Inhibition of nuclear protein import by a monoclonal antibody against a novel class of nuclear pore proteins

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab Ea-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS-albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small nonnuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex. (C) 1994 Academic Press, Inc.

Metallo-beta-lactamase-Catalyzed Hydrolysis of Cephalosporins: Some Mechanistic Insights into the Effect of Heterocyclic Thiones on Enzyme Activity

The hydrolysis of beta-lactam antibiotics using zinc-containing metallo-beta-lactamases (m beta l) is one of the major bacterial defense systems. These enzymes can catalyze the hydrolysis of a variety of antibiotics including the latest generation of cephalosporins, cephamycins, and imipenem. It is shown in this paper that the cephalosporins having heterocyclic - SR side chains are less prone to m beta l-mediated hydrolysis than the antibiotics that do not have such side chains. This is partly due to the inhibition of enzyme activity by the thione moieties eliminated during hydrolysis. When the enzymatic hydrolysis of oxacillin was carried out in the presence of heterocyclic thiones such as MU, MDT, DMETT, and MMA, the catalytic activity of the enzyme was inhibited significantly by these compounds. Although the heterocyclic - SR moieties eliminated from the beta-lactams upon hydrolysis undergo a rapid tautomerism between thione and thiol forms, these compounds act as thiolate ligands toward zinc(II) ions. The structural characterization of two model tetranuclear zinc(II) thiolate complexes indicates that the -SR side chains eliminated from the antibiotics may interact with the zinc(II) metal center of m beta l through their sulfur atoms.

Preparation of a soluble 58kDa-3-hydroxy-3-methylglutaryl CoA reductase from liver microsomes and its inhibition by ethoxysilatrane, a hypocholesterolemic compound

On repeated thawing at room temperature of frozen preparations of heavy microsomes from rat livers, HMGCoA reductase activity was solubilized due to limited proteolysis. This soluble enzyme was partially purified by fractionation with ammonium sulfate and filtration on Sephacryl S-200 column. The active enzyme was coeluted with a major 92 kDa-protein and was identified as a 58kDa-protein after separation by SDS-PAGE and immunoblotting. Ethoxysilatrane, a hypocholesterolemic compound, which decreased the liver-microsomal activity of HMGCoA reductase on intra-peritonial treatment of animals, showed little effect on the enzyme activity with isolated microsomes or the 50kDa-soluble enzyme when added in the assay. But it was able to inhibit the activity of the soluble 58kDa-enzyme in a concentration-dependent, reversible manner. Cholesterol and an oxycholesterol were without effect whereas chlorophenoxyisobutyrate and ubiquinone showed small inhibition under these conditions. The extra region that links the active site domain (50kDa protein) to the membrane, present in the 58kDa-protein appears to be involved in mediating the inhibition by silatrane.

Mechanism of growth inhibition of intraerythrocytic stages of Plasmodium falciparum by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)

Purine nucleotide synthesis in Plasmodium falciparum takes place solely by the purine salvage pathway in which preformed purine base(s) are salvaged from the host and acted upon by a battery of enzymes to generate AMP and GMP. Inhibitors of this pathway have a potent effect on the in vitro growth of P. falciparum and are hence, implicated as promising leads for the development of new generation anti-malarials. Here, we describe the mechanism of inhibition of the intraerythrocytic growth of P. falciparum by the purine nucleoside precursor, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Our results show that AICAR toxicity is mediated through the erythrocyte in which AICAR is phosphorylated to its nucleotide, ZMP. Further, purine metabolite labeling of the parasitized erythrocytes by H-3]-hypoxanthine, in the presence of AICAR, showed a significant decrease in radioactive counts in adenylate fractions but not in guanylate fractions. The most dramatic effect on parasite growth was observed when erythrocytes pretreated with AICAR were used in culture. Pretreatment of erythrocytes with AICAR led to significant intracellular accumulation of ZMP and these erythrocytes were incapable of supporting parasite growth. These results implicate that in addition to the purine salvage pathway in P. falciparum, AICAR alters the metabolic status of the erythrocytes, which inhibits parasite growth. As AICAR and ZMP are metabolites in the human serum and erythrocytes, our studies reported here throw light on their possible role in disease susceptibility, and also suggests the possibility of AICAR being a potential prophylactic or chemotherapeutic anti-malarial compound. (C) 2011 Elsevier B.V. All rights reserved.

Role of Water in the Enzymatic Catalysis: Study of ATP plus AMP -> 2ADP Conversion by Adenylate Kinase

The catalytic conversion ATP + AMP -> 2ADP by the enzyme adenylate kinase (ADK) involves the binding of one ATP. molecule to the LID domain and one AMP molecule to the NMP domain. The latter is followed by a. phosphate transfer and then the release of two ADP molecules. We have computed a novel two-dimensional configurational free energy surface (2DCFES), with one reaction coordinate each for the LID and the NMP domain motions, while considering explicit water interactions. Our computed 2DCFES clearly reveals the existence of a stable half-open half-closed (HOHC) intermediate stale of the enzyme. Cycling of the enzyme through the HOHC state reduces the conformational free energy barrier for. the reaction by about 20 kJ/mol. We find that the stability of the HOHC state (missed in all earlier studies with implicit solvent model) is largely because of the increase of specific interactions of the polar amino acid side chains with water, particularly with the arginine and the histidine residues. Free energy surface of the LID domain is rather rugged, which can conveniently slow down LID's conformational motion, thus facilitating a new substrate capture after the product release in the catalytic cycle.

Enzymatic resolution of dioxygenated dicyclopentadienes: Enantiopure hydrindanes, hydroisoquinolones, diquinanes and application to a synthesis of (+)-coronafacic acid

A 5, 10-dioxygenated-tricyclo[5.2.1.0(2,6)]decane derivative 6 has yielded to efficient enzymatic resolution to provide a range of chiral building blocks, whose absolute configuration has been determined through a total synthesis of naturally occuring (+)-coronafacic acid. (C) 1999 Elsevier Science Ltd. All rights reserved.

Inhibition of hepatitis C virus replication by herbal extract: Phyllanthus amarus as potent natural source

Hepatitis C virus infection is a major health problem worldwide. Developing effective antiviral therapy for HCV is the need of the hour. The viral enzymes NS3 protease and NS5B RNA dependent RNA polymerase are essential enzymes for polyprotein processing and viral RNA replication and thus can be potential targets for screening anti-HCV compounds. A large number of phytochemicals are present in plants, which are found to be promising antiviral agents. In this study, we have screened inhibitory effect of different plant extracts against the NS3 and NS5B enzymes of hepatitis C virus. Methanolic extracts were prepared from various plant materials and their inhibitory effects on the viral enzymes were determined by in vitro enzyme assays. Effect on viral RNA replication was investigated by using TaqMan Real time RT-PCR. Interestingly, Phyllanthus amarus root (PAR) extract showed significant inhibition of HCV-NS3 protease enzyme; whereas P. amarus leaf (PAL) extract showed considerable inhibition of NS5B in the in vitro assays. Further, the PAR and PAL extracts significantly inhibited replication of HCV monocistronic replicon RNA and HCV H77S viral RNA in HCV cell culture system. However, both PAR and PAL extracts did not show cytotoxicity in Huh7 cells in the MTT assay. Furthermore, addition of PAR together with IFN-alpha showed additive effect in the inhibition of HCV RNA replication. Results suggest the possible molecular basis of the inhibitory activity of PA extract against HCV which would help in optimization and subsequent development of specific antiviral agent using P. amarus as potent natural source. (C) 2011 Elsevier B.V. All rights reserved.

Synergy between the N-terminal and C-terminal domains of Mycobacterium tuberculosis HupB is essential for high-affinity binding, DNA supercoiling and inhibition of RecA-promoted strand exchange

The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN), HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair.

Lysozyme: A model protein for amyloid research

Ever since lysozyme was discovered by Fleming in 1922, this protein has emerged as a model for investigations on protein structure and function. Over the years, several high-resolution structures have yielded a wealth of structural data on this protein. Extensive studies on folding of lysozyme have shown how different regions of this protein dynamically interact with one another. Data is also available from numerous biotechnological studies wherein lysozyme has been employed as a model protein for recovering active recombinant protein from inclusion bodies using small molecules like L-arginine. A variety of conditions have been developed in vitro to induce fibrillation in hen lysozyme. They include (a) acidic pH at elevated temperature, (b) concentrated solutions of ethanol, (c) moderate concentrations of guanidinium hydrochloride at moderate temperature, and (d) alkaline pH at room temperature. This review aims to bring together similarities and differences in aggregation mechanisms, morphology of aggregates, and related issues that arise using the different conditions mentioned above to improve our understanding. The alkaline pH condition (pH 12.2), discovered and studied extensively in our lab, shall receive special attention. More than a decade ago, it was revealed that mutations in human lysozyme can cause accumulation of large quantities of amyloid in liver, kidney, and other regions of gastrointestinal tract. Understanding the mechanism of lysozyme aggregation will probably have therapeutic implications for the treatment of systemic nonneuropathic amyloidosis. Numerous studies have begun to focus attention on inhibition of lysozyme aggregation using antibody or small molecules. The enzymatic activity of lysozyme presents a convenient handle to quantify the native population of lysozyme in a sample where aggregation has been inhibited. The rich information available on lysozyme coupled with the multiple conditions that have been successful in inducing/inhibiting its aggregation in vitro makes lysozyme an ideal model protein to investigate amyloidogenesis.

Inhibition of peroxynitrite- and peroxidase-mediated protein tyrosine nitration by imidazole-based thiourea and selenourea derivatives

In the present study, the synthesis and characterization of a series of N-methylimidazole-based thiourea and selenourea derivatives are described. The new compounds were also studied for their ability to inhibit peroxynitrite (PN)- and peroxidase-mediated nitration of protein tyrosine residues. It has been observed that the selenourea derivatives are more efficient than the thiourea-based compounds in the inhibition of protein nitration. The higher activity of selenoureas as compared to that of the corresponding thioureas can be ascribed to the zwitterionic nature of the selenourea moiety. Single crystal X-ray diffraction studies on some of the thiourea and selenourea derivatives reveal that the C S bonds in thioureas possess more of double bond character than the C=Se bonds in the corresponding selenoureas. Therefore, the selenium compounds can react with PN or hydrogen peroxide much faster than their sulfur analogues. The reactions of thiourea and selenourea derivatives with PN or hydrogen peroxide produce the corresponding sulfinic or seleninic acid derivatives, which upon elimination of sulfurous/selenous acids produce the corresponding N-methylimdazole derivatives.

Inhibition of type IA topoisomerase by a monoclonal antibody through perturbation of DNA cleavage-religation equilibrium

Type IA DNA topoisomerases, typically found in bacteria, are essential enzymes that catalyse the DNA relaxation of negative supercoils. DNA gyrase is the only type II topoisomerase that can carry out the opposite reaction (i.e. the introduction of the DNA supercoils). A number of diverse molecules target DNA gyrase. However, inhibitors that arrest the activity of bacterial topoisomerase I at low concentrations remain to be identified. Towards this end, as a proof of principle, monoclonal antibodies that inhibit Mycobacterium smegmatis topoisomerase I have been characterized and the specific inhibition of Mycobacterium smegmatis topoisomerase I by a monoclonal antibody, 2F3G4, at a nanomolar concentration is described. The enzyme-bound monoclonal antibody stimulated the first transesterification reaction leading to enhanced DNA cleavage, without significantly altering the religation activity of the enzyme. The stimulated DNA cleavage resulted in perturbation of the cleavagereligation equilibrium, increasing single-strand nicks and proteinDNA covalent adducts. Monoclonal antibodies with such a mechanism of inhibition can serve as invaluable tools for probing the structure and mechanism of the enzyme, as well as in the design of novel inhibitors that arrest enzyme activity.