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Volume 134 (2003)
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Volume 134, Issue 2
Displaying 1-17 of 17 articles from this issue
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Kazuhiro Iwai2003 Volume 134 Issue 2 Pages 175-182
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSProtein oxidation is a natural consequence of aerobic metabolism in cells. Oxidative modification of amino acid residues of proteins causes to lose activity or function of proteins. Organisms have thus developed pathways to remove oxidized proteins by rapid protein degradation. These pathways are important components in cellular quality control mechanisms. It has been suggested that oxidized proteins are degraded by the proteasome. However, whether ubiquitylation is necessary for the degradation of oxidized proteins remains a controversial issue. We have recently identified HOIL-1 (heme-oxidized IRP2 ubiquitin ligase-1) as an E3 ligase that recognizes a protein that has been oxidized by iron. This review describes the recent progress made in understanding the ubiquitin-proteolytic pathway and the regulation of iron metabolism. The process involved in eliminating oxidized proteins and the possible roles that HOIL-1 ubiquitin ligase may play in these processes are discussed.View full abstractDownload PDF (3864K) -
Yukiko Yoshida2003 Volume 134 Issue 2 Pages 183-190
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe endoplasmic reticulum (ER) provides a quality-control system for newly synthesized secretory and membrane proteins. Any improperly folded or incompletely assembled oligomers are retained in the ER, and they are retro-translocated into the cytosol when misfolding persists, where they are destroyed by the proteasome through ubiquitylation. This disposal process is called ER-associated degradation (ERAD). Although much is known about the fate of ERAD substrates near the point of degradation, little information is available about how these proteins are recognized, retained, and targeted for translocation and ubiquitylation machinery. Recent studies indicate that N-linked oligosaccharides attached to nascent proteins function as tags for several processes of a quality-control system, such as individual steps of ER-retention, selection for ERAD substrates, and ubiquitylation. In this review, I describe recent advances in the molecular basis of the ERAD system, particularly those mediated by N-glycan recognition molecules.View full abstractDownload PDF (5135K) -
Eriko Tomitsuka, Hiroko Hirawake, Yu-ichi Goto, Masafumi Taniwaki, Shi ...2003 Volume 134 Issue 2 Pages 191-195
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSSuccinate-ubiquinone reductase (complex II) is an important enzyme complex in both the tricarboxylic acid cycle and aerobic respiration. A recent study showed that defects in human complex II are associated with cancers as well as mitochondrial diseases. Mutations in the four subunits of human complex II are associated with a wide spectrum of clinical presentations. Such tissue-specific clinical symptoms suggest the presence of multiple isoforms of the subunits, but subunit isoforms have not been previously reported. In the present study, we identified two distinct cDNAs for the human flavoprotein subunit (Fp) from a single individual, and demonstrated expression of these two isoforms in skeletal muscle, liver, brain, heart and kidney. Interestingly, one of the Fp isoforms was encoded as an intronless gene.View full abstractDownload PDF (3241K) -
Yasuo Shiota, Masatoshi Ikeda, Fumie Hashimoto, Hidenori Hayashi2003 Volume 134 Issue 2 Pages 197-202
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe effects of two peroxisome proliferators, gemfibrozil and clofibrate, on syntheses of dolichol and cholesterol in rat liver were investigated. Gemfibrozil did not affect the overall content of dolichyl phosphate, but it changed the chain-length distribution of dolichyl phosphate, increasing the levels of species with shorter isoprene units. Gemfibrozil suppressed synthesis of dolichyl phosphate from [3H] mevalonate and [3H] farnesyl pyrophosphate in rat liver. In contrast, clofibrate increased the content of dolichol (free and acyl ester forms). It remarkably enhanced dolichol synthesis from mevalonate, but did not affect dolichol synthesis from farnesyl pyrophosphate. Gemfibrozil elevated cholesterol synthesis from [14C] acetate, but did not affect the synthesis from mevalonate. Clofibrate suppressed cholesterol synthesis from acetate, but did not affect cholesterol synthesis from mevalonate. These results suggest that gemfibrozil suppresses synthesis of dolichyl phosphate by inhibiting, at the least, the pathway from farnesyl pyrophosphate to dolichyl phosphate. As a result, the chain-length pattern of dolichyl phosphate may show an increase in shorter isoprene units. Clofibrate may increase the content of dolichol by enhancing dolichol synthesis from mevalonate. Gemfibrozil may increase cholesterol synthesis by activating the pathway from acetate to mevalonate. Unlike gemfibrozil, clofibrate may decrease cholesterol synthesis by inhibiting the pathway from acetate to mevalonate.View full abstractDownload PDF (759K) -
Michito Hamada, Takashi Moriguchi, Tomomasa Yokomizo, Naoki Morito, Ch ...2003 Volume 134 Issue 2 Pages 203-210
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe b-Zip transcription factor MafB is an essential determinant of neural development and an inducer of monocytic differentiation. The MafB protein is expressed in a variety of tissues including the developing spinal cord, retina, myelomonocytic line-ages of hematopoietic cells, and peritoneal macrophages. However, the tissue-specific regulatory mechanism of mafB gene expression and its biological relevance have not been examined in detail. Here, we report, for the first time, analysis of the regulatory mechanism and tissue-specific expression of the mafB gene in vivo using transgenic mice. A transgene, containing the 8.2-kb sequence flanking the 5' end of the mafB exon, directed the expression of a GFP reporter gene specifically in the retina, myelo-monocytic lineages of hematopoietic cells, peritoneal macrophages and the ventral spinal cord. In situ hybridization analysis showed that the reporter gene expression specifically recapitulates the endogenous expression profile of mafB in the retina and spinal cord. FACS analysis revealed that the Gr-1, Mac-1 and F4/80 antigens were present on most of the GFP-positive hematopoietic cells from transgenic adult bone marrow and spleen. On the other hand, B220 CD4, 8, and Ter119 cells were almost absent from among the GFP-positive cells examined. These observations suggest that gene regulatory regions located in the 8.2-kb upstream region of mafB are responsible for directing mafB expression in the retina, myelomonocytic lineages, peritoneal macrophages and the ventral spinal cord.View full abstractDownload PDF (3467K) -
Kozo Morimoto, Shusei Sato, Satoshi Tabata, Masato Nakai2003 Volume 134 Issue 2 Pages 211-217
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIscA homologues are involved in iron-sulfur cluster biosynthesis. In the non-nitrogen-fixing cyanobacterium Synechocystis PCC 6803, there are two IscA homologues, SLR1417 and SLR1565 (designated IscA1 and IscA2), of which only IscA2 exists as a protein complex with the HEAT-repeat-containing protein, SLR1098 (IaiH). We observed that the absorption spectrum of the recombinant IscA2/IaiH complex resembles that of IscA2 alone, although it is sharper. In the presence of dithiothreitol, the [2Fe-2S] cluster of IscA2 alone, but not of the IscA2/IaiH complex, became reductively labile upon the addition of sodium dithionite. This implies that the IscA2 moiety of the [2Fe-2S] cluster is stabilized by the presence of IaiH. The [2Fe-2S] cluster of the IscA2/IaiH complex was destabilized by sodium dithionite in the absence of dithiothreitol, suggesting that the in vivo stability of the iron-sulfur cluster in the IscA2/IaiH complex is influenced by the redox state of cellular thiols. When any one of three conserved cysteine residues in IscA2, potential ligands for the [2Fe-2S] cluster, was replaced with serine, the amount of assembled [2Fe-2S] cluster and protein complex was significantly reduced in E. coli cells. The cysteine mutated IscA2/IaiH complexes that were present all contained a [2Fe-2S]-like cluster suggesting that the assembly of a stable iron-sulfur cluster bound to IscA2 is required for efficient and stable complex formation. Truncated IaiH proteins were analyzed using the yeast two-hybrid assay to identify the essential domain of IaiH that interacts physically with IscA2. At least 2 of the 5 N-terminal HEAT repeats of IaiH were found to be required for interaction with IscA2.View full abstractDownload PDF (3053K) -
Yukihiro Umebayashi, Yuuichi Miyamoto, Masayoshi Wakita, Akiko Kobayas ...2003 Volume 134 Issue 2 Pages 219-224
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIn this report, we describe a laser-latex combination system that enables membrane-impermeable molecules to penetrate cell membranes. Laser light (Q-switched Nd: YAG laser, 532.5 nm) was used to irradiate a mixture of commercial latex particles (blue dyed, 1 μm in diameter) and mouse fibrosarcoma (Meth-A) cells. After irradiation, membrane permeability was evaluated by flow cytometric assaying using propidium iodide (PI) and fluorescein diacetate (FDA). The proportion of permeabilized-resealed cells was affected by changes in the light intensity (_??_780 mW/cm2), the irradiation time (_??_240 s), and/or the particle concentration (_??_109 particles/ml). The permea-bility persisted up to 20min after light irradiation. Near the sites of individual particles, the permeability of the cell membrane is modified, probably due to localized temperature changes. These results suggest that this laser-induced permeabilization strategy constitutes a new means of delivering exogenous materials into living cells.View full abstractDownload PDF (1699K) -
Mizuho Une, Yusuke Iguchi, Tomoko Sakamoto, Takashi Tomita, Yasuyuki S ...2003 Volume 134 Issue 2 Pages 225-230
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe bile acid intermediate 3α, 7α, 12α-trihydroxy-5β-cholestanoic acid (THCA) is converted to cholic acid exclusively in peroxisomes by the oxidative cleavage of the side chain. To investigate the mechanism by which the biosynthetic intermediates of bile acids are transported into peroxisomes, we incubated THCA or its CoA ester (THC-CoA) with isolated intact rat liver peroxisomes and analyzed their oxidation products, cholic acid and 3α, 7α, 12α-trihydroxy-5β-cholest-24-enoic acid. The oxidation of both THCA and THC-CoA was dependent on incubation time and peroxisomal proteins, and was stimulated by ATP. THC-CoA was efficiently oxidized to cholic acid and 3α, 7α, 12α-trihydroxy-50-cholest-24-enoic acid as compared with THCA, suggesting that THC-CoA is the preferred substrate for transport into peroxisomes. The oxidation of THC-CoA was significantly inhibited by sodium azide, verapamile, and N-ethylmaleimide. Furthermore, the stimulatory effect of ATP on the oxidation was not replaced by GTP or AMP. In addition, the ATP-dependent oxidation of THC-CoA was markedly inhibited by pretreatment of peroxisomes with proteinase K when peroxi-somal matrix proteins were not degraded. These results suggest that an ATP-dependent transport system for THC-CoA exists on peroxisomal membranes.View full abstractDownload PDF (1911K) -
Hiroyuki Nomoto, Tomoko Yonezawa, Kouichi Itoh, Katsuhiko Ono, Kiyotak ...2003 Volume 134 Issue 2 Pages 231-238
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA novel oligodendrocyte (OL)-specific cDNA was isolated from brain capillary endothelial cells and characterized. The cDNA encodes a protein of 1099 amino acids that contains a signal peptide and a transmembrane domain. The protein was expressed in mature OLs in vivo and in vitro cell cultures and was thus designated as mature OL transmembrane protein (MOLT). RT-PCR analysis showed that MOLT mRNA was expressed in brain, lung, pancreas, and testis. A polyclonal antibody raised against a part of the mouse MOLT reacted specifically with multipolar OLs possessing radially oriented processes that penetrated into the gray matter. More cells were detected in the white matter, and these had longitudinally oriented processes. In a rat OL lineage culture system, oligodendrocyte precursor cells did not initially produce MOLT mRNA and protein, but when they begun to differentiate into mature OLs, they started expressing MOLT. Consequently, MOLT may function as OLs become mature and may serve as a cell-surface marker for OL differentiation.View full abstractDownload PDF (4440K) -
Kei Wada, Toshiji Tada, Yoshihiro Nakamura, Takahiro Ishikawa, Yukinor ...2003 Volume 134 Issue 2 Pages 239-244
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSAscorbate peroxidase (APX) is a heme-containing protein that plays a central role in scavenging H2O2 in higher plants. The structure of stromal APX (sAPX) was determined at 1.6 Å to an R-factor of 19.1% and an R-free-factor of 22.3%. The electrostatic potential of the γ-channel that connects the molecular surface of sAPX to the γ-edge of heme was more positive than that of cytosolic APX (cAPX) from pea, so sAPX might bind more easily with ascorbate than cAPX. The overall structure of sAPX was similar to those of cAPX from pea and cytochrome c peroxidase (CCP) from yeast, with a substantial difference in a loop structure located in the vicinity of the heme. The side chain of Arg169 in sAPX corresponding to His169 in cAPX and His181 in CCP extended in the opposite direction from the heme, forming two hydrogen bonds with carbonyl groups in the loop structure. The rapid inactivation of sAPX might be due to the characteristic conformation of Arg169 owing to the loop structure of sAPX.View full abstractDownload PDF (4207K) -
Hiroshi Kuwahara, Jun-ichi Kamei, Norihiro Nakamura, Miho Matsumoto, H ...2003 Volume 134 Issue 2 Pages 245-250
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCalcineurin homologous protein (CHP) is an EF-hand Ca2+-binding protein capable of interacting with various cellular proteins including Na+/H+ exchangers, kinesinrelated proteins, and apoptosis-inducing protein kinase DRAK2. We investigated the role of CHP on the DRAK2 protein kinase in vitro. CHP significantly reduced (_??_85% inhibition) the kinase activity of DRAK2 for both autophosphorylation and phosphorylation of exogenous substrate (myosin light chain). The inhibitory effect of CHP was dependent on the presence of Ca2+, whereas the interaction between CHP and DRAK2 was not Ca2+-dependent. These observations suggest that CHP negatively regulates the apoptosis-inducing protein kinase DRAK2 in a manner that depends on intracellular Ca2+-concentration.View full abstractDownload PDF (2882K) -
Yasuo Yamauchi, Yukinori Ejiri, Yasuyuki Toyoda, Kiyoshi Tanaka2003 Volume 134 Issue 2 Pages 251-257
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPlant acylamino acid-releasing enzyme (AARE) catalyzing the N-terminal hydrolysis of Na-acylpeptides to release Na-acylated amino acids, was biochemically characterized using recombinant and native AAREs. A cDNA encoding a deduced Arabidopsis thaliana AARE (AtAARE) was cloned and sequenced. The deduced amino acid sequence encoded a 764 amino acid protein of 83.9 kDa, which was 31.8% identical with that of rat AARE. In particular, the proposed catalytic residues (Ser, Asp, and His) of AARE, called the “catalytic triad residues, ” were completely conserved. Recombinant AtAARE was expressed in Escherichia coli and confirmed to be a functional AARE. Native AAREs were prepared from A. thaliana and cucumber (Cucumis sativus, L.) plants. Both native AAREs were tetrameric proteins of 350 kDa comprising four subunits of 82 kDa, and showed typical enzymological properties of other AAREs, i.e. sensitivity to diisopropyl fluorophosphate, an optimum pH of around 7.0, and an optimum temperature of 37°C. Both the native and recombinant AAREs were immunochemically homologous. Intracelluar fractionation analysis showed that the AARE was mainly present in the stroma of chloroplasts. Native AARE degraded the glycated ribulose-1, 5-bisphoshate carboxylase/oxygenase protein but not the native protein. Thus, plant AARE might be involved in not only catalysis of the N-terminal hydrolysis of Na-acylpeptides but also the elimination of glycated proteins.View full abstractDownload PDF (2999K) -
Takefumi Morizumi, Hiroo Imai, Yoshinori Shichida2003 Volume 134 Issue 2 Pages 259-267
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSRhodopsin is a prototypical G-protein-coupled receptor that contains 11-cis-retinal as a light-absorbing chromophore. Light causes conformational changes in the protein moiety through cis-trans isomerization of the chromophore, which leads to the formation of G-protein-interacting states. Our previous studies indicated that there are two intermediate states of rhodopsin, Meta Ib and Meta II, which interact differently with retinal G-protein transducin (Gt) [S. Tachibanaki, H. Imai, T. Mizukami, T. Okada, Y. Imamoto, T. Matsuda, Y. Fukada, A. Terakita, and Y. Shichida (1997) Biochemistry 36, 14173-14180]. Here we demonstrate that the interactions of Gt with these intermediates in the absence of GTPγS can be mimicked by the C-terminus 11-amino acid peptide (340-350) of the α-subunit of Gt (Gta), suggesting that the C-terminal region of Gta plays important roles in the interaction with rhodopsin intermediates. Replacement of either of the two leucine residues (Leu344 and Leu349) in the peptide with alanine caused the loss of the interaction with Meta II. However, the interaction with Meta Ib was abolished only when both residues were replaced. These results indicate that rearrangement of the C-terminal region of Gta after the binding of a rhodopsin intermediate is necessary for the GDP-GTP exchange reaction on Gta.View full abstractDownload PDF (1114K) -
Shinya Mitsuhashi, Naohiro Saito, Keiko Watano, Keiichi Igarashi, Seii ...2003 Volume 134 Issue 2 Pages 269-276
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIt has been shown that calcineurin (CN), a serine/threonine protein phosphatase type 2B (PP2B), plays an important role in the development and diseases of cardiac muscles. However, reports on CN activity in dilated cardiomyopathy (DCM) are inconsistent, since there are few good disease models and the measurement of the amount of CN is difficult. Previously, we developed a novel line of DCM hamster, J2N-k, and its healthy control counterpart, J2N-n, by crossbreeding cardiomyopathy (CM) hamsters, Bio 14.6, and Golden hamsters followed by consecutive sib mating. In this study, we identified the DCM-causative gene in J2N-k by analysis of F2 of these two lines, and then we analyzed the change in CN gene expression in the course of the disease, and the change in CN activity using a newly developed method. We show that: (i) the DCM gene of J2N-k hamster is the δ- sarcoglycan (SG) gene, (ii) CN expression and potential CN activities (CN activity fully activated with Ca2+ and calmodulin) in the hearts of J2N-k and J2N-n hamsters are the same levels, (iii) transcription levels of natriuretic peptides, which are augmented by activation of Ca2+/calmodulin-dependent enzyme including CN, are significantly increased in the DCM stage in J2N-k hamster. J2N-k and J2N-n hamsters will be a useful tool for studying the pathogenesis, therapy, and prevention of human DCM. Although the total amount and potential activity of CN did not change in the cell extracts, targets of CN in vivo were activated in cardiomyocytes of DCM, suggesting that CN activity in the cells is activated by the raising of Ca2+ concentration in cardiomyocytes of DCM, which is caused by the defect in the δ-SG gene. Our results reveal the complexity of CN regulation in the heart and indicate the need for additional experimentation.View full abstractDownload PDF (4731K) -
Mohammad Mainul Islam, Hideyuki Hayashi, Hiroyuki Kagamiyama2003 Volume 134 Issue 2 Pages 277-285
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe reaction of Escherichia coli aspartate aminotransferase (AspAT) with glutamate and other C5-dicarboxylates was analyzed in order to compare its mechanism of action toward C5 substrates with that toward C4 substrates, which had been exten-sively characterized. The association of the amino-group protonated and unprotonated forms of glutamate (SH+ and S, respectively) with the Schiff-base protonated and unprotonated forms of the enzyme (ELH+ and EL, respectively) yields at least three forms of the Michaelis complex, whereas in the case of aspartate, only two species of this complex exist, EL•SH+ and ELH+•S. The reaction of AspAT with 2-methyl-glutamate can be explained only when we consider all the protonation states of the Michaelis complex. Based on the previous crystallographic studies [Miyahara et al. (1994) J. Biochem. 116, 1001-1012], we consider that glutamate binds to the open form of AspAT and takes an extended conformation in the Michaelis complex, with the α-amino group of glutamate oriented in the opposite direction to the Schiff base. This is in contrast to the Michaelis complex of aspartate, in which a strong interaction of the α-amino group of aspartate and the Schiff base excludes the presence of the species ELH+•SH+. It is concluded that AspAT recognizes the two types of dicarboxylates with different chain lengths by changing the gross conformation of the enzyme protein.View full abstractDownload PDF (3586K) -
Shou Takashima, Shuichi Tsuji, Masafumi Tsujimoto2003 Volume 134 Issue 2 Pages 287-296
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe cDNA encoding a second type of mouse β-galactoside α2, 6-sialyltransferase (ST6Gal II) was cloned and characterized. The sequence of mouse ST6Gal II encoded a protein of 524 amino acids and showed 77.1% amino acid sequence identity with human ST6Gal II. Recombinant ST6Gal II exhibited α2, 6-sialyltransferase activity toward oligosaccharides that have the Galβ1, 4GlcNAc sequence at the nonreducing end of their carbohydrate groups, but it exhibited relatively low and no activity toward some glycoproteins and glycolipids, respectively. On the other hand, ST6Gal I, which has been known as the sole member of the ST6Gal-family for more than ten years, exhibited broad substrate specificity toward oligosaccharides, glycoproteins, and a glycolipid, paragloboside. The ST6Gal II gene was mainly expressed in brain and embryo, whereas the ST6Gal I gene was ubiquitously expressed, and its expression levels were higher than those of the ST6Gal II gene. The ST6Gal II gene is located on chromosome 17 and spans over 70 kb of mouse genomic DNA consisting of at least 6 exons. The ST6Gal II gene has a similar genomic structure to the ST6Gal I gene. In this paper, we have shown that ST6Gal II is a counterpart of ST6Gal I.View full abstractDownload PDF (3599K) -
Atsuko Satoh, Yoshitaka Nakajima, Ikuko Miyahara, Ken Hirotsu, Takeyuk ...2003 Volume 134 Issue 2 Pages 297-304
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe flavoenzyme medium-chain acyl-CoA dehydrogenase (MCAD) eliminates the α-proton of the substrate analog, 3-thiaoctanoyl-CoA (3S-C8-CoA), to form a charge-transfer complex with deprotonated 3S-C8-CoA. This complex can simulate the metastable reaction intermediate immediately after the α-proton elimination of a substrate and before the β-hydrogen transfer as a hydride, and is therefore regarded as a transition-state analog. The crystalline complex was obtained by co-crystallizing MCAD in the oxidized form with 3S-C8-CoA. The three-dimensional structure of the complex was solved by X-ray crystallography. The deprotonated 3S-C8-CoA was clearly located within the active-site cleft of the enzyme. The arrangement between the flavin ring and deprotonated 3S-C8-CoA is consistent with a charge transfer interaction with the negatively charged acyl-chain of 3S-C8-CoA as an electron donor stacking on the pyrimidine moiety of the flavin ring as an electron acceptor. The structure of the model complex between lumiflavin and the deprotonated ethylthioester of 3-thiabutanoic acid was optimized by molecular orbital calculations. The obtained theoretical structure was essentially the same as that of the corresponding region of the X-ray structure. A considerable amount of negative charge is transferred to the flavin ring system to stabilize the complex by 9.2 kcal/mol. The large stabilization energy by charge transfer probably plays an important role in determining the alignment of the flavin ring with 3S-C8-CoA. The structure of the highest occupied molecular orbital of the complex revealed the electron flow pathway from a substrate to the flavin ring.View full abstractDownload PDF (4814K)
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