SKPDB: a structural database of shikimate pathway enzymes

Background  

The functional and structural characterisation of enzymes that belong to microbial metabolic pathways is very important for structure-based drug design. The main interest in studying shikimate pathway enzymes involves the fact that they are essential for bacteria but do not occur in humans, making them selective targets for design of drugs that do not directly impact humans.     

Formation of the ascidian epidermal sensory neurons: insights into the origin of the chordate peripheral nervous system

The vertebrate peripheral nervous system (PNS) originates from neural crest and placodes. While its developmental origin is the object of intense studies, little is known concerning its evolutionary history. To address this question, we analyzed the formation of the larval tail PNS in the ascidian Ciona intestinalis. The tail PNS of Ciona is made of sensory neurons located within the epidermis midlines and extending processes in the overlying tunic median fin. We show that each midline corresponds to a single longitudinal row of epidermal cells and neurons sharing common progenitors. This simple organization is observed throughout the tail epidermis, which is made of only eight single-cell rows, each expressing a specific genetic program. We next demonstrate that the epidermal neurons are specified in two consecutive steps. During cleavage and gastrula stages, the dorsal and ventral midlines are independently induced by FGF9/16/20 and the BMP ligand ADMP, respectively. Subsequently, Delta/Notch–mediated lateral inhibition controls the number of neurons formed within these neurogenic regions. These results provide a comprehensive overview of PNS formation in ascidian and uncover surprising similarities between the fate maps and embryological mechanisms underlying formation of ascidian neurogenic epidermis midlines and the vertebrate median fin.     

Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease

Sequence-specific endonucleases recognizing long target sequences are emerging as powerful tools for genome engineering. These endonucleases could be used to correct deleterious mutations or to inactivate viruses, in a new approach to molecular medicine. However, such applications are highly demanding in terms of safety. Mutations in the human RAG1 gene cause severe combined immunodeficiency (SCID). Using the I-CreI dimeric LAGLIDADG meganuclease as a scaffold, we describe here the engineering of a series of endonucleases cleaving the human RAG1 gene, including obligate heterodimers and single-chain molecules. We show that a novel single-chain design, in which two different monomers are linked to form a single molecule, can induce high levels of recombination while safeguarding more effectively against potential genotoxicity. We provide here the first demonstration that an engineered meganuclease can induce targeted recombination at an endogenous locus in up to 6% of transfected human cells. These properties rank this new generation of endonucleases among the best molecular scissors available for genome surgery strategies, potentially avoiding the deleterious effects of previous gene therapy approaches.     

Positive Allosteric Interaction of Structurally Diverse T-Type Calcium Channel Antagonists

Low-voltage-activated (T-type) calcium channels play a role in diverse physiological responses including neuronal burst firing, hormone secretion, and cell growth. To better understand the biological role and therapeutic potential of the target, a number of structurally diverse antagonists have been identified. Multiple drug interaction sites have been identified for L-type calcium channels, suggesting a similar possibility exists for the structurally related T-type channels. Here, we radiolabel a novel amide T-type calcium channel antagonist (TTA-A1) and show that several known antagonists, including mibefradil, flunarizine, and pimozide, displace binding in a concentration-dependent manner. Further, we identify a novel quinazolinone T-type antagonist (TTA-Q4) that enhanced amide radioligand binding, increased affinity in a saturable manner and slowed dissociation. Functional evaluation showed these compounds to be state-dependent antagonists which show a positive allosteric interaction. Consistent with slowing dissociation, the duration of efficacy was prolonged when compounds were co-administered to WAG/Rij rats, a genetic model of absence epilepsy. The development of a T-type calcium channel radioligand has been used to demonstrate structurally distinct TTAs interact at allosteric sites and to confirm the potential for synergistic inhibition of T-type calcium channels with structurally diverse antagonists.     

UV-induced downregulation of the CDC25B protein in human cells

The CDC25B phosphatase regulates the activation of CDK1-Cyclin B at the onset of mitosis, being a key target of the checkpoint pathways activated by cellular stress and DNA damage. Previous work has reported that checkpoint activation induces the sequestration of CDC25B in the cytoplasm. Here we show that in response to UV irradiation, the levels of CDC25B protein can be downregulated independently of classical checkpoints pathways such as p53, ATM/ATR and p38 MAPK. We also show that translational repression mediated by eIF2α phosphorylation regulates CDC25B expression levels. Taken together, our results illustrate a new mechanism of CDC25B regulation in response to stress.     

Biochemical characterization of glutaredoxins from Chlamydomonas reinhardtii: Kinetics and specificity in deglutathionylation reactions

Protein deglutathionylation is mainly catalyzed by glutaredoxins (GRXs). We have analyzed the biochemical properties of four of the six different GRXs of Chlamydomonas reinhardtii. Kinetic parameters were determined for disulfide and dehydroascorbate reduction but also for deglutathionylation of artificial and protein substrates. The results indicate that GRXs exhibit striking differences in their catalytic properties, mainly linked to the class of GRX considered but also to the pK_a of the N-terminal catalytic cysteine. Furthermore, glutathionylated proteins were found to exhibit distinct reactivities with GRXs. These results suggest that glutathionylation may allow a fine tuning of cell metabolism under stress conditions.

Structured summary

MINT-7761120: GRX6 (uniprotkb:A8HN52) and GRX6 (uniprotkb:A8HN52) bind (MI:0408) by comigration in non denaturing gel electrophoresis (MI:0404)MINT-7761098:GRX5 (uniprotkb:A8I7Q4) and GRX5 (uniprotkb:A8I7Q4) bind (MI:0408) by comigration in non denaturing gel electrophoresis (MI:0404)     

Overexpression and purification of the galactose operon enzymes from Escherichia coli

A convenient new procedure for the purification of galactokinase, galactose-1-phosphate uridyltransferase, and UDP-galactose 4-epimerase overexpressed in Escherichia coli is presented. The procedure is shorter than any other described in the literature and facilitates the purification of the three recombinant enzymes in considerable amounts and at high purity and specific activity. The purified gal operon enzymes were biochemically characterized by gel-filtration column chromatography and isoelectric focusing, and the Km values for their substrates were determined.     

Bombesin-like immunoreactivity in bovine adrenal medulla

The presence of immunoreactive (ir)-bombesin in bovine adrenal medulla, isolated adrenal chromaffin cells and subcellular fractions of the adrenal medulla was demonstrated using a specific antibody to the synthetic peptide. High levels of ir-bombesin were detected in acid (HCl) extracts of the adrenal tissue (27 pmol/g) and isolated cells (0.35 pmol per 10(6) cells). Subpopulations of adrenal chromaffin cells were also obtained by centrifugation of the original cell preparation through a stepwise bovine serum albumin gradient (cell layers I, II and III). The highest concentration of ir-bombesin (0.77 pmol/10(6) cells) was found in a cell population (cell layer I) enriched in noradrenaline (adrenaline/noradrenaline ratio of 0.6). At the subcellular level, ir-bombesin was mainly concentrated in the secretory granules (0.61 pmol/mg protein) along with catecholamines (1097 nmol/mg protein), but a relatively high concentration of ir-bombesin (0.26 pmol/mg protein) was also found in the microsomal fraction. Isolation and high performance liquid chromatography (HPLC) analysis of adrenomedullary ir-bombesin revealed the presence of four molecular forms, one of them corresponding to gastrin releasing peptide (GRP), another one (major peak) eluting closely to synthetic neuromedin B and another one coeluting with GRP-(18-27). HPLC analysis of the molecular forms of ir-bombesin in the microsomes and secretory granules indicated that GRP- and neuromedin B-like materials can be generated between the two fractions.