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Tumor necrosis factor-α (TNF) exerts its biological effect through two types of receptors, p55 TNF receptor (TNFR1) and p75 TNF receptor (TNFR2). An inflammatory response is known to be induced mainly by TNFR1, whereas an anti-inflammatory reaction is thought to be mediated by TNFR2 in some autoimmune diseases. We have been investigating the use of an antagonistic TNF mutant (TNFR1-selective antagonistic TNF mutant (R1antTNF)) to reveal the pharmacological effect of TNFR1-selective inhibition as a new therapeutic modality. Here, we aimed to further improve and optimize the activity and behavior of this mutant protein both in vitro and in vivo Specifically, we examined a trimeric structural fusion of R1antTNF, formed via the introduction of short peptide linkers, as a strategy to enhance bioactivity and molecular stability. By comparative analysis with R1antTNF, the trimeric fusion, referred to as single-chain R1antTNF (scR1antTNF), was found to retain in vitro molecular properties of receptor selectivity and antagonistic activity but displayed a marked increase in thermal stability. The residence time of scR1antTNF in vivo was also significantly prolonged. Furthermore, molecular modification using polyethylene glycol (PEG) was easily controlled by limiting the number of reactive sites. Taken together, our findings show that scR1antTNF displays enhanced molecular stability while maintaining biological activity compared with R1antTNF., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1-3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro(103). A molecular dynamics simulation and mutational analyses revealed that Arg(40) in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nm. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)·EPR on the known complex structure of EGF·EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)·EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Gata3 acts as a master regulator for T helper 2 (Th2) cell differentiation by inducing chromatin remodeling of the Th2 cytokine loci, accelerating Th2 cell proliferation, and repressing Th1 cell differentiation. Gata3 also directly transactivates the interleukin-5 (Il5) gene via additional mechanisms that have not been fully elucidated. We herein identified a mechanism whereby the methylation of Gata3 at Arg-261 regulates the transcriptional activation of the Il5 gene in Th2 cells. Although the methylation-mimicking Gata3 mutant retained the ability to induce IL-4 and repress IFNγ production, the IL-5 production was selectively impaired. We also demonstrated that heat shock protein (Hsp) 60 strongly associates with the methylation-mimicking Gata3 mutant and negatively regulates elongation of the Il5 transcript by RNA polymerase II. Thus, arginine methylation appears to play a pivotal role in the organization of Gata3 complexes and the target gene specificity of Gata3., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

To investigate the mechanism for low pH adaptation by a carboxylesterase, structural and biochemical analyses of EstFa_R (a recombinant, slightly acidophilic carboxylesterase from Ferroplasma acidiphilum) and SshEstI (an alkaliphilic carboxylesterase from Sulfolobus shibatae DSM5389) were performed. Although a previous proteomics study by another group showed that the enzyme purified from F. acidiphilum contained an iron atom, EstFa_R did not bind to iron as analyzed by inductively coupled plasma MS and isothermal titration calorimetry. The crystal structures of EstFa_R and SshEstI were determined at 1.6- and 1.5-Å resolutions, respectively. EstFa_R had a catalytic triad with an extended hydrogen bond network that was not observed in SshEstI. Quadruple mutants of both proteins were created to remove or introduce the extended hydrogen bond network. The mutation on EstFa_R enhanced its catalytic efficiency and gave it an alkaline pH optimum, whereas the mutation on SshEstI resulted in opposite effects (i.e. a decrease in the catalytic efficiency and a downward shift in the optimum pH). Our experimental results suggest that the low pH optimum of EstFa_R activity was a result of the unique extended hydrogen bond network in the catalytic triad and the highly negatively charged surface around the active site. The change in the pH optimum of EstFa_R happened simultaneously with a change in the catalytic efficiency, suggesting that the local flexibility of the active site in EstFa_R could be modified by quadruple mutation. These observations may provide a novel strategy to elucidate the low pH adaptation of serine hydrolases., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

By UV-irradiation, cells are subjected to DNA damage followed by mutation, cell death and/or carcinogenesis. DNA repair systems such as nucleotide excision repair (NER) and translesion DNA synthesis (TLS) protect cells against UV-irradiation. To understand the role of histone acetyltransferase GCN5 in regulation of DNA repair, we studied the sensitivity of GCN5-deficient DT40, GCN5(-/-), to various DNA-damaging agents including UV-irradiation, and effects of GCN5-deficiency on the expression of NER- and TLS-related genes. After UV-irradiation, cell death and DNA fragmentation of GCN5(-/-) were appreciably accelerated as compared with those of DT40. Interestingly, GCN5(-/-) showed a remarkable sensitivity to only UV-irradiation but not to other DNA-damaging agents tested. Semiquantitative RT-PCR showed that transcription of DNA polymerase η (POLH) gene whose deficiency is responsible for a variant form of xeroderma pigmentosum was drastically down-regulated in GCN5(-/-) (to ∼25%). In addition, ectopic expression of human POLH in GCN5(-/-) dramatically reversed the sensitivity to UV-irradiation of GCN5(-/-) to almost the same level of wild type DT40. Moreover, chromatin immunoprecipitation assay revealed that GCN5 binds to the chicken POLH gene 5'-flanking region that contains a typical CpG island and acetylates Lys-9 of histone H3, but not Lys-14 in vivo. These data suggest that GCN5 takes part in transcription regulation of POLH gene through alterations in the chromatin structure by direct interaction with its 5'-flanking region, and protects vertebrate cells against UV-induced DNA damage via controlling POLH gene expression.

Ordered nucleosome disassembly and reassembly are required for eukaryotic DNA replication. The facilitates chromatin transcription (FACT) complex, a histone chaperone comprising Spt16 and SSRP1, is involved in DNA replication as well as transcription. FACT associates with the MCM helicase, which is involved in DNA replication initiation and elongation. Although the FACT-MCM complex is reported to regulate DNA replication initiation, its functional role in DNA replication elongation remains elusive. To elucidate the functional role of FACT in replication fork progression during DNA elongation in the cells, we generated and analyzed conditional SSRP1 gene knock-out chicken (Gallus gallus) DT40 cells. SSRP1-depleted cells ceased to grow and exhibited a delay in S-phase cell cycle progression, although SSRP1 depletion did not affect the level of chromatin-bound DNA polymerase α or nucleosome reassembly on daughter strands. The tracking length of newly synthesized DNA, but not origin firing, was reduced in SSRP1-depleted cells, suggesting that the S-phase cell cycle delay is mainly due to the inhibition of replication fork progression rather than to defects in the initiation of DNA replication in these cells. We discuss the mechanisms of how FACT promotes replication fork progression in the cells.

Elucidating the regulatory mechanism for tissue-specific gene expression is key to understanding the differentiation process. The chondromodulin-I gene (ChM-I) is a cartilage-specific gene, the expression of which is regulated by the transcription factor, Sp3. The binding of Sp3 to the core-promoter region is regulated by the methylation status of the Sp3-binding motif as we reported previously. In this study, we have investigated the molecular mechanisms of the down-regulation of ChM-I expression in mesenchymal stem cells (MSCs) and normal mesenchymal tissues other than cartilage. The core-promoter region of cells in bone and peripheral nerve tissues was hypermethylated, whereas the methylation status in cells of other tissues including MSCs did not differ from that in cells of cartilage, suggesting the presence of inhibitory mechanisms other than DNA methylation. We found that a transcriptional repressor, YY1, negatively regulated the expression of ChM-I by recruiting histone deacetylase and thus inducing the deacetylation of associated histones. As for a positive regulator, we found that a transcriptional co-activator, p300, bound to the core-promoter region with Sp3, inducing the acetylation of histone. Inhibition of YY1 in combination with forced expression of p300 and Sp3 restored the expression of ChM-I in cells with a hypomethylated promoter region, but not in cells with hypermethylation. These results suggested that the expression of tissue-specific genes is regulated in two steps; reversible down-regulation by transcriptional repressor complex and tight down-regulation via DNA methylation.

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

The differentiation of naive CD4 T cells into Th2 cells requires the T cell receptor-mediated activation of the ERK MAPK cascade. Little is known, however, in regard to how the ERK MAPK cascade regulates Th2 cell differentiation. We herein identified Gfi1 (growth factor independent-1) as a downstream target of the ERK MAPK cascade for Th2 cell differentiation. In the absence of Gfi1, interleukin-5 production and the change of histone modification at the interleukin-5 gene locus were severely impaired. Furthermore, the interferon gamma gene showed a striking activation in the Gfi1(-/-) Th2 cells. An enhanced ubiquitin/proteasome-dependent degradation of GATA3 protein was observed in Gfi1(-/-) Th2 cells, and the overexpression of GATA3 eliminated the defect of Th2 cell function in Gfi1-deficient Th2 cells. These data suggest that the T cell receptor-mediated induction of Gfi1 controls Th2 cell differentiation through the regulation of GATA3 protein stability.

In acentriolar higher plant cells, the surface of the nucleus acts as a microtubule-organizing center, substituting for the centrosome. However, the protein factors responsible for this microtubule organization are unknown. The nuclear surfaces of cultured tobacco BY-2 cells possess particles that generate microtubules. We attempted to isolate the proteins in these particles to determine their role in microtubule organization. When incubated with plant or mammalian tubulin, some, but not all, of the isolated nuclei generated abundant microtubules radially from their surfaces. The substance to induce the formation of radial microtubules was confirmed by SDS-PAGE to be a protein with apparent molecular mass of 38 kDa. Partial analysis of the amino acid sequences of the peptide fragments suggested it was a histone H1-related protein. Cloning and cDNA sequence analysis confirmed this and revealed that when the recombinant protein was incubated with tubulin, it could organize microtubules as well as the 38-kDa protein. Histone H1 and tubulin formed complexes immediately, even on ice, and then clusters of these structures were formed. These clusters generated radial microtubules. This microtubule-organizing property was confined to histone H1; all other core histones failed to act as organizers. On immunoblot analysis, rabbit antibodies raised against the 38-kDa protein cross-reacted with histone H1 proteins from tobacco BY-2 cells. These antibodies virtually abolished the ability of the nucleus to organize radial microtubules. Indirect immunofluorescence showed that the antigen was distributed at the nuclear plasm and particularly at nuclear periphery independently from DNA.

Atrial fibrillation (AF) is characterized by decreased L-type calcium current (I(Ca,L)) in atrial myocytes and decreased atrial contractility. Oxidant stress and redox modulation of calcium channels are implicated in these pathologic changes. We evaluated the relationship between glutathione content (the primary cellular reducing moiety) and I(Ca,L) in atrial specimens from AF patients undergoing cardiac surgery. Left atrial glutathione content was significantly lower in patients with either paroxysmal or persistent AF relative to control patients with no history of AF. Incubation of atrial myocytes from AF patients (but not controls) with the glutathione precursor N-acetylcysteine caused a marked increase in I(Ca,L). To test the hypothesis that glutathione levels were mechanistically linked with the reduction in I(Ca,L), dogs were treated for 48 h with buthionine sulfoximine, an inhibitor of glutathione synthesis. Buthionine sulfoximine treatment resulted in a 24% reduction in canine atrial glutathione content, a reduction in atrial contractility, and an attenuation of I(Ca,L) in the canine atrial myocytes. Incubation of these myocytes with exogenous glutathione also restored I(Ca,L) to normal or greater than normal levels. To probe the mechanism linking decreased glutathione levels to down-regulation of I(Ca), the biotin switch technique was used to evaluate S-nitrosylation of calcium channels. S-Nitrosylation was apparent in left atrial tissues from AF patients; the extent of S-nitrosylation was inversely related to tissue glutathione content. S-Nitrosylation was also detectable in HEK cells expressing recombinant human cardiac calcium channel subunits following exposure to nitrosoglutathione. S-Nitrosylation may contribute to the glutathione-sensitive attenuation of I(Ca,L) observed in AF.

Isoflavones, a class of flavonoids, play very important roles in plant-microbe interactions in certain legumes such as soybeans (Glycine max L. Merr.). G. max UDP-glucose:isoflavone 7-O-glucosyltransferase (GmIF7GT) is a key enzyme in the synthesis of isoflavone conjugates, which accumulate in large amounts in vacuoles and serve as an isoflavonoid pool that allows for interaction with microorganisms. In this study, the 14,000-fold purification of GmIF7GT from the roots of G. max seedlings was accomplished. The purified enzyme is a monomeric protein of 46 kDa, catalyzing regiospecific glucosyl transfer from UDP-glucose to isoflavones to produce isoflavone 7-O-beta-D-glucosides (k(cat) = 0.74 s(-1), K(m) for genistein = 3.6 microM, and K(m) for UDP-glucose = 190 microM). The GmIF7GT cDNA was isolated based on the amino acid sequence of the purified enzyme. Phylogenetic analysis showed that GmIF7GT is a novel member of glycosyltransferase family 1 and is distantly related to Glycyrrhiza echinata UDP-glucose:isoflavonoid 7-O-glucosyltransferase. The purified enzyme was unexpectedly devoid of the N-terminal 49-residue segment and thus lacks the histidine residue corresponding to the proposed catalytic residue of glycosyltransferases from Medicago truncatula (UGT71G1) and Vitis vinifera (VvGT1). The results of kinetic studies of site-directed mutants of GmIF7GT showed that both His-15 and Asp-125, which correspond to the catalytic residues of UGT71G1 and VvGT1, are not important for GmIF7GT activity. The results also suggest that an acidic residue at position 392 is very important for primary catalysis of GmIF7GT. These results led to the proposal that GmIF7GT utilizes a strategy of catalysis that is distinct from those proposed for UGT71G1 and VvGT1.

The BAHD family is a class of acyl-CoA-dependent acyltransferases that are involved in plant secondary metabolism and show a diverse range of specificities for acyl acceptors. Anthocyanin acyltransferases make up an important class of the BAHD family and catalyze the acylation of anthocyanins that are responsible for most of the red-to-blue colors of flowers. Here, we describe crystallographic and mutational studies of three similar anthocyanin malonyltransferases from red chrysanthemum petals: anthocyanidin 3-O-glucoside-6''-O-malonyltransferase (Dm3MaT1), anthocyanidin 3-O-glucoside-3'', 6''-O-dimalonyltransferase (Dm3MaT2), and a homolog (Dm3MaT3). Mutational analyses revealed that seven amino acid residues in the N- and C-terminal regions are important for the differential acyl-acceptor specificity between Dm3MaT1 and Dm3MaT2. Crystallographic studies of Dm3MaT3 provided the first structure of a BAHD member, complexed with acyl-CoA, showing the detailed interactions between the enzyme and acyl-CoA molecules. The structure, combined with the results of mutational analyses, allowed us to identify the acyl-acceptor binding site of anthocyanin malonyltransferases, which is structurally different from the corresponding portion of vinorine synthase, another BAHD member, thus permitting the diversity of the acyl-acceptor specificity of BAHD family to be understood.

Synovial sarcoma, a soft tissue sarcoma that develops in adults, is pathologically subclassified into monophasic spindle synovial sarcoma and biphasic synovial sarcoma with epithelial components. The molecular mechanism building the epithelial components in biphasic synovial sarcoma is totally unknown. Here we investigated claudins, critical molecules in the tight junction, in biphasic synovial sarcoma. Expression profiles of 21 claudins in 17 synovial sarcoma tumor samples, including 9 biphasic tumors, identified claudin4, claudin7, and claudin10 as biphasic tumor-related claudins, and immunohistochemical analyses demonstrated the localization of these claudins in the epithelial component in biphasic tumors, with claudin7 the most closely associated with the epithelial component. The mRNA expression and protein localization of claudin7 coincided with those of the ELF3, an epithelia-specific member of the Ets family of transcription factors. Luciferase reporter assays demonstrated that the presence of the Ets-binding site at -150 in the promoter region of the claudin7 gene was critical for the transcriptional activity, and gel shift and chromatin immunoprecipitation assays confirmed the binding of ELF3 to the Ets site at -150. Inhibition of ELF3 expression by small interfering RNA simultaneously down-regulated the mRNA expression of the claudin7 gene, and the introduction of ELF3 expression in claudin7-negative cell lines induced mRNA expression of the claudin7 gene. Therefore, the induction of claudin7 expression by ELF3 appears critical to the formation of the epithelial structures in biphasic synovial sarcoma.

Soybeans (Glycine max (L.) Merr.) and certain other legumes excrete isoflavones from their roots, which participate in plantmicrobe interactions such as symbiosis and as a defense against infections by pathogens. In G. max, the release of free isoflavones from their conjugates, the latent forms, is mediated by an isoflavone conjugate-hydrolyzing beta-glucosidase. Here we report on the purification and cDNA cloning of this important beta-glucosidase from the roots of G. max seedlings as well as related phylogenetic and cellular localization studies. The purified enzyme, isoflavone conjugate-hydrolyzing beta-glucosidase from roots of G. max seedling (GmICHG), is a homodimeric glycoprotein with a subunit molecular mass of 58 kDa and is capable of directly hydrolyzing genistein 7-O-(6 ''-O-malonyl-beta-d-glucoside) to produce free genistein (k(cat), 98 s(-1); K(m), 25 microM at 30 degrees C, pH 7.0). GmICHG cDNA was isolated based on the amino acid sequence of the purified enzyme. GmICHG cDNA was abundantly expressed in the roots of G. max seedlings but only negligibly in the hypocotyl and cotyledon. An immunocytochemical analysis using anti-GmICHG antibodies, along with green fluorescent protein imaging analyses of Arabidopsis cultured cells transformed by the GmICHG:GFP fusion gene, revealed that the enzyme is exclusively localized in the cell wall and intercellular space of seedling roots, particularly in the cell wall of root hairs. A phylogenetic analysis revealed that GmICHG is a member of glycoside hydrolase family 1 and can be co-clustered with many other leguminous beta-glucosidases, the majority of which may also be involved in flavonoid-mediated interactions of legumes with microbes.

Asf1 (anti-silencing function 1), a well conserved protein from yeast to humans, acts as a histone chaperone and is predicted to participate in a variety of chromatin-mediated cellular processes. To investigate the physiological role of vertebrate Asf1 in vivo, we generated a conditional Asf1-deficient mutant from chicken DT40 cells. Induction of Asf1 depletion resulted in the accumulation of cells in S phase with decreased DNA replication and increased mitotic aberrancy forming multipolar spindles, leading to cell death. In addition, nascent chromatin in Asf1-depleted cells showed increased nuclease sensitivity, indicating impaired nucleosome assembly during DNA replication. Complementation analyses revealed that the functional domain of Asf1 for cell viability was confined to the N-terminal core domain (amino acids 1-155) that is a binding platform for histones H3/H4, CAF-1p60, and HIRA, whereas Asf1 mutant proteins, abolishing binding abilities with both p60 and HIRA, exhibit no effect on viability. These results together indicate that the vertebrate Asf1 plays a crucial role in replication-coupled chromatin assembly, cell cycle progression, and cellular viability and provide a clue of a possible role in a CAF-1- and HIRA-independent chromatin-modulating process for cell proliferation.

We reported previously that chicken HIRA, a homolog of Saccharomyces cerevisiae transcriptional co-repressors Hir1p and Hir2p, possesses seven WD dipeptide motifs and an LXXLL motif in its N-terminal and C-terminal halves, respectively, required for transcription regulations. Here, by using the gene targeting technique, we generated the homozygous HIRA-deficient DT40 mutant DeltaHIRA. The HIRA deficiency caused slightly delayed cell growth and affected the opposite transcriptions of cell cycle-related genes, i.e. repressions for P18, CDC25B, and BCL-2, activations for P19 and cyclin A, and histones H2A, H2B, H3, and H4. These altered expressions were completely revived by the artificial stable expression of hemagglutinin-tagged HIRA in DeltaHIRA. The ability to rescue the delayed growth rate was preferentially aided by the N-terminal half instead of the C-terminal half. We cloned the chicken P18 genomic DNA, and we established that its promoter was located surrounding the sequence GCGGGCGC at positions -1157 to -1150. Chromatin immunoprecipitation assay revealed that the N-terminal half interacted directly or indirectly with the putative promoter region of the p18 gene, resulting in up-regulation of the gene. These results indicated that the N-terminal half of HIRA should contribute positively to the growth rate via up-regulation of a set of cell cycle-related genes, whereas the C-terminal half down-regulated another set of them without exhibiting any effect on the cell growth.

Differentiation of naive CD4 T cells into Th2 cells requires protein expression of GATA3. Interleukin-4 induces STAT6 activation and subsequent GATA3 transcription. Little is known, however, on how T cell receptor-mediated signaling regulates GATA3 and Th2 cell differentiation. Here we demonstrated that T cell receptor-mediated activation of the Ras-ERK MAPK cascade stabilizes GATA3 protein in developing Th2 cells through the inhibition of the ubiquitin-proteasome pathway. Mdm2 was associated with GATA3 and induced ubiquitination on GATA3, suggesting its role as a ubiquitin-protein isopeptide ligase for GATA3 ubiquitination. Thus, the Ras-ERK MAPK cascade controls GATA3 protein stability by a post-transcriptional mechanism and facilitates GATA3-mediated chromatin remodeling at Th2 cytokine gene loci leading to successful Th2 cell differentiation.

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) Ca(2+) channel plays pivotal roles in many aspects of physiological and pathological events. It was previously reported that IP(3)R forms clusters on the endoplasmic reticulum when cytosolic Ca(2+) concentration ([Ca(2+)](C)) is elevated. However, the molecular mechanism of IP(3)R clustering remains largely unknown, and thus its physiological significance is far from clear. In this study we found that the time course of clustering of green fluorescent protein-tagged IP(3)R type 1 (GFP-IP(3)R1), evoked by IP(3)-generating agonists, did not correlate with [Ca(2+)](C) but seemed compatible with cytoplasmic IP(3) concentration. IP(3) production alone induced GFP-IP(3)R1 clustering in the absence of a significant increase in [Ca(2+)](C) but elevated [Ca(2+)](C) without IP(3) production did not. Moreover IP(3)R1 mutants that do not undergo an IP(3)-induced conformational change failed to form clusters. Thus, IP(3)R clustering is induced by its IP(3)-induced conformational change to the open state. We also found that GFP-IP(3)R1 clusters colocalized with ERp44, a luminal protein of endoplasmic reticulum that inhibits its channel activity. This is the first example of ligand-induced clustering of a ligand-gated channel protein.

In contrast to the wealth of biochemical and genetic information on vertebrate glucuronosyltransferases (UGATs), only limited information is available on the role and phylogenetics of plant UGATs. Here we report on the purification, characterization, and cDNA cloning of a novel UGAT involved in the biosynthesis of flower pigments in the red daisy (Bellis perennis). The purified enzyme, BpUGAT, was a soluble monomeric enzyme with a molecular mass of 54 kDa and catalyzed the regiospecific transfer of a glucuronosyl unit from UDP-glucuronate to the 2''-hydroxyl group of the 3-glucosyl moiety of cyanidin 3-O-6''-O-malonylglucoside with a kcat value of 34 s(-1) at pH 7.0 and 30 degrees C. BpUGAT was highlyspecific for cyanidin 3-O-glucosides (e.g. Km for cyanidin 3-O-6''-O-malonylglucoside, 19 microM) and UDP-glucuronate (Km, 476 microM). The BpUGAT cDNA was isolated on the basis of the amino acid sequence of the purified enzyme. Quantitative PCR analysis showed that transcripts of BpUGAT could be specifically detected in red petals, consistent with the temporal and spatial distributions of enzyme activity in the plant and also consistent with the role of the enzyme in pigment biosynthesis. A sequence analysis revealed that BpUGAT is related to the glycosyltransferase 1 (GT1) family of the glycosyltransferase superfamily (according to the Carbohydrate-Active Enzymes (CAZy) data base). Among GT1 family members that encompass vertebrate UGATs and plant secondary product glycosyltransferases, the highest sequence similarity was found with flavonoid rhamnosyltransferases of plants (28-40% identity). Although the biological role (pigment biosynthesis) and enzymatic properties of BpUGAT are significantly different from those of vertebrate UGATs, both of these UGATs share a similarity in that the products produced by these enzymes are more water-soluble, thus facilitating their accumulation in vacuoles (in BpUGAT) or their excretion from cells (in vertebrate UGATs), corroborating the proposed general significance of GT1 family members in the metabolism of small lipophilic molecules.

The core structure of membrane lipids of archaea have some unique properties that permit archaea to be distinguished from the others, i.e. bacteria and eukaryotes. (S)-2,3-Di-O-geranylgeranylglyceryl phosphate synthase, which catalyzes the transfer of a geranylgeranyl group from geranylgeranyl diphosphate to (S)-3-O-geranylgeranylglyceryl phosphate, is involved in the biosynthesis of archaeal membrane lipids. Enzymes of the UbiA prenyltransferase family are known to catalyze the transfer of a prenyl group to various acceptors with hydrophobic ring structures in the biosynthesis of respiratory quinones, hemes, chlorophylls, vitamin E, and shikonin. The thermoacidophilic archaeon Sulfolobus solfataricus was found to encode three homologues of UbiA prenyltransferase in its genome. One of the homologues encoded by SSO0583 was expressed in Escherichia coli, purified, and characterized. Radio-assay and mass spectrometry analysis data indicated that the enzyme specifically catalyzes the biosynthesis of (S)-2,3-di-O-geranylgeranylglyceryl phosphate. The fact that the orthologues of the enzyme are encoded in almost all archaeal genomes clearly indicates the importance of their functions. A phylogenetic tree constructed using the amino acid sequences of some typical members of the UbiA prenyltransferase family and their homologues from S. solfataricus suggests that the two other S. solfataricus homologues, excluding the (S)-2,3-di-O-geranylgeranylglyceryl phosphate synthase, are involved in the production of respiratory quinone and heme, respectively. We propose here that archaeal prenyltransferases involved in membrane lipid biosynthesis might be prototypes of the protein family and that archaea might have played an important role in the molecular evolution of prenyltransferases.

Interleukin (IL)-4-induced STAT6 activation and the subsequent up-regulation of GATA3 are crucial for the induction of chromatin remodeling of the Th2 cytokine gene loci as Th2 cells undergo development. This study probes the role of these molecules in the maintenance of memory Th2 cells. IL-4 was not required to maintain the capability for Th2 cytokine production in in vivo generated antigen-specific memory Th2 cells. Histone H3-K9/14 hyperacetylation and intergenic transcripts associated with the IL-4 gene locus were preserved in the absence of IL-4, but those associated with the IL-13 gene were partially IL-4-dependent. Histone H3-K4 methylation of the IL-13 and IL-4 gene loci was fully preserved in memory Th2 cells and accompanied by memory cell-specific accumulation of Pol II complex to highly restricted sites. Thus, memory Th2 cells maintain a unique Th2-specific remodeled chromatin in the IL-4 and IL-13 gene loci by active molecular events that are IL-4-independent.

Transcriptional regulation of cell- and stage-specific genes is a crucial process in the development of mesenchymal tissues. Here we have investigated the regulatory mechanism of the expression of the chondromodulin-I (ChM-I) gene, one of the chondrocyte-specific genes, in osteogenic cells using osteosarcoma (OS) cells as a model. Methylation-specific sequence analyses revealed that the extent of methylation in the core-promoter region of the ChM-I gene was correlated inversely with the expression of the ChM-I gene in OS primary tumors and cell lines. 5-Aza-deoxycytidine treatment induced the expression of the ChM-I gene in ChM-I-negative OS cell lines, and the induction of expression was associated tightly with the demethylation of cytosine at -52 (C(-52)) in the middle of an Sp1/3 binding site to which the Sp3, but not Sp1, bound. The replacement of C(-52) with methyl-cytosine or thymine abrogated Sp3 binding and also the transcription activity of the genomic fragment including C(-52). The inhibition of Sp3 expression by small interfering RNA reduced the expression of the ChM-I gene in ChM-I-positive normal chondrocytes, indicating Sp3 as a physiological transcriptional activator of the ChM-I gene. These results suggest that the methylation status of the core-promoter region is one of the mechanisms to determine the cell-specific expression of the ChM-I gene through the regulation of the binding of Sp3.

Chemokines are a family of cytokines that induce directed migration of various types of leukocytes through specific interactions with a group of seven transmembrane receptors. Scavenger receptors are a heterogenous family of transmembrane molecules that commonly bind and uptake oxidized low density lipoprotein and bacteria. Here, we show that not only CXC chemokine 16 (CXCL16)/SR-PSOX, a transmembrane chemokine with scavenger receptor activity, but also 12 out of 15 chemokines examined efficiently bound scavenger receptor ligands in competition with cells expressing their specific chemokine receptors. Furthermore both the chemotactic and scavenger receptor activities of SR-PSOX/CXCL16 were similarly impaired in a series of mutants altered in the chemokine domain, indicating that SR-PSOX/CXCL16 binds scavenger receptor ligands as well as CXCR6 using highly overlapping binding motifs. Taken together, chemokines generally have scavenger receptor-like activity through their receptor-binding domain, suggesting a close evolutionary relationship between chemokines and scavenger receptors.

GATA3 expression is essential for type-2 helper T (Th2) cell differentiation. GATA3-mediated chromatin remodeling at the Th2 cytokine gene loci, including Th2-specific long range histone hyperacetylation of the interleukin (IL)-13/IL-4 gene loci, occurs in developing Th2 cells. However, little is known about the role of GATA3, if any, in the maintenance of established remodeled chromatin at the Th2 cytokine gene loci. Here, we established a Cre/LoxP-based site-specific recombination system in cultured CD4 T cells using a unique adenovirus-mediated gene transfer technique. This system allowed us to investigate the effect of loss of GATA3 expression in in vitro differentiated Th2 cells. After ablation of GATA3, we detected reduced production of all Th2 cytokines, increased DNA methylation at the IL-4 gene locus, and decreased histone hyperacetylation at the IL-5 gene locus but not significantly so at the IL-13/IL-4 gene loci. Thus, GATA3 plays important roles in the maintenance of the Th2 phenotype and continuous chromatin remodeling of the specific Th2 cytokine gene locus through cell division.

Syntaxin 1C is an alternative splice variant lacking the transmembrane domain of HPC-1/syntaxin 1A. We found previously that syntaxin 1C is expressed as a soluble protein in human astroglioma (T98G) cells, and syntaxin 1C expression is enhanced by stimulation with phorbol 12-myristate 13-acetate (PMA). However, the physiological function of syntaxin 1C is not known. In this study, we examined the relationship between syntaxin 1C and glucose transport. First, we discovered that glucose transporter-1 (GLUT-1) was the primary isoform in T98G cells. Second, we demonstrated that glucose uptake in T98G cells was suppressed following an increase in endogenous syntaxin 1C after stimulation with PMA, which did not alter the expression levels of other plasma membrane syntaxins. We further examined glucose uptake and intracellular localization of GLUT-1 in cells that overexpressed exogenous syntaxin 1C; glucose uptake via GLUT-1 was inhibited without affecting sodium-dependent glucose transport. The value of Vmax for the dose-dependent uptake of glucose was reduced in syntaxin 1C-expressing cells, whereas there was no change in Km. Immunofluorescence studies revealed a reduction in the amount of GLUT-1 in the plasma membrane in cells that expressed syntaxin 1C. Based on these results, we postulate that syntaxin 1C regulates glucose transport in astroglioma cells by changing the intracellular trafficking of GLUT-1. This is the first report to indicate that a syntaxin isoform that lacks a transmembrane domain can regulate the intracellular transport of a plasma membrane protein.

Interleukin 5 (IL-5) plays a unique role in allergic inflammatory responses, and the understanding of molecular mechanisms underlying the generation of IL-5-producing cells is crucial for the regulation of allergic disorders. Differentiation of naive CD4 T cells into type-2 helper (Th2) cells is accompanied by chromatin remodeling including hyperacetylation of histones H3 and H4 in the nucleosomes associated with the IL-4, IL-13, and IL-5 genes. Histone hyperacetylation of the IL-5 gene displayed a delayed kinetics compared with that of the IL-4 and IL-13 genes, suggesting a distinct remodeling mechanism for the IL-5-gene locus. Here we studied the role of CD28 costimulation in the generation of IL-5-producing cells and the histone hyperacetylation of the IL-5 gene locus. CD28-costimulation selectively enhanced histone hyperacetylation of the IL-5 gene locus that appeared to be mediated through NF-kappaB activation and subsequent up-regulation of GATA3. The CD28 costimulation-sensitive histone hyperacetylation spanned almost the entire intergenic region between the IL-5 and RAD50 accompanied with intergenic transcript. Thus, this is the first demonstration that CD28 costimulation controls a chromatin-remodeling process during Th2 cell differentiation.

Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low pH and in elevated temperatures. In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser(278)-Glu(78)-Asp(82)) for enzymatic activity. Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges. Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1', showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme. All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen. A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur. The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser(278), His(78), and Asp(82) do not interact with each other; thus, the canonical catalytic triad is disrupted.

Matrix metalloproteinases (MMPs) are thought to be responsible for dermal photoaging in human skin. In the present study, we evaluated the involvement of macrophage migration inhibitory factor (MIF) in MMP-1 expression under ultraviolet A (UVA) irradiation in cultured human dermal fibroblasts. UVA (20 J/cm(2)) up-regulates MIF production, and UVA-induced MMP-1 mRNA production is inhibited by an anti-MIF antibody. MIF (100 ng/ml) was shown to induce MMP-1 in cultured human dermal fibroblasts. We found that MIF (100 ng/ml) enhanced MMP-1 activity in cultured fibroblasts assessed by zymography. Moreover, we observed that fibroblasts obtained from MIF-deficient mice were much less sensitive to UVA regarding MMP-13 expression than those from wild-type BALB/c mice. Furthermore, after UVA irradiation (10 J/cm(2)), dermal fibroblasts of MIF-deficient mice produced significantly decreased levels of MMP-13 compared with fibroblasts of wild-type mice. Next we investigated the signal transduction pathway of MIF. The up-regulation of MMP-1 mRNA by MIF stimulation was found to be inhibited by a PKC inhibitor (GF109203X), a Src-family tyrosine kinase inhibitor (herbimycin A), a tyrosine kinase inhibitor (genistein), a PKA inhibitor (H89), a MEK inhibitor (PD98089), and a JNK inhibitor (SP600125). In contrast, the p38 inhibitor (SB203580) was found to have little effect on expression of MMP-1 mRNA. We found that PKC-pan, PKC alpha/beta II, PKC delta (Thr505), PKC delta (Ser(643)), Raf, and MAPK were phosphorylated by MIF. Moreover, we demonstrated that phosphorylation of PKC alpha/beta II and MAPK in response to MIF was suppressed by genistein, and herbimycin A as well as by transfection of the plasmid of C-terminal Src kinase. The DNA binding activity of AP-1 was significantly up-regulated 2 h after MIF stimulation. Taken together, these results suggest that MIF is involved in the up-regulation of UVA-induced MMP-1 in dermal fibroblasts through PKC-, PKA-, Src family tyrosine kinase-, MAPK-, c-Jun-, and AP-1-dependent pathways.

Acrolein, a representative carcinogenic aldehyde, that could be ubiquitously generated in biological systems under oxidative stress shows facile reactivity with a nucleophile such as a protein. In this study, to gain a better understanding of the molecular basis of acrolein modification of protein, we characterized the acrolein modification of a model peptide (the oxidized B chain of insulin) by electrospray ionization-liquid chromatography/mass spectrometry method and established a novel acrolein-lysine condensation reaction. In addition, we found that this condensation adduct represented the major antigenic adduct generated in acrolein-modified protein. To identify the modification site and structures of adducts generated in the acrolein-modified insulin B chain, both the acrolein-pretreated and untreated peptides were digested with V8 protease and the resulting peptides were subjected to electrospray ionization-liquid chromatography/mass spectrometry. This technique identified nine peptides, which contained the acrolein adducts at Lys-29 and the N terminus, and revealed that the reaction of the insulin B chain with acrolein gave multiple adducts, including an unknown adduct containing two molecules of acrolein per lysine. To identify this adduct, we incubated N(alpha)-acetyllysine with acrolein and isolated a product having the same molecular mass as the unknown acrolein-lysine adduct. On the basis of the chemical and spectroscopic evidence, the adduct was determined to be a novel pyridinium-type lysine adduct, N(epsilon)-(3-methylpyridinium)lysine (MP-lysine). The formation of MP-lysine was confirmed by amino acid analysis of proteins treated with acrolein. More notably, this condensation adduct appeared to be an intrinsic epitope of a monoclonal antibody 5F6 that had been raised against acrolein-modified protein.

During the mating of yeast Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus. This process requires the functions of BiP/Kar2p, a member of the Hsp70 family in the endoplasmic reticulum, and its partner protein, Jem1p. To investigate further the role of BiP and Jem1p in nuclear fusion, we screened for partner proteins for Jem1p by the yeast two-hybrid system and identified Nep98p. Nep98p is an essential integral membrane protein of the nuclear envelope and is enriched in the spindle pole body (SPB), the sole microtubule-organizing center in yeast. Temperature-sensitive nep98 mutant cells contain abnormal SPBs lacking the half-bridge, suggesting the essential role of Nep98p in the organization of the normal SPB. Additionally, nep98 mutant cells show defects in mitotic nuclear division and nuclear fusion during mating. Because Jem1p is not required for nuclear division, Nep98p probably has dual functions in Jem1p-dependent karyogamy and in Jem1p-independent nuclear division.

Protein 4.1N was identified as a binding molecule for the C-terminal cytoplasmic tail of inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) using a yeast two-hybrid system. 4.1N and IP(3)R1 associate in both subconfluent and confluent Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell line. In subconfluent MDCK cells, 4.1N is distributed in the cytoplasm and the nucleus; IP(3)R1 is localized in the cytoplasm. In confluent MDCK cells, both 4.1N and IP(3)R1 are predominantly translocated to the basolateral membrane domain, whereas 4.1R, the prototypical homologue of 4.1N, is localized at the tight junctions (Mattagajasingh, S. N., Huang, S. C., Hartenstein, J. S., and Benz, E. J., Jr. (2000) J. Biol. Chem. 275, 30573-30585), and other endoplasmic reticulum marker proteins are still present in the cytoplasm. Moreover, the 4.1N-binding region of IP(3)R1 is necessary and sufficient for the localization of IP(3)R1 at the basolateral membrane domain. A fragment of the IP(3)R1-binding region of 4.1N blocks the localization of co-expressed IP(3)R1 at the basolateral membrane domain. These data indicate that 4.1N is required for IP(3)R1 translocation to the basolateral membrane domain in polarized MDCK cells.