Journal of Biochemistry - current issue

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.