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.

Lignin biosynthesis in Norway spruce: from a model system to the tree

Lignin is a hydrophobic polymer that is synthesised in the secondary cell walls of all vascular plants. It enables water conduction through the stem, supports the upright growth habit and protects against invading pathogens. In addition, lignin hinders the utilisation of the cellulosic cell walls of plants in pulp and paper industry and as forage. Lignin precursors are synthesised in the cytoplasm through the phenylpropanoid pathway, transported into the cell wall and oxidised by peroxidases or laccases to phenoxy radicals that couple to form the lignin polymer. This study was conducted to characterise the lignin biosynthetic pathway in Norway spruce (Picea abies (L.) Karst.). We focused on the less well-known polymerisation stage, to identify the enzymes and the regulatory mechanisms that are involved. Available data for lignin biosynthesis in gymnosperms is scarce and, for example, the latest improvements in precursor biosynthesis have only been verified in herbaceous plants. Therefore, we also wanted to study in detail the roles of individual gene family members during developmental and stress-induced lignification, using EST sequencing and real-time RT-PCR. We used, as a model, a Norway spruce tissue culture line that produces extracellular lignin into the culture medium, and showed that lignin polymerisation in the tissue culture depends on peroxidase activity. We identified in the culture medium a significant NADH oxidase activity that could generate H2O2 for peroxidases. Two basic culture medium peroxidases were shown to have high affinity to coniferyl alcohol. Conservation of the putative substrate-binding amino acids was observed when the spruce peroxidase sequences were compared with other peroxidases with high affinity to coniferyl alcohol. We also used different peroxidase fractions to produce synthetic in vitro lignins from coniferyl alcohol; however, the linkage pattern of the suspension culture lignin could not be reproduced in vitro with the purified peroxidases, nor with the full complement of culture medium proteins. This emphasised the importance of the precursor radical concentration in the reaction zone, which is controlled by the cells through the secretion of both the lignin precursors and the oxidative enzymes to the apoplast. In addition, we identified basic peroxidases that were reversibly bound to the lignin precipitate. They could be involved, for example, in the oxidation of polymeric lignin, which is required for polymer growth. The dibenzodioxocin substructure was used as a marker for polymer oxidation in the in vitro polymerisation studies, as it is a typical substructure in wood lignin and in the suspension culture lignin. Using immunolocalisation, we found the structure mainly in the S2+S3 layers of the secondary cell walls of Norway spruce tracheids. The structure was primarily formed during the late phases of lignification. Contrary to the earlier assumptions, it appears to be a terminal structure in the lignin macromolecule. Most lignin biosynthetic enzymes are encoded for by several genes, all of which may not participate in lignin biosynthesis. In order to identify the gene family members that are responsible for developmental lignification, ESTs were sequenced from the lignin-forming tissue culture and developing xylem of spruce. Expression of the identified lignin biosynthetic genes was studied using real-time RT-PCR. Candidate genes for developmental lignification were identified by a coordinated, high expression of certain genes within the gene families in all lignin-forming tissues. However, such coordinated expression was not found for peroxidase genes. We also studied stress-induced lignification either during compression wood formation by bending the stems or after Heterobasidion annosum infection. Based on gene expression profiles, stress-induced monolignol biosynthesis appeared similar to the developmental process, and only single PAL and C3H genes were specifically up-regulated by stress. On the contrary, the up-regulated peroxidase genes differed between developmental and stress-induced lignification, indicating specific responses.

De novo synthesis of acetylcholinesterase in roots of Pisum sativum

Pisum sativum seeds contain a conserved acetylcholinesterase (AChE) which is active during the early stages of germination. The enzyme activity soon disappears and reappears after 72 hr of germination. A protein devoid of catalytic ability, but exhibiting similar chromatographic and electrophoretic properties as the active AChE, could be detected after 24 hr of germination. The pattern of incorporation of labelled amino acids into AChE and the influence of cycloheximide revealed that the AChE found in the roots from 72 hr onwards was entirely new. During this period of growth, the AChE protein accounts for 4–10% of the total proteins in the root tissue.

Substrate Selectivity and Molecular Adaptation in the Outer Membrane Proteases of Yersinia pestis and Salmonella enterica

Proteolysis is important in bacterial pathogenesis and colonization of animal and plant hosts. In this work I have investigated the functions of the bacterial outer membrane proteases, omptins, of Yersinia pestis and Salmonella enterica. Y. pestis is a zoonotic pathogen that causes plague and has evolved from gastroenteritis-causing Yersinia pseudotuberculosis about 13 000 years ago. S. enterica causes gastroenteritis and typhoid fever in humans. Omptins are transmembrane β-barrels with ten antiparallel β-strands and five surface-exposed loops. The loops are important in substrate recognition, and variation in the loop sequences leads to different substrate selectivities between omptins, which makes omptins an ideal platform to investigate functional adaptation and to alter their polypeptide substrate preferences. The omptins Pla of Y. pestis and PgtE of S. enterica are 75% identical in their amino acid sequences. Pla is a multifunctional protein with proteolytic and non-proteolytic functions, and it increases bacterial penetration and proliferation in the host. Functions of PgtE increase migration of S. enterica in vivo and bacterial survival in mouse macrophages, thus enhancing bacterial spread within the host. Mammalian plasminogen/fibrinolytic system maintains the balance between coagulation and fibrinolysis and participates in several cellular processes, e.g., cell migration and degradation of extracellular matrix proteins. This system consists of activation cascades, which are strictly controlled by several regulators, such as plasminogen activator inhibitor 1 (PAI-1), α2-antiplasmin (α2AP), and thrombin-activatable fibrinolysis inhibitor (TAFI). This work reveals novel interactions of the omptins of Y. pestis and S. enterica with the regulators of the plasminogen/fibrinolytic system: Pla and PgtE inactivate PAI-1 by cleavage at the reactive site peptide bond, and degrade TAFI, preventing its activation to TAFIa. Structure-function relationship studies with Pla showed that threonine 259 of Pla is crucial in plasminogen activation, as it prevents degradation of the plasmin catalytic domain by the omptin and thus maintains plasmin stability. In this work I constructed chimeric proteins between Pla and Epo of Erwinia pyrifoliae that share 78% sequence identity to find out which amino acids and regions in Pla are important for its functions. Epo is neither a plasminogen activator nor an invasin, but it degrades α2AP and PAI-1. Cumulative substitutions towards Pla sequence turned Epo into a Pla-like protein. In addition to threonine 259, loops 3 and 5 are critical in plasminogen activation by Pla. Turning Epo into an invasin required substitution of 31 residues located at the extracellular side of the Epo protein above the lipid bilayer, and also of the β1-strand in the N-terminal transmembrane region of the protein. These studies give an example of how omptins adapt to novel functions that advantage their host bacteria in different ecological niches.

The C-terminal domain is sufficient for endonuclease activity of Neisseria gonorrhoeae MutL

The mutL gene of Neisseria gonorrhoeae has been cloned and the gene product purified. We have found that the homodimeric N. gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular DNA in the presence of Mn2+, Mg2+ or Ca2+ ions, unlike human MutL alpha which shows endonuclease activity only in the presence of Mn2+. We report in the present paper that the C-terminal domain of N. gonorrhoeae MutL (NgoL-CTD) consisting of amino acids 460-658 exhibits Mn2+-dependent endonuclease activity. Sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments show NgoL-CTD to be a dimer. The probable endonucleolytic active site is localized to a metal-binding motif, DMHAX(2)EX(4)E, and the nicking endonuclease activity is dependent on the integrity of this motif. By in vitro comparison of wild-type and it mutant NgoL-CTD protein, we show that the latter protein exhibits highly reduced endonuclease activity. We therefore suggest that the mode of excision initiation in DNA mismatch repair may be different in organisms that lack MutH protein, but have MutL proteins that harbour the D[M/Q]HAX(2)EX(4)E motif.

Synthesis and Cytotoxic Evaluation of Novel 2-(4-(2,2,2-Trifluoroethoxy)-3-methylpyridin-2-ylthio)-1H-benzo[d]imid azole Derivatives

The present work deals with the anticancer effect of benzimidazole derivatives associated with the pyridine framework. By varying the functional group at N-terminal of the benzimidazole by different L-amino acids, several 2-(4-(2,2,2-trifluoroethoxy)-3-methylpyridin-2-ylthio)-1H-benzo[d]imid azole derivatives 9(a-j) were synthesized. Their chemical structures were confirmed by H-1 NMR, IR and mass spectroscopic techniques. The synthesized compounds were examined for their antiproliferative effects against human leukemia cell lines, K562 and CEM. The preliminary results showed most of the derivatives had moderate antitumor activity. Compound 9j containing cysteine residue exhibited good inhibition compared to other amino acid resides. In addition DNA fragmentation results suggest that 9j is more cytotoxic and able to induce apoptosis.

Convenient Synthesis of Ferrocene Conjugates Mediated by Benzyltriethylammonium Tetrathiomolybdate in a Multi-Step Tandem Process

The synthesis of a wide range of ferrocene-derived sulfur-linked mono- and disubstituted Michael adducts and conjugates mediated by benzyltriethylammonium tetrathiomolybdate (1) in a tandem process is reported. New route to access acryloylferrocene (4) and 1,1'-diacryloylferrocene (5) is discussed. Conjugation of amino acids to ferrocene is established via their N and C termini and also via side chains employing conjugate addition as key step to furnish mono-and divalent conjugates. This methodology has also been extended to access several ferrocene-carbohydrate conjugates. The electrochemical behavior of some selected ferrocene conjugates was studied by cyclic voltammetry.

Nuclear Import Mechanisms of STAT and NF-kB Transcription Factors

The eukaryotic cell nucleoplasm is separated from the cytoplasm by the nuclear envelope. This compartmentation of eukaryotic cells requires that all nuclear proteins must be transported from the cytoplasm into the nucleus. Transport of macromolecules between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs). Proteins to be targeted into the nucleus by the classical nuclear import system contain nuclear localization signals (NLSs), which are recognized by importin alpha, the NLS receptor. Importin alpha binds to importin beta, which docks the importin-cargo complex on the cytoplasmic side of the NPC and mediates the movement of the complex into the nucleus. Presently six human importin alpha isoforms have been identified. Transcription factors are among the most important regulators of gene expression in eukaryotic organisms. Transcription factors bind to specific DNA sequences on target genes and modulate the activity of the target gene. Many transcription factors, including signal transducers and activators of transcription (STAT) and nuclear factor kB (NF-kB), reside in the cytoplasm in an inactive form, and upon activation they are rapidly transported into the nucleus. In the nucleus STATs and NF-kB regulate the activity of genes whose products are critical in controlling numerous cellular and organismal processes, such as inflammatory and immune responses, cell growth, differentiation and survival. The aim of this study was to investigate the nuclear import mechanisms of STAT and NF-kB transcription factors. This work shows that STAT1 homodimers and STAT1/STAT2 heterodimers bind specifically and directly to importin alpha5 molecule via unconventional dimer-specific NLSs. Importin alpha molecules have two regions, which have been shown to directly interact with the amino acids in the NLS of the cargo molecule. The Arm repeats 2-4 comprise the N-terminal NLS binding site and Arm repeats 7-8 the C-terminal NLS binding site. In this work it is shown that the binding site for STAT1 homodimers and STAT1/STAT2 heterodimers is composed of Arm repeats 8 and 9 of importin alpha5 molecule. This work demonstrates that all NF-kB proteins are transported into the nucleus by importin alpha molecules. In addition, NLS was identified in RelB protein. The interactions between NF-kB proteins and importin alpha molecules were found to be directly mediated by the NLSs of NF-kB proteins. Moreover, we found that p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin alpha3. The results from this thesis work identify previously uncharacterized mechanisms in nuclear import of STAT and NF-kB. These findings provide new insights into the molecular mechanisms regulating the signalling cascades of these important transcription factors from the cytoplasm into the nucleus to the target genes.

Hyperexpression of chicken riboflavin carrier protein: Antibodies to the recombinant protein curtail pregnancy in rodents

Chicken riboflavin carrier protein (RCP) is a phosphoglycoprotein present in the egg white and yolk of egg-laying animals and in the sera of laying hens and of estrogenized chicks. The RCP cDNA, encoding a protein of predictedMr27,000, has been cloned into a T7 polymerase-driven vector, and high-level expression was observed on induction with IPTG inEscherichia coli.The protein was largely localized in inclusion bodies when expressed at 37°C but was present in the cytosolic fraction when induced at 22°C. At 37°C, two major bands were detected in whole-cell lysates of the strain expressing the protein. N-terminal sequence analysis indicated that the two proteins represented translated products with and without the pelB leader sequence encoded in the pET20b vector, but both included an additional 10 amino acids generated during cloning procedures. The inclusion body obtained at 37°C, on extraction with detergent, led to preferential solubilization of the protein without the pelB signal sequence. The solubilized recombinant RCP was recognized by polyclonal antisera to native RCP but radioimmunoassay revealed quantitative differences in the epitopes exhibited by the recombinant protein. Thus, sequence-specific monoclonal antibodies to chicken RCP also cross-reacted with the recombinant protein with almost equal efficiency, but antibodies which recognize conformation-dependent epitopes showed relatively reduced cross-reactivity with the recombinant protein. Polyclonal antibodies to recombinant RCP were able to recognize both the native and the denatured RCP. Administration of recombinant RCP antisera to pregnant mice led to embryonic resorption leading to early pregnancy termination. These findings reveal that the recombinant protein will be useful for investigations related to the mechanism of pregnancy termination on immunoneutralization of RCP in mammals, as well as in unraveling folding properties of RCP in terms of its ligand binding and antigenetic determinants exposed at its surface.

Positional cloning and pathway analysis of the asthma susceptibility gene, NPSR1

In the present study, we identified a novel asthma susceptibility gene, NPSR1 (neuropeptide S receptor 1) on chromosome 7p14.3 by the positional cloning strategy. An earlier significant linkage mapping result among Finnish Kainuu asthma families was confirmed in two independent cohorts: in asthma families from Quebec, Canada and in allergy families from North Karelia, Finland. The linkage region was narrowed down to a 133-kb segment by a hierarchial genotyping method. The observed 77-kb haplotype block showed 7 haplotypes and a similar risk and nonrisk pattern in all three populations studied. All seven haplotypes occur in all three populations at frequences > 2%. Significant elevated relative risks were detected for elevated total IgE (immunoglobulin E) or asthma. Risk effects of the gene variants varied from 1.4 to 2.5. NPSR1 belongs to the G protein-coupled receptor (GPCR) family with a topology of seven transmembrane domains. NPSR1 has 9 exons, with the two main transcripts, A and B, encoding proteins of 371 and 377 amino acids, respectively. We detected a low but ubiquitous expression level of NPSR1-B in various tissues and endogenous cell lines while NPSR1-A has a more restricted expression pattern. Both isoforms were expressed in the lung epithelium. We observed aberrant expression levels of NPSR1-B in smooth muscle in asthmatic bronchi as compared to healthy. In an experimental mouse model, the induced lung inflammation resulted in elevated Npsr1 levels. Furthermore, we demonstrated that the activation of NPSR1 with its endogenous agonist, neuropeptide S (NPS), resulted in a significant inhibition of the growth of NPSR1-A overexpressing stable cell lines (NPSR1-A cells). To determine which target genes were regulated by the NPS-NPSR1 pathway, NPSR1-A cells were stimulated with NPS, and differentially expressed genes were identified using the Affymetrix HGU133Plus2 GeneChip. A total of 104 genes were found significantly up-regulated and 42 down-regulated 6 h after NPS administration. The up-regulated genes included many neuronal genes and some putative susceptibility genes for respiratory disorders. By Gene Ontology enrichment analysis, the biological process terms, cell proliferation, morphogenesis and immune response were among the most altered. The expression of four up-regulated genes, matrix metallopeptidase 10 (MMP10), INHBA (activin A), interleukin 8 (IL8) and EPH receptor A2 (EPHA2), were verified and confirmed by quantitative reverse-transcriptase-PCR. In conclusion, we identified a novel asthma susceptibility gene, NPSR1, on chromosome 7p14.3. NPS-NPSR1 represents a novel pathway that regulates cell proliferation and immune responses, and thus may have functional relevance in the pathogenesis of asthma.

O6-Methylguanine Repair by O6-Alkylguanine-DNA Alkyltransferase

O6-Alkylguanine-DNA alkyltransferase (AGT) repairs O6-methylguanine (O6mG) in DNA that is known to cause Mutation and cancer. On the basis of Calculations performed using density functional theory involving the active site of AGT, a mechanism for catalytic demethylation of O6mG to guanine has been proposed. In this mechanism, roles of six amino acids, i.e., Cys145, His 146, Glu172, Tyr114, Lys165, and Ser159 in catalytic demethylation of O6mG are involved. This mechanism has three steps as follows. At the first step, Cys145 in the Cys145-water-His146-Glu172 tetrad is converted to cysteine thiolate anion while at the second step, abstraction of the Tyr114 proton by the N3 site of O6mG occurs in a barrierless manner. In the third step, abstraction of Lys165 proton by deprotonated Tyr114 and transfer of the methyl group of O6mG to the thiolate group of Cys145 anion Occur simultaneously. As AGT is a major target in cancer therapy, identification of the roles of the different amino acids in demethylation of O6mG is expected to be useful in designing efficient AGT inhibitors.

Identification and Characterization of a Novel Deoxyhypusine Synthase in Leishmania donovani

Deoxyhypusine synthase, an NAD(+)-dependent enzyme, catalyzes the first step in the post-translational synthesis of an unusual amino acid, hypusine (N-epsilon-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic initiation factor 5A precursor protein. Two putative deoxyhypusine synthase (DHS) sequences have been identified in the Leishmania donovani genome, which are present on chromosomes 20: DHSL20 (DHS-like gene from chromosome 20) and DHS34 (DHS from chromosome 34). Although both sequences exhibit an overall conservation of key residues, DHSL20 protein lacks a critical lysine residue, and the recombinant protein showed no DHS activity in vitro. However, DHS34 contains the critical lysine residue, and the recombinant DHS34 effectively catalyzed deoxyhypusine synthesis. Furthermore, in vivo labeling confirmed that hypusination of eukaryotic initiation factor 5A occurs in intact Leishmania parasites. Interestingly, the DHS34 is much longer, with 601 amino acids, compared with the human DHS enzyme (369 amino acids) and contains several unique insertions. To study the physiological role of DHS34 in Leishmania, gene deletion mutations were attempted via targeted gene replacement. However, chromosomal null mutants of DHS34 could only be obtained in the presence of a DHS34-containing episome. The present data provide evidence that DHS34 is essential for L. donovani and that structural differences in the human and leishmanial DHS enzyme may be exploited for designing selective inhibitors against the parasite.

Allostery and conformational free energy changes in human tryptophanyl-tRNA synthetase from essential dynamics and structure networks

The interdependence of the concept of allostery and enzymatic catalysis, and they being guided by conformational mobility is gaining increased prominence. However, to gain a molecular level understanding of llostery and hence of enzymatic catalysis, it is of utter importance that the networks of amino acids participating in allostery be deciphered. Our lab has been exploring the methods of network analysis combined with molecular dynamics simulations to understand allostery at molecular level. Earlier we had outlined methods to obtain communication paths and then to map the rigid/flexible regions of proteins through network parameters like the shortest correlated paths, cliques, and communities. In this article, we advance the methodology to estimate the conformational populations in terms of cliques/communities formed by interactions including the side-chains and then to compute the ligand-induced population shift. Finally, we obtain the free-energy landscape of the protein in equilibrium, characterizing the free-energy minima accessed by the protein complexes. We have chosen human tryptophanyl-tRNA synthetase (hTrpRS), a protein esponsible for charging tryptophan to its cognate tRNA during protein biosynthesis for this investigation. This is a multidomain protein exhibiting excellent allosteric communication. Our approach has provided valuable structural as well as functional insights into the protein. The methodology adopted here is highly generalized to illuminate the linkage between protein structure networks and conformational mobility involved in the allosteric mechanism in any protein with known structure.

Random network behaviour of protein structures

Geometric and structural constraints greatly restrict the selection of folds adapted by protein backbones, and yet, folded proteins show an astounding diversity in functionality. For structure to have any bearing on function, it is thus imperative that, apart from the protein backbone, other tunable degrees of freedom be accountable. Here, we focus on side-chain interactions, which non-covalently link amino acids in folded proteins to form a network structure. At a coarse-grained level, we show that the network conforms remarkably well to realizations of random graphs and displays associated percolation behavior. Thus, within the rigid framework of the protein backbone that restricts the structure space, the side-chain interactions exhibit an element of randomness, which account for the functional flexibility and diversity shown by proteins. However, at a finer level, the network exhibits deviations from these random graphs which, as we demonstrate for a few specific examples, reflect the intrinsic uniqueness in the structure and stability, and perhaps specificity in the functioning of biological proteins.

Anomalous reaction-diffusion as a model of nonexponential DNA escape kinetics

We show that data from recent experiments carried out on the kinetics of DNA escape from alpha-hemolysin nanopores [M. Wiggin, C. Tropini, C. T. Cossa, N. N. Jetha, and A. Marziali, Biophys. J. 95, 5317 (2008)] may be rationalized by a model of chain dynamics based on the anomalous diffusion of a particle moving in a harmonic well in the presence of a delta function sink. The experiments of Wiggin found, among other things, that the occasional occurrence of unusually long escape times in the distribution of chain trapping events led to nonexponential decays in the survival probability, S(t), of the DNA molecules within the nanopore. Wiggin ascribed this nonexponentiality to the existence of a distribution of trapping potentials, which they suggested was theresult of stochastic interactions between the bases of the DNA and the amino acids located on the surface of the nanopore. Based on this idea, they showed that the experimentally determined S(t) could be well fit in both the short and long time regimes by a function of the form (1+t/tau)(-alpha) (the so called Becquerel function). In our model, S(t) is found to be given by a Mittag-Leffler function at short times and by a generalized Mittag-Leffler function at long times. By suitable choice of certain parameter values, these functions are found to fit the experimental S(t) even better than the Becquerel function. Anomalous diffusion of DNA within the trap prior to escape over a barrier of fixed height may therefore provide a second, plausible explanation of the data, and may offer fresh perspectives on similar trapping and escape problems.