Journal of Biochemistry - Advance Access

Disulfide bond formation system in Eschericia coli

Inaba, K. — 2009-06-29

The biological kingdoms have evolved elaborate systems that ensure the catalysis of protein disulfide bond formation in the cell. Coexisting in the periplasm of E. coli are the DsbA-DsbB disulfide-introducing and DsbC-DsbD disulfide-isomerizing pathways, which promote the oxidative folding of secreted proteins. Recent structural studies of DsbB have illuminated conformational dynamics involved in the effective oxidation of the extremely reduction-prone oxidase, DsbA, as well as the structure of the reaction center involved in protein disulfide bond formation de novo in conjunction with ubiquinone. Extensive genetic and biochemical analysis has recently provided insight into how DsbD transports electrons from cytosolic thioredoxin to periplasmic DsbC. To a great extent, the molecular mechanisms of the Dsb enzyme system in E. coli have been elucidated, and are applicable to the study of protein disulfide formation systems in other organisms.

Identification of radicals formed in the reaction mixture of bovine kidney microsomes with NADPH

Kumamoto, K., Hirai, T., Kishioka, S., Iwahashi, H. — 2009-06-29

In order to explore the mechanism of myoglobinuric renal toxicity, detection and identification of free radicals was performed for the reaction mixtures of bovine kidney microsomes. EPR measurements showed prominent signals for the control reaction mixture containing 2.0 mg protein/ml bovine kidney microsomes, 5 mM NADPH, 0.1 M 4-POBN and 29 mM phosphate buffer (pH 7.4). Addition of myoglobin (Mb) to the control reaction mixture resulted in increase of EPR peak height. The result indicates that Mb enhances the radical formation. An HPLC-EPR measurement showed three peaks with retention times of 29.4 min (P₁), 32.4 min (P₂) and 46.6 min (P₃). HPLC-EPR-MS analyses of the P₁ and P₂ gave ions at m/z 282. The results show that 4-POBN/hydroxypentyl radical adducts form in the reaction mixture. An HPLC-EPR-MS analysis of the P₃ gave ions at m/z 266, indicating that 4-POBN/pentyl radical adduct forms in the reaction mixture.

Structural Insights into the Enzymatic Mechanism of Serine Palmitoyltransferase from Sphingobacterium multivorum

2009-06-29

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyzes the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of l-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The crystal structure of SPT from Sphingobacterium multivorum GTC97 complexed with l-serine was determined at 2.3 Å resolution. The electron density map showed the Schiff base formation between l-serine and PLP in the crystal. Because of the hydrogen bond formation with His138, the orientation of the C-H bond of the PLP-l-serine-aldimine was not perpendicular to the PLP-Schiff base plane. This conformation is unfavorable for the a-proton abstraction by Lys244 and the reaction is expected to stop at the PLP-l-serine-aldimine. Structural modeling of the following intermediates indicated that His138 changes its hydrogen bond partner from the carboxyl group of l-serine to the carbonyl group of palmitoyl-CoA upon the binding of palmitoyl-CoA, making the l-serine C-H bond perpendicular to the PLP-Schiff base plane. These crystal and model structures well explained the observations on bacterial SPTs that the -deprotonation of l-serine occurs only in the presence of palmitoyl-CoA. This study provides the structural evidence that directly supports our proposed mechanism of the substrate synergism in the SPT reaction.

Enzymatic characterization and comparison of various Poaceae UDP-GlcA 4-Epimerase Isoforms

Gu, X., Wages, C. J, Davis, K. E., Guyett, P. J., Bar-Peled, M. — 2009-06-29

UDP--d galacturonic acid (UDP-GalA) is a key precursor for the synthesis of various bacterial and plant polysaccharides. UDP-glucuronic acid 4-epimerase (UGlcAE) catalyzes the reversible conversion of UDP--d-glucuronic acid to UDP-GalA. UGlcAEs isolated from bacterial species have different biochemical properties when compared with the isoenzymes from the plant dicot species, Arabidopsis. However, little is known about the specificity of UGlcAE in Poaceae species. Therefore, we cloned and expressed in E. coli several maize and rice UGlcAE genes, and compared their enzymatic properties with dicot homologs from Arabidopsis. Our data show that UGlcAE isoforms in different plant species have different enzymatic properties. For example, the Poaceae UGlcAE enzymes from rice and maize have significantly lower K_i for UDP-xylose when compared with the Arabidopsis enzymes. The epimerases from different plant species are very specific and unlike their bacterial homolog in Klebsiella pneumoniae, can only use UDP-GlcA or UDP-GalA as their substrate. This study demonstrates that although members of plant UGlcAE isoforms are highly conserved, the in vitro enzymatic activity of specific Poaceae isoform(s) may be regulated differently by specific nucleotide or nucleotide-sugar.

Crystal Structure of Hypothetical Protein HP0062 (O24902_HELPY) from Helicobacter pylori at 1.65 A Resolution

Jang, S.-B., Kwon, A.-R., Son, W.-S., Park, S. J., Lee, B.-J. — 2009-06-29

The HP0062 gene encodes a small acidic protein of 86 amino acids with a theoretical pI of 4.6. The crystal structure of hypothetical protein HP0062 from Helicobacter pylori has been determined at 1.65 Å by molecular-replacement method. The crystallographic asymmetric unit contains dimer, in which HP0062 monomer folds into a helix-hairpin-helix structure. The two protomers are primarily held together by extensive hydrophobic interactions in an antiparallel arrangement, forming a four helix bundle. Aromatic residues located at a or g position in the heptad leucine zipper are not major contributor required for HP0062 dimerization but important for the thermostability of this protein.

Dynamic expression of peptidylarginine deiminase 2 in human monocytic leukemia THP-1 cells during macrophage differentiation

Hojo-Nakashima, I., Sato, R., Nakashima, K., Hagiwara, T., Yamada, M. — 2009-06-29

Peptidylarginine deiminases (PADs) consist of five enzymes which are widely distributed in human and rodent tissues. The two types of enzymes are found in human peripheral blood cells; PAD4 mainly in granulocytes and monocytes and PAD2 in lymphocytes and macrophages. Little is known about the regulation of PAD expression in macrophages. Here we report that PAD2 is expressed in human monocytic leukemia THP-1 cells during differentiation into macrophages by 12-O-tetradecanoylphorbol-13-acetate. During this differentiation the levels of PAD2 mRNA and protein increased concomitantly, indicating the transcriptional regulation of PAD2 gene expression in the cells. The treatment of THP-1-derived macrophages with calcium ionophore A23187 generated vimentin deimination and resulted in the disruption of vimentin filament organization. We discuss the possible role of vimentin deimination in cell physiology.

Polymyxin B identified as an inhibitor of alternative NADH dehydrogenase and malate: quinone oxidoreductase from the Gram-positive bacterium Mycobacterium smegmatis

2009-06-29

Tuberculosis is the leading cause of death due to a single infectious agent in the world and the emergence of mutidrug-resistant strains prompted us to develop new drugs with novel targets and mechanism. Here we screened a natural antibiotics library with Mycobacterium smegmatis membrane-bound dehydrogenases and identified polymyxin B (cationic decapeptide) and nanaomycin A (naphtoquinone derivative) as inhibitors of alternative NADH dehydrogenase (IC₅₀ values of 1.6 and 31 µg/ml, respectively) and malate: quinone oxidoreductase (IC₅₀ values of 4.2 and 49 µg/ml, respectively). Kinetic analysis on inhibition by polymyxin B showed that the primary site of action was the quinone-binding site. Because of the similarity in K_m value for ubiquinone-1 and inhibitor sensitivity, we examined amino acid sequences of actinobacterial enzymes and found possible binding sites for L-malate and quinones. Proposed mechanisms of polymyxin B and nanaomycin A for the bacteriocidal activity were the destruction of bacterial membranes and production of reactive oxygen species, respectively, while this study revealed their inhibitory activity on bacterial membrane-bound dehydrogenases. Screening of the library with bacterial respiratory enzymes resulted in unprecedented findings, so we are hoping that continuing efforts could identify lead compounds for new drugs targeting to mycobacterial respiratory enzymes.

Retraction: Cloning, Expression and Characterization of UDP-Glucose Pyrophosphorylase from Shoots of Bambusa oldhamii

Weng, C.-J., Deng, J.-Y., Lin, D.-G., Jeang, C.-L. — 2009-06-26

The paper has been published online in Journal of Biochemistry Advance Access and had been submitted without agreement from the co-authors. They therefore retract this paper and discourage citations of it

Changes in the conformation of the Vsr endonuclease amino-terminal domain accompany DNA cleavage.

Polosina, Y. Y., Cupples, C. G. — 2009-06-24

In Escherichia coli, T/G mismatches arising from deamination of 5-methylcytosine to thymine are converted to CG base pairs by the very short patch repair pathway. DNA polymerase I removes and resynthesizes the mismatched T starting from a 5' nick created by the Vsr endonuclease. We used limited trypsinolysis to probe conformational changes in the N-terminal domain of Vsr in response to DNA binding, DNA cleavage and interaction with the polymerase. Our data show that the domain becomes trypsin resistant only under conditions that allow DNA cleavage, while interaction with the polymerase restores trypsin sensitivity. We suggest that the domain changes its conformation as a result of DNA nicking, and that DNA PolI releases Vsr from the nick by reversing that conformational change.

Insights into the Enzyme-Substrate Interaction in the Norovirus 3C-like Protease

Someya, Y., Takeda, N. — 2009-06-24

The Glu54 residue of the norovirus 3C-like protease was implicated in proteolysis as a third-member carboxylate of the catalytic triad. The E54L mutant protease cleaved the sequence ¹³³LSFE/AP between the 3B and 3C regions of norovirus polyprotein, but did not cleave the sequence ¹⁹⁸ATSE/GK between the 3A and 3B. The 3BC junction mutation (3B-L133A or 3B-F135S) hampered the cleavage by the E54L protease, whereas the 3AB junction mutation (3A-A198L,S200F) allowed the E54L protease to digest. These results indicate that the E54L mutant protease is a substrate-specificity mutant and requires large hydrophobic amino acid residues at both P4 and P2 positions of the substrate. It was notable that the 3A-S200F P2 position mutation caused tight interaction between the wild-type protease and the C-terminus of the 3A protein, hence a decreased release rate of the product from the enzyme. This tight binding was dependent on the hydrophobicity of amino acid residues introduced at position 200 of the 3A region and was affected by the mutation in the bII-cII loop of the protease or the mutation of position 198 of 3A corresponding to the P4 position of the substrate. These results suggest that the protease and the substrate sense each other in the process of the proteolysis, being supported by crystal structures.

RNA interference targeted to the conserved dimerization initiation site (DIS) of HIV-1 restricts virus escape mutation

Sugiyama, R., Habu, Y., Ohnari, A., Miyano-Kurosaki, N., Takaku, H. — 2009-06-24

Short hairpin RNAs (shRNA) targeting viral or cellular genes can effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication. This inhibition, however, may induce mutations in the targeted gene, leading to rapid escape from the shRNA-induced inhibition. We generated a lymphoid cell line that stably expressed a 19-bp shRNA targeting a well-conserved dimerization initiation site (DIS) of HIV-1, which strongly inhibited viral replication, thereby delaying virus escape. Furthermore, treatment of HIV-1 infection with DIS- and vif-shRNA combination therapy resulted in superior antiviral responses compared to vif-shRNA monotherapy. Continuous challenge with HIV-1, however, generated virus mutants that could overcome the RNA interference restriction. Such anti-genes may be promising tools for HIV-1 gene therapy for HIV/acquired immunodeficiency syndrome.

Overexpression of Plk3 causes morphological change and cell growth suppression in Ras pathway-activated cells

Iida, M., Sasaki, T., Komatani, H. — 2009-06-24

To unravel the growth inhibition mechanism of Plk3, the effect of overexpression of Plk3 was examined in 293T cells. Cell rounding, changes in actin organization, and cellular detachment were induced by Plk3 transfection in a kinase activity-dependent manner. Although apoptosis was not observed, Plk3 overexpression suppressed cellular growth in a long-term colony-forming assay. Because both Plk3 and Ras affect f-actin organization, the effect of co-transfection of Plk3 and Ras was evaluated. Adhesion was synergistically lost by co-transfection of these two genes, compared with transfection of Plk3 alone. Furthermore, overexpression of Plk3 caused long-term growth suppression in Ras-transformed NIH3T3. Collectively, Plk3 activation might cause cytoskeleton re-organization and result in growth suppression more pronouncedly in Ras pathway-activated cells.

Storage of gangliosides GM2 and fucosyl GM1 in the kidney of MCC strain of mastomys (Praomys coucha)

Takimoto, K., Kawamura, N., Kasama, T. — 2009-06-24

Previously, we histochemically examined the kidney of the MCC strain of mastomys (Praomys coucha) and found the storage of gangliosides. In the present studies, the lipid-bound sialic acid content of gangliosides in the MCC kidney was about 9- to 14-fold higher than that of the control (MWC strain). In the MCC kidney, sialic acids of male gangliosides were composed of N-acetylneuraminic acid at 91.5%; sialic acids of female gangliosides, however, were composed almost entirely of N-glycolylneuraminic acid. TLC of gangliosides showed that the MCC kidney contained four abundant gangliosides (two gangliosides each in males and females). These gangliosides isolated by HPLC were identified to be GM2(NeuAc) and fucosyl GM1(NeuAc) in the male MCC kidney and GM2(NeuGc) and fucosyl GM1(NeuGc) in the female MCC kidney by secondary ion mass spectrometry, TLC/immunostaining, and TLC after enzyme treatments. Although the MCC kidney contained control levels of the activities of β-N-acetylhexosaminidase, a-L-fucosidase, N-acetylgalactosaminyltransferase, and fucosyltransferase, the activity of β-galactosidase in the MCC kidney was increased to 400-500% of that in the MWC kidney. Therefore we discussed the possibility that in the MCC kidney, GM2 was abundantly produced by the effect of increased β-galactosidase activity.

Subtilisin-like proprotein convertase PACE4 is required for skeletal muscle differentiation

Yuasa, K., Masuda, T., Yoshikawa, C., Nagahama, M., Matsuda, Y., Tsuji, A. — 2009-06-15

Most growth factors stimulate myoblast proliferation and prevent differentiation, whereas insulin-like growth factors (IGFs) promote myoblast differentiation through the phosphatidylinositol 3-kinase (PI3K) pathway. Subtilisin-like proprotein convertases (SPCs) are involved in cell growth and differentiation via activation of pro-growth factors. However, the role of SPCs in myogenesis remains poorly understood. Here we show that PACE4, a member of the SPC family, plays a critical role in myogenic differentiation of C2C12 cells. PACE4 mRNA levels increased markedly during myogenesis, whereas the expression of other member of SPC family, furin and PC6, remained unchanged. The expression pattern of pro-IGF-II, which is processed extracellularly by SPCs, was similar to that of PACE4. The expression of shRNA targeting PACE4, but not furin, suppressed the expression of the muscle-specific myosin light chain (MLC). Interestingly, reduced expression of MLC was restored following treatment with recombinant mature IGF-II. Finally, we demonstrated that the PI3K inhibitor LY294002 blocked the induction of PACE4 mRNA, a result not observed when another myogenic differentiation inhibitor, SB203580 (p38 MAP kinase inhibitor), was employed, indicating the presence of a positive feedback loop regulating PACE4 expression. These results suggest that PACE4 plays an important role in myogenic differentiation through its association with the IGF-II pathway.

CDK11p58 Phosphorylation of PAK1 Ser174 Promotes DLC2 Binding and Roles on Cell Cycle Progression

2009-06-11

CDK11^p58, a CDK11 family Ser/Thr kinase, is a G2/M specific protein and contributed to regulation of cell cycle, transcription and apoptotic signal transduction. Recently, CDK11^p58 has been reported to exert important functions in mitotic process, such as the regulation of bipolar spindle formation and sister chomatid cohesion. Here we identified p21 activated kinase 1 (PAK1) as a new CDK11^p58 substrate and we mapped a new phosphorylation site of Ser174 on PAK1. By mutagenesis we created PAK1^174A and PAK1^174E, which mimic the dephosphorylated and phosphorylated form of PAK1, further analysis showed PAK1^174Ecould be recruited to myosin V motor complex through binding to dynein light chain 2 (DLC2). PAK1^174Ecould accelerate the mitosis progression in a nocodazole blocked cell model, while PAK1^174A exhibited an opposite role. Our results indicated PAK1 may serve as a downstream effector of CDK11^p58 during mitosis progression.

Identification and characterization of a selective radioligand for ELOVL6

2009-06-08

ELOVL6, a member of the elongation of very long chain fatty acids (ELOVL) family, has recently been identified as the rate-limiting enzyme for the elongation of palmitoyl-CoA. ELOVL6 deficient mice are protected from high-fat diet induced insulin resistance, suggesting that ELOVL6 might be a promising target for the treatment of metabolic disorders. Despite the increasing interest in Elovl6 as a therapeutic target, the lack of chemical tools for this enzyme has limited further elucidation of the biochemical and pharmacological properties of ELOVL6. We have identified Compound-A, a potent inhibitor for ELOVL6, by screening our company library and subsequently optimizing hit compounds. Compound-A potently inhibited human and mouse ELOVL6 and displayed more than 100-fold greater selectivity for ELOVL6 over other ELOVL family members. Consistent with its potent and selective inhibitory activity toward ELOVL6, [³H]Compound-A bound to ELOVL6 with high affinity while showing no specific binding to other ELOVL enzymes. The observation that [³H]Compound-A bound to ELOVL6 in a palmitoyl-CoA dependent manner in the absence of malonyl-CoA and NADPH suggests that Compound-A might recognize an enzyme-substrate complex e.g. an acyl-enzyme intermediate. Collectively, these observations demonstrate that Compound-A and its tritiated form are useful tools for biochemical and pharmacological characterization of ELOVL6.

Molecular Cloning, Expression, and Characterization of A Novel Mouse SULT6 Cytosolic Sulfotransferase

2009-06-08

By searching the mouse EST database, we identified a novel mouse cytosolic sulfotransferase (SULT) cDNA (RIKEN cDNA 2410078J06). Sequence analysis revealed that this new SULT belongs to the cytosolic SULT6 gene family. The recombinant form of this newly identified SULT, designated SULT6B1, was expressed using the pGEX-4T-1 glutathione S-transferase fusion system and purified from transformed BL21 Escherichia coli cells. Purified mouse SULT6B1 exhibited sulfonating activity toward thyroxine and bithionol among a variety of endogenous and xenobiotic compounds tested as substrates. pH optimum of purified mouse SULT6B1 was determined to be 8.0. Tissue-specific expression of mouse and human SULT6B1 was examined by RT-PCR. While human SULT6B1 was specifically expressed in kidney and testis, mouse SULT6B1 was detected in brain, heart, kidney, thymus, lung, liver and testis. Further studies are needed in order to clarify the role of SULT6B1 in the metabolism of thyroxine and possibly some xenobiotics in mouse.

Crystallographic and mutational analyses of substrate recognition of endo-{alpha}-N-acetylgalactosaminidase from Bifidobacterium longum

2009-06-08

Endo--N-acetylgalactosaminidase (endo--GalNAc-ase), a member of the glycoside hydrolase (GH) family 101, hydrolyzes the O-glycosidic bonds in mucin-type O-glycan between -GalNAc and Ser/Thr. Endo--GalNAc-ase from Bifidobacterium longum JCM1217 (EngBF) is highly specific for the core 1-type O-glycan to release the disaccharide Galβ1-3GalNAc (GNB), whereas endo--GalNAc-ase from Clostridium perfringens (EngCP) exhibits broader substrate specificity. We determined the crystal structure of EngBF at 2.0 Å resolution and performed automated docking analysis to investigate possible binding modes of GNB. Mutational analysis revealed important residues for substrate binding, and two Trp residues (Trp748 and Trp750) appeared to form stacking interactions with the β-faces of sugar rings of GNB by substrate-induced fit. The difference in substrate specificities between EngBF and EngCP is attributed to variations in amino acid sequences in the regions forming the substrate binding pocket. Our results provide a structural basis for substrate recognition by GH101 endo--GalNAc-ases and will help structure-based engineering of these enzymes to produce various kinds of neo-glycoconjugates.

Siccanin rediscovered as a species-selective succinate dehydrogenase inhibitor

2009-06-08

To identify antibiotics targeting to respiratory enzymes, we carried out matrix screening of a structurally varied natural compound library with Pseudomonas aeruginosa membrane-bound respiratory enzymes. We identified a succinate dehydrogenase inhibitor, siccanin (IC₅₀, 0.9 µM), which is a potent antibiotic against some pathogenic fungi like Trichophyton mentagrophytes and inhibits their mitochondrial succinate dehydrogenase. We found that siccanin was effective against enzymes from P. aeruginosa, Pseudomonas putida, rat and mouse mitochondria but ineffective or less effective against Escherichia coli, Corynebacterium glutamicum, and porcine mitochondria enzyme. Action mode was mixed-type for quinone-dependent activity and noncompetitive for succinate-dependent activity, indicating the proximity of the inhibitor-binding site to the quinone-binding site. Species-selective inhibition by siccanin is unique among succinate dehydrogenase inhibitors, and thus siccanin is a potential lead compound for new chemotherapeutics.

TRAF-interacting protein with a forkhead-associated domain B (TIFAB) is a negative regulator of the TRAF6-induced cellular functions

Matsumura, T., Kawamura-Tsuzuku, J., Yamamoto, T., Semba, K., Inoue, J.-i. — 2009-05-26

Tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with a forkhead-associated domain (TIFA) activates TRAF6 to induce NF-B activation. TIFA-related protein, TIFAB, is highly expressed in spleen and inhibits TIFA-mediated TRAF6 activation. However, little is known about cell types that express TIFAB and its function in those cells. Here, we show that TIFAB is mainly expressed in B cells rather than T cells in spleen and that expression level was much higher in dendritic cells (DCs) and macrophages than splenic lymphocytes. TIFAB expression was downregulated when B cells, DCs or macrophages were stimulated by TRAF6-mediated proliferative or maturation signals including those emanating from CD40, sIgM and TLRs. Furthermore, microinjection experiments using NIH3T3 cells revealed that TIFAB inhibited entry into S phase of the cell cycle. Our results suggest that TIFAB could acts as a negative regulator of the TRAF6-induced cellular function such as B cell proliferation and maturation of DCs and macrophages.

Human C21orf63 is a heparin-binding protein

2009-05-26

Human C21orf63 is a type-1 transmembrane protein of hitherto unknown function, with two repeats of putative "galactose binding lectin domains". By using glycan microarray analysis and other assays, we found that human C21orf63 interacts with heparin and to a lesser extent with heparan sulfate. The C-terminal galactose binding lectin domain of C21orf63 is necessary for heparin binding. The inability of other human proteins with galactose binding lectin domains to interact with heparin suggests that heparin binding is a unique property of C21orf63. Results of real-time PCR and tissue immunostaining imply that C21orf63 is expressed on epithelia of various human tissues.

Domain-dependent interaction of eukaryotic initiation factor eIF4A for binding to middle and C-terminal domains of eIF4G

2009-05-26

The interactions of recombinant human eIF4A (4A) and its N- and C-terminal side domains (AN and AC, respectively) with the middle- and C-terminal-domain-linked fragment (GMC) of eIF4G and its middle and C-terminal domains (GM and GC, respectively) were investigated by surface plasmon resonance (SPR) analysis and isothermal titration calorimetry (ITC). It is remarkable that the kinetic parameter-dependent SPR profile observed for the 4A-GMC pair was quite different from the steady affinity profiles of the 4A-GM/GC pairs, suggesting the simultaneous contribution of the middle and C-terminal domains of eIF4G for the binding with eIF4A. On the other hand, ITC yielded the enthalpy energies of -1.5 ~ -2.5 x104 J/mol for the domain-domain interactions of 4A with GMC. Although the ITC profile of the 4A-GM pair reflects well the structural feature shown previously by NMR and X-ray analyses, it was essentially different from that of the 4A-GMC pair. The present results suggest that the intimate interaction between the eIF4A N- and C-terminal domains and the eIF4G middle and C-terminal domains is necessary to reveal the biologically active function of the eIF4A-eIF4G complex.

FT-IR Spectroscopic Studies on the Molecular Mechanism for Substrate Specificity/Activation of Medium-Chain Acyl-CoA Dehydrogenase

Nishina, Y., Sato, K., Tamaoki, H., Setoyama, C., Miura, R., Shiga, K. — 2009-05-26

The interactions of acyl-CoA with medium-chain acyl-CoA dehydrogenases (MCADs) reconstituted with artificial FADs, i.e., 8-CN-, 7,8-Cl2-, 8-Cl-, 8-OCH3-, and 8-NH2-FAD were investigated by UV-visible absorption and FT-IR measurments. 8-NH2-FAD-MCAD bound acyl-CoA but did not oxidize acyl-CoA and the wavelength of the absorption maximum of the flavin was altered by acyl-CoAs. Thus 8-NH2-FAD-MCAD is one of attractive materials for investigation of enzyme-substrate interaction in ES-complex (the complex of oxidized MCAD with acyl-CoA). FT-IR difference spectra between non-labeled and [1-13C]-labeled acyl-CoA free in solution and bound to oxidized 8-NH2-FAD-MCAD were obtained. The broad 1668-cm-1 band of free octanoyl-CoA assigned to the C(1)=O stretching vibration appeared as a sharp signal at 1626 cm-1 in the case of the complex. The downward shift indicates a large polarization of C(1)=O, and the sharpness suggests that the orientation of the C(1)=O in the active site cavity is fairly limited. The hydrogen-bond enthalpy change responsible for the polarization on the transfer of the substrate from aqueous solution to the active site of MCAD was estimated to be ca. 15 kcal/mol. The 1626-cm-1 band is noticeably weakened in the case of acyl-CoA with acyl-chains longer than C12 which are poor substrates for MCAD, suggesting that C(1)=O is likely to exist in multiple orientations in the active site cavity, whence the band becomes obscured. A band identical to that of bound C8-CoA was observed in the case of C4-CoA which is a poor substrate, indicating the strong hydrogen bond at C(1)=O.

Fatty acids bound to recombinant tear lipocalin and their role in structural stabilization

Tsukamoto, S., Fujiwara, K., Ikeguchi, M. — 2009-05-26

A variant of human tear lipocalin was expressed in Escherichia coli, and the bound fatty acids were analyzed by gas chromatography, mass spectroscopy, and nuclear magnetic resonance spectroscopy. Five major fatty acids were identified as hexadecanoic acid (palmitic acid, PA), cis-9-hexadecenoic acid (palmitoleic acid), 9,10-methylenehexadecanoic acid, cis-11-octadecenoic acid (vaccenic acid), and 11,12-methyleneoctadecanoic acid (lactobacillic acid). The composition of the bound fatty acids was similar to the fatty acid composition of Escherichia coli extract, suggesting that the binding affinities are similar for these fatty acids. The urea-induced and thermal unfolding transitions of the holoprotein (nondelipidated), apoprotein (delipidated), and PA-bound protein were observed by circular dichroism. Holoproteins and PA-bound proteins showed the same stability against urea and heat, and were more stable than apoprotein. These results show that each bound fatty acid stabilizes recombinant tear lipocalin to a similar extent.

Quality control of cytoplasmic membrane proteins in Escherichia coli

Akiyama, Y. — 2009-05-19

In Escherichia coli, like in any organism, the cytoplasmic (inner or plasma) membrane proteins play essential roles in transport of small and macro-molecules as well as in transmission of environmental signals across the membrane. Their quality control is critically important for growth and survival of the cell. However, our knowledge about the players and mechanisms of the system is still limited. This review focuses on proteolytic quality control of membrane proteins, in which two membrane-integrated proteases, FtsH and HtpX, with different modes of action, play central roles. The prohibitin family membrane protein complexes (HflKC and QmcA) contribute to the quality control system as a regulatory factor of FtsH and also as a possible membrane-chaperone. Failure of the quality control system to function normally leads to accumulation of malfolded cytoplasmic membrane proteins, which in turn activate the stress response pathways previously believed to be specialized for sensing protein abnormalities outside the cytoplasmic membrane. In fact, many of the cytoplasmic membrane quality control factors are stress-induced. Further characterization of them as well as of the stress-sensing mechanisms would prove useful to obtain an integrated picture of the membrane protein quality control system.

NMR structure of the heterodimer of Bem1 and Cdc24 PB1 domains from Saccharomyces cerevisiae

2009-05-18

Bem1 and Cdc24 of the budding yeast Saccharomyces cerevisiae interact with each other through PB1-PB1 heterodimer formation to regulate the establishment of cell polarity. Here we present the tertiary structure of the heterodimer of Bem1 and Cdc24 PB1 domains determined by NMR spectroscopy. To avoid ambiguity in the NMR spectral analysis, we first prepared a mutant of the Cdc24 PB1 domain that had truncated loops. The mutant provided well dispersed spectra without spectral overlapping, thus allowing unambiguous spectral assignments for structure determination. We confirmed that the loop deletion-mutant was quite similar to the wild type in both three-dimensional structure and binding affinity. The NMR structure of the heterodimer of the deletion-mutant of Cdc24 PB1 and Bem1 PB1 was determined using a variety of isotope labeled samples including perdeuteration.?The interface between the Bem1/Cdc24 PB1 heterodimer was analyzed at atomic resolution. Through a comparison with the tertiary structures of other PB1-PB1 heterodimers, we found that conserved electrostatic properties on the molecular surface were commonly used for PB1-PB1 interaction, but hydrophobic interactions were important for cognate interaction in Bem1/Cdc24 PB1 heterodimer formation.

DNA helicase activity in purified human RECQL4 protein

Suzuki, T., Kohno, T., Ishimi, Y. — 2009-05-18

Human RECQL4 protein was expressed in insect cells using a baculovirus protein expression system and it was purified to near homogeneity. The protein sedimented at a position between catalase (230 kDa) and ferritin (440 kDa) in glycerol gradient centrifugation, suggesting that it forms homo-multimers. Activity to displace annealed 17-mer oligonucleotide in the presence of ATP was co-sedimented with hRECQL4 protein. In ion-exchange chromatography, both DNA helicase activity and single-stranded DNA-dependent ATPase activity were co-eluted with hRECQL4 protein. The requirements of ATP and Mg for the helicase activity were different from those for the ATPase activity. The data suggest that the helicase migrates on single-stranded DNA in a 3'-5' direction. These results suggest that the hRECQL4 protein exhibits DNA helicase activity.

Protein Quality Control in Chloroplasts: a Current Model of D1 Protein Degradation in the Photosystem II Repair Cycle

Kato, Y., Sakamoto, W. — 2009-05-18

The chloroplast originated from endosymbiosis of photosynthetic bacteria. Thus, mechanisms essential for chloroplast biogenesis/homeostasis (protein synthesis, import from cytosol, assembly, and degradation) are predominantly governed by prokaryotic systems. Among these, the quality control system is crucial, because light energy constantly damages photosynthetic proteins and excessive light often limits plant growth by irreversibly inactivating the photosynthetic apparatuses. Here, we overview prokaryotic proteases (FtsH and Deg) which are two enzymes that play critical roles in this system. We particularly focus on Photosystem II (PSII) in thylakoid membranes, which is composed by more than 20 subunits. Among the subunits is one of the intrinsic reaction center proteins (D1) which is considered to be the target of photodamage. Its rapid and specific turnover suggests that photodamaged D1 is degraded by these proteases and replaced with a de novo synthesized one in a system which is termed the PSII repair cycle. We discuss a current model of D1 degradation which is executed by a concerted action of particular FtsH and Deg isoforms.

Nestin serves as a prosurvival determinant that is linked to the cytoprotective effect of epidermal growth factor in rat vascular smooth muscle cells

2009-05-18

Nestin is an intermediate filament protein mainly expressed in muscle and neural progenitors. Recently, we reported that nestin is expressed in rat vascular smooth muscle cells (VSMCs), disappears after serum-deprivation, and then is re-expressed again following EGF stimulation. As the function of nestin in VSMCs remains unknown, its anti-apoptotic function was investigated in this study. We first showed that cell viability of nestin-depleted cells following H₂O₂treatments decreased by nestin RNAi. Further DNA laddering analysis and flow cytometry results demonstrated that this loss of cell viability was mediated through apoptosis. In addition, caspase-9, caspase-3, and PARP were activated in nestin-depleted VSMCs following H₂O₂treatments, indicating that nestin has an upstream inhibitory effect on caspase activation. It is well-known that EGF serves as a survival factor in rat VSMCs. Herein, we show that the cytoprotective effect of EGF was prevented by nestin RNAi. In addition, the inhibition of Cdk5 prevented Bcl-2 phosphorylation and enhanced H₂O₂-induced caspase-3 activation as well as subsequent DNA fragmentation. Taken together, these results provide evidence for another cytoprotective role of EGF in that it is mediated through its stimulation of nestin expression which leads to the prevention of caspase activation by Cdk-5-induced Bcl-2 phosphorylation in rat VSMCs.

RecX is involved in the switch between DNA damage response and normal metabolism in D. radiodurans

Sheng, D., Jao, J., Li, M., Xu, P., Zhang, J. — 2009-05-14

Apart from inhibiting RecA activity through protein-protein interactions, Deinococcus radiodurans RecX inhibits the expression of RecA and two other antioxidant proteins. To identify the repertoire of proteins regulated by RecX, comparative proteomic studies were undertaken on a wild-type strain (R1) and recX null mutant (RecX^–). Two-dimensional electrophoresis followed by MALDI-TOF identification revealed 35 differentially expressed proteins, including 12 upregulated and 23 downregulated proteins in the mutant. The 12 upregulated proteins are DNA repair proteins, stress response proteins, and metabolism-related proteins. Most of these have been previously characterized as ionizing radiation (IR)-induced proteins. The 23 downregulated proteins are mainly involved in cellular metabolism, and some of these are key enzymes in the metabolic pathway. Thus, RecX is suggested to be involved in the switch between DNA damage response and normal metabolism in D. radiodurans.

Common skate (Raja kenojei) secretes pentraxin into the cutaneous secretion: The first skin mucus lectin in cartilaginous fish

Tsutsui, S., Yamaguchi, M., Hirasawa, A., Nakamura, O., Watanabe, T. — 2009-05-04

A lactose-specific lectin with a molecular mass of about 25 kDa was purified from the skin mucus of a cartilaginous fish—the common skate (Raja kenojei). The complementary DNA sequence of the lectin was 1540 bp long and contained a reading frame encoding 226 amino acids, which showed ~38% identity to pentraxins of mammals and teleosts. Gene expression was observed in the skin, gill, stomach, and intestine in the healthy skate. We also identified an isotype gene from the liver whose deduced amino acid sequence shared 69.0% identity with the skin type gene. The antiserum detected protein in the skin, in which the lectin is localized in the epidermal cells, and in the blood plasma. The lectin genes are multicopied in the common skate genome. Although pentraxins are acute phase proteins, mRNAs of both the isotypes were not upregulated after in vivo challenge with formalin-killed Escherichia coli, which suggests that they are constantly present in the skin mucus and blood plasma to protect against pathogenic invasion. This lectin is the fifth type of lectin found in the cutaneous secretions of fish, demonstrating that skin mucus lectins have evolved with marked molecular diversity in fish.

Lysophosphatidylmethanol is a pan lysophosphatidic acid receptor agonist and is produced by autotaxin in blood

2009-05-04

Lysophosphatidic acid (LPA) is a simple phospholipid but has numerous biological effects through a series of G protein-coupled receptors specific to LPA. LPA in general is short-lived when applied in vivo, which hinders most pharmacological experiments. In our continuing study to identify stable LPA analogs capable of in vivo applications, we identified here lysophosphatidylmethanol (LPM) as a stable and pan-LPA receptor agonist. A synthetic LPM activated all five LPA receptors (LPA_1-5), and stimulates both cell proliferation and LPA receptor-dependent cell motility. In addition, LPM showed a hypertensive effect in rodent when applied in vivo. We found that, when fetal calf serum was incubated in the presence of methanol, formation of LPM occurred rapidly, whereas it was completely blocked by depletion of autotaxin (ATX), a plasma enzyme that converts lysophosphatidylcholine (LPC) to LPA. When recombinant ATX was incubated with LPC in the presence of methanol, both LPM and LPA were produced with a ratio of 1:10, showing that ATX has transphosphatidylation activity in addition to its lysophospholipase D activity. Administration of methanol in mice resulted in the formation of several µM of LPM in plasma, which is much higher than that of LPA. The present study identified LPM as a novel and stable lysophospholipid mediator with LPA-like activities and ATX as a potential synthetic enzyme for LPM.

Biochemical and spectroscopic properties of cyanide-insensitive quinol oxidase from Gluconobacter oxydans

2009-05-04

Cyanide-insensitive quinol oxidase (CioAB), a relative of cytochrome bd, has no spectroscopic features of hemes b₅₉₅ and d in the wild-type bacteria and is difficult to purify for detailed characterization. Here we studied enzymatic and spectroscopic properties of CioAB from the acetic acid bacterium Gluconobacter oxydans. G. oxydans CioAB showed the K_m value for ubiquinol-1 comparable to that of Escherichia coli cytochrome bd but it was more resistant to KCN and quinone-analog inhibitors except piericidin A and LL-Z1272. We obtained the spectroscopic evidence for the presence of hemes b₅₉₅ and d. Heme b₅₉₅ showed the peak at 587 nm in the reduced state and a rhombic high-spin signal at g = 6.3 and 5.5 in the air-oxidized state. Heme d showed the peak at 626 and 644 nm in the reduced and air-oxidized state, respectively, and an axial high-spin signal at g = 6.0 and low-spin signals at g = 2.63, 2.37 and 2.32. We found also a broad low-spin signal at g = 3.2, attributable to heme b₅₅₈. Further, we identified the presence of heme D by mass spectrometry. In conclusion, CioAB binds all three heme species present in cytochrome bd quinol oxidase.

Activation of a Membrane-bound Serine Protease Matriptase on the Cell Surface

Miyake, Y., Yasumoto, M., Tsuzuki, S., Fushiki, T., Inouye, K. — 2009-04-26

Matriptase is a type II transmembrane serine protease. The activation (i.e., conversion of the single-chain pro-form to the disulfide-linked-two-chain active form) of this enzyme is known to occur via a mechanism requiring its catalytic triad. We reported previously that the activated enzyme was produced in the conditioned medium when full-length rat matriptase was expressed in monkey kidney COS-1 cells. The present study aimed to address when and where the matriptase activation occurs. COS-1 cells expressing matriptase were labeled with a membrane-impermeable biotin derivative and then solubilized with Triton. Both activated and non-activated matriptase molecules were detected in the avidin precipitants of Triton extracts, whereas only the non-activated molecules were detected in the flow-through fraction of avidin-precipitation procedure. Single-chain matriptase has been thought to have an inherent activity. Indeed, a secreted single-chain variant of recombinant matriptase bearing mutation at the activation-cleavage site was found to exhibit the activity in hydrolyzing a synthetic peptide substrate at pH 7.5. However, the variant had little activity at pH 5.5, as found in the lumen of post-Golgi secretory vesicles. Altogether, it is concluded that the activation of matriptase may occur when the enzyme reaches the cell surface.

Fast binding kinetics and conserved 3D structure underlie the antagonistic activity of mutant TNF: useful information for designing artificial proteo-antagonists.

2009-04-22

Tumor necrosis factor (TNF) is an important cytokine that induces an inflammatory response predominantly through the TNF receptor-1 (TNFR1). A crucial strategy for the treatment of many autoimmune diseases, therefore, is to block the binding of TNF to TNFR1. We previously identified a TNFR1-selective antagonistic mutant TNF (R1antTNF) from a phage library containing 6 randomized amino acid residues at the receptor binding site (amino acids 84-89). Two R1antTNFs, R1antTNF-T2 (A84S, V85T, S86T, Y87H, Q88N, and T89Q) and R1antTNF-T8 (A84T, V85P, S86A, Y87I, Q88N, and T89R), were successfully isolated from this library.

Here, we analyzed R1antTNF-T8 using surface plasmon resonance spectroscopy and X-ray crystallography to determine the mechanism underlying the antagonistic activity of R1antTNF. The kinetic association/dissociation parameters of R1antTNF-T8 were higher than those of wild-type TNF, indicating more rapid bond dissociation. X-ray crystallographic analysis suggested that the binding mode of the T89R mutation changed from a hydrophobic to an electrostatic interaction, which may be responsible for the antagonistic behavior of R1antTNF. Knowledge of these structure-function relationships will facilitate the design of novel TNF inhibitors based on cytokine structure.

A Novel Virtual Spectrometry: Visualized Regulatory Motifs on ADM, rPol{beta}, and CD83 mRNAs in Human-Friendly Manners.

Nakamura, S. — 2009-04-22

Recently, riboswitches and other structures discovered on mRNAs have been reported as examples of functional RNA structures, motifs. Such motifs were shown to be present as single-form valid structures but they are obscured among other less-valid structures. Here, I present a novel, practical virtual spectrometry (The GenoPoemics^TM Spectrometry) visualizing motifs on mRNA strands as spectra at-a-glance. Every motif along with validity of their existences could be observed on the spectra in human-friendly manners, and whole structures of mRNAs could be overviewed. Therefore, the spectra helped distinguish valid and less valid motifs. The spectrometry was applied to variety of mRNAs such as ADM, rPolβ and CD83 to identify structures of high validity on them, previously reported functional motifs were successfully revealed. These findings indicate that the structures of mRNAs that may be folded into multiple forms can be further discussed quantitatively based on the visual spectra to discover functional RNA motifs.

Analyses of conditions for KMSSS loop in Tyrosyl-tRNA synthetase by building a mutant library

Kamijo, S., Fujii, A., Onodera, K., Wakabayashi, K. — 2009-04-22

The KMSKS motif is the ATP binding motif for aminoacylation process of class I aminoacyl-tRNA synthetases. Although researches based on natural proteins inform us about the contribution of natural amino acid sequences for the catalysis, they have difficulties in discussing the other alternative sequences and prohibited sequences for the motif to maintain the catalytic ability. In order to reveal such the conditions for the alternative and prohibited sequences, it is important to investigate a library of various mutants for the motif. For that purpose, we build a library of more than two hundred mutants substituting the KMSSS loop, Lys204-Met205-Ser206-Ser207-Ser208, in tyrosyl-tRNA synthetase of Methanococcus jannaschii, and their catalytic abilities were examined by the Amber suppression method. Mutants of K204R and K204N still maintained catalytic abilities to a certain extent. On the other hand, a variety of alternative sequences for Ser206-Ser207-Ser208 were obtained, and some of those did not include either Ser or Thr which were regarded as necessary residues in the KMSKS motif in previous works. In this paper, catalytic activity of all the mutants are represented in detail and some suggestions for the condition of the motif are discussed.

The presence of OMP inclusion bodies in a Escherichia coli K-12 mutated strain is not related to lipopolysaccharide structure

2009-04-13

The role of lipopolysaccharides in the biogenesis of outer membrane proteins have been investigated in several studies. Some of these analyses showed that lipopolysaccharide is required for correct and efficient folding of outer membrane proteins; other studies support the idea of independence of outer membrane proteins biogenesis from lipopolysaccharide structure. In this paper we investigated the involvement of lipopolysaccharide structure in the anomalous aggregation of outer membrane proteins in a E. coli mutant strain (S17-1(pir)). To achieve this aim, the lipopolysaccharide structure of the mutant strain was carefully determined and compared with the E. coli K-12 one. It turned out that lipopolysaccharide of these two strains differs in the inner core for the absence of a heptose residue (HepIII). We demonstrated that this difference is due to a mutation in waaQ, a gene encoding the transferase for the branch heptose HepIII residue. The mutation was complemented to find out if the restoration of lipopolysaccharide structure influenced the observed outer membrane proteins aggregation. Data reported in this work demonstrated that, in E. coli S17-1(pir) there is no an influence of lipopolysaccharides structure on the outer membrane proteins inclusion bodies formation.

The peptidoglycan-binding (PGB) domain of the Escherichia coli Pal protein can also function as the PGB domain in E. coli flagellar motor protein MotB

Hizukuri, Y., Morton, J. F., Yakushi, T., Kojima, S., Homma, M. — 2009-04-13

The bacterial flagellar stator proteins, MotA and MotB, form a complex and are thought to be anchored to the peptidoglycan by the C-terminal conserved peptidoglycan-binding (PGB) motif of MotB. To clarify the role of the C-terminal region, we performed systematic cysteine mutagenesis and constructed a chimeric MotB protein which was replaced with the peptidoglycan-associated lipoprotein Pal. Although this chimera could not restore motility to a motB strain, we were able to isolate two motile revertants. One was F172V in the Pal region and the other was P159L in the MotB region. Furthermore, we attempted to map the MotB Cys mutations in the crystal structure of E. coli Pal. We found that the MotB mutations that affected motility nearly overlapped with the predicted PG-binding residues of Pal. Our results indicate that, although the functions of MotB and Pal are very different, the PGB region of Pal is interchangeable with the PGB region of MotB.

Reinvestigation of the molecular influence of hypoxanthine on the DNA cleavage efficiency of restriction endonucleases BglII, EcoRI and BamHI

Doi, A., Pack, S. P., Kodaki, T., Makino, K. — 2009-04-13

Hypoxanthine (Hyp), a deaminated base of adenine (Ade), can be employed as a good probe molecule to reveal the significance of the minor groove of guanine (Gua) in biomolecular interactions because Hyp possesses a similar structure to Gua lacking its 2-amino group. In this study, we examined cleavage efficiencies of restriction endonuclease enzymes on DNA substrates with Hyp in their recognition sequences. As a substrate for BglII, EcoRI and BamHI, 24-mer DNA oligomer with Hyp (in place of Gua) was prepared together with its complementary sequences with cytosine (Cyt) or thymine (Thy) as the counter base. At 37°C incubation for 1 h, BglII and EcoRI showed higher DNA cleavage reactivity on Hyp-containing DNA substrates than on normal ones, whereas BamHI showed lower values on Hyp-containing substrates. Such high cleavage performance of BglII and EcoRI on Hyp-containing DNA substrates is in contrast to the results obtained 20 years ago, in which short DNA substrates (8 or 10- mer) and low reaction temperatures (15–20°C) were employed. These new results suggest that the lack of the exocyclic 2-amino group of Gua could contribute to enhanced recognition access of BglII and EcoRI to DNA substrates.

Thermodynamic and Kinetic Effects of Morpholino Modification on Pyrimidine Motif Triplex Nucleic Acid Formation under Physiological Condition

Torigoe, H., Sasaki, K., Katayama, T. — 2009-04-07

Due to instability of pyrimidine motif triplex nucleic acid under physiological pH and low magnesium ion concentration, stabilization of the triplex under the physiological condition is crucial in improving its therapeutic potential to artificially control gene expression in vivo. To this end, we investigated the thermodynamic and kinetic effects of morpholino (MOR) modification of triplex-forming oligonucleotide (TFO) on the triplex formation under the physiological condition. The thermodynamic analyses indicated that the MOR modification of TFO not only significantly increased the thermal stability of the triplex but also increased the binding constant for the triplex formation by nearly 2 orders of magnitude. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the MOR-modified TFO in the free state relative to the corresponding unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. This information will be valuable for designing novel chemically modified TFO with higher binding affinity in the triplex formation under physiological conditions, leading to progress in therapeutic applications of the antigene strategy in vivo.

Distribution of Receptor Glycolipids for Lactobacilli in Murine Digestive Tract and Production of Antibodies Crossreactive with them by Immunization of Rabbits with Lactobacilli

Iwamori, M., Shibagaki, T., Nakata, Y., Adachi, S., Nomura, T. — 2009-04-07

In the digestive tract of mice (HR-1 strain), glycolipids belonging to the ganglio-series were revealed to be expressed in region-specific manners, i.e. FGA1 and FGM1 in the stomach, GA1 in the small intestine, and FGA1 and sulfatides in the cecum. The amount of GA1 as a receptor glycolipid for lactobacilli was especially higher in the small intestine than in the other regions, it comprising 1.6 to 2.8 µg per mg dry weight. On immunization of rabbits with Lactobacillus johnsonii and Lactobacillus intestinalis, both of which are murine intestinal bacteria, antibodies toward bacterial glycolipids, i.e. Gal1-2Glc1-3-DG, and tri- and tetrahexaosyl DGs, were effectively generated and, in addition, they were found to cross-react with GA1 and GalCer, but not with structurally related glycolipids such as Lc₄Cer, nLc₄Cer and IV³Gal-nLc₄Cer, indicating that GA1 is a preferable antigen for anti-lactobacillus antisera and suggesting the presence of epitopes common to both lactobacilli and the host. In fact, molecules reacting with anti-GA1 antibodies were detected among bacterial proteins on Western blotting, indicating a possible occurrence of the carbohydrate structure mimicking GA1 in bacterial proteins.

CEL-I, an N-Acetylgalactosamine (GalNAc)-Specific C-Type Lectin, Induces Nitric Oxide (NO) Production in RAW264.7 Mouse Macrophage Cell Line

Yamanishi, T., Hatakeyama, T., Yamaguchi, K., Oda, T. — 2009-04-07

We found that CEL-I, a GalNAc-specific C-type lectin isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), induces inducible nitric oxide synthase (iNOS) expression and NO production in RAW264.7 cells. The NO production was inhibited by an iNOS inhibitor, L-NAME, but was not by a lipopolysaccharide (LPS) inhibitor, polymyxin B. In the presence of 0.1 M GalNAc, increased NO production by CEL-I-treated RAW264.7 cells was observed rather than the inhibition. Bovine serum albumin (BSA) significantly inhibited the CEL-I-induced NO production as well as the binding of FITC-labeled CEL-I on RAW264.7 cells. Three MAP kinase inhibitors (specific to extracellular regulated kinase, c-jun NH₂-terminal kinase, and p38 MAP kinase) inhibited CEL-I-induced NO production with different extents. Heat-treatment of CEL-I resulted in a decreased activity of CEL-I depending on the temperature. These results suggest that CEL-I induces NO production in RAW264.7 cells through the protein-cell interaction rather than the binding to the specific carbohydrate chains on the cell surface.

A Fe deficiency responsive element (FeREs) with a core sequence of TGGCA regulates the expression of FEA1 in Chlamydomonas reinharditii

Fei, X., Eriksson, M., Yang, J., Deng, X. — 2009-04-07

Iron is essential to the unicellular green alga Chlamydomonas, but the molecular mechanism for response to iron deficiency remains largely unknown. In previous studies, we have identified FOX1 and ATX1 FEREs (Fe deficiency-responsive elements) as important regulation components of iron response in this organism. Here we present another iron regulated gene FEA1, which promoter was analyzed by using a 5' and 3' end deletion and a scanning mutagenesis assay. The results reveal that the co-existence of -273 /-188 and -118 /-49 regions from transcriptional start site of FEA1 were sufficient and necessary for Fe deficiency-induced expression. Further deletion analysis indicates both -273/-253 and -103 /-85 regions are essential for inducible expression. The scanning mutagenesis analysis of these regions identifies two cis-acting elements: the FeaFeRE1 at -273/-259(CTGCGGTGGCAAAGT) and FeaFeRE2 at -106/-85 (CCGCCGCNNNTGGCACCAGCCT). Sequence comparison of FeaFeRE1 and FeaFeRE2 reveals a core sequence of TGGCA, which had been found in our previously reported Fe-deficiency-inducible gene ATX1. Moreover, we show that the promoter region of several genes, including FRE1, IRT1,ISCA, ZRT1, ZRT5, NRAMP2and COPT1, also contains this core sequence, suggesting that at least two classes FeRE elements exist in Clamydomonas, one in FEA1and ATX1et al. the second in FOX1, FEA2, MTP4, NRAMP3 and RBOL1.

Effect of Metal Ion on the Structural Stability of Tumor Suppressor Protein p53 DNA Binding Domain

Xue, Y., Wang, S., Feng, X. — 2009-04-03

The tumor suppressor protein p53 is a sequence-specific transcription factor that coordinates one molecule of zinc in the core domain. In our recent study, magnesium can also bind to the p53DBD and enhance its DNA-binding activity. In this study, a systematic analysis of the conformation and stability changes induced by these two metal ions was reported. The spectra of protein intrinsic fluorescence were used to measure the equilibrium unfolding of the p53DBD protein. The stability against chemical denaturation increased in the order apo < Mg²⁺ < Zn²⁺. The thermal stability monitored by DSC scans showed that the binding of metal ions to p53DBD increased the thermal stability of the protein. To explore additional information of structural changes after the binding of metal ions, we used the fluorescent probes to evaluate the hydrophobic surface exposure. The results established that metal ions binding increased hydrophobic exposure on the surface of p53DBD. Analysis of acrylamide quenching experiments revealed that the binding of metal ions to p53DBD induced a structural modification of the protein and this change provided significant protection against acrylamide quenching. Overall, the present results indicated that p53DBD underwent a conformational change upon the binding of metal ions which was characterized by an increased stability of the protein.

Accurate determination of carboxyl-terminal fragment of Presenilin 1 in various tissues from rat and cell lines

Chai, H. L., Miura, S. — 2009-03-24

Presenilin 1 (PS1) has been identified as a causative gene for the early-onset of familial Alzheimer's disease, and it is mainly localized in the endoplasmic reticulum and the Golgi membrane as a multiple membrane-spanning protein. In the cell, PS1 is proteolytically processed to a 30 kDa N-terminal fragment and a 20 kDa C-terminal fragment (CTF), both of which exist as a stable high molecular weight protein complex, together with other components of -secretase. However, as there has been no report about the precise amount of PS1 expressed in mammalian tissues, the aim of this study was to quantitatively determine PS1-CTF amounts in various tissues such as liver, kidney, brain, and heart of rat by Western blotting using a [³⁵S]-methionine-labeled PS1-CTF as a standard synthesized in a wheat germ cell-free protein synthesizing system. PS1-CTF contents in kidney, liver, brain, and heart were 17.0, 6.6, 6.4, and 0.2 fmol/mg protein, respectively. PS1-CTF contents were also determined in cultured cell lines such as HeLa, HEK293, and COS-1.

Highly thermostable L-threonine dehydrogenase from the hyperthermophilic archaeon Thermococcus kodakaraensis

Bashir, Q., Rashid, N., Jamil, F., Imanaka, T., Akhtar, M. — 2009-03-23

L-Threonine dehydrogenase, a key enzyme in the L-threonine metabolism, catalyzes the NAD⁺-dependent conversion of L-threonine to 2-amino-3-ketobutyrate, that non-enzymically decarboxylates to aminoacetone. A search of the genome sequence of hyperthermophilic archaeon, Thermococcus kodakaraensis revealed the presence of a closely related orthologue (TK0916) of archaeal and bacterial L-threonine dehydrogenase genes. Expression in Escherichia coli, purification and characterization of the TK0916 gene product revealed that this gene actually coded for a protein with high levels of L-threonine dehydrogenase activity (7.26 U mg^-1). The enzyme exhibited highest activity at pH 12 and 90°C. The K_m values for L-threonine and NAD⁺ at 50°C were 1.6 mM and 0.028 mM, respectively. The enzyme activity was dependent on divalent cations. The half-life of the enzyme was more than two hours at 85°C and 24 min in boiling water. This is the most thermostable threonine dehydrogenase exhihibiting optimal activity at the highest pH (12) reported to date.This is the first report on the characterization of a TDH from genus Thermococcus.

DNA-Binding Property of the Novel DNA-Binding Domain STPR in FMBP-1 of the Silkworm Bombyx mori

2009-03-20

The STPR domain is a novel DNA-binding domain composed of repeats of 23 amino acid-long peptide found in the fibroin-modulator-binding protein-1 (FMBP-1) of the silkworm Bombyx mori. Theoretical proteins having the STPR domain are highly conserved, particularly in vertebrates, but the functions are mostly unknown. In this study, the DNA-binding property of the STPR domain in FMBP-1 was examined. Use of reagents selecting the DNA groove and an oligonucleotide in which the dA:dT pairs of the probe were replaced with dI:dC pairs in mobility shift assay demonstrated that FMBP-1 approaches DNA from the major groove. Permutation electrophoresis using probes of the same length but containing the FMBP-1-binding site at different positions showed that FMBP-1 bends DNA through its binding. To induce the sharp bend of DNA, the STPR domain alone was insufficient, and the long N-terminal extending region was necessary. Moreover, the basic region extending from the N-terminus of the STPR domain stabilized the DNA binding of the STPR domain. These results suggested that DNA-binding properties of the STPR domain are affected strongly by the structure of the flanking regions in the STPR domain-containing proteins.

Gene expression profiling identifies a role for CHOP during inhibition of the mitochondrial respiratory chain

2009-03-20

Mitochondrial dysfunction, in particular, interference in the respiratory chain, is often responsible for the toxicogenic effects of xenobiotics. In this study, changes in gene expression resulting from pharmacological inhibition of the respiratory chain were studied by DNA microarray analysis using cells treated with rotenone or antimycin A, which inhibit complexes I and III of the electron transport system, respectively. Forty-eight genes were either up- or down-regulated more than 3-fold. These included stress- and/or metabolic-related effector genes and several transcriptional regulators represented by CHOP-10. Further study using siRNA showed that among the 4 genes studied, upregulation of 3 was dependent on CHOP-10. C/EBPβ, a dimerizing partner of CHOP-10, was also involved in 2 of the 3 genes including Trib3, implying that CHOP-10, heterodimerizing with C/EBPβb or another partner played a key role in the expression of a set of genes under stress. Although CHOP-10 and Trib3 were both ER-stress response genes, signal inducing Trib3 during mitochondrial stress was distinct from that during ER stress. Cytotoxicity caused by inhibition of the respiratory chain was attenuated by treatment with siRNA for CHOP-10. This study demonstrated the importance of CHOP-10 in coordinating individual gene expression in response to the mitochondrial stress.

Oncogenic Functions of PTK6 Are Enhanced by Its Targeting to Plasma Membrane But Abolished by Its Targeting to Nucleus

Kim, H. I., Lee, S.-T. — 2009-03-20

PTK6 (also known as Brk) is an intracellular tyrosine kinase whose expression is upregulated in several tumor types. Because localization of protein tyrosine kinases plays an important role in the development of cancers, we investigated the relationship between subcellular localization of PTK6 and its oncogenic properties. PTK6 was targeted to the plasma membrane or the nucleus of HEK 293 cells using the Src myristoylation signal (Myr) or SV40 T-antigen nuclear localization signal (NLS), respectively. The profile of cellular proteins phosphorylated by Myr-PTK6 was quite different from those phosphorylated by NLS-PTK6. Localization of PTK6 to the plasma membrane enhanced the ability of PTK6 to promote proliferation, cell survival, and migration, and to permit anchorage-independent colony formation. In contrast, nuclear localization of PTK6 impaired these functions. Our results demonstrate that recruitment of PTK6 to the plasma membrane is required for oncogenic function.

Placenta Expressing the Greatest Quantity of Bisphenol A Receptor ERR{gamma} among the Human Reproductive Tissues: Predominant Expression of Type-1 ERR{gamma} Isoform

2009-03-20

Estrogen-related receptor (ERR), one of 48 human nuclear receptors, has a fully active conformation with no ligand. We recently demonstrated that ERR binds strongly bisphenol A (BPA), one of the nastiest endocrine disruptors, and thus retaining ERR’s high basal constitutive activity. A report that BPA accumulates in the human maternal-fetal placental unit has led us to hypothesize that a large amount of ERR might exist in the human placenta. Here we report evidence that placenta indeed expresses ERR exceptionally strongly. We first ascertained the presence of nine different ERR mRNA variants and the resulting three ERR protein isoforms. By real-time PCR, we estimated the relative amount of ERR mRNA using total RNA extracts from human reproductive tissues. Placenta was found to express ERR extremely highly. Among the three ERR protein isoforms, placenta exclusively expresses the type-1 isoform, which possesses additional 23-mer amino acid residues at the N-terminus of the ordinary ERR. This N-terminal elongation was found to elevate by approximately 50% the basal constitutive activity of ERR, as evidenced in the luciferase reporter gene assay. The present results suggest that BPA accumulates in the placenta by binding to ERR.

Characterization of Putative Tryptophan Monooxygenase from Ralstonia solanasearum

Kurosawa, N., Hirata, T., Suzuki, H. — 2009-03-20

The amino acid sequence of a putative tryptophan monooxygenase (PTMO) from Ralstonia solanacearum is homologous with that of proenzyme (proPAO) of L-Phe oxidase (deaminating and decarboxylating) (PAO) from Pseudomonas sp. P-501 in their overall sequences. PTMO was expressed in E. coli and purified, but had no catalytic activity to oxidize L-Phe. By treating PTMO with various proteases, the Pronase-treated PTMO (PTMOp) showed a relatively high activity to oxidize L-Phe, L-Trp, L-Tyr, and L-Met. Studies on the stoichiometry of the reaction showed that L-Phe and L-Tyr were mostly oxygenated, that L-Met was mostly oxidized, and both oxygenation and oxidation of L-Trp was observed. Initial velocity patterns were a ping-pong type with L-Phe and L-Tyr, and a sequential type with L-Trp and L-Met as substrate. The spectrum of enzymes with sufficient amounts of these substrates to reduce the enzyme showed a long wavelength species (purple complex) with L-Phe, but not with L-Tyr, L-Trp, and L-Met. These results lead to the conclusion that PTMO and PTMOp belong to proPAO and PAO, respectively.

Influence of Magnesium Ion on the Binding of p53 DNA Binding Domain to DNA Response Elements

Xue, Y., Wang, S., Feng, X. — 2009-03-18

Site-specific recognition and DNA-binding activity of p53 are crucial for its tumor suppressor function. Previous reports have shown that metal ions can affect the specific recognition and DNA-binding activity of p53DBD. Here we firstly report that magnesium ion can bind to the protein and influence its DNA-binding activity. To elucidate the nature and the effect of metal ions in the reaction chemistry, we utilized endogenous tryptophan fluorescence to quantitate the interaction between p53DBD and metal ions. The K_a value for the binding of Mg²⁺ to the protein is 1.88x10³ M^–1. Analysis of the CD data clearly suggested that the binding of magnesium ion induced a subtle conformational change rather than a radical modification of the overall protein architecture. Based on the results of electrophoretic mobility shift assays and fluorescence experiments, we concluded that the binding of Mg²⁺ significantly stimulated the binding of the protein to DNA in a sequence-independent manner, which differed from that of zinc ions in a sequence-specific manner. Based on these results and the fact that Mg2+ exists at relatively high concentration in the cell, we propose that Mg²⁺ is one of potential factors to affect or regulate the transactivation of p53.

High sensitivity analysis of naturally occurring sugar-chains, using a novel fluorescent linker molecule

Sato, M., Ito, Y., Arima, N., Baba, M., Sobel, M., Wakao, M., Suda, Y. — 2009-03-13

To analyze the binding of sugar-chains to proteins, viruses, and cells, the surface plasmon resonance (SPR) technique is very convenient and effective because it is a real time, nondestructive detection system. Key to this method is linker compounds for immobilization of the sugar-chains to the gold coated chip for SPR. Also, well-designed fluorescent labeling reagents are essential when analyzing the structure of trace amounts of sugar-chains derived from natural sources, such as glycoproteins on the surface of specific cells. In this report, we developed a novel linker molecule, named "f-mono", which has both of these properties: simple immobilization chemistry and a fluorescent label. Since the molecule contains a 2,5-diaminopyridyl group and a thioctic acid group, conjugation with sugar-chains can be achieved using the well-established reductive amination reaction. This conjugate of sugar-chain and fluorescent linker (fluorescent ligand-conjugate, FLC) has fluorescent properties (ex. 335 nm, em. 380 nm), and as little as 1 g of FLC can be easily purified using HPLC with a fluorescent detector. MS and MS/MS analysis of the FLC is also possible. Because a +2 Da larger MS peak ([M+H+2]+ ion) was always associated with the theoretical MS peak ([M+H]+) (due to the reduction of the thioctic acid moiety), the MS peaks of the FLC were easily found, even using unfractionated crude samples. Immobilization of the FLC onto gold coated chips, and their subsequent SPR analyses were successively accomplished, as had been performed previously using non-fluorescent ligand conjugates.

Crystallographic Snapshots of an Entire Reaction Cycle for a Retaining Xylanase from Streptomyces Olivaceoviridis E-86

2009-03-11

Retaining glycosyl hydrolases, which catalyze both glycosylation and deglycosylation in a concerted manner, are the most abundant hydrolases. To date, their visualization has tended to be focused on glycosylation because glycosylation reactions can be visualized by inactivating deglycosylation step and/or using substrate analogues to isolate covalent intermediates. Furthermore, during structural analyses of glycosyl hydrolases with hydrolytic reaction products by the conventional soaking method, mutarotation of an anomeric carbon in the reaction products promptly and certainly occurs. This undesirable structural alteration hinders visualization of the second step in the reaction. Here, we investigated X-ray crystallographic visualization as a possible method for visualizing the conformational itinerary of a retaining xylanase from Streptomyces olivaceoviridis E-86. To clearly define the stereochemistry at the anomeric carbon during the deglycosylation step, extraneous nucleophiles, such as azide, were adopted to substitute for the missing base catalyst in an appropriate mutant. The X-ray crystallographic visualization provided snapshots of the components of the entire reaction, including the E•S complex, the covalent intermediate, breakdown of the intermediate and the E•P complex.

Protein kinase C {delta} plays a key role in cellular senescence programs of human normal diploid cells

2009-03-11

In the present study, we clarified that transforming growth factor β(TGF-β) induces cellular senescence in human normal diploid cells, TIG-1, and identified protein kinase Cs (PKCs) as downstream mediators of TGF-β-induced cellular senescence. Among PKCs, we showed that PKC- induced cellular senescence in TIG-1 cells and was activated in replicatively and prematurely senescent TIG-1 cells. The causative role of PKC- in cellular senescence programs was demonstrated using a kinase negative PKC- and small interfering RNA against PKC-. Further, PKC- was shown to function in human telomerase reverse transcriptase (hTERT) gene repression. These results indicate that PKC- plays a key role in cellular senescence programs, and suggest that the induction of senescence and hTERT repression are coordinatedly regulated by PKC-.

Reevaluation of M-LAO, L-amino acid oxidase, from the venom of Gloydius blomhoffi as an anticoagulant protein

Fujisawa, D., Yamazaki, Y., Morita, T. — 2009-03-11

Many anticoagulant proteins have been found from snake venoms. Recently, _L-amino acid oxidase (LAO) from the venom of Gloydius blomhoffi, M-LAO, was reported to inhibit coagulation factor IX; however, the mechanism of its anticoagulant activity is still unclear. Here, we reevaluated the anticoagulant activity of M-LAO. We first purified M-LAO from the venom of G. blomhoffi, and examined the effect of LAO inhibitors and the hydrogen peroxide scavenger, catalase, on the anticoagulant activity of M-LAO. We found that the isolated M-LAO fraction prolongs the APTT, PT, and fibrinogen clotting time and cleaves the A-chain of fibrinogen. LAO inhibitors or catalase did not inhibit these effects. Detailed analysis revealed that the M-LAO fraction contained a small amount of 39 kDa metalloproteinase. The prolongation of clotting time and degradation of fibrinogen were inhibited by a metalloproteinase inhibitor. Therefore, we concluded that the anticoagulant activity of the M-LAO fraction was due to the 39 kDa metalloproteinase.

Sensitive Assay of Glycogen Phosphorylase Activity by Analyzing the Chain-Lengthening Action on a Fluologenic Maltooligosaccharide Derivative

Makino, Y., Omichi, K. — 2009-03-11

The action of glycogen phosphorylase (GP) is essentially reversible, although GP is generally classified as a glycogen-degrading enzyme. In this study, we developed a highly sensitive and convenient assay for GP activity by analyzing its chain-lengthening action on a fluorogenic maltooligosaccharide derivative in a glucose-1-phosphate-rich medium. Characterization of the substrate specificity of GP using pyridylaminated (PA-) maltooligosaccharides of various sizes revealed that a maltotetraosyl (Glc₄) residue comprising the nonreducing-end of a PA-maltooligosaccharide is indispensable for the chain-lengthening action of GP, and PA-maltohexaose is the most suitable substrate for the purpose of this study. By using a high-performance liquid chromatograph equipped with a fluorescence spectrophotometer, PA-maltoheptaose produced by the chain elongation of PA-maltohexaose could be isolated and quantified at 10 fmol. This method was used to measure the GP activities of crude and purified GP preparations, and was demonstrated to have about 1000 times greater sensitivity than the spectrophotometric orthophosphate assay.

PCNA Mono-ubiquitination and Activation of Translesion DNA Polymerases by DNA Polymerase {alpha}

2009-03-11

Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol , PCNA was spontaneously mono-ubiquitinated. Pol L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol errors, pol participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol ) suppressed this defect. These data suggest that nucleotide misincorporation by pol induces exposure of single-stranded DNA, PCNA mono-ubiquitination, and activates TLS pols.

Expression analysis of the aldo-keto reductases involved in the novel biosynthetic pathway of tetrahydrobiopterin in human and mouse tissues

2009-03-09

Tetrahydrobiopterin (BH4) acts as a cofactor of the aromatic amino acid hydroxylases, and its deficiency may result in hyperphenylalaninemia (HPA) and decreased production of the neurotransmitters. BH4 is synthesized by sepiapterin reductase (SPR) from 6-pyruvoyl-tetrahydropterin (PPH4). A patient with SPR deficiency shows no HPA; however, an SPR knockout mouse exhibits HPA. We have reported on the SPR-unrelated novel biosynthetic pathway from PPH4 to BH4 (salvage pathway II). in which 3 -hydroxysteroid dehydrogenase type 2 and aldose reductase work in concert. In this study, we performed the expression analysis of both proteins in humans and wild-type mice. The results of expression analysis indicated that salvage pathway II worked in human liver; however, it did not act in human brain or in mouse liver and brain. For this reason, a patient with SPR deficiency may show progressive neurological deterioration without HPA, and SPR knockout mice may exhibit HPA and abnormal locomotion activity.

This article has been published online in error and has been removed

2008-06-13

Retraction: One-step purification of a P. pseudoalcaligenes lipase induced by a specific substrate

Helmy, M, El-Adl, N — 2007-03-26

The authors, M Helmy and N El-Adl, found critical problems with some of the data they reported in the above paper and wish to withdraw it from publication. The paper has been published online in Journal of Biochemistry Advance Access and the authors hereby retract this paper and discourage citations of it.