Proinflammatory cytokine interleukin-1ß suppresses cold-induced thermogenesis in adipocytes.

In this study, we investigated the effects of interleukin-1ß (IL-1ß), a typical proinflammatory cytokine on the ß-adrenoreceptor-stimulated induction of uncoupling protein 1 (UCP1) expression in adipocytes. IL-1ß mRNA expression levels were upregulated in white adipose tissues of obese mice and in RAW264.7 macrophages under conditions designed to mimic obese adipose tissue. Isoproterenol-stimulated induction of UCP1 mRNA expression was significantly inhibited in C3H10T1/2 adipocytes by conditioned medium from lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages in comparison with control conditioned medium. This inhibition was significantly attenuated in the presence of recombinant IL-1 receptor antagonist and IL-1ß antibody, suggesting that activated macrophage-derived IL-1ß is an important cytokine for inhibition of ß-adrenoreceptor-stimulated UCP1 induction in adipocytes. IL-1ß suppressed isoproterenol-induced UCP1 mRNA expression in C3H10T1/2 adipocytes, and this effect was partially but significantly abrogated by inhibition of extracellular signal-regulated kinase (ERK). IL-1ß also suppressed the isoproterenol-induced activation of the UCP1 promoter and transcription factors binding to the cAMP response element. Moreover, intraperitoneal administration of IL-1ß suppressed cold-induced UCP1 expression in adipose tissues. These findings suggest that IL-1ß upregulated in obese adipose tissues suppresses ß-adrenoreceptor-stimulated induction of UCP1 expression through ERK activation in adipocytes.

Comparative analyses of the two proliferating cell nuclear antigens from the hyperthermophilic archaeon, Thermococcus kodakarensis.

The DNA sliding clamp is a multifunctional protein involved in cellular DNA transactions. In Archaea and Eukaryota, proliferating cell nuclear antigen (PCNA) is the sliding clamp. The ring-shaped PCNA encircles double-stranded DNA within its central hole and tethers other proteins on DNA. The majority of Crenarchaeota, a subdomain of Archaea, have multiple PCNA homologues, and they are capable of forming heterotrimeric rings for their functions. In contrast, most organisms in Euryarchaeota, the other major subdomain, have a single PCNA forming a homotrimeric ring structure. Among the Euryarchaeota whose genome is sequenced, Thermococcus kodakarensis is the only species with two genes encoding PCNA homologues on its genome. We cloned the two genes from the T. kodakarensis genome, and the gene products, PCNA1 and PCNA2, were characterized. PCNA1 stimulated the DNA synthesis reactions of the two DNA polymerases, PolB and PolD, from T. kodakarensis in vitro. PCNA2, however, only had an effect on PolB. We were able to disrupt the gene for PCNA2, whereas gene disruption for PCNA1 was not possible, suggesting that PCNA1 is essential for DNA replication. The sensitivities of the Δpcna2 mutant strain to ultraviolet irradiation (UV), methyl methanesulfonate (MMS) and mitomycin C (MMC) were indistinguishable from those of the wild-type strain.

Biochemical and genetical analyses of the three mcm genes from the hyperthermophilic archaeon, Thermococcus kodakarensis.

In eukaryotes, the replicative DNA helicase 'core' is the minichromosome maintenance (Mcm) complex (MCM), forming a heterohexameric complex consisting of six subunits (Mcm2-7). Recent studies showed that the CMG (Cdc45-MCM-GINS) complex is the actual helicase body in the replication fork progression complex. In Archaea, Thermococcus kodakarensis harbors three genes encoding the Mcm homologs on its genome, contrary to most archaea, which have only one homolog. It is thus, of high interest, whether and how these three Mcms share their functions in DNA metabolism in this hyperthermophile. Here, we report the biochemical properties of two of these proteins, TkoMcm1 and TkoMcm3. In addition, their physical and functional interactions with GINS, possibly an essential factor for the initiation and elongation process of DNA replication, are presented through in vitro ATPase and helicase assays, and an in vivo immunoprecipitation assay. Gene disruption and product quantification analyses suggested that TkoMcm3 is essential for cell growth and plays a key role as the main DNA helicase in DNA replication, whereas TkoMcm1 also shares some function in the cells.

Functional expression of single-chain antibody to leukotriene C(4).

Leukotriene C(4) (LTC(4)) is synthesized by binding of glutathione to LTA(4), an epoxide derived from arachidonic acid, and further metabolized to LTD(4) and LTE(4). We previously prepared a monoclonal antibody with a high affinity and specificity to LTC(4). To explore the structure of the antigen-binding site of a monoclonal antibody against LTC(4) (mAbLTC), we isolated full-length cDNAs for heavy and light chains of mAbLTC. The heavy and light chains consisted of 461 and 238 amino acids including a signal peptide with molecular weights of 51,089 and 26,340, respectively. An expression plasmid encoding a single-chain antibody comprising variable regions of mAbLTC heavy and light chains (scFvLTC) was constructed and expressed in COS-7 cells. The recombinant scFvLTC showed a high affinity with LTC(4) comparable to mAbLTC. The scFvLTC also bound to LTD(4) and LTE(4) with 48% and 17% reactivities, respectively, as compared with LTC(4) binding, whereas the antibody showed almost no affinity for LTB(4).

Recruitment of thioredoxin-like domains into prostaglandin synthases.

A variety of prostaglandin (PG) synthases with different evolutionary origins have been identified. These enzymes catalyze reduction and oxidation reactions. However, despite the similarity in their reactions, thioredoxin-like proteins were not found in the PG synthesis pathway until recently. We have identified two new enzymes, thioredoxin-type PGF synthase and membrane-associated PGE synthase-2, with thioredoxin-like domains. In addition, the N-terminal domain of hematopoietic PGD synthase is classified into the thioredoxin-like superfamily, based on structural similarity. The active sites of the former two enzymes have a CXXC motif, which is also critical for the thioredoxin activity. In contrast, hematopoietic PGD synthase lacks the motif, and the activity is carried out by glutathione. A phylogenetic tree of the thioredoxin-like domains suggests that they have been independently recruited into these PG synthases. We will discuss the functional meaning of the thioredoxin-like domains in the PG synthases from the viewpoint of the redox activity.

Computational and experimental analyses of furcatin hydrolase for substrate specificity studies of disaccharide-specific glycosidases.

Disaccharide-specific glycosidases (diglycosidases) are unique glycoside hydrolases, as their substrate specificities differ from those of monosaccharide-specific beta-glycosidases (monoglycosidases), in spite of similarities in their sequences and reaction mechanisms. Diglycosidases selectively hydrolyse the beta-glycosidic bond between glycone and aglycone of disaccharide glycosides, but do not cleave the bond between two saccharides, and barely hydrolyse monosaccharide glycosides. We analysed the substrate recognition mechanisms of diglycosidases by computational and experimental methods, using furcatin hydrolase (FH) (EC 3.2.1.161) derived from Viburnum furcatum. Amino acid sequence comparisons and model structure building revealed two residues, Ala419 and Ser504 of FH, as candidates determining the substrate specificity. These residues were specifically conserved in the diglycosidases. The model structure suggested that Ala419 is involved in the aglycone recognition, whereas Ser504 recognizes the external saccharide of the glycone. Mutations at these sites drastically decreased the diglycosidase activity. The mechanism by which the diglycosidases acquired their substrate specificity is discussed, based on these observations.

How does a topological inversion change the evolutionary constraints on membrane proteins?

The members of the aquaporin family and those of the ClC chloride ion channel family consist of two-fold tandem repeats. The orientation of the N-terminal domain against membrane is opposite to that of the C-terminal domain. Several lines of evidence suggest that the extracellular and the cytoplasmic environments impose different evolutionary constraints on proteins (e.g. positive-inside rule). Therefore, the different constraints would affect the corresponding regions of the two domains, which are exposed to the different environments. To examine this hypothesis, the N- and the C-terminal domains were aligned and the difference in residue composition or conservation pattern between the two domains was calculated at each alignment site by several methods. Then, the residues corresponding to the sites exhibiting significant difference were mapped onto the tertiary structure. In spite of the difference in the methods, the mapped residues clustered on the pore surface of the channel; in contrast, the number of the residues mapped on the extracellular or cytoplasmic sides of the proteins was small. A minor modification of the methods improved the sensitivity to detect sites related to the positive-inside rule. The results support our hypothesis about the relationship between the topological inversion and the different constraints.

Evolutionary Analysis of Functional Divergence among Chemokine Receptors, Decoy Receptors, and Viral Receptors.

Chemokine receptors (CKRs) function in the inflammatory response and in vertebrate homeostasis. Decoy and viral receptors are two types of CKR homologs with modified functions from those of the typical CKRs. The decoy receptors are able to bind ligands without signaling. On the other hand, the viral receptors show constitutive signaling without ligands. We examined the sites related to the functional difference. At first, the decoy and viral receptors were each classified into five groups, based on the molecular phylogenetic analysis. A multiple amino acid sequence alignment between each group and the CKRs was then constructed. The difference in the amino acid composition between the group and the CKRs was evaluated as the Kullback-Leibler (KL) information value at each alignment site. The KL information value is considered to reflect the difference in the functional constraints at the site. The sites with the top 5% of KL information values were selected and mapped on the structure of a CKR. The comparisons with decoy receptor groups revealed that the detected sites were biased on the intracellular side. In contrast, the sites detected from the comparisons with viral receptor groups were found on both the extracellular and intracellular sides. More sites were found in the ligand binding pocket in the analyses of the viral receptor groups, as compared to the decoy receptor groups. Some of the detected sites were located in the GPCR motifs. For example, the DRY motif of the decoy receptors was often degraded, although the motif of the viral receptors was basically conserved. The observations for the viral receptor groups suggested that the constraints in the pocket region are loose and that the sites on the intracellular side are different from those for the decoy receptors, which may be related to the constitutive signaling activity of the viral receptors.

Molecular characterization of a novel type of prostamide/prostaglandin F synthase, belonging to the thioredoxin-like superfamily.

Prostaglandin F (PGF) ethanolamide (prostamide F) synthase, which catalyzed the reduction of prostamide H(2) to prostamide F(2alpha), was found in mouse and swine brain. The enzyme was purified from swine brain, and its amino acid sequence was defined. The mouse enzyme consisted of a 603-bp open reading frame coding for a 201-amino acid polypeptide with a molecular weight of 21,669. The amino acid sequence placed the enzyme in the thioredoxin-like superfamily with Cys(44) being the active site. The enzyme expressed in Escherichia coli as well as the native enzyme catalyzed not only the reduction of prostamide H(2) to prostamide F(2alpha) but also that of PGH(2) to PGF(2alpha). The V(max) and K(m) values for prostamide H(2) were about 0.25 micromol/min.mg of protein and 7.6 microm, respectively, and those for PGH(2) were about 0.69 micromol/min.mg of protein and 6.9 microm, respectively. Neither PGE(2) nor PGD(2) served as a substrate for this synthase. Based on these data, we named the enzyme prostamide/PGF synthase. Although the enzyme showed a broad specificity for reductants, reduced thioredoxin preferentially served as a reducing equivalent donor for this enzyme. Moreover, Northern and Western blot analyses in addition to the prostamide F synthase activity showed that the enzyme was mainly distributed in the brain and spinal cord, and the immunohistochemical study in the spinal cord showed that the enzyme was found mainly in the cytosol. These results suggest that prostamide/PGF synthase may play an important functional role in the central nervous system.

A study of archaeal enzymes involved in polar lipid synthesis linking amino acid sequence information, genomic contexts and lipid composition.

Cellular membrane lipids, of which phospholipids are the major constituents, form one of the characteristic features that distinguish Archaea from other organisms. In this study, we focused on the steps in archaeal phospholipid synthetic pathways that generate polar lipids such as archaetidylserine, archaetidylglycerol, and archaetidylinositol. Only archaetidylserine synthase (ASS), from Methanothermobacter thermautotrophicus, has been experimentally identified. Other enzymes have not been fully examined. Through database searching, we detected many archaeal hypothetical proteins that show sequence similarity to members of the CDP alcohol phosphatidyltransferase family, such as phosphatidylserine synthase (PSS), phosphatidylglycerol synthase (PGS) and phosphatidylinositol synthase (PIS) derived from Bacteria and Eukarya. The archaeal hypothetical proteins were classified into two groups, based on the sequence similarity. Members of the first group, including ASS from M. thermautotrophicus, were closely related to PSS. The rough agreement between PSS homologue distribution within Archaea and the experimentally identified distribution of archaetidylserine suggested that the hypothetical proteins are ASSs. We found that an open reading frame (ORF) tends to be adjacent to that of ASS in the genome, and that the order of the two ORFs is conserved. The sequence similarity of phosphatidylserine decarboxylase to the product of the ORF next to the ASS gene, together with the genomic context conservation, suggests that the ORF encodes archaetidylserine decarboxylase, which may transform archaetidylserine to archaetidylethanolamine. The second group of archaeal hypothetical proteins was related to PGS and PIS. The members of this group were subjected to molecular phylogenetic analysis, together with PGSs and PISs and it was found that they formed two distinct clusters in the molecular phylogenetic tree. The distribution of members of each cluster within Archaea roughly corresponded to the experimentally identified distribution of archaetidylglycerol or archaetidylinositol. The molecular phylogenetic tree patterns and the correspondence to the membrane compositions suggest that the two clusters in this group correspond to archaetidylglycerol synthases and archaetidylinositol synthases. No archaeal hypothetical protein with sequence similarity to known phosphatidylcholine synthases was detected in this study.

Analysis of membrane stereochemistry with homology modeling of sn-glycerol-1-phosphate dehydrogenase.

Different enantiomeric isomers, sn-glycerol-1-phosphate and sn-glycerol-3-phosphate, are used as the glycerophosphate backbones of phospholipids in the cellular membranes of Archaea and the remaining two kingdoms, respectively. In Archaea, sn-glycerol-1-phosphate dehydrogenase is involved in the generation of sn-glycerol-1-phosphate, while sn-glycerol-3-phosphate dehydrogenase synthesizes the enantiomer in Eukarya and Bacteria. The coordinates of sn-glycerol-3-phosphate dehydrogenase are available, although neither the tertiary structure nor the reaction mechanism of sn-glycerol-1-phosphate dehydrogenase is known. Database searching revealed that the archaeal enzyme shows sequence similarity to glycerol dehydrogenase, dehydroquinate synthase and alcohol dehydrogenase IV. The glycerol dehydrogenase, with coordinates that are available today, is closely related to the archaeal enzyme. Using the structure of glycerol dehydrogenase as the template, we built a model structure of the Methanothermobacter thermautotrophicus sn-glycerol-1-phosphate dehydrogenase, which could explain the chirality of the product. Based on the model structure, we determined the following: (1) the enzyme requires a Zn(2+) ion for its activity; (2) the enzyme selectively uses the pro-R hydrogen of the NAD(P)H; (3) the putative active site and the reaction mechanism were predicted; and (4) the archaeal enzyme does not share its evolutionary origin with sn-glycerol-3-phosphate dehydrogenase.

Identification of cryptochrome DASH from vertebrates.

A new type of cryptochrome, CRY-DASH, has been recently identified. The CRY-DASH proteins constitute the fifth subfamily of the photolyase/cryptochrome family. CRY-DASHs have been identified from Synechocystis sp. PCC 6803, Vibrio cholerae, and Arabidopsis thaliana. The Synechocystis CRY-DASH was the first cryptochrome identified from bacteria, and its biochemical features and tertiary structure have been extensively investigated. To determine how broadly the subfamily is distributed within living organisms, we searched for new CRY-DASH candidates within several databases. We found five sequences as new CRY-DASH candidates, which are derived from four marine bacteria and Neurospora crassa. We also found many CRY-DASH candidates from the EST databases, which included sequences from fish and amphibians. We cloned and sequenced the cDNAs of the zebrafish and Xenopus laevis candidates, based on the EST sequences. The proteins encoded by the two genes were purified and characterized. Both proteins contained folate and flavin cofactors, and have a weak DNA photolyase activity. A phylogenetic analysis revealed that the seven candidates actually belong to the new type of cryptochrome subfamily. This is the first report of the CRY-DASH members from vertebrates and fungi.

Two novel genes expressed in Xenopus germ line: characteristic features of putative protein structures, their gene expression profiles and their possible roles in gametogenesis and embryogenesis.

We compared the secondary spermatogonia and the primary spermatocytes of Xenopus for the proteins in their microsomal fractions and identified a newly synthesized protein (94 kDa) and three other proteins (99, 85, and 72 kDa) which increased their amount after entering the meiotic phase. These four proteins were used as antigens to produce polyclonal antibody which was found to react with the four proteins as well as two other proteins (208 and 60 kDa). Immunoscreening of Xenopus testis cDNA library with this polyclonal antibody yielded two cDNA clones (Xmegs and Xtr) encoding novel proteins. Xmegs mRNA was specifically expressed in the spermatogenic cells from the mid-pachytene stage to completion of two meiotic divisions. The putative Xmegs protein contained 19 tandem repeats of 26 amino acid residues rich in proline as well as potential phosphorylation sites (i.e., serine and threonine residues). Around this repetitive area, we found five PEST sequences known as a proteolytic signal to target protein for degradation. The presence of PEST sequences was believed to allow protein levels to closely parallel mRNA abundance. These results suggested the possible role of this novel protein in the regulation of two meiotic divisions specific to the spermatogenesis in a phosphorylation- and/or dephosphorylation-dependent manner. On the other hand, Xtr mRNA was expressed in both spermatogenic and oogenic cells except for round spermatids and the later stage cells. This mRNA was also expressed in the early stage embryos and its amount was kept constant from the St. I oocyte to the gastrula stage and decreased thereafter. The putative Xtr protein contained four complete and one partial tudor-like domains that were discovered in Drosophila tudor protein which plays an important role in PGC differentiation and abdominal segmentation. The characteristic expression profile of Xtr and the protein structure similar to the Drosophila tudor protein suggested its possible role in the progression of meiosis and PGC differentiation.

Archaeal-type rhodopsins in Chlamydomonas: model structure and intracellular localization.

Phototaxis in the unicellular green alga Chlamydomonas reinhardtii is mediated by rhodopsin-type photoreceptor(s). Recent expressed sequence tag database from the Kazusa DNA Research Institute has provided the basis for unequivocal identification of two archaeal-type rhodopsins in it. Here we demonstrate that one is located near the eyespot, wherein the photoreceptor(s) has long been thought to be enriched, along with the results of bioinformatic analyses. Secondary structure prediction showed that the second putative transmembrane helices (helix B) of these rhodopsins are rich in glutamate residues, and homology modeling suggested that some additional intra- or intermolecular interactions are necessary for opsin-like folding of the N-terminal ca. 300-aa membrane spanning domains of 712 and 737-aa polypeptides. These results complement physiological and electrophysiological experiments combined with the manipulation of their expression [O.A. Sineshchekov, K.H. Jung, J.H. Spudich, Proc. Natl. Sci. USA 99 (2002) 8689; G. Nagel, D. Olig, M. Fuhrmann, S. Kateriya, A.M. Musti, E. Bamberg, P. Hegemann, Science 296 (2002) 2395].

Identification of a new cryptochrome class. Structure, function, and evolution.

Cryptochrome flavoproteins, which share sequence homology with light-dependent DNA repair photolyases, function as photoreceptors in plants and circadian clock components in animals. Here, we coupled sequencing of an Arabidopsis cryptochrome gene with phylogenetic, structural, and functional analyses to identify a new cryptochrome class (cryptochrome DASH) in bacteria and plants, suggesting that cryptochromes evolved before the divergence of eukaryotes and prokaryotes. The cryptochrome crystallographic structure, reported here for Synechocystis cryptochrome DASH, reveals commonalities with photolyases in DNA binding and redox-dependent function, despite distinct active-site and interaction surface features. Whole genome transcriptional profiling together with experimental confirmation of DNA binding indicated that Synechocystis cryptochrome DASH functions as a transcriptional repressor.