Chlororespiratory reduction 6 is a novel factor required for accumulation of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis

The chloroplast NAD(P)H dehydrogenase (NDH) complex is involved in photosystem I cyclic electron transport and chlororespiration in higher plants. An Arabidopsis (Arabidopsis thaliana) chlororespiratory reduction 6 (crr6) mutant lacking NDH activity was identified by means of chlorophyll fluorescence imaging. Accumulation of the NDH complex was impaired in crr6. Physiological characterization of photosynthetic electron transport indicated the specific defect of the NDH complex in crr6. In contrast to the CRR7 protein that was recently identified as a potential novel subunit of the NDH complex by means of the same screening, the CRR6 protein was stable under the crr2 mutant background in which the NDH complex does not accumulate. The CRR6 gene (At2g47910) encodes a novel protein without any known motif. Although CRR6 does not have any transmembrane domains, it is localized in the thylakoid membrane fraction of the chloroplast. CRR6 is conserved in phototrophs, including cyanobacteria, from which the chloroplast NDH complex has evolutionally originated, but not in Chlamydomonas reinhardtii, in which the NDH complex is absent. We believe that CRR6 is a novel specific factor for the assembly or stabilization of the NDH complex.     

Effect of Carbon Source on Production of Lytic Enzymes by the Sclerotial Parasites Trichoderma atroviride and Coniothyrium minitans

Three isolates of Trichoderma atroviride and two isolates of Coniothyrium minitans known to efficiently degrade sclerotia of Sclerotinia sclerotiorum were cultured on minimal medium with sucrose, carboxymethyl cellulose (CMC), xylan, laminarin, colloidal chitin or powdered sclerotia as carbon source. The activity of endochitinase, endo‐β‐1,3‐glucanase, endoxylanase and endocellulase in culture filtrates was determined after 7 and 15 days of culture using dye‐labelled substrates. The strongest inducers of chitinase were colloidal chitin and sclerotia powder. Chitinase activity appeared to be faster induced in the isolates of T. atroviride than in the isolates of C. minitans, but the maximum level of activity present in culture filtrates of the two species was similar. With CMC and xylan as carbon source, concurrent production of the corresponding enzymes was observed for the Trichoderma isolates. The isolates of C. minitans produced cellulase on xylan but not on CMC, whereas xylanase was produced on both carbon sources. Laminarin induced the formation of glucanases in the three isolates of T. atroviride but not the isolates of C. minitans. However, in the sclerotia‐containing cultures of C. minitans glucanase activity was even higher than in the respective cultures of the Trichoderma isolates. During the 31‐day study period, the pattern of enzyme production in shake cultures containing sclerotia powder was very similar for the isolates of T. atroviride and C. minitans. Glucanase activity generally reached a peak 24 days after inoculation of the flasks, whereas the activity of chitinase, cellulase and xylanase remained fairly constant throughout the experiment.     

Structure guided design of a series of selective pyrrolopyrimidinone MARK inhibitors

The initial structure activity relationships around an isoindoline uHTS hit will be described. Information gleaned from ligand co-crystal structures allowed for rapid refinements in both MARK potency and kinase selectivity. These efforts allowed for the identification of a compound with properties suitable for use as an in vitro tool compound for validation studies on MARK as a viable target for Alzheimer’s disease.

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Essential residues for the enzyme activity of ATP-dependent MurE ligase from Mycobacterium tuberculosis

The emergence of total drug-resistant tuberculosis (TDR-TB) has made the discovery of new therapies for tuberculosis urgent. The cytoplasmic enzymes of peptidoglycan biosynthesis have generated renewed interest as attractive targets for the development of new antimycobacterials. One of the cytoplasmic enzymes, uridine diphosphate (UDP)-MurNAc-tripeptide ligase (MurE), catalyses the addition of meso-diaminopimelic acid (m-DAP) into peptidoglycan in Mycobacterium tuberculosis coupled to the hydrolysis of ATP. Mutants of M. tuberculosis MurE were generated by replacing K157, E220, D392, R451 with alanine and N449 with aspartate, and truncating the first 24 amino acid residues at the N-terminus of the enzyme. Analysis of the specific activity of these proteins suggested that apart from the 24 N-terminal residues, the other mutated residues are essential for catalysis. Variations in K_m values for one or more substrates were observed for all mutants, except the N-terminal truncation mutant, indicating that these residues are involved in binding substrates and form part of the active site structure. These mutant proteins were also tested for their specificity for a wide range of substrates. Interestingly, the mutations K157A, E220A and D392A showed hydrolysis of ATP uncoupled from catalysis. The ATP hydrolysis rate was enhanced by at least partial occupation of the uridine nucleotide dipeptide binding site. This study provides an insight into the residues essential for the catalytic activity and substrate binding of the ATP-dependent MurE ligase. Since ATP-dependent MurE ligase is a novel drug target, the understanding of its function may lead to development of novel inhibitors against resistant forms of M. tuberculosis.     

Characterization of the active site of ADP-ribosyl cyclase.

ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and that Glu(179) may indeed be the catalytic residue.     

Binding sites for gonadotropins in the ovary of immature and adult mice

Binding sites for gonadotropins in the mouse ovary were studied using the immunohistochemical technique. Binding sites for LH were localized in the interstitial cells and theca as well as granulosa cells of large follicles. Binding sites for FSH were demonstrated in the interstitial cells and granulosa cells of small follicles. In immature mice very few binding sites for both the gonadotropins were observed until day 21 of age. An intense staining reaction for peroxidase was observed in the ovary of 24-day old mouse. In adult mice, maximum number of binding sites for FSH were demonstrated in the ovary in estrus and metestrus stage of the cycle, respectively. A good correlation between the circulating levels of gonadotropins and binding sites for them in the ovary could be noted in the cycling but not in the prepubertal mice.     

Replication of Nuclei from Cycling and Quiescent Mammalian Cells in 6-DMAP-TreatedXenopusEgg Extract

Nuclear membrane permeabilization is required for replication of quiescent (G0) cell nuclei inXenopusegg extract. We now demonstrate that establishment of replication competence in G0 nuclei is dependent upon a positive activity present in the soluble egg extract. Our hypothesis is that G0 nuclei lose the license to replicate following growth arrest and that this positive activity is required for relicensing DNA for replication. To determine if G0 nuclei contain licensed DNA, we used the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), to prepare egg extracts that are devoid of licensing activity. Intact nuclei, isolated from mammalian cells synchronized in G1-phase (licensed), G2-phase (unlicensed), and G0 were permeabilized and assayed for replication in 6-DMAP-treated and untreated extracts supplemented with [α-³²P]dATP or biotinylated-dUTP. Very little radioactivity was incorporated into nascent DNA in each nuclear population; however, nearly all nuclei in each population incorporated biotin in 6-DMAP extract. The pattern of biotin incorporation within these nuclei was strikingly similar to the punctate pattern observed within nuclei incubated in aphidicolin-treated extract, suggesting that initiation events occur within most replication factories in 6-DMAP extract. However, density substitution and alkaline gel analyses indicate that the incorporated biotin within these nuclei arises from a small number of active origins which escape 6-DMAP inhibition. We conclude that 6-DMAP-treated egg extract cannot differentiate licensed from unlicensed mammalian somatic cell nuclei and, therefore, cannot be used to determine the “licensed state” of G0 nuclei using the assays described here.     

Evolution of the relaxin-like peptide family

Background  

The relaxin-like peptide family belongs in the insulin superfamily and consists of 7 peptides of high structural but low sequence similarity; relaxin-1, 2 and 3, and the insulin-like (INSL) peptides, INSL3, INSL4, INSL5 and INSL6. The functions of relaxin-3, INSL4, INSL5, INSL6 remain uncharacterised. The evolution of this family has been contentious; high sequence variability is seen between closely related species, while distantly related species show high similarity; an invertebrate relaxin sequence has been reported, while a relaxin gene has not been found in the avian and ruminant lineages.     

Biochemical and structural characterization of a novel class of inhibitors of the type 1 insulin-like growth factor and insulin receptor kinases

The type 1 insulin-like growth factor receptor (IGF-1R) is often overexpressed on tumor cells and is believed to play an important role in anchorage-independent proliferation. Additionally, cell culture studies have indicated that IGF-1R confers increased resistance to apoptosis caused by radiation or chemotherapeutic agents. Thus, inhibitors of the intracellular kinase domain of this receptor may have utility for the clinical treatment of cancer. As part of an effort to develop clinically useful inhibitors of IGF-1R kinase, a novel class of pyrrole-5-carboxaldehyde compounds was investigated. The compounds exhibited selectivity against the closely related insulin receptor kinase intrinsically and in cell-based assays. The inhibitors formed a reversible, covalent adduct at the kinase active site, and treatment of such adducts with sodium borohydride irreversibly inactivated the enzyme. Analysis of a tryptic digest of a covalently modified IGF-1R kinase fragment revealed that the active site Lys1003 had been reductively alkylated with the aldehyde inhibitor. Reductive alkylation of the insulin receptor kinase with one of these inhibitors led to a similarly inactivated enzyme which was examined by X-ray crystallography. The crystal structure confirmed the modification of the active site lysine side chain and revealed details of the key interactions between the inhibitor and enzyme.