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BBA - General Subjects (v.1780, #9)

Editorial Board (pp. ii).

A mannose/glucose-specific lectin from Chinese evergreen chinkapin ( Castanopsis chinensis) by Jack H. Wong; H.Y. Edwin Chan; T.B. Ng (pp. 1017-1022).

A mannose/glucose-specific lectin has been purified from Chinese evergreen chinkapin ( Castanopsis chinensis) seeds, one of the most popular foods in East Asia. This lectin, designated as CCL, exhibited hemagglutinating activity in mouse and rabbit erythrocytes. It displayed a single band with a molecular mass of 29 kDa in SDS-PAGE and a 120-kDa peak in gel-filtration on Superdex-200. Its hemagglutinating activity was stable in the pH range 6–12 and at temperatures below 60 °C. The N-terminal amino acid sequence of CCL differed from those of other lectins in the same family. CCL inhibited the proliferation of HepG2 cells and adult emergence in fruitflies. CCL exhibited mitogenic activity toward mouse splenocytes, and induced nitric oxide production from mouse peritoneal macrophages but was devoid of inhibitory activity toward mycelial growth and HIV-1 reverse transcriptase.

Keywords: Lectin; Castanopsis chinensis; Isolation

Functional characterization of the histidine kinase of the E. coli two-component signal transduction system AtoS–AtoC by Panagiota S. Filippou; Lucy D. Kasemian; Christos A. Panagiotidis; Dimitrios A. Kyriakidis (pp. 1023-1031).

The Escherichia coli AtoS–AtoC two-component signal transduction system regulates the expression of the atoDAEB operon genes, whose products are required for short-chain fatty acid catabolism. In this study purified his-tagged wild-type and mutant AtoS proteins were used to prove that these proteins are true sensor kinases. The phosphorylated residue was identified as the histidine-398, which was located in a conserved Η-box since AtoS carrying a mutation at this site failed to phosphorylate. This inability to phosphorylate was not due to gross structural alterations of AtoS since the H398L mutant retained its capability to bind ATP. Furthermore, the H398L mutant AtoS was competent to catalyze the trans-phosphorylation of an AtoS G-box (G565A) mutant protein which otherwise failed to autophosphorylate due to its inability to bind ATP. The formation of homodimers between the various AtoS proteins was also shown by cross-linking experiments both in vitro and in vivo.

Keywords: Abbreviations; Az; antizyme; ODC; ornithine decarboxylase; TCS; two-component system; HK; histidine kinase; RR; response regulator; atoSC; genetic locus encoding the AtoS and AtoC proteins; AcAc; acetoacetateAtoS; AtoC; Antizyme; atoDAEB; Two-component system; Histidine kinase; Phosphorylation

Chymotrypsin–poly vinyl sulfonate interaction studied by dynamic light scattering and turbidimetric approaches by Valeria Boeris; Darío Spelzini; José Peleteiro Salgado; Guillemo Picó; Diana Romanini; Beatriz Farruggia (pp. 1032-1037).

The formation of non-soluble complexes between a positively charged protein and a strong anionic polyelectrolyte, chymotrypsin, and poly vinyl sulfonate, respectively, was studied under different experimental conditions such as pH (1–3.5), protein concentration, temperature, ionic strength, and the presence of anions that modifies the water structure. Turbidimetric titration and dynamic light scattering approaches were used as study methods. When low protein–polyelectrolyte ratio was used, the formation of a soluble complex was observed. The increase in poly vinyl sulfonate concentration produced the interaction between the soluble complex particules, thus inducing macro-aggregate formation and precipitation. Stoichiometry ratios of 500 to 780 protein molecules were found in the precipitate per polyelectrolyte molecule when the medium pH varied from 1.0 to 3.5. The kinetic of the aggregation process showed to be of first order with a low activation energy value of 4.2±0.2 kcal/mol. Electrostatic forces were found in the primary formation of the soluble complex, while the formation of the insoluble macro aggregate was a process driven by the disorder of the ordered water around the hydrophobic chain of the polymer.

Keywords: Abbreviations; ChTRP; chymotrypsin; PVS; poly vinyl sulfonate sodium salt; DLS; dynamic light scatteringChymotrypsin; Poly vinyl sulfonate; Protein-precipitation; Light scattering

Monitoring of viral cancer progression using FTIR microscopy: A comparative study of intact cells and tissues by Evgeny Bogomolny; Shmuel Argov; Shaul Mordechai; Mahmoud Huleihel (pp. 1038-1046).

Fourier transform infrared microspectroscopy (FTIR-MSP) is an analytical method with a promising potential for detecting the spectral changes due to cancerous changes in cells.The purpose of the present study is monitoring biochemical spectral changes accompanying viral cancer progression in cells and tissues using FTIR-MSP. As a model system, we used cells in culture which were transformed to malignant cells by infection with murine sarcoma virus (MuSV) and cervical tissues at different neoplastic stages.In order to devise a systematic follow-up of the cancer progression, it was essential first to determine and validate consistent and significant spectral biomarkers, which can evidently discriminate between normal and cancerous cells/tissues. Then these biomarkers were used for the characterization and classification of early stages of malignant transformation utilizing discriminant classification function techniques. Our study points out that malignancy progression can be eminently graded for both cell lines and tissues. For example, using the array of four biomarkers:A2958A2852+A2923,A1121/A1015,A1171/A1152and|A1082−A1056|A1028, we attained that the classification accuracies of different premalignant stages of cell lines and tissues were varied between 89.5 and 97.4%. These results strongly support the potential of developing FTIR microspectroscopy as a simple, reagent free method for early detection and accurate differentiation of premalignant stages.

Keywords: Fourier transform infrared microspectroscopy; Malignant transformation; Retroviruses; Cervical cancer; Discriminant classification function

Sulfated galactan is a catalyst of antithrombin-mediated inactivation of α-thrombin by Fábio R. Melo; Mariana S. Pereira; Robson Q. Monteiro; Débora Foguel; Mourao Paulo A.S. Mourão (pp. 1047-1053).

Novel compounds presenting anticoagulant activity, such as sulfated polysaccharides, open new perspectives in medicine. Elucidation of the molecular mechanism behind this activity is desirable by itself, as well as because it allows for the design of novel compounds. In the present study, we investigated the action of an algal sulfated galactan, which potentiates α-thrombin inactivation by antithrombin. Our results indicate the following: 1) both the sulfated galactan and heparin potentiate protease inactivation by antithrombin at similar molar concentrations, however they differ markedly in the molecular size required for their activities; 2) this galactan interacts predominantly with exosite II on α-thrombin and, similar to heparin, catalyzes the formation of a covalent complex between antithrombin and the protease; 3) the sulfated galactan has a higher affinity for α-thrombin than for antithrombin. We propose that the preferred pathway of sulfated galactan-induced inactivation of α-thrombin by antithrombin starts with the polysaccharide binding to the protease through a high-affinity interaction. Antithrombin is then added to the complex and the protease is inactivated by covalent interactions. Finally, the antithrombin–α-thrombin covalent complex dissociates from the polysaccharide chain. This mechanism resembles the action of heparin with low affinity for antithrombin, as opposed to heparin with high affinity for serpin.

Keywords: Abbreviations; APTT; activated partial thrombosplastin time; FPLC; fast protein liquid chromatography; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPLC; high pressure liquid chromatographyAnticoagulant activity; Algal polysaccharide; Exosite II of thrombin; Antithrombotic agent

Berberine–DNA complexation: New insights into the cooperative binding and energetic aspects by Kakali Bhadra; Motilal Maiti; Gopinatha Suresh Kumar (pp. 1054-1061).

The equilibrium binding of the cytotoxic plant alkaloid berberine to various DNAs and energetics of the interaction have been studied. At low ratios of bound alkaloid to base pair, the binding exhibited cooperativity to natural DNAs having almost equal proportions of AT and GC sequences. In contrast, the binding was non-cooperative to DNAs with predominantly high AT or GC sequences. Among the synthetic DNAs, cooperative binding was observed with poly(dA).poly(dT) and poly(dG).poly(dC) while non-cooperative binding was seen with poly(dA–dT).poly(dA–dT) and poly(dG–dC).poly(dG–dC). Both cooperative and non-cooperative bindings were remarkably dependent on the salt concentration of the media. Linear plots of ln K_a versus [Na⁺] for poly(dA).poly(dT) and poly(dA–dT).poly(dA–dT) showed the release of 0.56 and 0.75 sodium ions respectively per bound alkaloid. Isothermal titration calorimetry results revealed the binding to be exothermic and favoured by both enthalpy and entropy changes in all DNAs except the two AT polymers and AT rich DNA, where the same was predominantly entropy driven. Heat capacity values (ΔCp^o) of berberine binding to poly(dA).poly(dT), poly(dA–dT).poly(dA–dT), Clostridium perfringens and calf thymus DNA were −98, −140, −120 and −110 cal/mol K respectively. This study presents new insights into the binding dependent base pair heterogeneity in DNA conformation and the first complete thermodynamic profile of berberine binding to DNAs.

Keywords: Abbreviations; DNA; deoxyribonucleic acid; CP; Citrate–Phosphate; ITC; isothermal titration calorimetryBerberine; Cooperative binding; Non-cooperative binding; Heterogeneity; Calorimetry; Thermodynamics

PRMT3 inhibits ubiquitination of ribosomal protein S2 and together forms an active enzyme complex by Seeyoung Choi; Cho-Rok Jung; Jin-Young Kim; Dong-Soo Im (pp. 1062-1069).

Protein arginine methyltransferase 3 (PRMT3) comprises a region not required for catalytic activity in its amino-terminus and the core domain catalyzing protein arginine methylation. PRMT3 has been shown to interact with the 40S ribosomal protein S2 (rpS2) and methylate arginine residues in the arginine–glycine (RG) repeat region in the amino-terminus of rpS2. We investigated the biological implications of this interaction by delineating the domains that mediate binding between PRMT3 and rpS2. The rpS2 (100–293 amino acids) domain, but not the amino-terminus of rpS2 that includes the RG repeat region was essential for binding to PRMT3 and was susceptible to degradation. The amino-terminus of PRMT3, but not its catalytic core was required for binding to and the stability of rpS2. Overexpressed rpS2 was ubiquitinated in cells, but expression of PRMT3 reduced this ubiquitination and stabilized the rpS2 protein. Recombinant PRMT3 formed an active enzyme complex with endogenous rpS2 in vitro. Recombinant rpS2 in molar excess modestly increased the enzymatic activity of PRMT3 in vitro. Our results suggest that in addition to its catalytic function, PRMT3 may control the level of rpS2 protein in cells by inhibiting ubiquitin-mediated proteolysis of rpS2, while rpS2 may regulate the enzymatic activity of PRMT3 as a likely non-catalytic subunit.

Keywords: PRMT3; Arginine methylation; Ribosomal protein S2; Ubiquitin–proteasome

In silico description of differential enantioselectivity in methoxychlor O-demethylation by CYP2C enzymes by Zsolt Bikádi; Eszter Hazai (pp. 1070-1079).

Methoxychlor undergoes metabolism by cytochrome P450 (CYP) enzymes forming a chiral mono-phenolic derivative (Mono-OH-M) as main metabolite. In the current study, members of the CYP2C family were examined for their chiral preference in Mono-OH-M formation. CYP2C9 and CYP2C19 possessed high enantioselectivity favoring the formation of S-Mono-OH-M; CYP2C3 showed no enantioselectivity, whereas CYP2C5 slightly favored the formation of R-Mono-OH-M. Molecular modeling calculations were utilized in order to explain the observed differences in chiral preference of CYP2C enzymes. Molecular docking calculations could describe neither the existence of chiral preference in metabolism, nor the enantiomer which is preferentially formed. Molecular dynamic calculations were also carried out and were found to be useful for accurate description of chiral preference in biotransformation of methoxychlor by CYP2C enzymes. An in silico model capable of predicting chiral preference in cytochrome P450 enzymes in general can be developed based on the analysis of the stability and rigidity parameters of interacting partners during molecular dynamic simulation.

Keywords: Abbreviations; MXC; methoxychlor; Mono-OH-M; 1,1,1-trichloro-2-(4-hydroxyphenyl)-2′-(4-methoxyphenyl)ethane Heme protein; CYP2C; Stereoselective; metabolism; Molecular dynamic; Molecular docking; Homology model

Functional properties and active-site topographies of factor X Gla- and prothrombin Gla-domain chimeras of activated protein C by Shabir H. Qureshi; Likui Yang; Chandrashekhara Manithody; Jong-Sup Bae; Alireza R. Rezaie (pp. 1080-1086).

Substitution of the Gla-domain of activated protein C (APC) with the Gla-domain of prothrombin (APC-PTGla) improves the anticoagulant activity of APC independent of protein S. Previous FRET studies showed that this substitution alters the active-site topography of this mutant, rendering it identical to the active site of the APC–protein S complex. In this study, we characterized the functional properties and the active-site topography of another APC chimera containing the Gla-domain of factor X (APC-FXGla). We discovered that the anticoagulant activity of this mutant was similarly improved independent of protein S. The average distance of the closest approach ( L) between the donor dye fluorescein attached to the active site of APC derivatives and the acceptor dye octadecylrhodamine incorporated into PC/PS vesicles was determined to be 99 Å for APC and 84–86 Å for both APC-PTGla and APC-FXGla. Protein S minimally influenced the L values of the APC chimeras, however, it lowered this value to 87 Å for wild-type APC. Further studies revealed that neither chimera elicits a protective signaling response in the TNF-α-activated endothelial cells. These results suggest that unique structural features within the Gla-domain of APC enable the protease to interact with endothelial protein C receptor in the antiinflammatory pathway, while the same features also cause an inherently lower specific activity for APC in the anticoagulant pathway. This adaptation has made APC a cofactor-dependent protease, requiring the cofactor function of protein S for its optimal anticoagulant function, which appears to involve the alteration of the active-site topography of APC above the membrane surface.

Keywords: Abbreviations; FX; factor X; FVa; activated factor V; Gla; γ-carboxyglutamic acid; APC; activated protein C; APC-PTGla; a chimeric APC in which the Gla-domain and hydrophobic stack (residues 1–46) of the molecule has been replaced with the corresponding regions of prothrombin; APC-FXGla; a chimeric APC in which the Gla-domain and hydrophobic stack (residues 1–46) of the molecule has been replaced with the corresponding regions of factor X; EPCR; endothelial protein C receptor; Fl; fluorescein; OR; octadecylrhodamine; FPR; (D-Phe)-Pro-Arg; PC; dioleoylphosphatidylcholine; PS; dioleoylphosphatidylserine; PC/PS; phospholipid vesicles containing 80% PC and 20% PSFRET; APC; Prothrombin; Factor X; Gla-domain

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BBA - General Subjects (v.1780, #9)