Importance of COMT in the regulation of prefrontal dopamine

The prefrontal cortex (PFC), located in the anterior region of the frontal lobe, is considered to have several key roles in higher cognitive and executive functions. In general, the PFC can be seen as a coordinator of thought and action allowing subjects to behave in a goal-directed manner. Due to its anatomical connections with a variety of cortical and subcortical structures, several neurotransmitters, including dopamine, are involved in the regulation of PFC activity. In general, the majority of released dopamine is cleared by the dopamine transporter (DAT). In the PFC however, the number of presynaptic DAT is diminished, emphasizing the relative importance of catechol-O-methyltransferase (COMT) in dopamine metabolism. As a result, the role of COMT in the etiology of psychotic disorders is under constant debate. The present study investigated the role of COMT in prefrontal cortical dopamine metabolism by different neurochemical methods in COMT knockout (COMT-KO) mice. Pharmacological tools to inhibit other dopamine clearing mechanisms were also used for a more comprehensive and collective picture. In addition, this study investigated how a lack of the soluble (S-) COMT isoform affects the total COMT activity as well as the pharmacokinetics of orally administered L-dopa using mutant mice expressing only the membrane-bound (MB-) COMT isoform. Also the role of COMT in striatal and accumbal dopamine turnover during Δ9-tetrahydrocannabinol (THC) challenge was studied. We found markedly increased basal dopamine concentrations in the PFC, but not the striatum or nucleus accumbens (NAcc), of mice lacking COMT. Pharmacological inhibition of the noradrenaline transporter (NET) and monoamine oxidase (MAO) elevated prefrontal cortical dopamine levels several-fold, whereas inhibition of DAT did not. The lack of COMT doubled the dopamine raising effects of NET and MAO inhibition. No compensatory expression of either DAT or NET was found in the COMT-KO mice. The lack of S-COMT decreased the total COMT activity by 50-70 % and modified dopamine transmission and the pharmacokinetics of exogenous Ldopa in a sex and tissue specific manner. Finally, we found that subsequent tolcapone and THC increased dopamine levels in the NAcc, but not in the striatum. Conclusively, this study presents neurochemical evidence for the important role of COMT in the PFC and shows that COMT is responsible for about half of prefrontal cortical dopamine metabolism. This study also highlights the previously underestimated proportional role of MB-COMT and supports the clinical evidence of a gene x environment interaction between COMT and cannabis.

Diversity in Functional Organization of Class I and Class II Biotin Protein Ligase

The cell envelope of Mycobacterium tuberculosis (M. tuberculosis) is composed of a variety of lipids including mycolic acids, sulpholipids, lipoarabinomannans, etc., which impart rigidity crucial for its survival and pathogenesis. Acyl CoA carboxylase (ACC) provides malonyl-CoA and methylmalonyl-CoA, committed precursors for fatty acid and essential for mycolic acid synthesis respectively. Biotin Protein Ligase (BPL/BirA) activates apo-biotin carboxyl carrier protein (BCCP) by biotinylating it to an active holo-BCCP. A minimal peptide (Schatz), an efficient substrate for Escherichia coli BirA, failed to serve as substrate for M. tuberculosis Biotin Protein Ligase (MtBPL). MtBPL specifically biotinylates homologous BCCP domain, MtBCCP87, but not EcBCCP87. This is a unique feature of MtBPL as EcBirA lacks such a stringent substrate specificity. This feature is also reflected in the lack of self/promiscuous biotinylation by MtBPL. The N-terminus/HTH domain of EcBirA has the selfbiotinable lysine residue that is inhibited in the presence of Schatz peptide, a peptide designed to act as a universal acceptor for EcBirA. This suggests that when biotin is limiting, EcBirA preferentially catalyzes, biotinylation of BCCP over selfbiotinylation. R118G mutant of EcBirA showed enhanced self and promiscuous biotinylation but its homologue, R69A MtBPL did not exhibit these properties. The catalytic domain of MtBPL was characterized further by limited proteolysis. Holo-MtBPL is protected from proteolysis by biotinyl-59 AMP, an intermediate of MtBPL catalyzed reaction. In contrast, apo-MtBPL is completely digested by trypsin within 20 min of co-incubation. Substrate selectivity and inability to promote self biotinylation are exquisite features of MtBPL and are a consequence of the unique molecular mechanism of an enzyme adapted for the high turnover of fatty acid biosynthesis.

Rapid purification of tRNA(Lys) from rat liver

Fast protein liquid chromatography (FPLC) system using Mono Q (HR 5/5) anion-exchange column chromatography followed by highly cross-linked urea-polyacrylamide gel electrophoresis (urea-PAGE) was used for the purification of lysine-specific tRNA (tRNA(Lys)) from rat liver. Crude tRNA from rat liver was fractionated with a linear gradient of NaCl (0.3-0.8 M) in triethanolamine-HCl buffer, pH 4.5, and the activity of tRNA(Lys) was found to elute between 0.51 and 0.57 M NaCl. Using this concentration range of NaCl, tRNA(Lys) was refractionated on the same column with a shallow gradient, where a single peak of tRNA(Lys) activity was obtained. tRNA(Lys)-rich fractions recovered from the second run were electrophoretically separated on 16% polyacrylamide-7 M urea gel into one major band and three minor bands. The major band showed a specific activity of 997 pmols/A260 U for tRNALys with a 43-fold purification and approximately 17% recovery. The minor bands displayed negligible or no activity for lysine. tRNA(Lys) obtained by this method was found to be homogeneous by competitive aminoacylation. The advantages of FPLC followed by urea-PAGE in the purification of an amino acid-specific tRNA over conventional column chromatography are discussed.

Analysis of a conformation-specific epitope of the alpha subunit of human chorionic gonadotropin: Study using monoclonal antibody probes

Monoclonal antibodies (MAbs) have been used extensively for identification of sequence-specific epitopes using either the ELISA or/and IRMA methods, However, attempts to use MAbs for identification of conformation-specific epitopes have been very few as they are considered very labile. We have investigated the stability of conformation-specific epitopes of human chorionic gonadotropin (hCG) using a quantitative solid-phase radioimmnunoassay (SPRIA) technique. Several epitopes are stable to mild modification (chemical and proteolytic) conditions, and epitopes show differential stability for these modifications. Based on these observations, a monoclonal antibody (MAb 16) for an a-subunit-specific epitope of hCG has been used to monitor changes at the epitopic site (identified as epitope 16) on modification of hCG, using SPRIA with immobilized MAb 16. Modifications of amino groups, hydroxyl group of tyrosine as well as carboxyl group of Asp/Glu all bring about sufficient changes in the epitope integrity. Peptide bond hydrolysis at lysine residues damages the epitope, but not at arginine residues, Hydrolysis at tyrosine does not affect the epitope, though modification of the side-chain of tyrosine inactivates the epitope. Destruction of the epitope occurs on reduction of the disulphide bonds. Partial retention of the epitope activity is seen on modification of carboxyl or the epsilon-amino groups of lysine. Based on these results four to six amino acids have been identified to be at the epitopic site, and the data suggest that two peptide segments are brought together by the disulphide bond Cys10-Cys60 to form the epitope.

Important role of the amino acid attached to tRNA in formylation and in initiation of protein synthesis in Escherichia coli

In attempts to convert an elongator tRNA to an initiator tRNA, we previously generated a mutant elongator methionine tRNA carrying an anticodon sequence change from CAU to CUA along with the two features important for activity of Escherichia coli initiator tRNA in initiation. This mutant tRNA (Mi:2 tRNA) was active in initiation in vivo but only when aminoacylated with methionine by overproduction of methionyl-tRNA synthetase. Here we show that the Mi:2 tRNA is normally aminoacylated in vivo with lysine and that the tRNA aminoacylated with lysine is a very poor substrate for formylation compared with the same tRNA aminoacylated with methionine. By introducing further changes at base pairs 4:69 and 5:68 in the acceptor stem of the Mi:2 tRNA to those found in the E. coli initiator tRNA, we show that change of the U4:A69 base pair to G4:C69 and overproduction of lysyl-tRNA synthetase and methionyl-tRNA transformylase results in partial formylation of the mutant tRNA and activity of the formyllysyl-tRNAs in initiation of protein synthesis. Thus, the G4:C69 base pair contributes toward formylation of the tRNA and protein synthesis in E. coli can be initiated with formyllysine. We also discuss the implications of these and other results on recognition of tRNAs by E. coli lysyl-tRNA synthetase and on competition in cells among aminoacyl-tRNA synthetases.

DNA recognition by the EcoP15I and EcoPI modification methyltransferases

The DNA-binding properties of the EcoP15I DNA methyltransferase (M . EcoP15I; MTase) were studied using electrophoretic mobility shift assays. We show by molecular size-exclusion chromatography and dimethyl suberimidate crosslinking that M . EcoP15I is a dimer in solution. While M . EcoP15I binds approx. threefold more tightly to its recognition sequence, 5'-CAGCAG-3', than to non-specific sequences in the presence of AdoMet or its analogs, the discrimination between specific and non-specific sequences significantly increases in presence of ATP. These results suggest for the first time a role for ATP in DNA recognition by type-III restriction-modification enzymes. Furthermore, we show that although c2 EcoPI mutant MTases are defective in AdoMet binding, they are still able to bind DNA in a sequence-specific manner.

Characterization of RNA polymerase III transcription factor TFIIIC from the mulberry silkworm, Bombyx mori

Fractionation of nuclear extracts from posterior silk glands of mulberry silkworm Bombyx mori. resolved the transcription factor TFIIIC into two components (designated here as TFIIIC and TFIIIC1) as in HeLa cell nuclear extracts. The reconstituted transcription of tRNA genes required the presence of both components. The affinity purified TFIIIC is a heteromeric complex comprising of five subunits ranging from 44 to 240 kDa. Of these, the 51-kDa subunit could be specifically crosslinked to the B box of tRNA(1)(Gly). Purified swTFIIIC binds to the B box sequences with an affinity in the same range as of yTFIIIC or hTFIIIC2. Although an histone acetyl transferase (HAT) activity was associated with the TFIIIC fractions during the initial stages of purification. the HAT activity, unlike the human TFIIIC preparations, was separated at the final DNA affinity step. The tRNA transcription from DNA template was independent of HAT activity but the repressed transcription from chromatin template could be partially restored by external supplementation of the dissociated HAT activity. This is the first report on the purification and characterization of TFIIIC from insect systems.

Structure, function, and mechanism of HhaI DNA methyltransferases

A vast amount of literature has accumulated on the characterization of DNA methyltransferases. The HhaI DNA methyltransferase, a C5-cytosine methyltransferase, has been the subject of investigation for the last 2 decades. Biochemical and kinetic characterization have led to an understanding of the catalytic and kinetic mechanism of the methyltransfer reaction. The HhaI methyltransferase has also been subjected to extensive structural analysis, with the availability of 12 structures with or without a cofactor and a variety of DNA substrates. The mechanism of base flipping, first described for the HhaI methyltransferase, is conserved among all DNA methyltransferases and is also found to occur in numerous DNA repair enzymes. Studies with other methyltransferase reveal a significant structural and functional similarity among different types of methyltransferases. This review aims to summarize the available information on the HhaI DNA methyltransferase.

Structure and nucleotide specificity of Staphylococcus aureus dihydrodipicolinate reductase (DapB)

Lysine biosynthesis proceeds by the nucleotide-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) by dihydrodipicolinate reductase (DHDPR). The S. aureus DHDPR structure reveals different conformational states of this enzyme even in the absence of a substrate or nucleotide-cofactor. Despite lacking a conserved basic residue essential for NADPH interaction, S. aureus DHDPR differs from other homologues as NADPH is a more preferred co-factor than NADH. The structure provides a rationale-Lys35 compensates for the co-factor site mutation. These observations are significant for bi-ligand inhibitor design that relies on ligand-induced conformational changes as well as co-factor specificity for this important drug target. Structured summary of protein interactions: DHDPR binds to DHDPR by molecular sieving (View interaction). DHDPR binds to DHDPR by dynamic light scattering (View interaction). DHDPR binds to DHDPR by X-ray crystallography (View interaction). (C) 2011 Federation of European Biochemical Societies. Published by 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.

Molecular profile of oligodendrogliomas in young patients

Several studies on molecular profiling of oligodendrogliomas (OGs) in adults have shown a distinctive genetic pattern characterized by combined deletions of chromosome arms 1 p and 19q, O6-methylguanine-methyltransferase (MGMT) methylation, and isocitrate dehydrogenase 1 (IDH1) mutation, which have potential diagnostic, prognostic, and even therapeutic relevance. OGs in pediatric and young adult patients are rare and have been poorly characterized on a molecular and biological basis, and it remains uncertain whether markers with prognostic significance in adults also have predictive value in these patients. Fourteen cases of OGs in young patients (age, <= 25 years) who received a diagnosis over 7 years were selected (7 pediatric patients age <= 18 years and 7 young adults aged 19-25 years). The cases were evaluated for 1p/19q status, MGMT promoter methylation, p53 mutation, and IDH1 mutation. None of the pediatric cases showed 1p/19q deletion. In young adults, combined 1p/19q loss was observed in 57% and isolated 1p loss in 14% of cases. The majority of cases in both subgroups (71% in each) harbored MGMT gene promoter methylation. TP53 and IDH1 mutations were not seen in any of the cases in both the groups. To our knowledge, this is the first study to show that molecular profile of OGs in pediatric and young adult patients is distinct. Further large-scale studies are required to identify additional clinically relevant genetic alterations in this group of patients.

15-deoxyspergualin hinders physical interaction between basic residues of transit peptide in PfENR and Hsp70-1

The apicoplast of Plasmodium harbors several metabolic pathways. The enzymes required to perform these reactions are all nuclearly encoded and apicoplast targeted (NEAT) proteins. Plasmodium falciparum Enoyl-ACP Reductase (PfENR) is one such NEAT protein. The NEAT proteins have a transit peptide which is required for crossing the membranes of apicoplast. We studied the importance of basic residues like Arginine and Lysine within the transit peptide. Previous studies have suggested that all basic residues are essential for apicoplast trafficking. In this study, we demonstrate that only some of these residues are essential (K44, R48, K51, and R52), whereas others are dispensable (R40, K42, and K49). On mutating these specific residues, PfENR is not imported into the apicoplast and is mislocalized to the cytoplasm. We also demonstrate that these residues are also crucial for interaction with Hsp70-1, implying that interactions of Lysine 44, Arginine 48, Lysine 51, and Arginine 52 of the transit peptide with PfHsp70-1 are required for apicoplast trafficking. 15-Deoxyspergualin, which has earlier been proposed to interact with EEVD motif of PfHsp70-1 hinders the physical interaction between these cationic residues of PfENR and Hsp70-1. Hence, we propose that in the transport competent state of NEAT proteins some specific positively charged amino acids in the transit peptide interact with PfHsp70-1, and this interaction is essential for apicoplast targeting.

A Unique Modification of the Eukaryotic Initiation Factor 5A Shows the Presence of the Complete Hypusine Pathway in Leishmania donovani

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of an unusual amino acid hypusine (N-(sic)-(4-amino-2-hydroxybutyl) lysine), which is present on only one cellular protein, eukaryotic initiation factor 5A (eIF5A). We present here the molecular and structural basis of the function of DOHH from the protozoan parasite, Leishmania donovani, which causes visceral leishmaniasis. The L. donovani DOHH gene is 981 bp and encodes a putative polypeptide of 326 amino acids. DOHH is a HEAT-repeat protein with eight tandem repeats of alpha-helical pairs. Four conserved histidine-glutamate sequences have been identified that may act as metal coordination sites. A similar to 42 kDa recombinant protein with a His-tag was obtained by heterologous expression of DOHH in Escherichia coli. Purified recombinant DOHH effectively catalyzed the hydroxylation of the intermediate, eIF5A-deoxyhypusine (eIF5A-Dhp), in vitro. L. donovani DOHH (LdDOHH) showed similar to 40.6% sequence identity with its human homolog. The alignment of L. donovani DOHH with the human homolog shows that there are two significant insertions in the former, corresponding to the alignment positions 159-162 (four amino acid residues) and 174-183 (ten amino acid residues) which are present in the variable loop connecting the N- and C-terminal halves of the protein, the latter being present near the substrate binding site. Deletion of the ten-amino-acid-long insertion decreased LdDOHH activity to 14% of the wild type recombinant LdDOHH. Metal chelators like ciclopirox olamine (CPX) and mimosine significantly inhibited the growth of L. donovani and DOHH activity in vitro. These inhibitors were more effective against the parasite enzyme than the human enzyme. This report, for the first time, confirms the presence of a complete hypusine pathway in a kinetoplastid unlike eubacteria and archaea. The structural differences between the L. donovani DOHH and the human homolog may be exploited for structure based design of selective inhibitors against the parasite.

Biochemical characterization of a recombinant Swainsona canescens calcium-dependent protein kinase (ScCPK1)

Calcium-dependent protein kinases (CPKs) constitute a unique family of kinases involved in many physiological responses in plants. Biochemical and kinetic properties of a recombinant Swainsona canescens calcium-dependent protein kinase (ScCPK1) were examined in this study. The optimum pH and temperature for activity were pH 7.5 and 37 degrees C, respectively. Substrate phosphorylation activity of ScCPK1 was calmodulin (CaM) independent. Yet CaM antagonists, W7 N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and calmidazolium inhibited the activity with IC50 values of 750 nM and 350 pM, respectively. Both serine and threonine residues were found to be phosphorylated in auto-phosphorylated ScCPK1 and in histone III-S phosphorylated by ScCPK1. The Ca2+] for half maximal activity (K-0.5) was found to be 0.4 mu M for ScCPK1 with histone III-S as substrate. Kinetic analysis showed that Km of ScCPK1 for histone III-S was 4.8 mu M. These data suggest that ScCPK1 is a functional Ser/Thr kinase, regulated by calcium, and may have a role in Ca2+-mediated signaling in S. canescens. (C) 2012 Elsevier Masson SAS. All rights reserved.

MicroRNA-155 Is Required for Mycobacterium bovis BCG-Mediated Apoptosis of Macrophages

Pathogenic rnycobacteria, including Mycobacterium tuberculosis and Mycobacterium bovis, cause significant morbidity and mortality worldwide. However, the vaccine strain Mycobacterium bovis BCG, unlike virulent strains, triggers extensive apoptosis of infected macrophages, a step necessary for the elicitation of robust protective immunity. We here demonstrate that M. bovis BCG triggers Toll-like receptor 2 (TLR2)-dependent microRNA-155 (miR-155) expression, which involves signaling cross talk among phosphatidylinositol 3-kinase (PI3K), protein kinase C delta (PKC delta), and mitogen-activated protein kinases (MAPKs) and recruitment of NF-kappa B and c-ETS to miR-155 promoter. Genetic and signaling perturbations presented the evidence that miR-155 regulates PKA signaling by directly targeting a negative regulator of PKA, protein kinase inhibitor alpha (PKI-alpha). Enhanced activation of PKA signaling resulted in the generation of PKA C-alpha; phosphorylation of MSK1, cyclic AMP response element binding protein (CREB), and histone H3; and recruitment of phospho-CREB to the apoptotic gene promoters. The miR-155-triggered activation of caspase-3, BAK1, and cytochrome c translocation involved signaling integration of MAPKs and epigenetic or posttranslational modification of histones or CREB. Importantly, M. bovis BCG infection-induced apoptosis was severely compromised in macrophages derived from miR-155 knockout mice. Gain-of-function and loss-of-function studies validated the requirement of miR-155 for M. bovis BCG's ability to trigger apoptosis. Overall, M. bovis BCG-driven miR-155 dictates cell fate decisions of infected macrophages, strongly implicating a novel role for miR-155 in orchestrating cellular reprogramming during immune responses to mycobacterial infection.