Journal of Biochemistry - recent issues

Disulfide Bond Formation System in Escherichia coli

Inaba, K. — Wed, 04 Nov 2009 06:24:50 PST

The biological kingdoms have evolved elaborate systems that ensure the catalysis of protein disulfide bond (dsb) formation in the cell. Coexisting in the periplasm of Escherichia 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 centre involved in protein Dsb 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.

Disulphide Bond Formation in the Intermembrane Space of Mitochondria

Deponte, M., Hell, K. — Wed, 04 Nov 2009 06:24:50 PST

Proteins of the intermembrane space (IMS) of mitochondria fulfil crucial functions in cellular processes, such as transport of proteins and metal ions, ATP production and apoptotic cell death. All IMS proteins are synthesized in the cytosol and then transported across the mitochondrial outer membrane. A subset of these proteins contains disulphide bonds. For their import into the IMS, they employ a disulphide relay system, made up of two essential proteins, Mia40/Tim40 and the flavin-dependent sulfhydryl-electron transferase Erv1. The disulphide relay system introduces disulphide bonds in substrate proteins triggering their folding. The oxidative folding traps substrates in the IMS and thereby drives their net import into the IMS. Thus, protein import is coupled to oxidative protein folding, maybe providing a first control of protein quality. Here, we review the current knowledge about the Erv1-Mia40 system and address aspects that require further consideration.

Quality Control Against Misfolded Proteins in the Cytosol: A Network for Cell Survival

Kubota, H. — Wed, 04 Nov 2009 06:24:50 PST

Misfolded proteins are toxic to cells and the accumulation of toxic species can lead to protein misfolding diseases, such as neurodegenerative disorders. The toxicity of misfolded proteins is thought to result from the presence of exposed hydrophobic surfaces, which mediate unnecessary binding to normal proteins, interrupting essential interactions between cellular proteins. To prevent toxicity, quality control systems monitor protein folding and remove misfolded species in the cytosol. Molecular chaperones recognize and mask hydrophobic surfaces of misfolded monomers, and transfer them to the ubiquitin–proteasome system and chaperone-mediated autophagy. To eliminate soluble aggregates of misfolded proteins, the macroautophagy–lysosome system is thought to degrade proteasome-resistant toxic species. In addition, the microtubule-dependent transport system sequesters soluble oligomers/aggregates into inclusion bodies. These systems are regulated by stress-inducible transcription factors, cochaperones and other cofactors for the effective removal of toxic monomers and oligomers. This review explores the roles of protein quality control pathways and networks that control quality control activities in the cytosol, particularly focusing on recent progress in this field.

Functional Enhancement by Single-residue Substitution of Streptomyces coelicolor A3(2) H+-translocating Pyrophosphatase

Hirono, M., Maeshima, M. — Wed, 04 Nov 2009 06:24:50 PST

H⁺-translocating pyrophosphatase converts energy from hydrolysis of pyrophosphate to active H⁺ transport across biomembranes. Mutational analysis of Streptomyces coelicolor A3(2) enzyme revealed that amino acid substitution of Phe-388 and Ala-514 altered the enzyme activity. Both residues are located at the interface between the transmembrane domains and cytosolic loops, in which the catalytic domain exists. Systematic amino acid substitution was carried out using the Escherichia coli heterologous expression system. Two of the 38 mutant enzymes, F388Y and A514S, showed a high ratio of H⁺-pump to substrate hydrolysis without decrease in the substrate hydrolysis activity, indicating high energy-coupling efficiency.

Vascular Endothelium Expresses 3-Mercaptopyruvate Sulfurtransferase and Produces Hydrogen Sulfide

Shibuya, N., Mikami, Y., Kimura, Y., Nagahara, N., Kimura, H. — Wed, 04 Nov 2009 06:24:50 PST

Hydrogen sulfide (H₂S) has been recognized as a smooth muscle relaxant. Cystathionine -lyase, which is localized to smooth muscle, is thought to be the major H₂S-producing enzyme in the thoracic aorta. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) and cysteine aminotransferase (CAT) are localized to vascular endothelium in the thoracic aorta and produce H₂S. Both 3MST and CAT were localized to endothelium. Lysates of vascular endothelial cells produced H₂S from cysteine and -ketoglutarate. The present study provides a new insight into the production and release of H₂S as a smooth muscle relaxant from vascular endothelium.

Vimentin Intermediate Filaments as a Template for Silica Nanotube Preparation

Gohara, R., Liu, D., Nakashima, K., Takasaki, Y., Ando, S. — Wed, 04 Nov 2009 06:24:50 PST

Organic compounds are used as templates to regulate the morphology of inorganic nanostructures. In the present study, we used intermediate filaments (IFs), the major cytoskeleton component of most eukaryotic cells, as a template for hollow silica nanotube preparation. Sol–gel polymerization of tetraethoxysilane proceeded preferentially on the surface of IFs assembled from vimentin protein in vitro, resulting in silica-coated fibres. After removing IFs by calcination, electron microscopy revealed hollow silica nanotubes several micrometers long, with outer diameters of 35–55 nm and an average inner diameter of 10 nm (comparable to that of IFs). Furthermore, the silica nanotubes exhibited a gnarled surface structure with an 18–26 nm repeating pattern (comparable to the 21-nm beading pattern along IFs). Thus, the characteristic morphology of IFs were well replicated into hollow silica nanotubes, suggesting that IFs maybe useful as an organic template.

Characterization and Application of Carbohydrate-binding Modules of {beta}-1,3-xylanase XYL4

Kiyohara, M., Sakaguchi, K., Yamaguchi, K., Araki, T., Ito, M. — Wed, 04 Nov 2009 06:24:50 PST

β-1,3-Xylanase from Vibrio sp. strain AX-4 (XYL4) is a modular enzyme composed of an N-terminal catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules (CBMs) belonging to family 31 in the C-terminal region. To investigate the functions of these three modules, five deletion mutants lacking individual modules were constructed. The binding assay of these mutants showed that a repeating unit of the CBM was a non-catalytic β-1,3-xylan-binding module, while the catalytic module per se was not likely to contribute to the binding activity when insoluble β-1,3-xylan was used for the assay. The repeating CBMs were found to specifically bind to insoluble β-1,3-xylan, but not to β-1,4-xylan, Avicel, β-1,4-mannan, curdlan, chitin or soluble glycol-β-1,3-xylan. Both the enzyme and the binding activities for insoluble β-1,3-xylan but not soluble glycol-β-1,3-xylan were enhanced by NaCl in a concentration-dependent manner, indicating that the CBMs of XYL4 bound to β-1,3-xylan through hydrophobic interaction. This property of the CBMs was successfully applied to the purification of a recombinant XYL4 from the cell extracts of Escherichia coli transformed with the xyl4 gene and the detection of β-1,3-xylan-binding proteins including β-1,3-xylanase from the extract of a turban shell, Turbo cornutus.

Mutational Analysis of the GTP-binding Motif of FlhF which Regulates the Number and Placement of the Polar Flagellum in Vibrio alginolyticus

Kusumoto, A., Nishioka, N., Kojima, S., Homma, M. — Wed, 04 Nov 2009 06:24:50 PST

Precise regulation of the number and placement of flagella is critical for the mono-flagellated bacterium Vibrio alginolyticus to swim efficiently. We previously proposed a model in which the putative GTPase FlhF determines the polar location and generation of the flagellum, the putative ATPase FlhG interacts with FlhF to prevent FlhF from localizing to the pole, and thus FlhG negatively regulates the flagellar number in V. alginolyticus cells. To investigate the role of the GTP-binding motif of FlhF, we generated a series of alanine-replacement mutations at the positions that are highly conserved among homologous proteins. The results indicate that there is a correlation between the polar localization and the ability to produce flagella in the mutants. We investigated whether the mutations in the GTP-binding motif affected the ability to interact with FlhG. In contrast to our prediction, no significant difference was detected in the interaction with FlhG between the wild-type and mutant FlhFs. We showed that the GTP-binding motif of FlhF is important for polar localization of the flagellum but not for the interaction with FlhG.

Importance of the Hydrogen Bonding Network Including Asp52 for Catalysis, as Revealed by Asn59 Mutant Hen Egg-white Lysozymes

Ose, T., Kuroki, K., Matsushima, M., Maenaka, K., Kumagai, I. — Wed, 04 Nov 2009 06:24:50 PST

In the catalysis of sugar hydrolysis by hen egg-white lysozyme, Asp52 is thought to stabilize the reaction intermediate. This residue is involved in the well-ordered hydrogen bonding network including Asn46, Asp48, Ser50 and Asn59 on the anti-parallel β-sheet, designated as a ‘platform’, on which the substrate sugar sits. To reveal the role of this hydrogen bonding network in the hydrolysis, we characterized Asn59 mutants by biochemical and crystallographic studies. Surprisingly, the introduction of only a methylene group by the Asn59Gln mutation markedly reduced the bacteriolytic activity and abolished the hydrolytic activity towards the synthetic substrate, PNP-(GlcNAc)₅. A similar result was also obtained with the Asn59Asp mutant. The crystal structure of the Asn59Asp mutant in complex with the substrate analogue revealed that, as in the wild-type, the (GlcNAc)₃ was bound in the A–B–C subsites. The reduced activity would be caused by subtle changes in the side-chain orientations as well as the electrostatic characteristics of Asp59, resulting in the rearrangement of the hydrogen bonding network of the platform. These results suggest that the precise locations of these ‘platform’ residues, maintained by the well-ordered hydrogen bonding network, are crucial for efficient hydrolysis.

Mycolyltransferase from Mycobacterium leprae Excludes Mycolate-containing Glycolipid Substrates

Wed, 04 Nov 2009 06:24:50 PST

Trehalose dimycolate (TDM) is a major surface-exposed mycolyl glycolipid that contributes to the hydrophobic cell wall architecture of mycobacteria. Nevertheless, because of its potent adjuvant functions, pathogenic mycobacteria appear to have evolved an evasive maneuver to down-regulate TDM expression within the host. We have shown previously that Mycobacterium tuberculosis (M.tb) and Mycobacterium avium (M.av), replace TDM with glucose monomycolate (GMM) by borrowing host-derived glucose as an alternative substrate for the FbpA mycolyltransferase. Mycobacterium leprae (M.le), the causative microorganism of human leprosy, is also known to down-regulate TDM expression in infected tissues, but the function of its mycolyltransferases has been poorly analysed. We found that, unlike M.tb and M.av FbpA enzymes, M.av FbpA was unexpectedly inefficient in transferring -branched mycolates, resulting in impaired production of both TDM and GMM. Molecular modelling and mutational analysis indicated that a bulky side chain of leucine at position 130 of M.le FbpA obstructed the intramolecular tunnel that was proposed to accommodate the -branch portion of the substrates. Notably, even after a highly reductive evolution, M.le FbpA remained functional in terms of transferring unbranched acyl chains, suggesting a role that is distinct from that as a mycolyltransferase.

NMR Solution Structure of HP0827 (O25501_HELPY) from Helicobacter pylori: Model of the Possible RNA-binding Site

Wed, 04 Nov 2009 06:24:50 PST

The HP0827 protein is an 82-residue protein identified as a putative ss-DNA-binding protein 12RNP2 Precursor from Helicobacter pylori. Here, we have determined 3D structure of HP0827 using Nuclear Magnetic Resonance. It has a ferredoxin-like fold, β1–1–β2–β3–2–β4 (; -helix and β; β-sheet) and ribonucleoprotein (RNP) motifs which are thought to be important in RNA binding. By using structural homologues search and analyzing electrostatic potential of surface, we could compared HP0827 with other RNA-binding proteins (sex-lethal, T-cell restricted intracellular antigen-1, U1A) to predict RNA-binding sites of HP0827. We could predict that β sheets of HP0827, especially β1 and β3, are primary region for RNA binding. Consequently, similar to other RNA-binding proteins, RNP motifs (Y5, F45, F47), positively charged and hydrophobic regions (K32, R37, K40, K41, K43, R70, R73) are proposed as a putative RNA-binding sites. In addition, differences in amino acids composition of RNP motifs, N, C-terminal residues, loop-region fold and the orientation of 1-helix with other RNA recognition motif proteins could give specific biological functions to HP0827. Finally, the study on natural RNA target is also important to completely understand the biological function of HP0827.

Production of n-octanoyl-modified Ghrelin in Cultured Cells Requires Prohormone Processing Protease and Ghrelin O-acyltransferase, as well as n-octanoic Acid

Wed, 04 Nov 2009 06:24:50 PST

Ghrelin was originally isolated from rat stomach as an endogenous ligand for the GH secretagogue receptor. The major active form of ghrelin is a 28-amino acid peptide modified by an n-octanoic acid on the serine 3 residue, and this lipid modification is essential for the biological activity of ghrelin. However, it is not clear whether prohormone convertase (PC) and ghrelin O-acyltransferase (GOAT) are the minimal requirements for synthesis of acyl-modified ghrelin in cultured cells. By using three cultured cell lines, TT, AtT20 and COS-7, in which the expression levels of processing proteases and GOAT vary, we examined the processing patterns of ghrelin precursor. We found that not only PC1/3 but also both PC2 and furin could process proghrelin to the 28-amino acid ghrelin. Moreover, the presence of PC and GOAT in the cells, as well as n-octanoic acid in the culture medium, was necessary to produce n-octanoyl ghrelin.

Angiotensin II Stimulates KLF5 Phosphorylation and its Interaction with c-Jun Leading to Suppression of p21 Expression in Vascular Smooth Muscle Cells

He, M., Han, M., Zheng, B., Shu, Y.-N., Wen, J.-K. — Wed, 04 Nov 2009 06:24:50 PST

Krüppel-like factor 5 (KLF5) and c-Jun are involved in angiotensin II (Ang II)-induced cell proliferation and play an important role in p21 expression. But the direct and functional implications of KLF5 and c-Jun in regulating p21 expression in vascular smooth muscle cells (VSMCs) are unclear. Here, we show that Ang II upregulated KLF5 and c-Jun expression and inhibited p21 expression in VSMCs, and silencing of KLF5 expression by KLF5-specific small interfering RNA (siRNA) neutralized the inhibitory effects of Ang II on p21 expression. Exposure of VSMCs to Ang II rapidly and strongly stimulated KLF5 phosphorylation, which results in an increase of the interaction of KLF5 with c-Jun. Treating VSMCs with PD98059, the ERK inhibitor, inhibited ERK activation and KLF5 phosphorylation as well as the interaction between KLF5 and c-Jun. Reporter analysis showed that both KLF5 and c-Jun cooperatively repressed the promoter of p21. Furthermore, KLF5 bound to its cis-elements in the p21 promoter, and meanwhile interacted with c-Jun in Ang II-induced VSMCs. These results suggest that Ang II induces KLF5 phosphorylation mediated by the ERK signalling in VSMCs, which in turn stimulates the interaction of KLF5 with c-Jun, subsequently leads to the suppression of p21 expression.

Differential Usage of the Transport Systems for Folic acid and Methotrexate in Normal Human T-Lymphocytes and Leukemic Cells

Biswal, B. K., Verma, R. S. — Wed, 04 Nov 2009 06:24:50 PST

Methotrexate (MTX) has been used as an effective anti-cancer drug for a long time. Conceptually, it is accepted that MTX and folic acid are transported by folate receptors (FRs) in cancerous cells, but the exact mechanism of MTX uptake in human leukemia is unknown. The objective of this study was to investigate different transport systems for FA and MTX, and to delineate their uptake mechanism in MOLT4, K562, Hut78 leukemia cells and normal human T cells. In MOLT4, uptake of MTX was higher than FA, similar to that of K562, Hut78 and normal T cells. In MOLT4 cells, MTX uptake was maximum at pH 7.4 whereas FA uptake was maximum at pH 4.5. Uptake of FA and MTX was significantly inhibited by anions, suggesting anion-dependent transport system. FA uptake was found to be energy dependent whereas MTX uptake was energy independent. RT-PCR and immunofluorescence results demonstrated the presence of reduced folate carrier as well as proton coupled folate transporter and absence of FR in MOLT4 and normal T cells. These data suggest the existence of two separate and independent carrier-mediated transport systems for the uptake of FA and MTX in normal and leukemic human T cells.

Importance of Polarisome Proteins in Reorganization of Actin Cytoskeleton at Low pH in Saccharomyces cerevisiae

Motizuki, M., Xu, Z. — Wed, 04 Nov 2009 06:24:50 PST

The actin cytoskeleton of the yeast Saccharomyces cerevisiae can be altered rapidly in response to external cues. We reported previously that S. cerevisiae responds to low-pH stress by transiently depolarizing its actin cytoskeleton, and that this step requires a mitogen-activated protein kinase, high osmolarity glycerol 1 (Hog1p). This study further investigated the components involved in this actin reorganization at pH 3.0. Gene deletions on the Sln1p branch of the HOG pathway completely blocked actin depolarization, suggesting that Hog1p activation depends mainly on the osmosensor Sln1p. The protein-synthesis inhibitor cycloheximide did not influence the time course of actin depolarization, suggesting that the depolarization is a direct effect of the HOG pathway. Deletion of the scaffolding protein, Spa2p, or the Spa2p-interacting protein Pea2p, markedly inhibited the depolarization, and further deletion of the formin protein, Bni1p, notably delayed actin repolarization. Our results suggest the involvement of polarisome proteins, such as Spa2p, Pea2p and Bni1p, but not Bud6p, in Hog1p-dependent reorganization of the yeast actin cytoskeleton at low pH.

Expression, Purification and Characterization of Soluble Recombinant Periostin Protein Produced by Escherichia coli

Takayama, I., Kii, I., Kudo, A. — Wed, 04 Nov 2009 06:24:50 PST

Periostin is a matricellular protein participating in the tissue remodelling of damaged cardiac tissue after acute myocardial infarction and of the periodontal ligament in mice. However, further studies on the periostin protein have been limited by the intrinsic difficulty of purifying this protein produced in Escherichia coli due to its insolubility. Here, we demonstrate the expression of recombinant periostin protein with high solubility and monodispersity in E. coli. Periostin is composed of an amino-terminal EMI domain, a tandem repeat of 4 fas1 domains (RD1-4), and a carboxyl-terminal region (CTR). We expressed the RD4-CTR region tagged with GST at amino-terminal and 6x Histidine at carboxyl-terminal end in E. coli. The recombinant protein was purified by using GSH-Sepharose and nickel chelation affinity chromatography, followed by gel filtration chromatography. The RD4-CTR protein exhibited high solubility and monodispersity. On average, 9.1 mg of purified RD4-CTR was routinely obtained from 1 L of culture media. Furthermore, the RD4-CTR was biochemically active, because it bound to the RD1-4, the same as intact periostin protein that had been purified from mammalian cells. Our results should enable us to produce the periostin recombinant protein in large quantities and facilitate future studies on functional and structural analyses of periostin.

Association of a Novel Mitochondrial Protein M19 with Mitochondrial Nucleoids

Sumitani, M., Kasashima, K., Ohta, E., Kang, D., Endo, H. — Wed, 04 Nov 2009 06:24:50 PST

We have identified a novel mitochondrial protein, termed M19, by proteomic analysis of mitochondrial membrane proteins from HeLa cells. M19 is highly conserved among vertebrates, and possesses no homologous domains with other known proteins. By northern and western blotting, mouse M19 was shown to be expressed in various tissues, and to be especially abundant in the brain. Human M19 (hM19) is present in mitochondria, and protease-protection experiment showed it to be sublocalized in the matrix space. Carboxy-terminally tagged hM19 appeared as spotted signals within mitochondria and co-localized with signals arising from mitochondrial DNA (mtDNA), suggesting the inclusion of M19 in the mtDNA–protein complex (mitochondrial nucleoids). Fractionation of mitochondrial nucleoids from HeLa cells revealed that hM19 has a similar distribution pattern like that of known nucleoid components, such as mtSSB and PHBs, and surely exists in the nucleoid fraction. Furthermore, expression of M19 is closely related to the amount of mtDNA, because it was down-regulated in mtDNA-depleted ⁰ HeLa cells. These results indicate that M19 associates with the nucleoid and likely regulates the organization and metabolism of mtDNA.

Differential Assembly Properties of Escherichia coli FtsZ and Mycobacterium tuberculosis FtsZ: An Analysis Using Divalent Calcium

Jaiswal, R., Panda, D. — Wed, 04 Nov 2009 06:24:50 PST

The assembly of FtsZ is considered to be a fundamental process during the bacterial cytokinesis. We used several complimentary techniques to probe the assembly of recombinant Escherichia coli FtsZ (EcFtsZ) and Mycobacterium tuberculosis FtsZ (MtbFtsZ) proteins in vitro. As documented earlier, EcFtsZ was found to polymerize at much faster rate than MtbFtsZ. Interestingly, we found that MtbFtsZ produced higher sedimentable polymerized mass than that of the EcFtsZ and that MtbFtsZ formed thicker protofilaments than that of the EcFtsZ. The results indicated that the EcFtsZ polymers are more labile than the MtbFtsZ polymers. Further, divalent calcium exerted strikingly different effects on the assembly of EcFtsZ and MtbFtsZ. Divalent calcium strongly enhanced the assembly of EcFtsZ and promoted bundling and stability of the protofilaments. In contrast, it had no detectable effect on the assembly of MtbFtsZ. In vitro, divalent calcium bound to EcFtsZ with much stronger affinity than to MtbFtsZ and significantly affected the secondary structure of EcFtsZ whereas it did not cause any detectable change in the secondary structure of MtbFtsZ. The results suggested that the assembly characteristics of EcFtsZ and MtbFtsZ are different and indicated that the assembly dynamics of these proteins are regulated by different mechanisms.

Quality Control of Cytoplasmic Membrane Proteins in Escherichia coli

Akiyama, Y. — Mon, 05 Oct 2009 22:50:51 PDT

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.

Protein Quality Control in Mitochondria

Tatsuta, T. — Mon, 05 Oct 2009 22:50:51 PDT

Mitochondria are crucial for both life and death of eukaryotic cells. Compromised mitochondrial integrity has severe cellular consequences and is linked to senescence and neurodegenerative disorders in humans. To maintain the functionality of proteins in mitochondria, quality-control mechanisms including signal transduction pathways counteracting mitochondrial stress have evolved. A network of molecular chaperones and proteases monitors protein integrity and prevents accumulation of damaged proteins. In this review, the current knowledge of elaborate defence strategies within mitochondria is summarized.

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

Kato, Y., Sakamoto, W. — Mon, 05 Oct 2009 22:50:51 PDT

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 of more than 20 subunits. Among the subunits is one of the intrinsic reaction centre 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.

Dynamic Expression of Peptidylarginine Deiminase 2 in Human Monocytic Leukaemia THP-1 Cells During Macrophage Differentiation

Hojo-Nakashima, I., Sato, R., Nakashima, K., Hagiwara, T., Yamada, M. — Mon, 05 Oct 2009 22:50:51 PDT

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 leukaemia 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.

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. — Mon, 05 Oct 2009 22:50:51 PDT

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 anti-viral 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.

Polymyxin B Identified as an Inhibitor of Alternative NADH Dehydrogenase and Malate: Quinone Oxidoreductase from the Gram-positive Bacterium Mycobacterium smegmatis

Mon, 05 Oct 2009 22:50:51 PDT

Tuberculosis is the leading cause of death due to a single infectious agent in the world and the emergence of multidrug-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 [50% inhibitory concentration (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.

Overexpression of Plk3 causes Morphological Change and Cell Growth Suppression in Ras Pathway-activated Cells

Iida, M., Sasaki, T., Komatani, H. — Mon, 05 Oct 2009 22:50:51 PDT

To unravel the growth inhibition mechanism of Polo-like kinase 3 (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.

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

Someya, Y., Takeda, N. — Mon, 05 Oct 2009 22:50:51 PDT

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.

Changes in the Conformation of the Vsr Endonuclease Amino-terminal Domain Accompany DNA Cleavage

Polosina, Y. Y., Cupples, C. G. — Mon, 05 Oct 2009 22:50:51 PDT

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 (VSP) 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 Pol I releases Vsr from the nick by reversing that conformational change.

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. — Mon, 05 Oct 2009 22:50:51 PDT

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) catalyses 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 Escherichia 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 the 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. — Mon, 05 Oct 2009 22:50:51 PDT

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.

Membrane Localization of Protein-Tyrosine Phosphatase 1B is Essential for its Activation of Sterol Regulatory Element-Binding Protein-1 Gene Expression and Consequent Hypertriglyceridaemia

Mon, 05 Oct 2009 22:50:51 PDT

Protein-tyrosine phosphatase 1B (PTP1B) is a major regulator of insulin sensitivity. We have described a novel action of PTP1B in the induction of sterol regulatory element-binding protein-1 (SREBP-1) gene expression through activation of protein phosphatase 2A (PP2A). PTP1B is anchored to the endoplasmic reticulum membrane via its C-terminal tail. We have previously reported that membrane localization of PTP1B is essential for PP2A activation, which is crucial for enhancing SREBP-1 gene expression in in vitro experiments. In this study, we further investigated the physiological importance of membrane localization of PTP1B in vivo. We found that transient liver-specific overexpression of wild-type PTP1B (PTP1B-WT) using adenovirus-mediated gene transfer was associated with hypertriglyceridaemia and enhanced hepatic SREBP-1 gene expression in mice. However, overexpression of the C-terminal truncated PTP1B (PTP1BCT) failed to increase hepatic SREBP-1 expression or serum triglyceride levels, despite causing insulin resistance. Our results indicate that activation of PTP1B in the liver could induce hypertriglyceridaemia and that anchoring of PTP1B to the membrane is crucial for its action.

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

Mon, 05 Oct 2009 22:50:51 PDT

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyses 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 unfavourable for the -proton abstraction by Lys244 and the reaction is expected to stop at the PLP–l-serine aldimine. Structural modelling 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.

Glutamate64 to Glycine Substitution in G1 {beta}-bulge of Ubiquitin Impairs Function and Stabilizes Structure of the Protein

Mishra, P., Volety, S., Rao, Ch. M., Prabha, C. R. — Mon, 05 Oct 2009 22:50:51 PDT

Ubiquitin is a globular protein with a highly conserved sequence. Sequence conservation and compact structure make it an ideal protein for structure–function studies. One of the atypical secondary structural features found in ubiquitin is a parallel G1 β-bulge. Glutamate at 64 is the first residue of this β-bulge and the third residue in a type II turn. However, glycine is seen in these positions in several proteins. To understand the effects of substitution of glutamate64 by glycine on the structure, stability and function of ubiquitin, mutant UbE64G has been constructed and characterized in Saccharomyces cerevisiae. The secondary and tertiary structures of UbE64G mutant protein are only marginally different from wild-type protein (UbWt) and fluorescent form of ubiquitin (UbF45W). The earlier studies have shown that the structure and stability of UbWt and UbF45W were similar. However, UbE64G has less surface hydrophobicity than UbWt. UbE64G is found to be more stable compared with UbF45W towards guanidinium chloride induced denaturation. In vivo, complementation shows substrate proteins with Pro as the N-terminal residue, which undergo ubiquitination, have extended half-lives with UbE64G. This altered preference for Pro as opposed to Met might be related to natural preference of glutamate at 64th position in ubiquitin.

Identification of Radicals Formed in the Reaction Mixture of Bovine Kidney Microsomes with NADPH

Kumamoto, K., Hirai, T., Kishioka, S., Iwahashi, H. — Mon, 05 Oct 2009 22:50:51 PDT

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 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 P₃ gave ions at m/z 266, indicating that 4-POBN/pentyl radical adduct forms in the reaction mixture.

Photocontrol of Calmodulin Interaction with Target Peptides using Azobenzene Derivative

Shishido, H., Yamada, M. D., Kondo, K., Maruta, S. — Mon, 05 Oct 2009 22:50:51 PDT

Calmodulin (CaM), a physiologically important Ca²⁺-binding protein, participates in numerous cellular regulatory processes. It is dumbbell shaped and contains two globular domains connected by a short -helix. Each of the globular domains has two Ca²⁺-binding sites, the EF hands. CaM undergoes a conformational change upon binding to Ca²⁺, which enables it to bind to specific proteins for specific responses. Here, we successfully photocontrolled CaM binding to its target peptide using the photochromic compound N-(4-phenylazophenyl) maleimide (PAM), which reversibly undergoes cis–trans isomerization upon ultraviolet (UV) and visible (VIS) light irradiation. In order to specifically incorporate PAM, CaM mutants having reactive cysteine residues in the functional region were prepared; PAM was stoichiometrically incorporated into the cysteine residues in these mutants. Further, we prepared the target peptide, M13, fused with yellow fluorescent protein (YFP) to monitor the CaM–M13 peptide interaction. The binding of the PAM–CaM mutants, N60C, D64C and M124C, to M13–YFP was reversibly photocontrolled upon UV–VIS light irradiation at appropriate Ca²⁺ concentrations.

Nestin Serves as a Prosurvival Determinant that is Linked to the Cytoprotective Effect of Epidermal Growth Factor in Rat Vascular Smooth Muscle Cells

Wed, 26 Aug 2009 07:37:23 PDT

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 H₂O₂ treatments 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 H₂O₂ treatments, 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. Here, 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.

NMR Structure of the Heterodimer of Bem1 and Cdc24 PB1 Domains from Saccharomyces Cerevisiae

Wed, 26 Aug 2009 07:37:23 PDT

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 3D 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 labelled samples including perdeuteration. The interface between the Bem1/Cdc24 PB1 heterodimer was analysed 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. — Wed, 26 Aug 2009 07:37:23 PDT

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.

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. — Wed, 26 Aug 2009 07:37:23 PDT

Apart from inhibiting RecA activity through protein–protein interactions, Deinococcus radiodurans RecX inhibits the expression of RecA and two other anti-oxidant 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 up-regulated and 23 down-regulated proteins in the mutant. The 12 up-regulated proteins are DNA repair proteins, stress response proteins, and metabolism-related proteins. Most of these have been previously characterized as ionizing radiation-induced proteins. The 23 down-regulated 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.

Fatty Acids Bound to Recombinant Tear Lipocalin and Their Role in Structural Stabilization

Tsukamoto, S., Fujiwara, K., Ikeguchi, M. — Wed, 26 Aug 2009 07:37:23 PDT

A variant of human tear lipocalin was expressed in Escherichia coli, and the bound fatty acids were analysed 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 E. 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.

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. — Wed, 26 Aug 2009 07:37:24 PDT

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 measurements. Although 8-NH₂-FAD-MCAD did not oxidize acyl-CoA the wavelength of the absorption maximum of the flavin was altered by acyl-CoAs binding. Thus, 8-NH₂-FAD-MCAD is one of the attractive materials for investigation of enzyme–substrate (ES) interaction in ES complex (the complex of oxidized MCAD with acyl-CoA). FT-IR difference spectra between non-labelled and [1-¹³C]-labelled 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 ~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.

Domain-dependent Interaction of Eukaryotic Initiation Factor eIF4A for Binding to Middle and C-terminal Domains of eIF4G

Wed, 26 Aug 2009 07:37:24 PDT

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 x 10⁴ to –2.5 x 10⁴ 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.

Human C21orf63 is a Heparin-binding Protein

Wed, 26 Aug 2009 07:37:24 PDT

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 sulphate. 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 polymerase chain reaction and tissue immunostaining imply that C21orf63 is expressed on epithelia of various human tissues.

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. — Wed, 26 Aug 2009 07:37:24 PDT

Tumour 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 the 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 the spleen and that the expression level was much higher in dendritic cells (DCs) and macrophages than that in 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 the S phase of the cell cycle. Our results suggest that TIFAB could act as a negative regulator of the TRAF6-induced cellular function such as B cell proliferation and maturation of DCs and macrophages.

Siccanin Rediscovered as a Species-Selective Succinate Dehydrogenase Inhibitor

Wed, 26 Aug 2009 07:37:24 PDT

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, P. 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.

Crystallographic and Mutational Analyses of Substrate Recognition of Endo-{alpha}-N-acetylgalactosaminidase from Bifidobacterium longum

Wed, 26 Aug 2009 07:37:24 PDT

Endo--N-acetylgalactosaminidase (endo--GalNAc-ase), a member of the glycoside hydrolase (GH) family 101, hydrolyses 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 the 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.

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

Wed, 26 Aug 2009 07:37:24 PDT

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.

Subtilisin-like Proprotein Convertase PACE4 is Required for Skeletal Muscle Differentiation

Yuasa, K., Masuda, T., Yoshikawa, C., Nagahama, M., Matsuda, Y., Tsuji, A. — Wed, 26 Aug 2009 07:37:24 PDT

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

Wed, 26 Aug 2009 07:37:24 PDT

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 chromatid 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 PAK1^174E could be recruited to myosin V motor complex through binding to dynein light chain 2 (DLC2). PAK1^174E could 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

Wed, 26 Aug 2009 07:37:24 PDT

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 >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.

Storage of Gangliosides GM2 and Fucosyl GM1 in the Kidney of MCC Strain of Mastomys (Praomys coucha)

Takimoto, K., Kawamura, N., Kasama, T. — Wed, 26 Aug 2009 07:37:24 PDT

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, -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.