Ceramide kinase targeting and activity determined by its N-terminal pleckstrin homology domain

The N-terminus of ceramide kinase (CERK) is thought to be myristoylated and to contain a pleckstrin homology (PH) domain. We found that deletion of this region (DeltaPH-CERK) ablates activity. This is not due to prevention of N-terminal myristoylation since a G2A CERK mutant, which cannot be myristoylated, was active. CERK was able to bind liposomes, as well as the isolated unmyristoylated PH domain; DeltaPH-CERK was not. Upon analysis of EGFP-tagged proteins, CERK was found associated with the Golgi complex. Osmotic cell swelling induced translocation of CERK to the plasma membrane and formation of large vesicles displaying bound CERK. None of these features occurred with DeltaPH-CERK, which remained disseminated throughout the cytoplasm. These findings show that the PH domain of CERK is essential for localization, translocation, and activity of this lipid kinase.     

Structural insights into the human and avian IMP cyclohydrolase mechanism via crystal structures with the bound XMP inhibitor

Within de novo purine biosynthesis, the AICAR transformylase and IMP cyclohydrolase activities of the bifunctional enzyme ATIC convert the intermediate AICAR to the final product of the pathway, IMP. Identification of the AICAR transformylase active site and a proposed formyl transfer mechanism have already resulted from analysis of crystal structures of avian ATIC in complex with substrate and/or inhibitors. Herein, we focus on the IMPCH active site and the cyclohydrolase mechanism through comparison of crystal structures of XMP inhibitor complexes of human ATIC at 1.9 A resolution with the previously determined avian enzyme. This first human ATIC structure was also determined to ascertain whether any subtle structural differences, compared to the homologous avian enzyme, should be taken into account for structure-based inhibitor design. These structural comparisons, as well as comparative analyses with other IMP and XMP binding proteins, have enabled a catalytic mechanism to be formulated. The primary role of the IMPCH active site appears to be to induce a reconfiguration of the substrate FAICAR to a less energetically favorable, but more reactive, conformer. Backbone (Arg64 and Lys66) and side chain interactions (Thr67) in the IMPCH active site reorient the 4-carboxamide from the preferred conformer that binds to the AICAR Tfase active site to one that promotes intramolecular cyclization. Other backbone amides (Ile126 and Gly127) create an oxyanion hole that helps orient the formyl group for nucleophilic attack by the 4-carboxamide amine and then stabilize the anionic intermediate. Several other residues, including Lys66, Tyr104, Asp125, and Lys137', provide substrate specificity and likely enhance the catalytic rate through contributions to acid-base catalysis.     

Grass pollen immunotherapy induces mucosal and peripheral IL-10 responses and blocking IgG activity

T regulatory cells and IL-10 have been implicated in the mechanism of immunotherapy in patients with systemic anaphylaxis following bee stings. We studied the role of IL-10 in the induction of clinical, cellular, and humoral tolerance during immunotherapy for local mucosal allergy in subjects with seasonal pollinosis. Local and systemic IL-10 responses and serum Ab concentrations were measured before/after a double-blind trial of grass pollen (Phleum pratense, Phl P) immunotherapy. We observed local increases in IL-10 mRNA-positive cells in the nasal mucosa after 2 years of immunotherapy, but only during the pollen season. IL-10 protein-positive cells were also increased and correlated with IL-10 mRNA(+) cells. These changes were not observed in placebo-treated subjects or in healthy controls. Fifteen and 35% of IL-10 mRNA signals were colocalized to CD3(+) T cells and CD68(+) macrophages, respectively, whereas only 1-2% of total CD3(+) cells and 4% of macrophages expressed IL-10. Following immunotherapy, peripheral T cells cultured in the presence of grass pollen extract also produced IL-10. Immunotherapy resulted in blunting of seasonal increases in serum allergen Phl p 5-specific IgE, 60- to 80-fold increases in Phl p 5-specific IgG, and 100-fold increases in Phl p 5-specific IgG4. Post-immunotherapy serum exhibited inhibitory activity, which coeluted with IgG4, and blocked IgE-facilitated binding of allergen-IgE complexes to B cells. Both the increases in IgG and the IgG "blocking" activity correlated with the patients' overall assessment of improvement. Thus, grass pollen immunotherapy may induce allergen-specific, IL-10-dependent "protective" IgG4 responses.     

Nek2B stimulates zygotic centrosome assembly in Xenopus laevis in a kinase-independent manner

Pronuclear migration and formation of the first mitotic spindle depend upon assembly of a functional zygotic centrosome. For most animals, this involves both paternal and maternal contributions as sperm basal bodies are converted into centrosomes competent for microtubule nucleation through recruitment of egg proteins. Nek2B is a vertebrate NIMA-related protein kinase required for centrosome assembly, as its depletion from egg extracts delays microtubule aster formation from sperm basal bodies. Using Xenopus as a model system, we now show that protein expression of Nek2B begins during mid-oogenesis and increases further upon oocyte maturation. This is regulated, at least in part, at the level of protein translation. Nek2B protein is weakly phosphorylated in mitotic egg extracts but its recruitment to the sperm basal body, which occurs independently of its kinase activity, stimulates its phosphorylation, possibly through sequestration from a phosphatase present in mitotic egg cytoplasm. Importantly, although Nek2B is not required to organize acentrosomal microtubule asters, we show that addition of either active or kinase-dead recombinant Nek2B can restore centrosome assembly in a dose-dependent manner to a depleted extract. These results support a model in which maternal Nek2B acts to promote assembly of a functional zygotic centrosome in a kinase-independent manner.     

Characterization of glycerol trinitrate reductase (NerA) and the catalytic role of active-site residues

Glycerol trinitrate reductase (NerA) from Agrobacterium radiobacter, a member of the old yellow enzyme (OYE) family of oxidoreductases, was expressed in and purified from Escherichia coli. Denaturation of pure enzyme liberated flavin mononucleotide (FMN), and spectra of NerA during reduction and reoxidation confirmed its catalytic involvement. Binding of FMN to apoenzyme to form the holoenzyme occurred with a dissociation constant of ca. 10(-7) M and with restoration of activity. The NerA-dependent reduction of glycerol trinitrate (GTN; nitroglycerin) by NADH followed ping-pong kinetics. A structural model of NerA based on the known coordinates of OYE showed that His-178, Asn-181, and Tyr-183 were close to FMN in the active site. The NerA mutation H178A produced mutant protein with bound FMN but no activity toward GTN. The N181A mutation produced protein that did not bind FMN and was isolated in partly degraded form. The mutation Y183F produced active protein with the same k(cat) as that of wild-type enzyme but with altered K(m) values for GTN and NADH, indicating a role for this residue in substrate binding. Correlation of the ratio of K(m)(GTN) to K(m)(NAD(P)H), with sequence differences for NerA and several other members of the OYE family of oxidoreductases that reduce GTN, indicated that Asn-181 and a second Asn-238 that lies close to Tyr-183 in the NerA model structure may influence substrate specificity.     

A positive role for the Ku complex in DNA replication following strand break damage in mammals

Ku70-Ku80 complex is the regulatory subunit of DNA-dependent protein kinase (DNA-PK) and plays an essential role in double-strand break repair following ionizing radiation (IR). It preferentially interacts with chromosomal breaks and protects DNA ends from nuclease attack. Here we show evidence that cells defective in Ku80 exhibit a significantly slow S phase progression following DNA damage. IR-induced retardation in S phase progression in Ku80-/- cells was not due to the lack of DNA-PK kinase activity because both wild-type cells and DNA-PKcs-deficient cells showed no such symptom. Instead, proliferating cell nuclear antigen (PCNA) dissociated from chromosomes following IR in Ku80-deficient cells but not in wild-type or DNA-PKcs-deficient cells. Treatment of HeLa cells with IR induced colocalization of the Ku complex with PCNA on chromosomes. Together, these results suggest that binding of the Ku complex at chromosomal breaks may be necessary to maintain the sliding clamps (PCNA) on chromatin, which would allow cells to resume DNA replication without a major delay following IR.     

The mechanisms by which family 10 glycoside hydrolases bind decorated substrates

Endo-beta-1,4-xylanases (xylanases), which cleave beta-1,4 glycosidic bonds in the xylan backbone, are important components of the repertoire of enzymes that catalyze plant cell wall degradation. The mechanism by which these enzymes are able to hydrolyze a range of decorated xylans remains unclear. Here we reveal the three-dimensional structure, determined by x-ray crystallography, and the catalytic properties of the Cellvibrio mixtus enzyme Xyn10B (CmXyn10B), the most active GH10 xylanase described to date. The crystal structure of the enzyme in complex with xylopentaose reveals that at the +1 subsite the xylose moiety is sandwiched between hydrophobic residues, which is likely to mediate tighter binding than in other GH10 xylanases. The crystal structure of the xylanase in complex with a range of decorated xylooligosaccharides reveals how this enzyme is able to hydrolyze substituted xylan. Solvent exposure of the O-2 groups of xylose at the +4, +3, +1, and -3 subsites may allow accommodation of the alpha-1,2-linked 4-O-methyl-d-glucuronic acid side chain in glucuronoxylan at these locations. Furthermore, the uronic acid makes hydrogen bonds and hydrophobic interactions with the enzyme at the +1 subsite, indicating that the sugar decorations in glucuronoxylan are targeted to this proximal aglycone binding site. Accommodation of 3'-linked l-arabinofuranoside decorations is observed in the -2 subsite and could, most likely, be tolerated when bound to xylosides in -3 and +4. A notable feature of the binding mode of decorated substrates is the way in which the subsite specificities are tailored both to prevent the formation of "dead-end" reaction products and to facilitate synergy with the xylan degradation-accessory enzymes such as alpha-glucuronidase. The data described in this report and in the accompanying paper indicate that the complementarity in the binding of decorated substrates between the glycone and aglycone regions appears to be a conserved feature of GH10 xylanases.     

Partitioning of the serotonin transporter into lipid microdomains modulates transport of serotonin

The serotonin transporter (SERT) is an integral membrane protein responsible for the clearance of serotonin from the synaptic cleft following the release of the neurotransmitter. SERT plays a prominent role in the regulation of serotoninergic neurotransmission and is a molecular target for multiple antidepressants as well as substances of abuse. Here we show that SERT associates with lipid rafts in both heterologous expression systems and rat brain and that the inclusion of the transporter into lipid microdomains is critical for serotonin uptake activity. SERT is present in a subpopulation of lipid rafts, which is soluble in Triton X-100 but insoluble in other non-ionic detergents such as Brij 58. Disaggregation of lipid rafts upon depletion of cellular cholesterol results in a decrease of serotonin transport capacity (V(max)), due to the reduction of turnover number of serotonin transport. Our data suggest that the association of SERT with lipid rafts may represent a mechanism for regulating the transporter activity and, consequently, serotoninergic signaling in the central nervous system, through the modulation of the cholesterol content in the cell membrane. Furthermore, SERT-containing rafts are detected in both intracellular and cell surface fractions, suggesting that raft association may be important for trafficking and targeting of SERT.     

A role for the Fanconi anemia C protein in maintaining the DNA damage-induced G2 checkpoint

Fanconi anemia (FA) is a complex, heterogeneous genetic disorder composed of at least 11 complementation groups. The FA proteins have recently been found to functionally interact with the cell cycle regulatory proteins ATM and BRCA1; however, the function of the FA proteins in cell cycle control remains incompletely understood. Here we show that the Fanconi anemia complementation group C protein (Fancc) is necessary for proper function of the DNA damage-induced G2/M checkpoint in vitro and in vivo. Despite apparently normal induction of the G2/M checkpoint after ionizing radiation, murine and human cells lacking functional FANCC did not maintain the G2 checkpoint as compared with wild-type cells. The increased rate of mitotic entry seen in Fancc-/-mouse embryo fibroblasts correlated with decreased inhibitory phosphorylation of cdc2 kinase on tyrosine 15. An increased inability to maintain the DNA damage-induced G2 checkpoint was observed in Fancc -/-; Trp53 -/-cells compared with Fancc -/-cells, indicating that Fancc and p53 cooperated to maintain the G2 checkpoint. In contrast, genetic disruption of both Fancc and Atm did not cooperate in the G2 checkpoint. These data indicate that Fancc and p53 in separate pathways converge to regulate the G2 checkpoint. Finally, fibroblasts lacking FANCD2 were found to have a G2 checkpoint phenotype similar to FANCC-deficient cells, indicating that FANCD2, which is activated by the FA complex, was also required to maintain the G2 checkpoint. Because a proper checkpoint function is critical for the maintenance of genomic stability and is intricately related to the function and integrity of the DNA repair process, these data have implications in understanding both the function of FA proteins and the mechanism of genomic instability in FA.     

Effect of trophic status on the culturability and activity of bacteria from a range of lakes in the English Lake District

The bacterioplankton from a number of lakes that differed in nutrient status in the English Lake District was examined with a number of techniques for enumeration and activity assessment. Natural water samples showed a clear correlation between total counts and trophic status. Esterase activity measurements with Chemchrome B were able to distinguish high- and low-nutrient-status lakes, whereas tetrazolium salt (5-cyano-2,3-ditoyltetrazolium chloride) reduction, the direct viable count-cell elongation assay, and culturability measurements could not. Tetrazolium salt reduction and esterase activity measurements labeled a significant number of cells from water of all nutrient levels, whereas the direct viable count-cell elongation method was of use only in oligotrophic waters. Size fractionation of samples showed that the culturable cells were retained by the larger filters, especially in nutrient-rich waters. Esterase activity measurements also favored the larger cells. The differences observed between assays using water that differed in trophic status raise questions about the use of these tests as a definitive measure of viability.