Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia

T-protein, a component of the glycine cleavage system, catalyzes the formation of ammonia and 5,10-methylenetetrahydrofolate from the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein. Several mutations in the human T-protein gene cause non-ketotic hyperglycinemia. To gain insights into the effect of disease-causing mutations and the catalytic mechanism at the molecular level, crystal structures of human T-protein in free form and that bound to 5-methyltetrahydrofolate (5-CH3-H4folate) have been determined at 2.0 A and 2.6 A resolution, respectively. The overall structure consists of three domains arranged in a cloverleaf-like structure with the central cavity, where 5-CH3-H4folate is bound in a kinked shape with the pteridine group deeply buried into the hydrophobic pocket and the glutamyl group pointed to the C-terminal side surface. Most of the disease-related residues cluster around the cavity, forming extensive hydrogen bonding networks. These hydrogen bonding networks are employed in holding not only the folate-binding space but also the positions and the orientations of alpha-helix G and the following loop in the middle region, which seems to play a pivotal role in the T-protein catalysis. Structural and mutational analyses demonstrated that Arg292 interacts through water molecules with the folate polyglutamate tail, and that the invariant Asp101, located close to the N10 group of 5-CH3-H4folate, might play a key role in the initiation of the catalysis by increasing the nucleophilic character of the N10 atom of the folate substrate for the nucleophilic attack on the aminomethyl lipoate intermediate. A clever mechanism of recruiting the aminomethyl lipoate arm to the reaction site seems to function as a way of avoiding the release of toxic formaldehyde.     

Down-regulation of SMT3A gene expression in association with DNA synthesis induction after X-ray irradiation in nevoid basal cell carcinoma syndrome (NBCCS) cells

Fibroblast cells derived from nevoid basal carcinoma syndrome (NBCCS) patients show increased levels of DNA synthesis after X-ray irradiation. Genes, whose expression is modulated in association with the DNA synthesis induction, were searched by using PCR-based mRNA differential display analysis in one of the NBCCS cell lines, NBCCS1 cells. Decreased levels of SMT3A gene expression were found in X-ray-irradiated NBCCS1 cells. This decrease was also shown by RT-PCR analysis in another cell line, NBCCS3 cells. In addition to NBCCS cells, normal fibroblast cells showed the DNA synthesis induction after X-ray irradiation when they were treated with antisense oligonucleotides (AO) for SMT3A. However, treatment of normal fibroblasts with the random oligonucleotides (RO) resulted in decreased levels of DNA synthesis after X-ray irradiation. Thus, down-regulation of SMT3A gene expression may be involved in the DNA synthesis induction after X-ray irradiation in the NBCCS cells at least tested.     

Measurement of homocysteine thiolactone hydrolase activity using high-performance liquid chromatography with fluorescence detection and polymorphisms of paraoxonase in normal human serum

We developed a non-radioactive and sensitive assay method for measurement of the HTL hydrolase (HTLase) activity in biological samples, using OPA as a fluorescent post-labeling agent, l-homocysteine thiolactone (L-HTL) as the substrate, and HPLC to achieve rapid and selective separation of the substrate and product. The method was applied to measure the activity of HTLase in human, rabbit, rat and mouse serum samples. In addition, the correlation between the serum HTLase activity and PON1 polymorphisms in Japanese subjects was also investigated. The serum HTLase activity in humans, as determined by measurement of the enzyme activity in 22 subjects, was found to be in the range of 0.89-2.06 nmol/min mg protein, with a mean activity of 1.44 nmol/min mg protein.     

A specific microdomain ("glycosynapse 3") controls phenotypic conversion and reversion of bladder cancer cells through GM3-mediated interaction of alpha3beta1 integrin with CD9

Cell motility is highly dependent on the organization and function of microdomains composed of integrin, proteolipid/tetraspanin CD9, and ganglioside (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry 40, 6414-6421; Kawakami, Y., Kawakami, K., Steelant, W. F. A., Ono, M., Baek, R. C., Handa, K., Withers, D. A., and Hakomori, S. (2002) J. Biol. Chem. 277, 34349-34358), later termed "glycosynapse 3" (Hakomori, S., and Handa, K. (2002) FEBS Lett. 531, 88-92, 2002). Human bladder cancer cell lines KK47 (noninvasive and nonmetastatic) and YTS1 (highly invasive and metastatic), both derived from transitional bladder epithelia, are very similar in terms of integrin composition and levels of tetraspanin CD9. Tetraspanin CD82 is absent in both. The major difference is in the level of ganglioside GM3, which is several times higher in KK47 than in YTS1. We now report that the GM3 level reflects glycosynapse function as follows: (i) a stronger interaction of integrin alpha3 with CD9 in KK47 than in YTS1; (ii) conversion of benign, low motility KK47 to invasive, high motility cells by depletion of GM3 by P4 (D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol) treatment or by knockdown of CD9 by the RNA interference method; (iii) reversion of high motility YTS1 to low motility phenotype like that of KK47 by exogenous GM3 addition, whereby the alpha3-to-CD9 interaction was enhanced; (iv) low GM3 level activated c-Src in YTS1 or in P4-treated KK47, and high GM3 level by exogenous addition caused Csk translocation into glycosynapse, with subsequent inhibition of c-Src activation; (v) inhibition of c-Src by "PP2" in YTS1 greatly reduced cell motility. Thus, GM3 in glycosynapse 3 plays a dual role in defining glycosynapse 3 function. One is by modulating the interaction of alpha3 with CD9; the other is by activating or inhibiting the c-Src activity, possibly through Csk translocation. High GM3 level decreases tumor cell motility/invasiveness, whereas low GM3 level enhances tumor cell motility/invasiveness. Oncogenic transformation and its reversion can be explained through the difference in glycosynapse organization.     

Effects of free proline accumulation in petunias under drought stress

Petunias (Petunia hybrida cv. 'Mitchell') accumulate free proline (Pro) under drought-stress conditions. It is therefore believed that Pro acts as an osmoprotectant in plants subjected to drought conditions. Petunia plants were transformed by Delta(1)-pyrroline-5-carboxylate synthetase genes (AtP5CS from Arabidopsis thaliana L. or OsP5CS from Oryza sativa L.). The transgenic plants accumulated Pro and their drought tolerance was tested. The Pro content amounted to 0.57-1.01% of the total amino acids in the transgenic plants, or 1.5-2.6 times that in wild-type plants grown under normal conditions. The transgenic plant lines tolerated 14 d of drought stress, which confirms that both P5CS transgenes had full functionality. Exogenous L-Pro treatment caused the plants to accumulate Pro; plants treated with 5 mM L-Pro accumulated up to 18 times more free Pro than untreated plants. Exogenous L-Pro restricted the growth of wild-type petunias more than that of Arabidopsis plants. The capacity for free Pro accumulation might depend on the plant species. The growth of petunia plants was influenced not only by the Pro concentration in the plants, but by the ratio of the Pro content to the total amino acids, because the growth of the transgenic petunia plants appeared normal.     

Heat stable ssDNA/RNA-binding activity of a wheat cold shock domain protein

The cold-induced wheat WCSP1 protein belongs to the cold shock domain (CSD) protein family. In prokaryotes and eukaryotes, the CSD functions as a nucleic acid-binding domain. Here, we demonstrated that purified recombinant WCSP1 is boiling soluble and binds ss/dsDNA and mRNA. Furthermore, boiled-WCSP1 retained its characteristic nucleic acid-binding activity. A WCSP1 deletion mutant, containing only a CSD, lost ssDNA/RNA-binding activity; while a mutant containing the CSD and the first glycine-rich region (GR) displayed the activity. These data indicated that the first GR of WCSP1 is necessary for the binding activity but is not for the heat stability of the protein.     

Arabidopsis stress-inducible gene for arginine decarboxylase AtADC2 is required for accumulation of putrescine in salt tolerance

Arginine decarboxylase (ADC) catalyzes the first step of polyamine (PA) biosynthesis to produce putrescine (Put) from arginine (Arg). One of the 2 Arabidopsis ADC genes, AtADC2, is induced in response to salt stress causing the accumulation of free Put. To analyze the roles of stress-inducible AtADC2 gene and endogenous Put in stress tolerance, we isolated a Ds insertion mutant of AtADC2 gene (adc2-1) and characterized its phenotypes under salt stress. In the adc2-1 mutant, free Put content was reduced to about 25% of that in the control plants and did not increase under salt stress. Furthermore, the adc2-1 mutant was more sensitive to salt stress than the control plants. The stress sensitivity of adc2-1 was recovered by the addition of exogenous Put. These results indicate that endogenous Put plays an important role in salt tolerance in Arabidopsis. AtADC2 is a key gene for the production of Put under not only salinity conditions, but also normal conditions.     

Expression of mouse Coiled-coil-DIX1 (Ccd1), a positive regulator of Wnt signaling, during embryonic development

Wnt signaling plays an important role in cell growth, differentiation, polarity formation, and neural development. We have recently identified the Coiled-coil-DIX1 (Ccd1) gene encoding a third type of a DIX domain-containing protein. Ccd1 forms homomeric and heteromeric complexes with Dishevelled and Axin, and positively regulates the Wnt/beta-catenin pathway. Here, we examined the spatiotemporal expression pattern of Ccd1 mRNA in mouse embryos from embryonic day 6.5 (E6.5) to E17.5 by in situ hybridization. Ccd1 expression was detected in the node region in gastrula embryos, in the cephalic mesenchyme and tail bud at E8.5, and in the branchial arch and forelimb bud at E9.5. In the central nervous system, Ccd1 expression began and persisted in the regions where the neurons differentiated, so that it was observed throughout the brain and spinal cord at E17.5. Ccd1 expression was also strong in the peripheral nervous system, including sensory cranial ganglia (trigeminal, facial, and vestibulocochlear ganglia), dorsal root ganglia, and autonomic ganglia (sympathetic ganglia, celiac ganglion, and hypogastric plexus). Ccd1 was detected in the sensory organs, such as the inner nuclear layer of the neural retina, saccule and cochlea of the inner ear, and nasal epithelium. Outside the nervous system, Ccd1 mRNA was observed in the cartilage, tongue, lung bud, stomach, and gonad at E12.5-E14.5, and in the tooth bud, bronchial epithelium, and kidney at E17.5. Taken together, these findings demonstrate that Ccd1 expression is observed in all the neurons in the nervous system, closely associated with neural crest-derived tissues, and largely overlapping with the regions where several Wnt genes are reported to play a role.     

Two Golgi-resident 3′-Phosphoadenosine 5′-Phosphosulfate Transporters Play Distinct Roles in Heparan Sulfate Modifications and Embryonic and Larval Development in Caenorhabditis elegans

Synthesis of extracellular sulfated molecules requires active 3′-phosphoadenosine 5′-phosphosulfate (PAPS). For sulfation to occur, PAPS must pass through the Golgi membrane, which is facilitated by Golgi-resident PAPS transporters. Caenorhabditis elegans PAPS transporters are encoded by two genes, pst-1 and pst-2. Using the yeast heterologous expression system, we characterized PST-1 and PST-2 as PAPS transporters. We created deletion mutants to study the importance of PAPS transporter activity. The pst-1 deletion mutant exhibited defects in cuticle formation, post-embryonic seam cell development, vulval morphogenesis, cell migration, and embryogenesis. The pst-2 mutant exhibited a wild-type phenotype. The defects observed in the pst-1 mutant could be rescued by transgenic expression of pst-1 and hPAPST1 but not pst-2 or hPAPST2. Moreover, the phenotype of a pst-1;pst-2 double mutant were similar to those of the pst-1 single mutant, except that larval cuticle formation was more severely defected. Disaccharide analysis revealed that heparan sulfate from these mutants was undersulfated. Gene expression reporter analysis revealed that these PAPS transporters exhibited different tissue distributions and subcellular localizations. These data suggest that pst-1 and pst-2 play different physiological roles in heparan sulfate modification and development.     

Overproduction of the Membrane-bound Receptor-like Protein Kinase 1, RPK1, Enhances Abiotic Stress Tolerance in Arabidopsis

RPK1 (receptor-like protein kinase 1) localizes to the plasma membrane and functions as a regulator of abscisic acid (ABA) signaling in Arabidopsis. In our current study, we investigated the effect of RPK1 disruption and overproduction upon plant responses to drought stress. Transgenic Arabidopsis overexpressing the RPK1 protein showed increased ABA sensitivity in their root growth and stomatal closure and also displayed less transpirational water loss. In contrast, a mutant lacking RPK1 function, rpk1-1, was found to be resistant to ABA during these processes and showed increased water loss. RPK1 overproduction in these transgenic plants thus increased their tolerance to drought stress. We performed microarray analysis of RPK1 transgenic plants and observed enhanced expression of several stress-responsive genes, such as Cor15a, Cor15b, and rd29A, in addition to H₂O₂-responsive genes. Consistently, the expression levels of ABA/stress-responsive genes in rpk1-1 had decreased compared with wild type. The results suggest that the overproduction of RPK1 enhances both the ABA and drought stress signaling pathways. Furthermore, the leaves of the rpk1-1 plants exhibit higher sensitivity to oxidative stress upon ABA-pretreatment, whereas transgenic plants overproducing RPK1 manifest increased tolerance to this stress. Our current data suggest therefore that RPK1 overproduction controls reactive oxygen species homeostasis and enhances both water and oxidative stress tolerance in Arabidopsis.