The potential role of a late gene expression factor, lef2, from Bombyx mori nuclear polyhedrosis virus in very late gene transcription and DNA replication

Several late gene expression factors (Lefs) have been implicated in fostering high levels of transcription from the very late gene promoters of polyhedrin and p10 from baculoviruses. We cloned and characterized from Bombyx mori nuclear polyhedrosis virus a late gene expression factor (Bmlef2) that encodes a 209-amino-acid protein harboring a Cys-rich C-terminal domain. The temporal transcription profiles of lef2 revealed a 1.2-kb transcript in both delayed early and late periods after virus infection. Transcription start site mapping identified the presence of an aphidicolin-sensitive late transcript arising from a TAAG motif located at -352 nucleotides and an aphidicolin-insensitive early transcript originating from a TTGT motif located 35 nucleotides downstream to a TATA box at -312 nucleotides, with respect to the +1 ATG of lef2. BmLef2 trans-activated very late gene expression from both polyhedrin and p10 promoters in transient expression assays. Internal deletion of the Cys-rich domain from the C-terminal region abolished the transcriptional activation. Inactivation of Lef2 synthesis by antisense lef2 transcripts drastically reduced the very late gene transcription but showed little effect on the expression from immediate early promoter. Decrease in viral DNA synthesis and a reduction in virus titer were observed only when antisense lef2 was expressed under the immediate early (ie-1) promoter. Furthermore, the antisense experiments suggested that lef2 plays a direct role in very late gene transcription.

Synthesis of 2-(5-((5-(4-chlorophenyl)furan 2-yl)methylene)-4-oxo-2-thioxo-thiazolidi n-3-yl)acetic acid derivatives and evaluation of their cytotoxicity and induction of apoptosis in human leukemia cells

In order to explore the anticancer effect associated with the thiazolidinone framework, several 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid derivatives 5(a-1) were synthesized. Variation in the functional group at C-terminal of the thiazolidinone led to set of compounds bearing amide moiety. Their chemical structures were confirmed by H-1 NMR, IR and Mass Spectra analysis. These thiazolidinone compounds containing furan moiety exhibits moderate to strong antiproliferative activity in a cell cycle stage-dependent and dose dependent manner in two different human leukemia cell lines. The importance of the electron donating groups on thiazolidinone moiety was confirmed by MTT and Trypan blue assays and it was concluded that the 4th position of the substituted aryl ring plays a dominant role for its anticancer property. Among the synthesized compounds, 5e and 5f have shown potent anticancer activity on both the cell lines tested. To rationalize the role of electron donating group in the induction of cytotoxicity we have chosen two molecules (5e and 5k) having different electron donating group at different positions. LDH assay, Flow cytometric analysis and DNA fragmentation suggest that 5e is more cytotoxic and able to induce the apoptosis. (C) 2009 Elsevier Ltd. All rights reserved.

Orchestration of Haemophilus influenzae RecJ Exonuclease by Interaction with Single-Stranded DNA-Binding Protein

RecJ exonuclease plays crucial roles in several DNA repair and recombination pathways, and its ubiquity in bacterial species points to its ancient origin and vital cellular function. RecJ exonuclease from Haemophilus influenzae is a 575-amino-acid protein that harbors the characteristic motifs conserved among RecJ homologs. The purified protein exhibits a process 5'-3' single-stranded-DNA-specific exonuclease activity. The exonuclease activity of H. influenzae RecJ (HiRecJ) was supported by Mg2+ or Mn2+ and inhibited by Cd2+ suggesting a different mode of metal binding in HiRecJ as compared to Escherichia coli RecJ (EcoRecJ). Site-directed mutagenesis of highly conserved residues in HiRecJ abolished enzymatic activity. Interestingly, substitution of alanine for aspartate 77 resulted in a catalytically inactive enzyme that bound to DNA with a significantly higher affinity as compared to the wild-type enzyme. Noticeably, steady-state kinetic studies showed that H. influenzae single-stranded DNA-binding protein (HiSSB) increased the affinity of HiRecJ for single-stranded DNA and stimulated its exonuclease activity. HiSSB, whose C-terminal tail had been deleted, failed to enhance RecJ exonuclease activity. More importantly, HiRecJ was found to directly associate with its cognate single-stranded DNA-binding protein (SSB), as demonstrated by various in vitro assays, Interaction studies carried out with the truncated variants of HiRecJ and HiSSB revealed that the two proteins interact via the C-terminus of SSB protein and the core-catalytic domain of RecJ. Taken together, these results emphasize direct interactio between RecJ and SSB, which confers functional cooperativity to these two proteins. In addition, these results implicate SSB as being involved in the recruitment of RecJ to DNA and provide insights into the interplay between these proteins in repair and recombination pathways.

Identification of a discrete intermediate in the assembly disassembly of physalis mottle tymovirus through mutational analysis

Assembly intermediates of icosahedral viruses are usually transient and are difficult to identify. In the present investigation, site-specific and deletion mutants of the coat protein gene of physalis mottle tymovirus (PhMV) were used to delineate the role of specific amino acid residues in the assembly of the virus and to identify intermediates in this process. N-terminal 30, 34, 35 and 39 amino acid deletion and single C-terminal (N188) deletion mutant proteins of PhMV were expressed in Escherichia coli. Site-specific mutants H69A, C75A, W96A, D144N, D144N-T151A, K143E and N188A were also constructed and expressed. The mutant protein lacking 30 amino acid residues from the N terminus self-assembled to T = 3 particles in vivo while deletions of 34, 35 and 39 amino acid residues resulted in the mutant proteins that were insoluble. Interestingly, the coat protein (pR PhCP) expressed using pRSET B vector with an additional 41 amino acid residues at the N terminus also assembled into T = 3 particles that were more compact and had a smaller diameter. These results demonstrate that the amino-terminal segment is flexible and either the deletion or addition of amino acid residues at the N terminus does not affect T = 3 capsid assembly, in contrast, the deletion of even a single residue from the C terminus (PhN188 Delta 1) resulted in capsids that were unstable. These capsids disassembled to a discrete intermediate with a sedimentation coefficent of 19.4 S. However, the replacement of C-terminal asparagine 188 by alanine led to the formation of stable capsids. The C75A and D144N mutant proteins also assembled into capsids that were as stable as the pR PhCP, suggesting that C75A and D144 are not crucial for the T = 3 capsid assembly. pR PhW96A and pR PhD144N-T151A mutant proteins failed to form capsids and were present as heterogeneous aggregates. Interestingly, the pR PhK143E mutant protein behaved in a manner similar to the C-terminal deletion protein in forming unstable capsids. The intermediate with an s value of 19.4 S was the major assembly product of pR PhH69A mutant protein and could correspond to a 30mer. It is possible that the assembly or disassembly is arrested at a similar stage in pR PhN188 Delta 1, pR PhH69A and pR PhK143E mutant proteins.

Interaction Between Two Residues in the Inter-Domain Interface of Escherichia coli Peptidase N Modulates Catalytic Activity

The role of interaction between Asn259 (catalytic domain) with Gln821 (C-terminal domain) in PeptidaseN was investigated. The k(cat) of PeptidaseN containing Asn259Asp or Gln821Glu is enhanced whereas it is suppressed in Asn259AspGln821Glu. Structural analysis shows this interaction to change the relative disposition of active site residues, which modulates catalytic activity.

Immunological cross-reactivity of mycobacterial topoisomerase I and divergence from other bacteria

Mycobacterium smegmatis topoisomerase I exhibits several distinctive characteristics among all topoisomerases. The enzyme is devoid of Zn2+fingers found typically in other bacterial type I topoisomerases and binds DNA in a site-specific manner. Using polyclonal antibodies, we demonstrate the high degree of relatedness of the enzyme across mycobacteria but not other bacteria. This absence of cross-reactivity from other bacteria indicates that mycobacterial topoisomerase I has diverged from Escherichia coli and other bacteria. We have investigated further the immunological properties of the enzyme by raising a panel of monoclonal antibodies that recognises different antigenically active regions of the enzyme and binds it with widely varied affinity. Inhibition of a C-terminal domain-specific antibody binding by enzyme-specific and non-specific oligonucleotides suggests the possibility of using these monoclonal antibodies to probe the structure, function and in vivo role of the enzyme.

On the reactivity of carboxyl groups of ribonuclease-A in anhydrous methanol

The esterification of Ribonuclease-A in methanol/0.1 M hydrochloric acid has been studied by measuring the decrease in the number of titratable groups of the protein and estimating the amount of methanol incorporated. Esterification of nearly five of the 11 free carboxyl groups of the protein resulted in almost complete inactivation of the enzyme. The initial products of esterification have been chromatographed on Amberlite columns, and five partially active methyl ester derivatives of Ribonuclease-A have been isolated. The dimethyl ester, the initial product of esterification with reduced catalytic activity, has the carboxyl groups of Glu-49 and Asp-53 modified. Even in the non-aqueous solvent, as in the native structure of the protein in aqueous solution, these carboxyl groups are the fast reacting ones. Subsquently, the esterification reaction appears to proceed preferentially at the C-terminal region of the molecule. Comparison of the reactivities of carboxyl groups of Ribonuclease-A in acidic methanol to that known in aqueous solutions (with carbodiimides) suggests that the structure of Ribonuclease-A in the non-aqueous solvent resembles, at least in part, the structure in aqueous environment.

A single point mutation disrupts the capsid assembly in Sesbania Mosaic Virus resulting in a stable isolated dimer

Protein-protein interactions play a Crucial role in Virus assembly and stability. With the view of disrupting capsid assembly and capturing smaller oligomers, interfacial residue mutations were carried Out in the coat protein gene of Sesbania Mosaic Virus, a T=3 ss (+) RNA plant virus. A single point mutation of a Trp 170 present at the five-fold interface of the virus to a charged residue (Glu or Lys) arrested assembly of virus like particles and resulted in stable Soluble dimers of the capsid Protein. The X-ray crystal structure of one of the isolated dimer mutants - rCP Delta N65W170K was determined to a resolution of 2.65 angstrom. Detailed analysis of the dimeric mutant protein structure revealed that a number of Structural changes take place, especially in the loop and interfacial regions during the course of assembly. The isolated chiller was ``more relaxed'' than the dimer found in the T=3 or T=1 capsids. The isolated dimer does not bind Ca2+ ion and consequently four C-terminal residues are disordered. The FG loop, which interacts with RNA in the Virus, has different conformations in the isolated dimer and the intact Virus Suggesting its flexible nature and the conformational changes that accompany assembly. The isolated choler mutant was much less stable when compared to the assembled capsids, suggesting the importance of inter-subunit interactions and Ca2+ mediated interactions in the stability of the capsids. With this study, SeMV becomes the first icosahedral virus for which X-ray crystal Structures of T=3, T=1 capsids as well as a smaller oligomer of the capsid protein have been determined.

Inhibition of Human Two-Pore Domain K+ Channel TREK1 by Local Anesthetic Lidocaine: Negative Cooperativity and Half-of-Sites Saturation Kinetics

TWIK-related K+ channel TREK1, a background leak K+ channel, has been strongly implicated as the target of several general and local anesthetics. Here, using the whole-cell and single-channel patch-clamp technique, we investigated the effect of lidocaine, a local anesthetic, on the human (h) TREK1 channel heterologously expressed in human embryonic kidney 293 cells by an adenoviral-mediated expression system. Lidocaine, at clinical concentrations, produced reversible, concentration-dependent inhibition of hTREK1 current, with IC50 value of 180 mu M, by reducing the single-channel open probability and stabilizing the closed state. We have identified a strategically placed unique aromatic couplet (Tyr352 and Phe355) in the vicinity of the protein kinase A phosphorylation site, Ser348, in the C-terminal domain (CTD) of hTREK1, that is critical for the action of lidocaine. Furthermore, the phosphorylation state of Ser348 was found to have a regulatory role in lidocaine-mediated inhibition of hTREK1. It is interesting that we observed strong intersubunit negative cooperativity (Hill coefficient = 0.49) and half-of-sites saturation binding stoichiometry (half-reaction order) for the binding of lidocaine to hTREK1. Studies with the heterodimer of wild-type (wt)-hTREK1 and Delta 119 C-terminal deletion mutant (hTREK1(wt)-Delta 119) revealed that single CTD of hTREK1 was capable of mediating partial inhibition by lidocaine, but complete inhibition necessitates the cooperative interaction between both the CTDs upon binding of lidocaine. Based on our observations, we propose a model that explains the unique kinetics and provides a plausible paradigm for the inhibitory action of lidocaine on hTREK1.

Mapping of assembled epitopic regions of human chorionic gonadotropin reveals proximity of CTP alpha to the determinant loop beta 93-100

Three overlapping assembled epitopes of beta hCG have been mapped using MAb probes and a single step solid phase radioimmunoassay. These epitopes have been shown to be at receptor binding region comprising of the loop region beta Cys93-Cys100. Importance of disulphide bonds in maintaining integrity of these epitopes is assessed. Two MAbs (INN 58 and INN 22) interact with the beta region as well as the alpha C-terminal peptide, while the other MAb INN 24 interacts with only the beta region. Cross-reactivity pattern with beta hCG and hLH as web as the reported crystal structure of hCG substantiates the epitope identification. The results demonstrate utility of MAbs as probes in investigations on three-dimensional structure of gonadatropins.

Primary structure of a Thomsen-Friedenreich-antigen-specific lectin, jacalin [Artocarpus integrifolia (jack fruit) agglutinin]. Evidence for the presence of an internal repeat

Jacalin [Artocarpus integrifolia (jack fruit) agglutinin] is made up of two types of chains, heavy and light, with M(r) values of 16,200 +/- 1200 and 2090 +/- 300 respectively (on the basis of gel-permeation chromatography under denaturing conditions). Its complete amino acid sequence was determined by manual degradation using a 4-dimethylaminoazobenzene 4'-isothiocyanate double-coupling method. Peptide fragments for sequence analysis were obtained by chemical cleavages of the heavy chain with CNBr, hydroxylamine hydrochloride and iodosobenzoic acid and enzymic cleavage with Staphylococcus aureus proteinase. The peptides were purified by a combination gel-permeation and reverse-phase chromatography. The light chains, being only 20 residues long, could be sequenced without fragmentation. Amino acid analyses and carboxypeptidase-Y-digestion C-terminal analyses of the subunits provided supportive evidence for their sequence. Computer-assisted alignment of the jacalin heavy-chain sequence failed to show sequence similarity to that of any lectin for which the complete sequence is known. Analyses of the sequence showed the presence of an internal repeat spanning residues 7-64 and 76-130. The internal repeat was found to be statistically significant.

Assembly of physalis mottle virus capsid protein in Escherichia coli and the role of amino and carboxy termini in the formation of the icosahedral particles

The coat protein gene of physalis mottle tymovirus (PhMV) was over expressed in Escherichia coli using pET-3d vector. The recombinant protein was found to self assemble into capsids in vivo. The purified recombinant capsids had an apparent s value of 56.5 S and a diameter of 29(±2) nm. In order to establish the role of amino and carboxy-terminal regions in capsid assembly, two amino-terminal deletions clones lacking the first 11 and 26 amino acid residues and two carboxy-terminal deletions lacking the last five and ten amino acid residues were constructed and overexpressed. The proteins lacking N-terminal 11 (PhCPN1) and 26 (PhCPN2) amino acid residues self assembled into T = 3 capsids in vivo, as evident from electron microscopy, ultracentrifugation and agarose gel electrophoresis. The recombinant, PhCPN1 and PhCPN2 capsids were as stable as the empty capsids formed in vivo and encapsidated a small amount of mRNA. The monoclonal antibody PA3B2, which recognizes the epitope within region 22 to 36, failed to react with PhCPN2 capsids while it recognized the recombinant and PhCPN1 capsids. Disassembly of the capsids upon treatment with urea showed that PhCPN2 capsids were most stable. These results demonstrate that the N-terminal 26 amino acid residues are not essential for T = 3 capsid assembly in PhMV. In contrast, both the proteins lacking the C-terminal five and ten amino acid residues were present only in the insoluble fraction and could not assemble into capsids, suggesting that these residues are crucial for folding and assembly of the particles.

Structural Modes of Stabilization of Permissive Phosphorylation Sites in Protein Kinases: Distinct Strategies in Ser/Thr and Tyr Kinases

Protein kinases phosphorylate several cellular proteins providing control mechanisms for various signalling processes. Their activity is impeded in a number of ways and restored by alteration in their structural properties leading to a catalytically active state. Most protein kinases are subjected to positive and negative regulation by phosphorylation of Ser/Thr/Tyr residues at specific sites within and outside the catalytic core. The current review describes the analysis on 3D structures of protein kinases that revealed features distinct to active states of Ser/Thr and Tyr kinases. The nature and extent of interactions among well-conserved residues surrounding the permissive phosphorylation sites differ among the two classes of enzymes. The network of interactions of highly conserved Arg preceding the catalytic base that mediates stabilization of the activation segment exemplifies such diverse interactions in the two groups of kinases. The N-terminal and the C-terminal lobes of various groups of protein kinases further show variations in their extent of coupling as suggested from the extent of interactions between key functional residues in activation segment and the N-terminal αC-helix. We observe higher similarity in the conformations of ATP bound to active forms of protein kinases compared to ATP conformations in the inactive forms of kinases. The extent of structural variations accompanying phosphorylation of protein kinases is widely varied. The comparison of their crystal structures and the distinct features observed are hoped to aid in the understanding of mechanisms underlying the control of the catalytic activity of distinct subgroups of protein kinases.

Genetic studies of the PRP17 gene of Saccharomyces cerevisiae: a domain essential for function maps to a nonconserved region of the protein

The PRP17 gene product is required for the second step of pre-mRNA splicing reactions. The C-terminal half of this protein bears four repeat units with homology to the beta transducin repeat. Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein. We have generated, in vitro, 11 missense alleles at the beta transducin repeat units and find that only one affects function in vivo. A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains. Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units. Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction. A mutually allele-specific interaction between Prp17 and snr7, with mutations in U5 snRNA, was observed. We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is the N terminal region of the protein.

Acetylation of Transition Protein 2 (TP2) by KAT3B (p300) Alters Its DNA Condensation Property and Interaction with Putative Histone Chaperone NPM3

The hallmark of mammalian spermiogenesis is the dramatic chromatin remodeling process wherein the nucleosomal histones are replaced by the transition proteins TP1, TP2, and TP4. Subsequently these transition proteins are replaced by the protamines P1 and P2. Hyperacetylation of histone H4 is linked to their replacement by transition proteins. Here we report that TP2 is acetylated in vivo as detected by anti-acetylated lysine antibody and mass spectrometric analysis. Further, recombinant TP2 is acetylated in vitro by acetyltransferase KAT3B (p300) more efficiently than by KAT2B (PCAF). In vivo p300 was demonstrated to acetylate TP2. p300 acetylates TP2 in its C-terminal domain, which is highly basic in nature and possesses chromatin-condensing properties. Mass spectrometric analysis showed that p300 acetylates four lysine residues in the C-terminal domain of TP2. Acetylation of TP2 by p300 leads to significant reduction in its DNA condensation property as studied by circular dichroism and atomic force microscopy analysis. TP2 also interacts with a putative histone chaperone, NPM3, wherein expression is elevated in haploid spermatids.Interestingly, acetylation of TP2 impedes its interaction with NPM3. Thus, acetylation of TP2 adds a new dimension to its role in the dynamic reorganization of chromatin during mammalian spermiogenesis.