The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
132 巻, 5 号
選択された号の論文の23件中1~23を表示しています
  • Yasuhito Shirai, Naoaki Saito
    2002 年 132 巻 5 号 p. 663-668
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    The biological function of protein kinase C (PKC) depends on its catalytic activity and spatial localization. Its catalytic competence and localization in the resting state are regulated by serine/threonine phosphorylations, i.e., “maturation.” Upon stimulation of various receptors, PKC is catalytically activated by several activators including diacylglycerol. In addition, PKC often translocates to particular subcellular compartments including the plasma membrane and Golgi complex, and such translation is here referred to as “targeting.” In short, the physiological function of PKC is controlled by the three events: maturation, catalytic activation, and targeting. Catalytic activation and targeting contribute to temporal, spatial, and isotype-specific regulation of PKC. This review summarizes the evidence for the role of these three events in the isotype-specific activation of PKC, with particular emphasis on catalytic activation and targeting by lipid mediators.
  • Shigeru Nakashima
    2002 年 132 巻 5 号 p. 669-675
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    Protein kinase Cα (PKCα) is a serine/threonine kinase and a member of the conventional (classical) PKCs (cPKCs), which have four conserved (C1 to C4) regions. This ubiquitously expressed PKC isotype is activated in response to many different kinds of stimuli and translocates from cytosol to the specialized cellular compartments (nucleus, focal adhesion, caveolae, etc.) where it is presumed to work. Therefore, PKCα has been implicated in a variety of cellular functions including proliferation, apoptosis, differentiation, motility, and inflammation. However, the responses induced by activation or overexpression of PKCα vary depending on the types, and sometimes conditions, of cells. For example, in some types of cells, PKCα is implicated in cell growth. In contrast, it may play a role in cell cycle arrest and differentiation in other types of cells. Therefore, alterations of cell responses induced by PKCα are not an intrinsic property of this isoform. The responses are modulated by dynamic interactions with cell-type specific factors: substrates, modulators and anchoring proteins.
  • Toshiaki Kawakami, Yuko Kawakami, Jiro Kitaura
    2002 年 132 巻 5 号 p. 677-682
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    PKCβI and PKCβII are DAG- and Ca2+-dependent conventional or classical isoforms of protein kinase C. Generated by alternative splicing from a single gene, they differ at their C-terminal 50 (βI) or 52 (βII) residues. They are expressed as major PKC isoforms in a variety of tissues, and thus the functions ascribed to “PKC” based on early studies using phorbol esters and PKC inhibitors could be attributed to them. As tools to probe into isoform-specific functions have recently become available, our understanding of the normal functions of these isoforms has dramatically increased. This minireview will focus mainly on two areas of signal transduction where the roles of PKCβI and PKCβII are relatively well-characterized: immunoreceptor and insulin receptor systems. Their involvement in disorders due to pertubations in these signaling systems, i.e., immunodeficiencies and diabetes, is also reviewed. Finally, patterns of PKC action in these and other biologic systems are discussed.
  • Naoaki Saito, Yasuhito Shirai
    2002 年 132 巻 5 号 p. 683-687
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    The gamma isotype of protein kinase C (PKCγ) is a member of the classical PKC (cPKC) subfamily which is activated by Ca2+ and diacyiglycerol in the presence of phosphatidylserine. Physiologically, PKCγ is activated by a mechanism coupled with receptor-mediated breakdown of inositol phospholipid as other cPKC isotypes such as PKCα and PKCβ. PKCγ is expressed solely in the brain and spinal cord and its localization is restricted to neurons, while PKCα and PKCβ are expressed in many tissues in addition to the brain. Within the brain, PKCγ is the most abundant in the cerebellum, hippocampus and cerebral cortex, where the existence of neuronal plasticity has been demonstrated. Pharmacological and electrophysiological studies have shown that several neuronal functions, including long term potentiation (LTP) and long term depression (LTD), specifically require PKCγ. Generation of mice deficient in PKCγ provided more information regarding the physiological functions of this isotype. PKCγ deficient mice (i) have modified long term potentiation (LTP) in hippocampus, (ii) exhibit mild deficits in spatial and contextual learning (iii) exhibit impaired motor coordination due to persistent multiple innervations of climbing fibers on Purkinje cells, (iv) show attenuation of opioid receptor activation, and (v) show decreased effects of ethanol on type A of γ-aminobutyric acid (GABA) receptor. Furthermore, a point mutation in the PKCγ gene may contribute to retinitis pigmentosa and Parkinsonian syndrome. This article reviews the specific functions of this neuron-specific isotype of PKC in neuronal signal transduction.
  • Kiyotaka Shiba, Tomoko Hatada, Yuki Takahashi, Tetsuo Noda
    2002 年 132 巻 5 号 p. 689-696
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    Faster and more efficient searches of a huge protein sequence space for the purpose of conducting experiments in protein evolution can be achieved through the development of a block shuffling-based evolution system. One of the key components of such a system is the accurate and efficient linkage of gene units. Here we introduce a new method that allows accurate and controllable linkage of microgene blocks. This method employs a thermostable DNA ligase that links two single-stranded microgene blocks when they hybridize a complementary guide oligonucleotide. At high temperature, the ligation of the microgene units is fully dependent on the guide oligonucleotide, which can exclude undesired polymer formation, including the incorporation of microgenes having illegitimate sizes and “head-to-head” and “tail-to-tail” ligation of blocks. We were also able to assemble three microgene units using two guide oligonucleotides. Using this method of controllable linkage should facilitate further development of a step-by-step system for the polymerization of gene blocks, leading to a versatile block shuffling-based protein evolution system.
  • Chie Sakanaka
    2002 年 132 巻 5 号 p. 697-703
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    β-Catenin transduces cytosolic signals to the nucleus in the Wnt pathway. The Wnt ligand stabilizes cytosolic β-catenin protein, preventing its phosphorylation by inhibiting glycogen synthase kinase 3 (GSK3). Serine-33 and -37 of β-catenin are GSK3 phosphorylation sites that serve as recognition sites for the β-TRCP-ubiquitin ligase complex, which ultimately triggers β-catenin degradation. Mutations at those two sites, as well as in Ser-45, stabilize β-catenin. Recently, casein kinase Iε (CKIε) has been shown to be a positive regulator of the Wnt pathway. Its action mechanism, however, remains unknown. Here I show that Ser-45 is phosphorylated not by GSK3 but by CKIε. Axin, a scaffold protein that binds CKIε and β-catenin, enhances this CKIε-mediated phosphorylation. Overexpression of CKIε in cells increases the amount of β-catenin phosphorylated at Ser-45. Ser-45 phosphorylated β-catenin is a better substrate for GSK3, which suggests that CKIε and GSK3 may co-operate in destabilizing β-catenin. In spite of the fact that CKIε was found as a positive regulator of the Wnt pathway, mutational analysis suggests that mutation of Ser-45 regulates β-catenin stability by inhibiting the ability of GSK3 to phosphorylate Ser-33 and -37, thereby disrupting the interaction between β-catenin, β-TRCP and Axin. I propose that phosphorylation of Ser-45 by CKIε plays an important role in regulating β-catenin stability.
  • Yoshiaki Takahashi, Takashi Mitsuma, Shigeru Hirayama, Shoji Odani
    2002 年 132 巻 5 号 p. 705-711
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    The interaction of ribosomal proteins with mRNA in the 40S initiation complex was examined by chemical cross-linking. 40S initiation complexes were formed by incubating rat liver [3H]Met-tRNAi, rat liver 40S ribosomal subunits, rabbit globin mRNA, and partially purified initiation factors of rabbit reticulocytes in the presence of guanylyl (β, γ-methylene)-diphosphonate. The initiation complexes were then treated with 1, 3-butadiene diepoxide to introduce crosslinks between the mRNA and proteins. The covalent mRNA-protein conjugates were isolated by chromatography on an oligo (dT) cellulose column in the presence of sodium dodecyl sulfate, followed by sucrose density gradient centrifugation. Proteins cross-linked to the mRNA were labeled with Na125I, extracted by extensive ribonuclease digestion, and analyzed by two-dimensional and diagonal polyacrylamide gel electrophoresis. Three ribosomal proteins, S6, S8, and S23/S24, together with small amounts of S3/S3a, S27, and S30, were identified as the protein components cross-linked to the globin mRNA protein complex, and were shown to attach directly to the mRNA. It is suggested that these proteins constitute the ribosomal binding site for mRNA in the 40S initiation complex.
  • Yasushi Ohki, Yoshiya Ikawa, Hideaki Shiraishi, Tan Inoue
    2002 年 132 巻 5 号 p. 713-718
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    The Tetrahymena group I intron ribozyme folds into a complex three dimensional structure for performing the self-splicing reaction. Catalysis depends on its core structure comprising two helical domains, P4-P6 and P3-P7. The two domains are joined by three sets of conserved base-triple (s) and other tertiary interactions. We found that the disruption of J8/7 X P4, one such conserved base-triple, causes the catalytic ability to deteriorate without altering the folding rate. This suggests that the base-triple stabilizes the active structure of the ribozyme but plays no significant role in RNA folding. By combining the present and previous results, it can be concluded that three sets of conserved base-triples play distinct roles in the Tetrahymena ribozyme.
  • Aya Fukui, Tomoko Yuasa-Nakagawa, Yusuke Murakami, Kenji Funami, Natue ...
    2002 年 132 巻 5 号 p. 719-728
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    Human C4b-binding protein (C4bp) facilitates the factor I-mediated proteolytic cleavage of the active forms of complement effectors C3b and C4b into their inactive forms. C4bp comprises a disulfide-linked heptamer of α-chains with complement (C) regulatory activity and a β-chain. Each α-chain contains 8 short consensus repeat (SCR) domains. Using SCR-deletion mutants of recombinant multimeric C4bp, we identified the domains responsible for the C3b/C4b-binding and C3b/C4b-inactivating cofactor activity. The C4bp mutant with deletion of SCR2 lost the C4b-binding ability, as judged on C3b/ C4b-Sepharose binding assaying and ELISA. In contrast, the essential domains for C3b-binding extended more to the C-terminus, exceeding SCR4. Using fluid phase cofactor assaying and deletion mutants of C4bp, SCR2 and 3 were found to be indispensable for C4b cleavage by factor I, and SCR1 contributed to full expression of the factor I-mediated C4b cleaving activity. On the other hand, SCR1, 2, 3, 4, and 5 participated in the factor I-cofactor activity for C3b cleavage, and SCR2, 3, and 4 were absolutely required for C3b inactivation. Thus, different sets of SCRs participate in C3b and C4b inactivation, and the domain repertoire supporting C3b cofactor activity is broader than that supporting C4b inactivation by C4bp and factor I. Furthermore, the domains participating in C3b/C4b binding are not always identical to those responsible for cofactor activity. The necessity of the wide range of SCRs in C3b inactivation compared to C4b inactivation by C4bp and factor I may reflect the physiological properties of C4bp, which is mainly directed to C4b rather than C3b.
  • Taishin Takuma, Toshiya Arakawa, Miki Okayama, Itaru Mizoguchi, Akihik ...
    2002 年 132 巻 5 号 p. 729-735
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    SNARE proteins are widely accepted to be involved in the docking and fusion process of intracellular vesicle trafficking. VAMP-2, syntaxin-4, and SNAP-23 are plausible candidate SNARE proteins for non-neuronal exocytosis. Thus, we examined the localization, protein-protein interaction, and intracellular trafficking of these proteins by expressing them as green fluorescent protein (GFP)- and FLAG-tagged fusion proteins in various cells, including HSY cells, a human parotid epithelial cell line. GFP-VAMP-2 was expressed strongly in the Golgi area and weakly on the plasma membrane. Although GFP-SNAP-23 seemed to be expressed universally in the cytosol, the GFP signal was clearly seen on the plasma membrane, when soluble GFP-SNAP-23 was removed by treatment with saponin. GFP-syntaxin-4 was undetectable on the plasma membrane but was strongly expressed on unidentified unusually large vesicles. GFP-syntaxin-4 without its transmembrane domain was still incompletely soluble and observed as aggregates. When syntaxin-4 and munc18c were coexpressed, syntaxin-4 was translocated at least in part to the plasma membrane. The protein-protein interaction between syntaxin-4 and VAMP-2 with their transmembrane domains was markedly inhibited on coexpression of munc18c. These results suggest that munc18c plays an important role in the trafficking of syntaxin-4 to its proper destination by preventing premature interactions with other proteins, including SNARE proteins.
  • Takashi Maeda, Kazutoshi Mahara, Midori Kitazoe, Junichiro Futami, Aik ...
    2002 年 132 巻 5 号 p. 737-742
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    There have been some attempts to develop immunotoxins utilizing human RNase as a cytotoxic domain of antitumor agents. We have recently shown that only human RNase 3 (eosinophil cationic protein, ECP) among five human pancreatic-type RNases excels in binding to the cell surface and has a growth inhibition effect on several cancer cell lines, even though the RNase activity of RNase 3 is completely inhibited by the ubiquitously expressed cytosolic RNase inhibitor. This phenomenon may be explained by that RNase 3 is very stable against proteolytic degradation because RNase 3 internalized through endocytosis could have a longer life time in the cytosol, resulting in the accumulation of enough of it to exceed the concentration of RNase inhibitor, which allows the degradation of cytosolic RNA molecules. Thus, we compared the stabilities of human pancreatic-type RNases (RNases 1-5) and bovine RNase A by means of guanidium chloride-induced denaturation experiments based on the assumption of a two-state transition for unfolding. It was demonstrated that RNase 3 is extraordinarily stabler than either RNase A or the other human RNases (by more than 25kJ/mol). Thus, our data suggest that in addition to its specific affinity for certain cancer cell lines, the stability of RNase 3 contributes to its unique cytotoxic effect and that it is important to stabilize a human RNase moiety through protein engineering for the design of human RNasebased immunotoxins.
  • Masaaki Narita, David M. Holtzman, Anne M. Fagan, Mary Jo LaDu, Li Yu, ...
    2002 年 132 巻 5 号 p. 743-749
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    Apolipoprotein E (apoE), an apoprotein involved in lipid transport in both the plasma and within the brain, mediates the binding of lipoproteins to members of the low density lipoprotein (LDL) receptor family including the LDL receptor and the LDL receptor-related protein (LRP). ApoE/LRP interactions may be particularly important in brain where both are expressed at high levels, and polymorphisms in the apoE and LRP genes have been linked to AD. To date, only apoE-enriched lipoproteins have been shown to be LRP ligands. To investigate further whether other, more lipid-poor forms of apoE interact with LRP, we tested whether lipid-free apoE in the absence of lipoprotein particles interacts with its cell-surface receptors. No detectable lipid was found associated with bacterially expressed and purified apoE either prior to or following incubation with cells when analyzed by electrospray ionization mass spectrometry. We found that the degradation of lipid-poor 125I-apoE was significantly higher in wild type as compared to LRP-deficient cells, and was inhibited by receptor-associated protein (RAP). In contrast, 125I-apoE-enriched β-VLDL was degraded by both LRP and the LDL receptor. When analyzed via a single cycle of endocytosis, 125I-apoE was internalized prior to its subsequent intracellular degradation with kinetics typical of receptor-mediated endocytosis. Thus, we conclude that a very lipid-poor form of apoE can be catabolized via cell surface LRP, suggesting that the conformation of apoE necessary for recognition by LRP can be imposed by situations other than an apoE-enriched lipoprotein.
  • Yuya Yokozawa, Hitomi Tamai, Shuntaro Tatewaki, Takaho Tajima, Takahid ...
    2002 年 132 巻 5 号 p. 751-758
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    We have cloned four cDNAs encoding astacin-like squid metalloproteases (ALSMs)-I and -II from the Japanese common squid and AlSMs-I and -III from the spear squid. Analysis of the deduced amino acid sequences revealed that ALSMs possess a signal peptide and a pro-sequence followed by an astacin-like catalytic domain and an MAM (_??_eprin, _??_5 protein, receptor protein-tyrosine phosphatase _??_) domain. Phylogenetic analysis revealed that ALSM corresponds to a new cluster of astacins. To analyze the function of the MAM domain, wild-type ALSM and an MAM-truncated mutant were expressed in a baculovirus expression system. The expressed protein encoding full-length ALSM hydrolyzed myosin heavy chain as effectively as native ALSM, whereas the MAM-truncated mutant possessed no protease activity, suggesting that the MAM domain contributes to substrate recognition. ALSM has been isolated from squid liver and mantle muscle. However, analysis with a specific antibody generated against ALSM indicated the presence of ALSM in a wide variety of tissues. ALSM was located in the extracellular matrix of mantle muscle cells. Thus, ALSM is a secreted protease, as are other members of the astacin family. The extracellular localization raises the possibility of substrates other than myosin. The physiological role of ALSM remains unknown, at this time.
  • Rie Omi, Hiroyuki Mizuguchi, Masaru Goto, Ikuko Miyahara, Hideyuki Hay ...
    2002 年 132 巻 5 号 p. 759-765
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    Imidazole glycerol phosphate synthase (IGPs) catalyzes the fifth step in the histidine biosynthetic pathway located at the branch point to de novo purine biosynthesis. IGPs is a multienzyme comprising glutaminase and synthase subunits. The glutaminase activity, which hydrolyzes glutamine to give ammonia, is coupled with substrate binding to the synthase subunit. The three-dimensional structure of the IGPs from Thermus thermophilus HB8 has been determined at 2.3 Â resolution, and compared with the previously determined structures for the yeast and Thermotoga maritima enzymes. The structure of each subunit is similar to that of the corresponding domain in the yeast enzyme or subunit in the T. maritima enzyme. However, the overall structure is significantly different from the yeast and T. maritima enzymes, indicating that IGPs may change the relative orientation between the two subunits and close the glutaminase site upon glutamine binding. The putative ammonia tunnel, which carries nascent ammonia from glutaminase to the synthase site, has a closed gate comprising a cyclic salt bridge formed by four charged residues of the synthase subunit. The side chain of Lys100 in the cyclic salt bridge might change its side chain direction to form new interactions with the main chain carbonyl group of glutamine from the synthase subunit and the hydoxyl group of tyrosine from the glutaminase subunit, resulting in the opening of the gate for ammonia transfer.
  • Noriko Takahashi, Toshihiro Ohba, Shin-ichi Togashi, Tetsuya Fukui
    2002 年 132 巻 5 号 p. 767-774
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    Fenretinide, N-(4-hydroxyphenyl) retinamide (4-HPR), is a synthetic amide of all-trans-retinoic acid (RA), which inhibits cell growth, induces apoptosis, and is an antioxidant, and cancer chemopreventive and antiproliferative agent. These findings led us to investigate which structural component of 4-HPR contributes to these potent activities. Our approach was to examine 4-aminophenol (4-AP), p-methylaminophenol (p-MAP), and p-acetaminophen (p-AAP). It was found that vitamin E, 4-AP and p-MAP scavenge α, α-diphenyl-β-picrylhydrazyl (DPPH) radicals in a 1:2 ratio, in contrast to 4-HPR and p-AAP, for which 1:1 and 1:0.5 ratios were observed relative to DPPH radicals. However, RA was inactive. Lipid peroxidation in rat liver microsomes was reduced by compounds (RA>p-MAP=4-HPR>4-AP) in a dose-dependent manner, while p-AAP was inactive. In addition, both p-MAP and 4-HPR are potent inhibitors of cell growth and inducers of apoptosis in HL60 cells. p-MAP exhibits the same level of antiproliferative activity as 4-HPR against HL60R cells, which are a resistant clone against RA, and it inhibits the growth of various cancer cell lines (MCF-7, MCF-7/AdrR, HepG2, and DU-145) to an extent greater than 4-AP and p-AAP, but is less potent than 4-HPR. Thus, although the antioxidant activity of p-MAP is more potent than that of 4-HPR, p-MAP is less potent than 4-HPR in anticancer activity. These results suggest that both the anticancer and antioxidative activities shown by 4-HPR are due to the structure of p-MAP. The retinoyl residue or long alkyl chain substituent attached to an aminophenol may be significant for anticancer properties.
  • Takahide Aburatani, Hiroshi Ueda, Teruyuki Nagamune
    2002 年 132 巻 5 号 p. 775-782
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    Although the cooperativity of the VH and VL domains of an antibody in antigen binding has been extensively studied, the interaction between the VH and VL domains had not received sufficient attention. To systematically investigate the relationship between the amino acid sequence and VH/VL interaction strength, we here used a set of anti-bovine serum albumin antibodies having a single human framework for VH (V3-23/DP-47 and JH4b) and Vk (O12/O2/DPK9 and Jk1), but with different VH/VL interaction strengths. By phage display of a VH mini-library and analysis of the interaction of amino acids with immobilized VL fragments, the residue at H95 (Kabat numbering) at the beginning of seven CDR H3 residues was found to play a key role in determining the VH/VL interaction. On saturation mutagenesis of H95, Gly showed the strongest interaction, while Asp, Asn, and Glu showed lesser interaction in that order. The generality of the rule was confirmed by the test with urine-derived human L chain instead of a particular VL. The results demonstrate that H95 plays a central role in deciding the VH/VL interaction of human Fvs that have most commonly found frameworks.
  • Masaru Hamaoki, Kyoko Hiramatsu, Setsuo Suzuki, Atsuo Nagata, Masao Ka ...
    2002 年 132 巻 5 号 p. 783-788
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    We obtained monoclonal antibodies against N1, N12-diacetylspermine (DiAcSpm) and N1, N8-diacetylspermidine (DiAcSpd), and developed two systems of competitive ELISA that utilize the antibodies and a common enzyme-labeled antigen to measure these diacetylpolyamines. Cross-reactions with N1-acetylspermidine in the assay of DiAcSpm and with N8-acetylspermidine in the assay of DiAcSpd were as low as 0.26 and 0.6%, respectively, and were judged to be insignificant in clinical use for measuring urinary diacetylpolyamines. These assays were used to assess diurnal variations in diacetylpolyamine excretion in urine to show that the excretion of diacetylpolyamines after normalization for the concentration of creatinine is stable over a day with only minimal diurnal variation.
  • Takeshi Murata, Yasushi Yoshikawa, Toshiaki Hosaka, Kazuma Takase, Yos ...
    2002 年 132 巻 5 号 p. 789-794
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    Enterococcus hirae V-ATPase, in contrast to most V-type ATPases, is resistant to N-ethylmaleimide (NEM). Alignment of the amino acid sequences of NtpA suggests that the NEM-sensitive Cys of V-type ATPases is replaced by Ala in E. hirae V-ATPase. Consistent with this prediction, the V-ATPase became sensitive upon substitution of the Ala with Cys. The three-dimensional structure of the NtpB subunit of V-ATPase was modeled based on the structure of the corresponding subunit (α subunit) of bovine F1-ATPase by homology modeling. Overall, the 3D structure of the subunit resembled that of α subunit of bovine F1-ATPase. The NtpB subunit, which lacks the P-loop consensus sequence for nucleotide binding, was predicted to bind a nucleotide at the modeled nucleotide-binding site. Experimental data supported the prediction that the E. hirae V-ATPase had about six nucleotide-binding sites.
  • Shigeki Shimba, Kazuo Komiyama, Itaru Moro, Masakatsu Tezuka
    2002 年 132 巻 5 号 p. 795-802
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    The arylhydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxic and biological effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although the physiological ligand for the AhR has not yet been identified, several reports have suggested that the AhR may play important roles not only in the regulation of xenobiotic metabolism but also in the maintenance of homeostatic functions [Singh et al. (1996) Arch. Biochem. Biophys. 329, 47-55; Crawford et al. (1997) Mol. Pharmacol. 52, 921-927; Chang et al. (1998) Mol. Cell. Biol. 18, 525-535]. Several lines of evidence suggest that one of the possible physiological roles of the AhR is regulation of cell proliferation. In this study, we first showed that treatment of A549 cells with the AhR agonist stimulates cell proliferation. The effect was antagonized by co-treatment with α-naphthoflavone. To obtain direct evidence that the AhR regulates cell proliferation, we isolated the clones that overexpress the AhR. These clones grow faster than control cells, and the rate of growth is proportional to the amount of the AhR. Cell cycle analysis revealed that the acceleration of cell growth by overexpression of the AhR is most probably due to shortening of the late M to S phases. Studies on the expression profiles of cell cycle regulators showed that the AhR or AhR ligand induces the expression of DP2, PCNA, and RFC38. DP2 is the transcription factor that forms the functional dimer with E2F and regulates the expression of several genes involved in DNA synthesis. Interestingly, both PCNA and RFC38 are target genes of E2F and the DP complex. Also, both of these factors are involved in regulating DNA polymerase δ activity. E2F activity was substantially increased in both the AhR-overexpressing cells and the AhR-agonist treated cells, suggesting that AhR-activated E2F/DP2 may induce the expression of PCNA and RFC38 and subsequent DNA synthesis. Down-regulation of the expression of the Arnt by RNAi diminished the effects of the AhR on the cell proliferation of the A549 cells. Consequently, we conclude that the AhR, presumably in collaboration with the Arnt, activates the DNA synthesis and the subsequent cell proliferation in A549 cells.
  • Yuki Jitsuhara, Teruko Toyoda, Tomokazu Itai, Haruki Yamaguchi
    2002 年 132 巻 5 号 p. 803-811
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    It has recently become apparent that high-mannose type N-glycans directly promote protein folding, whereas complex-type ones play a crucial role in the stabilization of protein functional conformations through hydrophobic interactions with the hydrophobic protein surfaces. Here an attempt was made to understand more deeply the molecular basis of these chaperone-like functions with the aid of information obtained from spacefill models of N-glycans. The promotion of protein folding by high-mannose N-glycans seemed to be based on their unique structure, which includes a hydrophobic region similar to the cyclodextrin cavity. The promotive features of high-mannose N-glycans newly observed under various conditions furnished strong support for the view that both intra- and extramolecular high-mannose N-glycans are directly involved in the promotion of protein folding in the endoplasmic reticulum. Further, it was revealed that the N-acetyllactosamine units in complex-type N-glycans have an amphiphilic structure and greatly contribute to the formation of extensive hydrophobic surfaces and, consequently, to the N-glycan-protein hydrophobic interactions. The processing of high-mannose type N-glycans to complex-type ones seems to be an ingenious device to enable the N-glycans to perform these two chaperone-like functions.
  • Hung-Ming Huang, Ming F. Tam, Tsuey-Chyi S. Tam, Da-Huang Chen, Mingli ...
    2002 年 132 巻 5 号 p. 813-818
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    We investigated the global distribution of methylaccepting proteins in lymphoblastoid cells by two-dimensional (2-D) gel electrophoresis. The 2-D electrophoreograms of normal and hypo-methylation (cells grown with a methyltransferase inhibitor adenosine dialdehyde) protein extracts did not exhibit significant differences. However, in vitro methylation of the hypomethylated extracts in the presence of the methyl-group donor S-adenosyl-[methyl-3H]-methionine revealed close to a hundred signals. Less than one-fifth of the signals could be correlated with protein stains, indicating that most of the methylaccepting proteins are low abundant ones. We analyzed six of the spots that can be correlated with protein stains and suggested their identities. Among these putative protein methyl acceptors, three are heterogeneous nuclear ribonucleoproteins (hnRNPA2/B1 and hnRNP K) that are reportedly methylated in their arginine- and glycine-rich RGG motifs.
  • Nobuyuki Kasai, Yoshiyuki Mizushina, Fumio Sugawara, Kengo Sakaguchi
    2002 年 132 巻 5 号 p. 819-828
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
    Previously, we reported the three-dimensional molecular interactions of nervonic acid (NA) with mammalian DNA polymerase β (pol β) [Mizushina et al. (1998) J. Biol. Chem. 274, 25599-25607]. By three-dimensional structural model analysis and comparison with the spatial positioning of specific amino acids binding to NA on pol β (Leu11, Lys35, His51, and Thr79), we obtained supplementary information that allowed us to build a structural model of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT). In HIV-1 RT, Leu100, Lys65, His235, and Thr386 corresponded to these four amino acid residues. These results suggested that the NA binding domains of pol β and HIV-1 RT are three-dimensionally very similar. The effects of NA on HIV-1 RT are thought to be same as those on pol β in binding to the rhombus of the four amino acid residues. NA dose-dependently inhibited the HIV-1 RT activity. For binding to pol β, the kinetics were competitive when the rhombus was present on the DNA binding site. However, as the rhombus in HIV-1 RT was not present in the DNA binding site, the three-dimensional structure of the DNA binding site must be distorted, and subsequently the enzyme is inhibited non-competitively.
  • 2002 年 132 巻 5 号 p. 829
    発行日: 2002年
    公開日: 2008/06/30
    ジャーナル フリー
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