231. The CLA-mix, made up of the isomers CLA-9cis 11trans and CLA-10trans 12cis, was compared to three purified isomers, i.e., the CLA-9cis 11cis, CLA-9cis 11trans, and CLA-10trans 12cis. Using the apoptotic marker YO-PRO-1, the CLA-9cis 11cis at 50 micro mol/L turned out to be the best apoptotic inducer leading to a 10-fold increase in MCF-7 cells and a 2,5-fold increase in MDA-MB-231 cells, comparatively to the CLA-mix. Contrary to previous studies on colorectal and prostate cancer cells, CLA-10trans 12cis does not lead to an apoptotic response on breast cancer cell lines MCF-7 and MDA-MB-231. Our results also suggest that the main components of the CLA-mix (CLA-9cis 11trans and CLA-10trans 12cis) are not involved in the induction of apoptosis in the breast cancer cells studied. A dose of 5 Gy did not induce apoptosis in MCF-7 and MDA-MB-231 cells. The addition of CLA-9cis 11cis or CLA-mix has allowed us to observe a radiation-induced apoptosis, with the CLA-9cis 11cis being about 8-fold better than the CLA-mix. CLA-9cis 11cis turned out to be the best radiosensitizer, although the isomers CLA-9cis 11trans and CLA-10trans 12cis have also reduced the cell survival following irradiation, but using a mechanism not related to apoptosis. In conclusion, the radiosensitizing property of CLA-9cis 11cis supports its potential as an agent to improve radiotherapy against breast carcinoma.     

Ultrafast conformational dynamics in cyclic azobenzene peptides of increased flexibility

Structural changes of peptides containing the azobenzene dye 4-aminomethyl-phenylazobenzoic acid (AMPB) are studied with ultrafast spectroscopy. AMPB peptides are a new class of molecules where the photoisomerizable dye azobenzene is linked to the peptide moiety via a flexible methylene spacer. The ultrafast reactions in the femtosecond to nanosecond time domain are investigated for the optical switch AMPB, a linear and cyclic octapeptide, and a bicyclic octapeptide containing an additional disulfide bridge. These molecules with increasing conformational constraints are studied for the cis to trans and the trans to cis photoreactions. For the cis to trans reaction the isomerization of the chromophore occurs fast in the 1-ps range, whereas it is slower (10-ps range) in the trans to cis reaction. In all peptides the structural changes of the chromophore lead to modifications in the peptide structure in the 10-ps-1-ns time range. The results indicate that the chromophore AMPB acts simultaneously as a fast molecular switch and as a sensor for initial conformational dynamics in the peptide. Experiments in the mid-infrared range where the structural changes of the peptide backbone are directly observed demonstrate that the essential part of the structural dynamics in the bicyclic AMPB peptide occurs faster than 10 ns.     

Catalysis of proline-directed protein phosphorylation by peptidyl-prolyl cis/trans isomerases

Proline-directed protein phosphorylation was shown to depend on the capacity of the targeted Ser(Thr)-Pro bond to exhibit conformational polymorphism. The cis/trans isomer specificity underlying ERK2-catalyzed phosphate transfer leads to a complete discrimination of the cis Ser(Thr)-Pro conformer of oligopeptide substrates. We investigated in vitro the ERK2-catalyzed phosphorylation of Aspergillus oryzae RNase T1 containing two Ser-Pro bonds both of which share high stabilization energy in their respective native state conformation, the cis Ser54-Pro and the trans Ser72-Pro moiety. Despite trans isomer specificity of ERK2, a doubly phosphorylated RNase T1 was found as the final reaction product. Similarly, the RNase T1 S54G/P55N and RNase T1 P73V variants, which retain the prolyl bond conformations of the RNase T1-wt, were both monophosphorylated with a catalytic efficiency kcat/KM of 425 M(-1) s(-1) and 1228 M(-1) s(-1), respectively. However, initial phosphorylation rates did not depend linearly on the ERK2 concentration. The phosphorylation rate of the resulting plateau region at high ERK2 concentrations can be increased up to threefold for the RNase T1 P73V variant in the presence of the peptidyl-prolyl cis/trans isomerase Cyclophilin 18, indicating a conformational interconversion as the rate limiting step in the catalyzed phosphate group transfer. Using peptidyl-prolyl cis/trans isomerases with different substrate specificity, we identified a native state conformational equilibrium of the Ser54-Pro bond with the minor trans Ser54-Pro bond as the phosphorylation-sensitive moiety. This technique can therefore be used for a determination of the ratio and the interconversion rates of prolyl bond isomers in the native state of proteins.     

Effect of Environmental Factors on the trans/cis Ratio of Unsaturated Fatty Acids in Pseudomonas putida S12

The membrane reactions of Pseudomonas putida S12 to environmental stress were investigated. Cells reacted to the addition of six different heavy metals with an increase in the ratio of trans to cis unsaturated fatty acids. A correlation among the increase in the trans/cis ratio, the toxic effects of the heavy metals, and nonspecific permeabilization of the cytoplasmic membrane, as indicated by an efflux of potassium ions, was measured. Cells previously adapted to toxic concentrations of toluene exhibited increased tolerance to all applied concentrations of zinc compared with nonadapted cells. Cells exposed to different temperatures grew optimally at 30(deg)C. The degree of saturation of the membrane fatty acids of these cells decreased with decreasing temperature. An increase in the trans/cis ratio of unsaturated fatty acids took place only at higher temperatures. Osmotic stress, expressed as reduced water activity, was obtained by using different types of solutes. Only in the presence of toxic concentrations of sodium chloride or sucrose did the trans/cis ratio increase. At no applied water activity a significant effect of glycerol on the trans/cis ratio was measured. When cells were exposed to different pHs, a distinct optimum cis/trans isomerase activity was measured at pHs between 4.0 and 5.0, whereas at higher or lower pHs no reaction occurred. This optimum coincided with a loss of viability between pH 4 and 5.     

Examination of a reaction intermediate in the active site of riboflavin synthase

The riboflavin synthase catalyzed reaction proceeds through a pentacyclic intermediate of undetermined stereochemistry. Calculations at the B3LYP/6-31G(d) level of theory indicate that the trans pentacyclic structure is favored over the cis by 3.3kcal/mol. A model of the the trans, but not the cis, pentacycle in the enzyme active site shows good fitness and the availability of highly conserved protein residues for catalytic interactions. The model of the trans intermediate complements the model of the two substrates in the active site and allows for a hypothetical mechanism of the roles of specific protein residues in catalysis to be proposed.     

Bradykinin and its Gly6 analogue are substrates of cyclophilin: a fluorine-19 magnetization transfer study

Fluorine-19 magnetization transfer experiments have been used to determine the rates of cis/trans isomerization about the X-Pro7 peptide bond in [p-fluoro-Phe8]bradykinin (cis/trans ratio approximately 0.1) and its Gly6 analogue (cis/trans ratio approximately 0.4). The measurements were carried out both prior to and after the addition of cyclophilin, which has recently been shown to have peptidyl-proline cis/trans isomerase activity and is the apparent target enzyme of the immunosuppressive agent cyclosporin A. Magnetization transfer measurements over the temperature range 40-75 degrees C in the absence of enzyme give activation energies of 22.8 and 23.0 kcal/mol for [p-fluoro-Phe8]bradykinin and its Gly6 analogue, respectively. The values for the uncatalyzed cis----trans rate constant, kc, are determined by extrapolation to be 4.8 x 10(-2) and 2.1 x 10(-2) s-1 for the two peptides at 25 degrees C. The enzyme-catalyzed enhancement of the cis/trans interconversion rate was proportional to added cyclophilin concentration and was strongly sequence specific, with bradykinin a much better substrate than [Gly6]bradykinin. At a peptide concentration of 2.2 mM, the catalytic activity expressed as kc per micromolar cyclophilin was determined to be 1.2 s-1/microM for [p-fluoro-Phe8]bradykinin and 0.13 s-1/microM for the Gly6 analogue. The increased cis----trans interconversion rates were strongly inhibited by cyclosporin A and the 6-(methylalanine) derivative, which bind to cyclophilin, but not by the 1-(tetrahydrofurfuryl) derivative of cyclosporin that binds weakly.     

Activity regulation of tyrosinase by using photoisomerizable inhibitors

Enzymatic activity of tyrosinase was controlled on the basis of cis-trans photoisomerization of inhibitors, 4-azobenzene carboxylic acid (ACA) and 4,4'-azobenzene dicarboxylic acid (ADCA). In the case of ACA, the cis form inhibited tyrosinase-catalyzed oxidation of L-tyrosine more strongly than the trans form. On the contrary, in the case of ADCA, the cis form was less inhibitory. The oxidation rate was controlled reversibly by light irradiation in the course of the reaction. In the presence of ACA, UV light irradiation, which isomerized trans to cis form, decelerated the tyrosine oxidation, while visible light irradiation, which isomerized backward, accelerated the reaction. In contrast, in the presence of ADCA, UV light accelerated and visible light decelerated the reaction.     

NMR investigations of proline and its derivatives. 4-Proton magnetic resonance parameters and structure of acetyl-proline amide.

The proton magnetic resonance (PMR) spectrum of acetyl-proline amide in D2O solution has been analysed by computer simulation. The spectra of the cis and the trans isomers have been separated and their PMR parameters (chemical shift and coupling constants) are given. Vicinal coupling constants of the pyrrolidine ring are interpreted by means of a Karplus zone relation. The chemical shift effect of the anisotropy of both peptide planes is considered. It follows that both isomers are puckered with Cgamma in an endo position, but the cis isomer is more rigid than the trans isomer, which moreover undergoes a small interconversion of the Cgamma and Cdelta atoms between two extreme spatial positions. The dihedral angle phi has different values in both isomers. Thus, the dihedral angle between the two peptide planes is smaller in the trans isomer than in the cis isomer.