Development of an enzyme-linked immunosorbent assay for measurement of activity of myristoyl-coenzyme A:protein N-myristoyltransferase

Myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal glycine residue of various proteins. To develop a high-throughput assay for NMT, the principle of enzyme-linked immunosorbent assay (ELISA) is used, in which anti-N-myristoylglycine (anti-N-Myr-Gly) monoclonal antibody is utilized for the detection of the N-myristoylglycine moiety of the product of NMT catalysis. Enzyme-catalyzed reaction was performed using recombinant NMT expressed in Escherichia coli, myristoyl-CoA, and an octapeptide substrate that is biotinylated at its C terminus. The mixture of the products of the reaction was added to immunoplate wells precoated with anti-N-Myr-Gly monoclonal antibody. Then, the N-myristoyl-biotinylated octapeptide product was specifically captured by the antibody and stained with streptavidin-biotinylated peroxidase and tetramethylbenzidine substrate. This was followed by absorbance measurement (lambda(450)-lambda(630)). In this ELISA, the calibration curve showed a strong correlation between the concentration of the synthetic N-myristoyl-biotinylated octapeptide and the absorbance, indicating that this system may be useful for enzyme kinetics studies. Using this ELISA system, we assayed for serinal derivatives to determine their NMT inhibitory activity and found that serinal bisulfite inhibits yeast NMT activity. This is the first report of the measurement of NMT activity by the ELISA system.     

Two-state conformational changes in inositol 1,4,5-trisphosphate receptor regulated by calcium

Inositol 1,4,5-trisphosphate receptor (IP3R) is a highly controlled calcium (Ca2+) channel gated by inositol 1,4,5-trisphosphate (IP3). Multiple regulators modulate IP3-triggered pore opening by binding to discrete allosteric sites within IP3R. Accordingly we have postulated that these regulators structurally control ligand gating behavior; however, no structural evidence has been available. Here we show that Ca2+, the most pivotal regulator, induced marked structural changes in the tetrameric IP3R purified from mouse cerebella. Electron microscopy of the IP3R particles revealed two distinct structures with 4-fold symmetry: a windmill structure and a square structure. Ca2+ reversibly promoted a transition from the square to the windmill with relocations of four peripheral IP3-binding domains, assigned by binding to heparin-gold. Ca2+-dependent susceptibilities to limited digestion strongly support the notion that these alterations exist. Thus, Ca2+ appeared to regulate IP3 gating activity through the rearrangement of functional domains.     

Involvement of vertebrate polkappa in Rad18-independent postreplication repair of UV damage

DNA damage, which is left unrepaired by excision repair pathways, often blocks replication, leading to lesions such as breaks and gaps on the sister chromatids. These lesions may be processed by either homologous recombination (HR) repair or translesion DNA synthesis (TLS). Vertebrate Polkappa belongs to the DNA polymerase Y family, as do most TLS polymerases. However, the role for Polkappa in vertebrate cells is unclear because of the lack of reverse genetic studies. Here, we generated cells deficient in Polkappa (polkappa cells) from the chicken B lymphocyte line DT40. Although purified Polkappa is unable to bypass ultraviolet (UV) damage, polkappa cells exhibited increased UV sensitivity, and the phenotype was suppressed by expression of human and chicken Polkappa, suggesting that Polkappa is involved in TLS of UV photoproduct. Defects in both Polkappa and Rad18, which regulates TLS in yeast, in DT40 showed an additive effect on UV sensitivity. Interestingly, the level of sister chromatid exchange, which reflects HR-mediated repair, was elevated in normally cycling polkappa cells. This implies functional redundancy between HR and Polkappa in maintaining chromosomal DNA. In conclusion, vertebrate Polkappa is involved in Rad18-independent TLS of UV damage and plays a role in maintaining genomic stability.     

Novel nuclear and mitochondrial glycosylases revealed by disruption of the mouse Nth1 gene encoding an endonuclease III homolog for repair of thymine glycols

Endonuclease III, encoded by nth in Escherichia coli, removes thymine glycols (Tg), a toxic oxidative DNA lesion. To determine the biological significance of this repair in mammals, we established a mouse model with mutated mNth1, a homolog of nth, by gene targeting. The homozygous mNth1 mutant mice showed no detectable phenotypical abnormality. Embryonic cells with or without wild-type mNth1 showed no difference in sensitivity to menadione or hydrogen peroxide. Tg produced in the mutant mouse liver DNA by X-ray irradiation disappeared with time, though more slowly than in the wild-type mouse. In extracts from mutant mouse liver, we found, instead of mNTH1 activity, at least two novel DNA glycosylase activities against Tg. One activity is significantly higher in the mutant than in wild-type mouse in mitochondria, while the other is another nuclear glycosylase for Tg. These results underscore the importance of base excision repair of Tg both in the nuclei and mitochondria in mammals.     

Crystal structure of the RuvA-RuvB complex: a structural basis for the Holliday junction migrating motor machinery

We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration. Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring. The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode.     

Increased binding and chemotactic capacities of PDGF-BB on fibroblasts in radiation pneumonitis

Although pulmonary fibrosis is a frequent and serious consequence of radiotherapy for thoracic malignant diseases such as lung cancer, the pathogenesis of this radiation-induced lung disorder remains unclear. To clarify the mechanisms underlying radiation pneumonitis and pulmonary fibrosis, we investigated the expression of platelet-derived growth factor receptor (PDGFR) on fibroblasts obtained from irradiated rat lungs and on control fibroblasts. Whole lungs of male Wistar rats were irradiated with a single dose of 15 Gy, and lung fibroblasts were isolated at 4 weeks after the irradiation. The chemotactic response of irradiated lung fibroblasts to PDGF-BB was significantly higher than that of control lung fibroblasts, whereas there was no significant difference between irradiated lung fibroblasts and control lung fibroblasts in the response to PDGF-AA. Receptor binding assay showed more specific binding sites for PDGF-BB on irradiated lung fibroblasts than on control lung fibroblasts, and the displacement of (125)I-labeled PDGF binding to fibroblasts by unlabeled PDGF showed that (125)I-labeled PDGF-BB was displaced by PDGF-BB but not by PDGF-AA. These results suggest that the increased binding sites for PDGF-BB on irradiated lung fibroblasts correspond mainly to PDGFRB. Scatchard analysis of the saturation data demonstrated an approximately twofold increase both in the number of PDGF-BB binding sites and in the binding affinity in irradiated lung fibroblasts compared to that in control lung fibroblasts. Those results suggest that the increased chemotactic response of irradiated lung fibroblasts to PDGF-BB is related to the overexpression of PDGFRB, which may have an important role in the pathogenesis of radiation-induced pneumonitis and pulmonary fibrosis.     

Ameliorating effect of anti-Fas ligand MAb on wasting disease in murine model of chronic colitis

Fas/Fas ligand (FasL) interaction has been implicated in the pathogenesis of various diseases. To clarify the involvement of Fas/FasL in the pathogenesis of intestinal inflammation, we investigated the preventive and therapeutic effects of neutralizing anti-FasL monoclonal antibody (MAb) on the development of chronic colitis induced by adaptive transfer of CD4+CD45RBhigh T cells to SCID mice. Administration of anti-FasL MAb from 1 day after T cell transfer (prevention study) resulted in a significant improvement of clinical manifestations such as wasting and diarrhea. However, histological examination showed that mucosal inflammation in the colon, such as infiltration of T cells and macrophages, was not improved by the anti-FasL MAb treatment. In vitro studies showed that anti-FasL MAb did not inhibit IFN-gamma production by anti-CD3/CD28-stimulated lamina propria CD4+ T cells but suppressed TNF-alpha and IL-1beta production by lamina propria mononuclear cells. Therapeutic administration of anti-FasL MAb from 3 wk after T cell transfer also improved ongoing wasting disease but not intestinal inflammation. These results suggest that the Fas/FasL interaction plays a critical role in regulating systemic wasting disease but not local intestinal inflammation.