Journal of Biochemistry - Advance Access

Mechanism and components of endoplasmic reticulum-associated degradation

Hoseki, J., Ushioda, R., Nagata, K. — Wed, 18 Nov 2009 06:43:57 PST

The folding of secretory and membrane proteins takes place in the endoplasmic reticulum (ER). The quality of the proteins folded in the ER is carefully monitored by an ER quality control mechanism that allows only correctly folded proteins to be transported to their final destination, and misfolded or unassembled proteins to be retained in the ER and subsequently degraded in a process termed ER-associated degradation (ERAD). The ERAD pathway is conserved from yeast to mammals, and plays an essential role in the maintenance of ER homeostasis, as well as in the prevention of various diseases that arise from the accumulation of aberrant proteins in the ER. In the ERAD pathway, molecular chaperones and lectin-like proteins are involved in the identification of misfolded proteins, ER-resident reductases cleave disulfide bonds in these proteins to facilitate retrograde transport to the cytosol, and AAA⁺ ATPase withdraws them from the retrotranslocation channel to the cytosol where they are degraded by the ubiquitin/proteasome system. The possible mechanisms that underlie ERAD and the various factors involved in this process are discussed in this review.

The extracellular domains of E- and N-cadherin determine the scattered punctate localization in epithelial cells and the cytoplasmic domains modulate the localization

Ozaki, C., Obata, S., Yamanaka, H., Tominaga, S., Suzuki, S. T. — Mon, 16 Nov 2009 23:36:36 PST

The accumulation of classical cadherins is essential for their function, but the mechanism is poorly understood. Hence, we investigated the accumulation of E- and N-cadherin and the formation of cell junctions in epithelial cells. Immunostaining revealed a scattered dot-like accumulation of E- and N-cadherin throughout the lateral membrane in MDCK II and other epithelial cells. Mutant E-cadherin lacking the ß-catenin binding site accumulated granularly at cell-cell contact sites and showed weak cell aggregation activity in cadherin-deficient epithelial cells, MIA PaCa2 cells. Mutant E-cadherin lacking the p120-catenin binding site exhibited scattered punctate accumulation and strong cell adhesion activity in MIA PaCa2 cells. Electron microscopy demonstrated that MIA PaCa2 transfectants of E-cadherin containing ß-catenin binding site formed adherens junction, whereas E-cadherin lacking the binding site did not. Mutant N-cadherins showed accumulation properties similar to those of corresponding mutant E-cadherins. Moreover, wild type and mutant N-cadherin lacking the p120-catenin binding site showed subapical accumulation in polarized DLD-1 cells, whereas mutant N-cadherin lacking ß-catenin binding site did not. These results indicate that the extracellular domains of E- and N-cadherin determines the basic localization pattern, whereas the cytoplasmic domains modulate it thereby affects the cell adhesion activity, subapical accumulation, and the formation of adherens junction.

Protein phosphatase 1{alpha} associates with protein tyrosine phosphatase-PEST inducing dephosphorylation of phospho-serine 39

Nakamura, K., Palmer, H. E.F., Ozawa, T., Mashima, K. — Mon, 16 Nov 2009 23:36:35 PST

Protein tyrosine phosphatase (PTP)-PEST is expressed in a wide variety of several cell types and is an efficient regulator of cell adhesion, spreading and migration. PTP-PEST-associating molecules are important in elucidating the function of PTP-PEST. Herein, we have identified protein phosphatase 1 (PP1) as a novel PTP-PEST binding protein, and then we aimed to determine how PP1 contributes to the phosphorylation at Ser39 of PTP-PEST, whose phosphorylation suppresses PTP-PEST enzymatic activity. The HEK 293 cells overexpressing exogenous PTP-PEST were stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and the phosphorylation of PTP-PEST at Ser39 was evaluated using an anti-phospho-Ser39 PTP-PEST specific antibody (anti-pS39-PEST Ab). It was demonstrated that the phosphorylation at Ser39 detected by anti-pS39-PEST Ab was dependent on TPA treatment and a significant inverse correlation between the PTP activity of PTP-PEST and anti-pS39-PEST Ab-immunoreactive band intensity. The phosphorylation of Ser39 was suppressed by co-transfection of a plasmid encoding wild-type PP1, but not by that of the dominant-negative PP1 mutant. Furthermore, TPA-induced phosphorylation could take place in PTP-PEST catalytic domain, but the phosphorylation of PTP-PEST catalytic domain could not be abrogated by co-transfection of a plasmid expressing wild-type PP1. In conclusion, PP1 associates with the non-catalytic domain of PTP-PEST and regulates PTP activity via dephosphorylation of phospho-Ser39.

The Optimal Activity of a Pseudozymogen Form of Recombinant Matriptase under the Mildly Acidic pH and Low Ionic Strength Conditions

Inouye, K., Yasumoto, M., Tsuzuki, S., Mochida, S., Fushiki, T. — Mon, 16 Nov 2009 23:36:35 PST

Matriptase is a transmembrane serine protease that is strongly expressed in epithelial cells. The single-chain zymogen of matriptase is considered to have inherent activity, leading to its own activation (i.e., conversion to the disulphide-linked-two-chain form by cleavage after Thr-Lys-Gln-Ala-Arg614). Also, there is growing evidence that the activation of zymogen occurs at the cell surface and in relation to the acidification and lowering of ionic strength within cell-surface microenvironments. The present study aimed to provide evidence for the involvement of zymogen activity in its activation in physiologically relevant cellular contexts. For this purpose, the activity of a pseudozymogen form of recombinant matriptase (HL-matriptase zymogen) was examined using acetyl-L-Lys-L-Thr-L-Lys-L-Gln-L-Leu-L-Arg-4-methyl-coumaryl -7-amide as a substrate. HL-matriptase zymogen exhibited optimal activity toward the substrate around pH 6.0. The substrate hydrolysis at the pH value was hardly detected when NaCl was present at a concentration of 145 mM. In a buffer of pH 6.0 containing 5 mM NaCl, the activity of HL-matriptase zymogen was only about 30-times lower than that of the respective two-chain form. These findings suggest that the in vivo activation of matriptase zymogen occurs via a mechanism involving the zymogen activity.

Molecular Mechanisms of the LPS-induced Non-apoptotic ER Stress-CHOP Pathway

Nakayama, Y., Endo, M., Tsukano, H., Mori, M., Oike, Y., Gotoh, T. — Mon, 16 Nov 2009 23:36:34 PST

The expression of C/EBP homologous protein (CHOP), which is an endoplasmic reticulum (ER) stress-induced transcription factor, induces apoptosis. Our previous study demonstrated that LPS-induced CHOP expression does not induce apoptosis, but activates a pro-IL-1β activation process. However, the mechanism by which CHOP activates different pathways depending on the difference in the inducing-stimuli remains to be clarified. The present study shows that LPS rapidly activates the ER function-protective pathway, but not the PERK pathway in macrophages. PERK plays a major role in CHOP induction, and other ER stress sensors-mediated pathways play minor roles. The induction of CHOP by LPS was delayed and weak, in comparison to CHOP induction by ER stress-inducer thapsigargin. In addition, LPS-pretreatment or overexpression of ER chaperone BiP, prevented ER stress-mediated apoptosis. LPS plus IFN-treated macrophages produce a larger amount of nitric oxide (NO) in comparison to LPS-treated cells. Treatment with the NO-donor, SNAP, induces CHOP at an earlier period than LPS treatment. The depletion of NO retards CHOP induction and prevents apoptosis in LPS plus IFN-treated cells. We concluded that apoptosis is prevented in LPS-treated macrophages, because the ER function-protective mechanisms are induced before CHOP expression and induction level of CHOP is low.

Identification of essential residues of CTLA-2{alpha} for inhibitory potency

Deshapriya, R.M.C, Yuhashi, S., Usui, M., Kageyama, T., Yamamoto, Y. — Wed, 11 Nov 2009 23:13:36 PST

To identify functionally essential sequences and residues of CTLA-2, in vitro mutagenesis was carried out. The coefficient of inhibition (Ki) was determined towards rabbit cathepsin L using Z-Phe-Arg-MCA as the substrate. Recombinant CTLA-2 inhibited the enzyme potently (Ki =15nM). A truncated mutant, lacking the N-and C-terminal Ala1 -Asp9 and Leu80 -Glu109 regions, was also a potent inhibitor (Ki=10nM). Subsequent short deletions in the central region (Asn10-Ser79) showed three functionally essential distinct regions, Asn10 -Phe19, His30 -Ala44, and Ser55 -Ser79. These regions cover sequences corresponding to three helices (1,2, and 3) and sequences that interact with the cognate enzyme. Alanine scanning showed that replacement of one of three conserved Trp residues increased the Ki by 15-20 fold; whereas, replacement of two/three Trp residues at once caused complete lost of potency, as did replacing Cys75 with Ala or Ser. The proteins from wild type (WT) CTLA-2 and mutant C75A were stable overnight when incubated with cathepsin L; whereas, proteins from mutants W12A, W15A, and W35A were quickly digested. Incubation of cathepsin L/WT CTLA-2 formed a complex; whereas, C75S did not form a complex. Our overall results point to a critical role of W12, W15, W35 and Cys75 residues in CTLA-2.

Ribosome-binding site interference caused by Shine-Dalgarno-like nucleotide sequences in Escherichia coli cells

Nishizawa, A., Nakayama, M., Uemura, T., Fukuda, Y., Kimura, S. — Wed, 11 Nov 2009 23:13:36 PST

Two-cistronic expression plasmids are useful for high-level expression of heterologous genes in Escherichia coli cells by preventing the inhibition of translational initiation. In the process of constructing a two-cistronic expression plasmid pCbSTCR-4 containing the fragments of the porcine cytochrome b₅ (Psb5) and NADPH-cytochrome P450 reductase (PsCPR) genes as the first and second cistrons, respectively, the presence of a specific region in the first cistron that lowered the accumulation level of the PsCPR was suggested (Kimura, S. et al. (2005) J. Biochem. 137, 523-533). In this study, a disturbing nucleotide sequence similar to a Shine-Dalgarno (SD) sequence (SD-like sequence), AGGAG, was identified at the 5'-upstream region near the SD-sequence for the second cistron. Silent mutations in the SD-like sequence that lowered the similarity to a typical SD-sequence, increased the accumulation level of PsCPR. SD-like sequences introduced into mono-cistronic expression plasmids for the Psb5 and PsCPR genes also decreased the accumulation level of these proteins. The SD-like sequence also decreased the accumulation level of the insoluble PsCPR protein. This type of ribosome-binding site interference is useful not only for precise control of protein accumulation, but also for increasing the soluble form of recombinant proteins in E. coli cells.

Antioxidation and DNA binding properties of binuclear Er(III) complexes with Schiff-base ligands derived from 8-hydroxyquinoline-2-carboxaldehyde and four aroylhydrazines

Liu, Y.-c., Yang, Z.-y. — Wed, 11 Nov 2009 23:13:36 PST

The Er(III) complexes are prepared from Er(NO₃)₃·6H₂O and Schiff-base ligands derived from 8-hydroxyquinoline-2-carboxaldehyde with four aroylhydrazines including benzoylhydrazine, 2-hydroxybenzoylhydrazine, 4-hydroxybenzoylhydrazine and isonicotinylhydrazine, respectively. X-ray crystal and other structural analyses indicate that Er(III) and every ligand can form a binuclear Er(III) complex with nine-coordination and 1:1 metal-to-ligand stoichiometry at the Er(III) center. All the Er(III) complexes can bind to Calf thymus DNA through intercalation with the binding constants at the order of magnitude 10⁶ M^-1, and they may be used as potential anticancer drugs. All the Er(III) complexes have strong scavenging effects for hydroxyl radicals and superoxide radicals, but complex containing active phenolic hydroxyl group shows stronger scavenging effects for hydroxyl radicals and complex containing N-heteroaromatic substituent shows stronger scavenging effects for superoxide radicals.

Transdermal delivery of a readthrough-inducing drug: a new approach of gentamicin administration for the treatment of nonsense mutation-mediated disorders

Wed, 11 Nov 2009 23:13:35 PST

To induce the readthrough of premature termination codons, aminoglycoside antibiotics such as gentamicin have attracted interest as potential therapeutic agents for diseases caused by nonsense mutations. The transdermal delivery of gentamicin is considered unfeasible because of its low permeability through the dermis. However, if the skin permeability of gentamicin could be improved, it would allow topical application without the need for systemic delivery. In this report, we demonstrated that the skin permeability of gentamicin increased with the use of a thioglycolate-based depilatory agent. After transdermal administration the readthrough activity in skeletal muscle, as determined using a lacZ/luc reporter system, was found to be equivalent to systemic administration when measured in transgenic mice. Transdermally applied gentamicin was detected by liquid chromatography-tandem mass spectrometry in the muscles and sera of mice only after depilatory agent-treatment. In addition, expansion of the intercellular gaps in the basal and prickle-cell layers was observed by electron microscopy only in the depilatory agent-treated mice. Depilatory agent-treatment may be useful for the topical delivery of readthough-inducing drugs for the rescue of nonsense mutation-mediated genetic disorders. This finding may also be applicable for the transdermal delivery of other pharmacologically active molecules.

Refolding of an unstable lysozyme by gradient removal of a solubilizer and gradient addition of a stabilizer

Kohyama, K., Matsumoto, T., Imoto, T. — Wed, 11 Nov 2009 07:16:08 PST

Previously we formally established an effective refolding procedure for a protein by gradient removal of a solubilizer such as urea [Maeda et al. (1995) Effective renaturation of reduced lysozyme by gentle removal of urea. Protein Eng. 8, 201-205]. However, this procedure was less effective for unstable proteins. We developed here an excellent method to add protein stabilizer so as to get reasonable amounts of folded protein under the concentration of solubilizer where the unstable protein does not form aggregate. We examined many stabilizers and found that 60% of a concentrated (2.5 mg/ml) unstable protein can be refolded using 40% glycerol as the best stabilizer. This procedure can be widely applicable for the refolding of unstable proteins.

Role of Nrf2 and p62/ZIP in the Neurite Outgrowth by Carnosic Acid in PC12h Cells.

Mon, 09 Nov 2009 04:20:11 PST

Neurotrophins such as NGF promote neuronal survival and differentiation via the cell surface TrkA neurotrophin receptor. Compounds with neurotrophic actions that are low in molecular weight and can permeate the blood-brain barrier are promising therapeutic agents against neurodegenerative diseases such as Alzheimer's disease. Carnosic acid (CA), an electrophilic compound in rosemary, activates antioxidant responsive element (ARE)-mediated transcription via activation of Nrf2. In the present study, we discovered that CA strongly promotes neurite outgrowth of PC12h cells. NGF as well as CA activated Nrf2, whereas CA and NGF-mediated neuronal differentiation was suppressed by Nrf2 knockdown. On the other hand, CA activated TrkA-downstream kinase Erk1/2 independently of Nrf2. CA induced p62/ZIP expression in an Nrf2-dependent manner, while the CA-induced neural differentiation was suppressed by p62/ZIP knockdown. Furthermore, CA-induced ARE activation was attenuated both by p62/ZIP knockdown and a Trk signal inhibitor. These results suggest that the CA induction of p62/ZIP by Nrf2 enhances TrkA signaling which subsequently potentiates Nrf2 pathway. This is the first demonstration that activation of the Nrf2-p62/ZIP pathway by a low-molecular natural electrophilic compound plays important roles in TrkA-mediated neural differentiation and may represent the common molecular mechanism for neurotrophic activities of electrophilic compounds.

Purification of Paracoccidioides brasiliensis catalase P; subsequent kinetic and stability studies.

Fri, 06 Nov 2009 06:03:29 PST

Catalases are essential components of the cellular equipment to cope with oxidative stress. Here we have purified a highly abundant catalase P of Paracoccidioides brasiliensis (PbCatP) that is preferentially expressed in the parasitic yeast phase. This oxidative stress-induced protein was isolated from yeast cells grown in the presence of 15 mM of hydrogen peroxide. We have used consecutive steps of protein precipitation and gel filtration chromatography to achieve the purified protein. Protein purification was validated using matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and bioinformatics analysis. The purified enzyme showed strong similarity to small-subunit catalases. Like most monofunctional catalases, PbCatP is a homotetramer, resistant to inactivation by acidic conditions, temperature, and denaturants. Furthermore, the kinetic behavior of catalase P was observed to be different at low compared to high H₂O₂ concentrations. The results demonstrated that a purified catalase P of P. brasiliensis is a homotetrameric enzyme, classified as a small subunit catalase.

Importance of Tyr310 residue in the third repeat of microtubule binding domain for filament formation of tau protein

Fri, 06 Nov 2009 06:03:29 PST

The inhibition of tau fibrillation is a potential therapeutic target for Alzheimer's and other neurodegenerative diseases. As a series of studies on inhibiting the transition of soluble monomeric tau into mature fibril, the effect of Tyr310 residue in the third repeat (R3) of the microtubule-binding domain (MBD) on the assembly of MBD was investigated using Tyr-substituted MBD mutants by fluorescence, circular dichroism spectroscopy, and electron microscopy. Consequently, the importance of the Tyr residue located at position 310, not at other positions, was clearly shown. The conformational comparison of the Tyr310Ala-substituted R3 repeat peptide with the unsubstituted one showed that the Tyr residue contributes to the rigid extended structure of the N terminal V³⁰⁶QIVYK³¹¹ sequence, and its replacement by Ala leads to the deformation of the extended structure, consequently losing its aggregation ability. The present results indicate that a compound that interacts specifically with the Tyr residue or an antibody recognizing the region containing the Tyr residue becomes a candidate for inhibiting tau fibrillation.

Characteristics of Nuclease Activity of the SbcCD Complex from Deinococcus radiodurans

Hu, Y., Tian, B., Xu, G., Yin, L., Hua, X., Lin, J., Hua, Y. — Fri, 06 Nov 2009 06:03:29 PST

The bacterium Deinococcus radiodurans is extremely resistant to the intense ionizing irradiation which causes extensive DNA double-strand breaks (DSBs). The deinococcal SbcCD complex (drSbcCD) is required for DSB repair. The drSbcC and drSbcD genes were cloned and overexpressed in Escherichia coli cells, respectively. The nearly homogeneous drSbcC and drSbcD proteins were purified and reconstituted to form a stable complex in vitro. The drSbcCD complex has an ATP-independent 3' to 5' exonuclease activity to cleave both dsDNA and ssDNA substrates in the presence of either Mn²⁺ or Mg²⁺ ion. The drSbcCD complex also has an ATP-independent endonuclease activity. It can cleave the circular ssDNA, nick the supercoiled circular dsDNA, cleave the 3' flap DNA substrate at the site of the single-strand branch adjacent to duplex DNA, and cleave the hairpin DNA taking no account of the DNA end free or not. It is a kind of secondary structure-specific endonuclease. The drSbcCD complex still has a 3' to 5' exonuclease activity when the DNA termini are blocked by the proteins. These results suggest that the drSbcCD complex takes part in the metabolism of DNA, and its nuclease activities may play important roles in DNA repair process.

Mimicking the evolution of a thermally stable monomeric four-helix bundle by fusion of four identical single-helix peptides

Akanuma, S., Matsuba, T., Ueno, E., Umeda, N., Yamagishi, A. — Wed, 04 Nov 2009 05:51:45 PST

Internal symmetry is a common feature of the tertiary structures of proteins and protein domains. Probably because the genes of homo-oligomeric proteins duplicated and fused, their evolutionary descendants are proteins with internal symmetry. To identify any advantages that cause monomeric proteins with internal symmetry to be selected evolutionarily, we characterized some of the physical properties of a recombinant protein with a sequence consisting of two tandemly fused copies of the Escherichia coli Lac repressor C-terminal -helix. This polypeptide exists in solution mainly as dimer that likely maintains a four-helix bundle motif. Thermal unfolding experiments demonstrate that the protein is considerably more stable at elevated temperatures than is a homotetramer consisting of four noncovalently associated copies of a 21-residue polypeptide similar in sequence to that of the Lac repressor C-terminal -helix. A tandem duplication of our helix-loop-helix polypeptide yields an even more thermally stable protein. Our results exemplify the concept that fusion of noncovalently assembled polypeptide chains leads to enhanced protein stability. Herein, we discuss how our work relates to the evolutionary selective-advantages realized when symmetrical homo-oligomers evolve into monomers. Moreover, our thermally stable single-chain four-helix bundle protein may provide a robust scaffold for development of new biomaterials.

The effect of PKA-Phosphorylation on the structure of inhibitor-1 studied by NMR spectroscopy

Tue, 03 Nov 2009 05:38:26 PST

Inhibitor-1 is an acid- and heat-stable protein. It can be turned into a potent inhibitor of protein phosphatase-1 (PP1) after phosphorylation at Thr³⁵ by c-AMP-dependent protein kinase (PKA). Although it has been known that pre-phosphorylation is essential for inhibition of PP1, the structure-function relationship of Thr³⁵-phosphorylated inhibitor-1, such as whether or not PKA-phosphorylation pre-triggers conformational changes in inhibitor-1, remains unclear. In this study, we performed structural characterization of Thr³⁵-phosphoroylated inhibitor-1 by using multi-dimensional heternuclear NMR spectroscopy. The result of structural comparison between Thr³⁵-phosphoroylated and non-phosphorylated inhibitor-1 indicated that PKA-phosphorylation has no significant effect on the global conformation of free state inhibitor-1. This finding may support the inference that regulation of the interactions between inhibitor-1 and PP1 through PKA-phosphorylation mainly depends on the phosphate group instead of phosphorylation-induced conformational change.

Identification of the Phr-dependent heat shock regulon in the hyperthermophilic archaeon, Thermococcus kodakaraensis

Kanai, T., Takedomi, S., Fujiwara, S., Atomi, H., Imanaka, T. — Tue, 03 Nov 2009 05:38:26 PST

The hyperthermophilic archaeon Thermococcus kodakaraensis harbors a putative transcriptional regulator (Tk-Phr) that is orthologous to the Pyrococcus furiosus Phr (Pf-Phr). Pf-Phr, a transcriptional regulator, represses genes encoding the small heat shock protein (sHSP), AAA⁺ ATPase, and Pf-Phr itself under normal growth temperatures. Here we constructed a gene disruption strain of Tk-Phr (strain KHR1). KHR1 cells showed similar specific growth rates with those of the wild-type strain under various temperatures. A whole genome microarray analysis was performed between KHR1 and wild-type cells grown at 80°C. Transcript levels of more than 20 genes were significantly higher in KHR1 cells. Most genes contained a sequence motif virtually identical to that of Pf-Phr in their 5'-flanking regions. The Tk-Phr regulon included genes encoding sHSP, AAA⁺ ATPase, prefoldin, RecA superfamily ATPase, and Tip49. On the other hand, more than half of the members in the regulon encoded conserved/hypothetical proteins, raising the possibility that these proteins participate in unidentified processes of the heat shock response. In contrast, Tk-Phr deletion did not lead to dramatic increases in transcript and protein levels of a chaperonin (CpkB) previously shown to respond to heat shock, suggesting the presence of a second, Phr-independent heat shock response mechanism in T. kodakaraensis.

Calcium-dependent cleavage of the Na+/Ca2+ exchanger by m-calpain in isolated endoplasmic reticulum

Samanta, K., Kar, P., Chakraborti, T., Chakraborti, S. — Mon, 02 Nov 2009 03:22:46 PST

We have recently demonstrated the localization of associated m-calpain and calpastatin in the endoplasmic reticulum (ER) of bovine pulmonary artery smooth muscle. Herein, we sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of Na⁺/Ca²⁺ exchanger (NCX) in the ER. Treatment of the ER with Ca²⁺ (5 mM) dissociates m-calpain-calpastatin association leading to the activation of m-calpain, which subsequently cleaves the ER integral trans-membrane protein NCX1 (116 kDa) to an 82 kDa fragment. Pretreatment of the ER with calpain inhibitors, calpeptin (10 µM) or MDL28170 (10 µM); or Ca²⁺ chelator, EGTA (10 mM) does not cleave NCX1. In vitro cleavage of the ER purified NCX1 by the ER purified m-calpain also supports our finding. Cleavage of NCX1 by m-calpain in the ER may be interpreted as the main cause of intracellular Ca²⁺ overload in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.

Multiple conformational state of human serum albumin around single tryptophan residue at various pH revealed by time-resolved fluorescence spectroscopy

Otosu, T., Nishimoto, E., Yamashita, S. — Mon, 02 Nov 2009 03:22:45 PST

Human serum albumin (HSA) plays important roles for transport of fatty acids and for binding a variety of drugs and organic compounds in the circulatory system. This protein experiences several conformational transitions by the change of pH, and the resulting conformations were essential for completing the physiological roles in vivo. Steady-state and time-resolved fluorescence spectroscopy was applied to single tryptophan residue solely arranged in HSA to study subtle conformational change around single tryptophan residue in HSA at various pH. The results showed the characteristic feature of local conformation around tryptophan residue in domain II responding to the change in entire structure. The study of time-resolved area-normalized fluorescence emission spectra (TRANES) also showed the peculiar dielectric property of water molecule trapped nearby tryptophan residue depending on pH. These results suggested that microenvironment around tryptophan residue was tightly packed at acidic and basic pH although entire conformation was loosened.

Expression and molecular characterization of the Mycobacterium tuberculosis PII protein

Mon, 02 Nov 2009 03:22:43 PST

The signal transduction protein PII plays an important role in cellular nitrogen assimilation and regulation. The molecular characteristics of the M. tuberculosis PII (Mtb PII) were investigated using biophysical experiments. The Mtb PII coding ORF Rv2919c was cloned and expressed in Escherichia coli. The binding characteristics of the purified protein with ATP and ADP were investigated using Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC). Mtb PII binds to ATP strongly with K_d in the range 1.93-6.44 µM. This binding strength was not significantly affected by the presence of 2-ketoglutarate even in molar concentrations of 66 (ITC) or 636 (SPR) fold excess of protein concentration. However, an additional enthalpy of 0.3 kcal/mol was released in presence of 2-ketoglutarate. Binding of Mtb PII to ADP was weaker by an order of magnitude. Binding of ATP and 2-ketoglutarate were analysed by docking studies on the Mtb PII crystal structure (PDB id 3BZQ). We observed that hydrogen bonds involving the -phosphate of ATP contribute to enhanced binding of ATP compared with ADP. Glutaraldehyde Crosslinking showed that Mtb PII exists in homotrimeric state which is consistent with other PII proteins. Phylogenetic analysis showed that Mtb PII consistently grouped with other actinobacterial PII proteins.

Kinesin-Calmodulin Fusion Protein as a Molecular Shuttle

Shishido, H., Nakazato, K., Katayama, E., Chaen, S., Maruta, S. — Fri, 30 Oct 2009 08:09:13 PDT

In this study, we developed a molecular shuttle with reversible cargo-loading system by using calmodulin (CaM) and M13 peptide. We designed a kinesin (K560) chimera protein with CaM fused at the C-terminal tail region of K560 (K560-CaM). K560-CaM was expressed using an Escherichia coli expression system and purified. Its ATPase activity and microtubule gliding velocity were almost in a similar range as those of the wild-type kinesin. Ca²⁺-dependent reversible binding of K560-CaM and M13 peptide was monitored by size-exclusion-HPLC. Rotary shadowing and electron microscopy revealed tetrameric configuration of K560-CaM in the absence of Ca²⁺, while both dimeric and tetrameric configurations in the presence of Ca²⁺. Further, Ca²⁺-dependent change in the configuration of K560-CaM was monitored by size-exclusion-HPLC and analytical ultracentrifugation. Finally, by total internal reflection fluorescence microscopy, we successfully observed that K560-CaM transported quantum dot-conjugated M13 peptide along the microtubule in the presence of Ca²⁺.

Highly Amphiphilic Manganese Porphyrin for the Mitochondrial Targeting Antioxidant

Haruyama, T., Asayama, S., Kawakami, H. — Fri, 30 Oct 2009 08:09:12 PDT

Here we communicate that the design of the antioxidant consisting of amphiphilic manganese porphyrin with one dimethylimidazolium group and three phenyl groups for mitochondrion targeting. The resulting Mn-porphyrin MnMImP₃P exhibited highly partition coefficient (logP_ow=+4.78) as well as significant superoxide dismutase and peroxynitrite decomposition activities. Accordingly, the MnMImP₃P exhibited a little increase in fluorescence intensity attributed to 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide [diS-C₃-(5)], a tracer dye to assess the mitochondrial membrane potential, which suggested the interaction of the MnMImP₃P, leading to the release of the fluorescence dye from the mitochondrial membrane, with the mitochondria. As a result, the MnMImP₃P rescued the cell death under oxidative stress concerned with mitochondrial damage.

Separation and quantification of sn-1 and sn-2 fatty acid positional isomers in phosphatidylcholine by RPLC-ESIMS/MS

Nakanishi, H., Iida, Y., Shimizu, T., Taguchi, R. — Fri, 30 Oct 2009 08:09:10 PDT

Endogenous phosphatidylcholine in biological membranes exists as isomers with acyl moieties at the sn-1 or sn-2 positions of the glycerol backbone. However, detailed biochemical information on these positional isomers is not generally available. This study is the first report on the separation and identification of positional isomers of endogenous phosphatidylcholine using reversed-phase LC-ESIMS/MS. The separation of positional isomers in PC was achieved by using ultra pressure LC (UPLC), which uses a high-resolution HPLC system. To identify positional isomers in individual PC species, their lyso-PC-related fragments and fatty acids, which were obtained by MS/MS analysis in the negative ion mode, were used. From the application results of biological samples, the lipid extracts of mouse brain were found to be abundant in PC containing 22:6 at the sn-1 position of the glycerol backbone. However, the lipid extracts from mouse heart and liver were not abundant in positional isomers. This achievement demonstrates that the relative amounts of positional isomers in various tissues or molecular species differ. These results will be useful for the clarification of the biological mechanisms of remodeling enzymes such as phospholipase and acyltransferase. Thus, our report provides a novel and critical milestone in understanding how molecular composition of phospholipids is established and their biological roles.

Signaling pathways in the unfolded protein response: development from yeast to mammals

Mori, K. — Fri, 30 Oct 2009 04:15:18 PDT

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) under ER stress conditions activates a series of homeostatic responses collectively termed the unfolded protein response (UPR). The UPR is unique in that the molecular mechanisms it uses to transmit signals from the ER lumen to the nucleus are completely different to those used for signaling from the plasma membrane. An ER stress signal is sensed and transmitted across the membrane by a transmembrane protein(s) in the ER. Interestingly, the number of such functional sensors/transducers ubiquitously expressed has increased with evolution, namely one in S. cerevisiae, two in C. elegans and D. melanogaster, and three in mammals. Accordingly, mammalian cells are able to cope with ER stress in a more sophisticated manner. Here, I summarize the mechanisms and activation consequences of UPR signaling pathways in yeast, worm, fly and mammalian cells. I also discuss how they have evolved to counteract ER stress effectively

Role of N-glycans in maintaining the activity of protein O-mannosyltransferases POMT1 and POMT2

Manya, H., Akasaka-Manya, K., Nakajima, A., Kawakita, M., Endo, T. — Thu, 29 Oct 2009 21:35:38 PDT

The complex of protein O-mannosyltransferase 1 (POMT1) and POMT2 catalyzes the initial step of O-mannosyl glycan biosynthesis. The mutations in either POMT1 or POMT2 can lead to Walker-Warburg syndrome, a congenital muscular dystrophy with abnormal neuronal migration. Here, we used three algorithms for predicting transmembrane helices to construct the secondary structural models of human POMT1 and POMT2. In these models, POMT1 and POMT2 have seven- and nine-transmembrane helices and contain four and five potential N-glycosylation sites, respectively. To determine whether these sites are actually glycosylated, we prepared mutant proteins that were defective in each site by site-directed mutagenesis. Three of the POMT1 sites and all of the POMT2 sites were found to be N-glycosylated, suggesting that these sites face the luminal side of the ER. Mutation of any single site did not significantly affect POMT activity, but mutations of all N-glycosylation sites of either POMT1 or POMT2 caused a loss of POMT activity. The loss of activity appeared to be due to the decreased hydrophilicity. These results suggest that the N-glycosylation of POMT1 and POMT2 is required for maintaining the conformation as well as the activity of the POMT1-POMT2 complex.

Suppression of AhR signaling pathway is associated with the downregulation of UDP-glucuronosyltransferases during BBN-induced urinary bladder carcinogenesis in mice

Thu, 29 Oct 2009 21:35:37 PDT

Downregulation of carcinogen detoxifying enzymes might be a critical factor in tumor formation by increasing the carcinogen concentration in the target organ. Previous reports revealed that the expression of UGT1A mRNA is either lost or decreased in certain human cancer tissues, including urinary bladder cancer. To elucidate this downregulation mechanism, we used an N-nitrosobutyl (4-hydroxybutyl) amine (BBN)-induced mouse urinary bladder carcinogenesis model. Similar to human cancer, the expressions of Ugt1a6, Ugt1a9 and total Ugt1a mRNA in the BBN-induced bladder cancer were markedly decreased compared with those of normal mice. BBN downregulated the basal Ugt1a mRNA expression in a time-dependent manner and this was reversible in the first 2 weeks of BBN treatment. However, after 4 weeks of BBN treatment the repression became persistent after the cessation of BBN treatment. AhR regulates the constitutive and inducible expression of Ugt1a mRNA. We found that the constitutive Ugt1a mRNA expression is decreased in the bladder of AhR knockout (KO) mice. Furthermore, BBN-induced Ugt1a downregulation was lost in AhR KO mice, and the canonical AhR target gene Cyp1a1 was similarly downregulated by BBN in the bladder. These results demonstrate that BBN repressed Ugt1a mRNA expression via suppression of AhR signaling pathway during BBN-induced carcinogenesis.

The third type III module of human fibronectin mediates cell adhesion and migration

Obara, M., Sakuma, T., Fujikawa, K. — Thu, 29 Oct 2009 21:35:37 PDT

Fibronectin (FN) is a major extracellular matrix protein involved in various biological events. This study demonstrated that the third FN type III repeat (FnIII3) and several fragments containing the repeat promote cell spreading and migration of human dermal fibroblasts (HDFs), whereas the fourth repeat (FnIII4) did not. A variety of cell types also spread on FnIII3 in a cell-type specific manner, but not on FnIII4. Immunofluorescence assays revealed that FnIII3 induced the organization of focal contacts and stress fibers in HDFs. Cyclic [RGDFV] peptides with a D-Phe residue, which are selective inhibitors of cell adhesion to vitronectin, inhibited HDF spreading on FnIII3 equally with GRGDS, indicating little involvement of V-integrins in FnIII3 spreading. An anti-β1 integrin mAb inhibited cell spreading on FnIII3 and FN. To our knowledge, this is the first demonstration that a novel domain of FnIII3 functions in cell spreading and migration through an interaction with unresolved β1 integrin(s) in an RGD-dependent manner.

Inhibition of Calcineurin by Quercetin in vitro and in Jurkat cells

Wang, H., Zhou, C.-L., Lei, H., Wei, Q. — Thu, 29 Oct 2009 21:35:36 PDT

Calcineurin (CN), the Ca²⁺/calmodulin (CaM)-dependant protein phosphatase, is an integral enzyme involved in activation of T cells. It is also the target of various inhibitors such as cyclosporine A (CsA) and FK506 both of which have been widely used as immunosuppressants. We show that the novel CN inhibitor, quercetin (QC), associates with CN both in vitro and in Jurkat cells, and that it causes non-competitive inhibition of phosphatase activity. Unlike CsA and FK506, QC does not require a matchmaker protein for CN inhibition. It acts directly on the catalytic domain and its inhibitory effect was increased by the presence of CNB. Using semi quantitative and real-time RT-PCR, we show that QC inhibits IL-2 gene expression in activated Jurkat cells. The physiological inhibitory activity of QC together with its hypotoxicity suggest that it may be an effective immunosuppressant.

Cellular signal transduction of the hypoxia response

Nakayama, K. — Wed, 28 Oct 2009 07:25:44 PDT

Cells induce the hypoxia responses to adapt to the environment when organisms are exposed to a low oxygen environment. The hypoxia response leads to the activation of multiple cellular signaling pathways involved in regulation of respiration, metabolism, cell survival and so forth. Hypoxia-Inducible-Factor (HIF) pathway plays a central role during the hypoxia response as its expression and activity are regulated in an oxygen-dependent manner and it also regulates the expression of multiple hypoxia responsive genes. The expression of HIF is regulated by proline hydroxylation, which is mediated by HIF prolyl-hydroxylase named PHD. The hydroxylated HIF-alpha subunit is degraded via the ubiquitin-proteasome pathway. The PHD activity needs to be strictly regulated to ensure the stabilization of HIF under hypoxic conditions, because PHD leads to HIF degradation. This review describes the regulatory mechanism of HIF stability and activity under normoxia and hypoxic conditions. Furthermore, the role of the HIF-independent pathways during the hypoxia response, which is as important as the HIF pathway, will also be described.

Topology of 4-Pyridoxic Acid Dehydrogenase in Transformed Escherichia coli Cells

Ge, F., Yagi, T. — Tue, 27 Oct 2009 06:05:11 PDT

The topology of 4-pyridoxic acid dehydrogenase in the Escherichia coli cell membrane was examined with transformed E. coli cells overexpressing the enzyme from Mesorhizobium loti. The recombinant enzymes with a His6-tag on N-terminal region or C-terminal were localized on the E. coli cell membrane like the wild-type enzyme without a His₆-tag. The His₆-tags were labeled with Ni-NTA AP conjugate only when the E. coli protoplast cells were broken. The membrane-bound enzyme in the intact protoplast cells was not digested by trypsin, although the one in the gently broken protoplast cells was almost totally digested. Thus, 4-pyridoxic acid dehydrogenase was an integral monotopic protein, protruding into a cytoplasm side from the bacterial membrane. The deletion or mutation of a deduced transmembrane segment in 4-pyridoxic acid dehydrogenase made it an inclusion body, and the enzyme protein was not found in the E. coli cell membrane. Thus, it was suggested that the intact deduced transmembrane segment was necessary for 4-pyridoxic acid dehydrogenase to be localized on the bacterial cell membrane.

Neutralization of Toxic Heme by Porphyromonas gingivalis Hemoglobin Receptor

Tue, 27 Oct 2009 06:05:10 PDT

Free heme is known to be toxic to organs, tissues and cells. It enhances permeability by binding to a cell membrane, which leads to cell death, and damages lipids, proteins and DNA through the generation of reactive oxygen species. Lysine- and arginine-specific gingipains (Kgp and RgpA/B) are major proteinases that play an important role in the pathogenicity of a black-pigmented periodontopathogen named Porphyromonas gingivalis. One of the adhesin domains of gingipain, HbR could bind heme as an iron nutrient source for Porphyromonas gingivalis. Using erythrocyte and its membrane as a model, results from the present study demonstrate that recombinant HbR expressed in Escherichia coli could inhibit heme-induced hemolysis, probably through removing heme from heme membrane complex and lowering free heme toxicity by mediating dimerization of heme molecules. The ability to protect a cell membrane from heme toxicity is a new function for HbR.

Circadian clock in Ciona intestinalis revealed by microarray analysis and oxygen consumption

Sun, 25 Oct 2009 17:06:14 PDT

The molecular mechanisms of the endogenous circadian clocks that allow most animals to adapt to environmental cycles have recently been uncovered. The draft genome of the ascidian, Ciona intestinalis, a model animal that is close to vertebrates, has been described. However, the C. intestinalis genome lacks the canonical clock genes such as Per, Bmal and Clock that are shared by vertebrates and insects. Here, we found the circadian rhythms at the physiological and molecular levels. The oxygen consumption rate was lower during the light phase and higher during the dark phase during a day, and the rhythm highly damped and continued under constant darkness. From the microarray analysis, the 396 spots (1.8% of the total; corresponding to 388 clones) were extracted as candidates for circadian expression. We confirmed the circadian expression of several candidate genes by Northern blotting. Furthermore, three of four rhythmic expressed genes showed phase-shifts to prolonged light period. However, most of known clock genes did not oscillate. These data suggest that C. intestinalis have a unique molecular circadian clock and suggest that the daily environmental change is not so strong effect for sea squirt in its evolution when compared to vertebrates and insects.

Site-directed Mutation at Residues near the Catalytic Site of Histamine Dehydrogenase from Nocardioides simplex and Its Effects on Substrate Inhibition

Tsutsumi, M., Tsuse, N., Fujieda, N., Kano, K. — Tue, 20 Oct 2009 23:42:12 PDT

Histamine dehydrogenase from Nocardioides simplex (nHmDH) is a homodimer containing one 6-S-cysteinyl FMN (CFMN) and one [4Fe-4S] cluster per monomer. nHmDH catalyzes the oxidative deamination of histamine to ammonia and imidazole acetaldehyde, but histamine inhibits its catalytic activity at high concentrations. We mutated gene encoded residues (Tyr180, Gly268 and Asp269) near CFMN to understand the biophysical meaning of the substrate inhibition. Three mutants Y180F, G268D/D269C and Y180F/G268D/D269C were expressed by considering the DNA sequence alignment of histamine dehydrogenase from Rhizobium sp. 4-9 (rHmDH), which does not suffer from the substrate inhibition. The Y180F/G268D/D269C mutation to mimic rHmDH successfully suppressed the inhibition, although the catalytic activity decreased. The substrate inhibition was weakened by the Y180F mutation, but G268D/D269C was still susceptible to the inhibition. It was found that also causes changes in the UV-vis absorption spectra of the substrate-reduced form and the redox potential of the enzymes. The characterization suggests that the thermodynamic preference of the semiquinone form of CFMN in the two-electron-reduced subunit of the enzyme is responsible for the substrate inhibition. However, destabilization of the semiquinone form leads to kinetic hindrance due to the uphill single electron transfer from the fully reduced CFMN to the [4Fe-4S] cluster.

Dppa2 Knockdown-induced Differentiation and Repressed Proliferation of Mouse Embryonic Stem Cells

Du, J., Chen, T., Zou, X., Xiong, B., Lu, G. — Tue, 20 Oct 2009 23:42:12 PDT

Developmental pluripotency-associated 2 (Dppa2) gene is one of the genes recently identified to be expressed specifically in pluripotent cells. To investigate the role of Dppa2 in mouse embryonic stem (ES) cells, we examined its expression during differentiation and performed knockdown of Dppa2 in mouse ES cells. Our results showed that the expression of Dppa2 decreased markedly in differentiated cells. Dppa2 knockdown induced the differentiation of mouse ES cells, as indicated by reduced alkaline phosphatase (AKP) activity, slightly downregulated expression of the putative pluripotency marker genes Oct4 and Nanog, and increased expression of early differentiation marker genes, such as Fst and Psx1. Moreover, reduced expression of Dppa2 also repressed cell proliferation activity as shown by the 5-bromo-2-deoxyuridine (BrdU) incorporation assay. Hence, Dppa2 might play a role in maintenance of the undifferentiated state and proliferation of ES cells.

Structural explanation for the acquisition of glycosynthase activity

Hidaka, M., Fushinobu, S., Honda, Y., Wakagi, T., Shoun, H., Kitaoka, M. — Fri, 09 Oct 2009 09:23:53 PDT

Glycosynthases are engineered glycoside hydrolases (GHs) that catalyse the synthesis of glycoside from glycosyl-fluoride donors and suitable acceptors. We have determined five crystal structures of the glycosynthase mutants reducing-end xylose-releasing exo-oligoxylanase (Rex), an inverting GH, that exhibit various levels of glycosynthetic activities. At the active site of the Y198F mutant, the most efficient glycosynthase, a water molecule is observed at the same position as nucleophilic water (NW) in the parent enzyme, and the loss of the fixation of the direction of the lone pair of water molecules in the mutant drastically decreases hydrolytic activity. Water molecules were also observed at each active site of the general base mutant, but they were shifted 1.0-3.0 Å from the NW in the wild type. Their positions exhibited a strong correlation with the strength of glycosynthase activity. Here, we propose that a structural prerequisite for the sufficient glycosynthase reaction is the presence of a water molecule at the NW position, and mutation at the NW holder provides a general strategy for inverting GHs. The idea on the position of a water molecule may also be applicable to the design of efficient glycosynthases from retaining GHs.

Differences in the P1' Substrate Specificities of Pepsin A and Chymosin

Fri, 09 Oct 2009 09:23:52 PDT

Porcine pepsin A and bovine chymosin are typical models of aspartic proteinases. The hydrolytic specificities of these proteinases, along with those of human pepsin A and monkey chymosin, were investigated with 29 peptide substrates that included various P1' variants of seven parent peptides. From these peptides, AFPLEFFREL was preferred by pepsin A and chymosin, while its P1' variant, AFPLEFEREL, was preferred by bovine chymosin. Porcine and human pepsin A showed similar hydrolytic specificities, strongly preferring a hydrophobic/aromatic residue at P1' of any type of peptide. This specificity is well explained by the very hydrophobic nature of the S1' subsite that consists of Tyr¹⁸⁹, Ile²¹³, Ile³⁰⁰, Met²⁸⁹, Val/Leu²⁹¹, and Leu²⁹⁸. The first three residues are well conserved in pepsin family enzymes. Although bovine and monkey chymosin showed similar P1' specificity, bovine chymosin preferred peptides having Glu at P1', while monkey chymosin preferred peptides having Lys at P1'. The dual characteristics of chymosin are due to the occurrence of polar/charged residues in the S1' subsite, such as Glu/Asp²⁸⁹, Gln²⁹⁸, and Lys/Gln²⁹⁹, which are different from the S1' subsite of pepsin A. Molecular models suggest that Glu in position 289 of bovine chymosin, and Asp in position 289 of monkey chymosin are responsible for the difference in P1' specificities between the chymosins.

Molecular identification and characterization of an acidic peptide:N-glycanase from tomato (Lycopersicum esculentum) fruits

Hossain, Md. A., Nakano, R., Nakamura, K., Kimura, Y. — Fri, 09 Oct 2009 09:23:51 PDT

Plant acidic peptide:N-glycanase (PNGase) is one of the deglycosylation enzymes and has been considered to be involved in the catabolism of glycoproteins in plant cells. However, the tangible physiological significance involved in plant differentiation or growth is yet unclear. In this study, as a first step to elucidate the physiological role of free N-glycans and the de-N-glycosylation machinery working in developing plant cells, we have succeeded in expressing a cDNA from tomato fruits in Pichia pastoris and identified an acidic peptide:N-glycanase in the culture supernatant. The PNGase-gene-encoded protein is a single polypeptide chain of 588 amino acids with a predicted molecular mass of 65.8 kDa. The deduced amino acid sequence showed 57.9% similarity with almond PNGase A. The recombinant tomato PNGase showed optimum activity at pH 4.5 and 40°C. It did not require any metal ions for full enzymatic activity and could release the complex-type N-glycan from glycopeptides. Our phylogenetic analysis reveals that the plant acidic PNGase is completely different from the ubiquitous cytosolic PNGase and is involved in a different de-N-glycosylation mechanism associated with plant growth and development.

A Critical Role for highly Conserved GLU610 Residue of Oligopeptidase B from Trypanosoma Brucei in Thermal Stability

Fri, 09 Oct 2009 09:23:50 PDT

Oligopeptidase B from Trypanosoma brucei (Tb OPB) is a virulence factor and therapeutic target in African sleeping sickness. Three glutamic acid residues at positions 607, 609 and 610 of the catalytic domain are highly conserved in the OPB subfamily. In this study, the roles of Glu⁶⁰⁷, Glu⁶⁰⁹ and Glu⁶¹⁰ in Tb OPB were investigated by site-directed mutagenesis. A striking effect on k_cat/Km was obtained following mutation of Glu⁶⁰⁷ to glutamine. In contrast, the heat stability of Tb OPB decreased markedly following the single mutation of Glu⁶¹⁰ to glutamine, although this mutation had significantly less effect on catalytic properties compared with the Glu⁶⁰⁷ mutation. Although no differences were found in the tertiary and secondary structures between wild-type OPB and the E610Q mutant prior to heat treatment, the E610Q mutant is inactivated more rapidly than wild-type OPB following heat treatment in a manner correlating with its attendant structural changes. Trypsin digestion showed that the boundary regions between the β-propeller and catalytic domain of the E610Q mutant are unfolded with heat treatment. It is concluded that Glu⁶⁰⁷ is essential for the catalytic activity of Tb OPB and that Glu⁶¹⁰ plays a critical role in stabilization rather than catalytic activity despite their close proximity.

Atypical kinetics of cytochromes P450 catalyzing 3'-hydroxylation of flavone from the white-rot fungus Phanerochaete chrysosporium

Fri, 09 Oct 2009 05:18:15 PDT

We cloned full-length cDNAs of 130 cytochrome P450s (P450s) derived from Phanerochaete chrysosporium, and successfully expressed 70 isoforms in Saccharomyces cerevisiae. To elucidate substrate specificity of Phanerochaete chrysosporium P450s, we examined various substrates including steroid hormones, several drugs, flavonoids, and polycyclic aromatic hydrocarbons using the recombinant S. cerevisiae cells. Of these P450s, two CYPs designated as PcCYP50c and PcCYP142c with 14% identity in their amino acid sequences catalyze 3’-hydroxylation of flavone and O-deethylation of 7-ethoxycoumarin. Kinetic data of both enzymes on both reactions fitted not to the Michaelis-Menten equation but to Hill's equation with a coefficient of 2, suggesting that two substrates bind to the active site. Molecular modeling of PcCYP50c and a docking study of flavone to its active site supported this hypothesis. The enzymatic properties of PcCYP50c and PcCYP142c resemble mammalian drug-metabolizing P450s, suggesting that their physiological roles are metabolism of xenobiotics. It is noted that these unique Phanerochaete chrysosporium P450s have a potential for production of useful flavonoids.

Crystal Structure of Peroxiredoxin from Aeropyrum pernix K1 Complexed with its Substrate, Hydrogen Peroxide

Nakamura, T., Kado, Y., Yamaguchi, T., Matsumura, H., Ishikawa, K., Inoue, T. — Fri, 09 Oct 2009 05:18:14 PDT

Peroxiredoxin reduces hydrogen peroxide and alkyl peroxides to water and corresponding alcohols, respectively. The reaction is dependent on a peroxidatic cysteine, whose sulfur atom nucleophilically attacks one of the oxygen atoms of the peroxide substrate. In spite of the many structural studies that have been carried out on this reaction, the tertiary structure of the hydrogen peroxide-bound form of peroxiredoxin has not been elucidated. In this paper, we report the crystal structure of peroxiredoxin from Aeropyrum pernix K1 in the peroxide-bound form. The conformation of the polypeptide chain is the same as that in the reduced apo-form. The hydrogen peroxide molecule is in close contact with the peroxidatic Cys50 and the neighboring Thr47 and Arg126 side chain atoms, as well as with the main chain nitrogen atoms of Val49 and Cys50. Bound peroxide was also observed in the mutant C50S, in which the peroxidatic cysteine was replaced by serine. Therefore, the sulfur atom of the peroxidatic cysteine is not essential for peroxide binding, although it enhances the binding affinity. Hydrogen peroxide binds to the protein so that it fills the active site pocket. This study provides insight into the early stage of the peroxiredoxin reaction.

Hypoxemia Induces Expression of Heme Oxygenase-1 and Heme Oxygenase-2 Proteins in the Mouse Myocardium

Fri, 09 Oct 2009 00:50:09 PDT

Heme oxygenase (HO) catalyzes oxidative breakdown of heme, and constitutes two isozymes, HO-1 and HO-2. Here, we explored the tissue-specific regulation of expression of HO-1 and HO-2 under hypoxemia. There was no significant change in the overall expression levels of HO-1 and HO-2 mRNAs and proteins in the lung during adaptation of C57BL/6 mice to normobaric hypoxia (10% O₂). However, immunohistochemical analysis revealed the increased expression of HO-1 and HO-2 proteins after 28 days of normobaric hypoxia in the pulmonary venous myocardium that is the extension of the left atrial myocardium into pulmonary venous walls. Moreover, the expression of HO-2 protein was increased in the subendocardial myocardium of ventricles under hypoxia, while HO-1 protein level was increased in the full-thickness walls. Thus, hypoxemia induces expression of both HO-1 and HO-2 proteins in the myocardium. Using C57BL/6 mice lacking HO-2 (HO-2^-/-), which manifest chronic hypoxemia, we also showed that the HO-1 protein level in the lung was similar between HO-2^-/- mice and wild-type mice. Unexpectedly, HO-1 protein level was lower by 35% in the HO-2^-/- mouse liver than the wild-type liver. These results indicate that the expression of HO-1 protein is regulated in a tissue-specific manner under hypoxemia.

Structure and Reaction Mechanism of Human Nicotinamide Phosphoribosyltransferase

Fri, 09 Oct 2009 00:50:08 PDT

Nicotinamide phosphoribosyltransferase (NMPRTase) catalyzes the reaction of nicotinamide (NM) and 5'-phosphoribosyl-1'-pyrophosphate (PRPP) to form nicotinamide mononucleotide (NMN) and pyrophosphate (PPi) in the pathway of NAD-biosynthesis. Monitoring the ¹H and ³¹P NMR spectra of the reaction mixture, we found that this reaction is reversible as dictated by the equilibrium constant K = [NMN][PPi]/([NM][PRPP]) = 0.14, which agreed well with the ratio of second-order rate constants for forward and backward reactions, K = 0.16. The crystal structures of this enzyme in the free form and bound to NM and PRPP at the resolution of 2.0 – 2.2 Å were essentially identical to that of the complex with NMN, except for some variations that could facilitate the substitution reaction by fixing the nucleophile and the leaving group for the requisite inversion of configuration at the C1' carbon of the ribose ring. In the active site near the C1' atom of the bound PRPP or NMN, there was neither negatively charged group nor waterproof environment necessary to support the feasibility of a ribo-oxocarbocation intermediate inherent in the S_N1 mechanism. The structures and catalytic mechanism thus revealed are also discussed in connection with the multiple biological functions of NMPRTase.

Generation of a recombinant single-chain variable fragment (scFv) targeting 5-methyl-2'-deoxcytidine

Ohshima, M., Tadakuma, T., Hayashi, H., Inoue, K., Itoh, K. — Thu, 08 Oct 2009 09:45:08 PDT

We generated a single-chain variable fragment (scFv) against 5-methyl-2'-deoxycytidine (m⁵dCyd) using phage display technology. The heavy and light chain variable region genes were amplified by the polymerase chain reaction (PCR) from hybridoma cell line FMC9 and assembled as an scFv fragment with a flexible linker (Gly₄-Ser)₃. The scFv DNA fragment was then cloned into pCANTAB-5E, and a phage displaying the scFv was produced. Antigen-positive phage clones were successfully selected by ELISA. The scFv was modified with FLAG and His tags for detection and purification. The scFv reacted strongly with m⁵dCyd and weakly with 5-methylcytidine (m⁵Cyd) but not with cytidine (Cyd) and 1-methyladenosine in a manner similar to the monoclonal antibody (MoAb). Although the specificities of scFv and MoAb were almost identical, the sensitivity of the scFv (IC₅₀ 0.054 mg/ml) was approximately 80 times higher than that of the parent MoAb (IC₅₀ 4.27 µ g/ml), determined by inhibition ELISA. As a biochemical application of this scFv, we quantified the m⁵dCyd content of genomic DNA by enzymatic hydrolysis using inhibition ELISA. The cancer cell lines HeLa, HeLa S3, and MDA-MB-453 contained approximately 1% of methylated DNA in total genomic DNA, as did peripheral blood cell genomic DNA from healthy volunteers, but HT29 and T-47D showed hypomethylation compared with the HeLa, HeLa S3, and MDA-MB-453 cell lines. The scFv generated here may be applicable to the assessment of cellular DNA methylation levels and is more sensitive than the MoAb.

Characteristic amino acid distribution around segments unique to allergens

Asakawa, N., Sakiyama, N., Teshima, R., Mitaku, S. — Thu, 08 Oct 2009 09:45:08 PDT

Epitopes are located at the surface of allergens with which antibodies specifically bind. On the assumption that fragments unique to allergens have common, characteristic amino acid sequences, we compared the amino acid sequences of allergens with those of non-allergens. Segments around fragments unique to allergens showed wavelet-like distributions for several amino acids. Charged residues, alanine and glycine had positive peaks at the center of the unique segments with small valleys on both sides, while aromatic residues, proline and cysteine showed the inverse distribution. Furthermore, the wavelet-like distribution of amino acids could be represented by a universal distribution function together with an index characterizing the intensity of the wavelet. Using the universal distribution function and the novel index of amino acids, we developed a simple method for extracting segments and fragments that are unique to allergens. The significance of the universal distribution function and the novel index is also discussed, by comparing the plot of the AUF index and dynamic fluctuation in the three-dimensional structure of birch pollen allergen as both a single molecule and a complex with the corresponding antibody.

Bone morphogenetic protein receptors and signal transduction

Miyazono, K., Kamiya, Y., Morikawa, M. — Sun, 27 Sep 2009 21:30:46 PDT

Bone morphogenetic proteins (BMPs) exhibit broad spectra of biological activities in various tissues, including bone, cartilage, blood vessels, heart, kidney, neurons, liver, and lung. BMPs are members of the transforming growth factor-β (TGF-β) family that bind to type II and type I serine-threonine kinase receptors, and transduce signals through Smad and non-Smad signaling pathways. Recent findings have revealed that BMP signaling is finely tuned by various mechanisms in both positive and negative fashions. Perturbations of BMP signaling pathways are linked to a wide variety of clinical disorders, including vascular diseases, skeletal diseases, and cancer. Administration of recombinant BMP ligands and increasing endogenous expression of BMPs provide therapeutic effects on some diseases. The recent development of BMP receptor inhibitors may also prove useful for some clinical diseases induced by hyperactivation of the BMP signaling pathways.

Antisense transcription occurs at the promoter of a mouse imprinted gene, Commd1, on the repressed paternal allele.

Joh, K., Yatsuki, H., Higashimoto, K., Mukai, T., Soejima, H. — Thu, 17 Sep 2009 00:56:07 PDT

The Commd1 gene is imprinted in the adult mouse brain and is predominantly expressed from the maternal allele. A paternally expressing imprinted gene, U2af1-rs1, resides in the first intron of Commd1 in an antisense orientation. We found that RNA polymerase II phosphorylated at serine 2 of the carboxyl-terminal domain repeats, a marker of transcription elongation, is enriched on the paternal allele than on the maternal allele of the Commd1 promoter. The Commd1 promoter harbors no allelic differences in DNA methylation and histone modifications. These results strongly suggested that imprinting of Commd1 is generated by interference with paternal Commd1 transcription by the oppositely directed U2af1-rs1 transcription.

Inhibition of electron acceptance from ascorbate by the specific N-carbethoxylations of maize cytochrome b561: A common mechanism for the transmembrane electron transfer in cytochrome b561 protein family5,6

Wed, 16 Sep 2009 23:38:02 PDT

Cytochromes b₅₆₁ constitute a novel class of proteins in eukaryotic cells with a number of highly relevant common features including six transmembrane -helices and two heme groups. Of particular interest is the presence of a large number of plant homologues having putative ascorbate- and monodehydroascorbate radical-binding sites. We conducted a diethylpyrocarbonate-modification study employing Zea mays cytochrome b₅₆₁ heterologously-expressed in Pichia pastoris cells. Pre-treatment of cytochrome b₅₆₁ with diethylpyrocarbonate in oxidized form caused N-carbethoxylation of His⁸⁶, His¹⁵⁹, and Lys⁸³, leading to a drastic inhibition of the electron transfer from ascorbate. The activity was protected by the inclusion of ascorbate during the treatment. However, midpoint potentials of two heme centers did show only slight decreases upon the treatment, suggesting that changes in the midpoint potentials were not the major cause of the inhibition. Present results indicated that Zea mays cytochrome b₅₆₁ conducted an ascorbate-specific transmembrane electron transfer by utilizing a concerted H⁺/e- transfer mechanism and that the specific _N-carbethoxylation of heme axial His⁸⁶ that would inhibit the removal of a proton from the bound ascorbate was a major cause of the inhibition. On the other hand, Lys⁸³ might be important for an initial step(s) of the fast electron acceptance from ascorbate.

Purification and Some Properties of Wild-type and N-terminal-truncated Ethanolamine Ammonia-lyase of Escherichia coli{dagger}

Wed, 16 Sep 2009 23:38:01 PDT

The methods of homologous high-level expression and simple large-scale purification for coenzyme B₁₂-dependent ethanolamine ammonia-lyase of Escherichia coli were developed. The eutB and eutC genes in the eut operon encoded the large and small subunits of the enzyme, respectively. The enzyme existed as the heterododecamer ₆β₆. Upon active-sites titration with adeninylpentylcobalamin, a strong competitive inhibitor for coenzyme B₁₂, the binding of 1 mol of the inhibitor per mol of the β unit caused complete inhibition of enzyme, in consistent with its subunit structure. EPR spectra indicated the formation of substrate-derived radicals during catalysis and the binding of cobalamin in the base-on mode, i.e., with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. The purified wild-type enzyme underwent aggregation and inactivation at high concentrations. Limited proteolysis with trypsin indicated that the N-terminal region is not essential for catalysis. His-tagged truncated enzymes were similar to the wild-type enzyme in catalytic properties, but more resistant to p-chloromercuribenzoate than the wild-type enzyme. A truncated enzyme was highly soluble even in the absence of detergent and resistant to aggregation and oxidative inactivation at high concentrations, indicating that a short N-terminal sequence is sufficient to change the solubility and stability of the enzyme.

A lentiviral vector encoding two fluorescent proteins enables imaging of adenoviral infection via adenovirus-encoded miRNAs in single living cells

Kato, Y., Sawata, S. Y., Inoue, A. — Wed, 16 Sep 2009 23:38:01 PDT

MicroRNAs (miRNAs) are non-coding small RNAs that have been found in various kinds of eukaryotic cells and viruses. Recently, adenovirus non-coding RNAs, VA RNAs, have been reported to generate miRNAs. Here we developed a lentiviral vector for monitoring adenovirus-derived miRNAs in living cells. By using red and green fluorescent proteins under the control of bidirectional two distinct promoters, adenoviral infection and consequent miRNA expression was successfully visualized and quantified by the reduction in green fluorescence when 3' untranslated regions were connected to the target sequences of the adenovirus-derived miRNAs. Our functional analysis using a lentiviral vector is a useful method to examine the activity of miRNA in living mammalian cells.

Protruding masticatory (superfast) myosin heads from staggered thick filaments of dog jaw muscle revealed by X-ray diffraction

Wed, 16 Sep 2009 23:37:59 PDT

To characterize the structure of jaw muscle fibers expressing masticatory (superfast) myosin, X-ray diffraction patterns of glycerinated fibers of dog masseter were compared with those of dog tibialis anterior in the relaxed state. Meridional reflections of masseter fibers were laterally broad, indicating that myosin filaments are staggered along the filament axis. Compared with tibialis anterior fibers, the peak of the first myosin layer line of masseter fibers was lower in intensity and shifted towards the meridian, while lattice spacings were larger at a similar sarcomere length. These suggest that the myosin heads of masticatory fibers are mobile, and tend to protrude from the filament shaft towards actin filaments. Lowering temperature or treating with N-phenylmaleimide shifted the peak of the first myosin layer line of tibialis anterior fibers towards the meridian and the resulting profile resembled that of masseter fibers. This suggests that the protruding mobile heads in the non-treated masticatory fibers are in the ATP-bound state. The increased population of weakly binding cross-bridges may contribute towards the high specific force of masticatory fibers during contraction. Electron micrographs confirmed the staggered alignment of thick filaments along the filament axis within sarcomeres of masticatory fibers, a feature that may confer efficient force development over a wide range of the sarcomere lengths.

Retinoic acids induce neurosteroid biosynthesis in human glial GI-1 cells via the induction of steroidogenic genes.

Kushida, A., Tamura, H. — Thu, 10 Sep 2009 04:55:20 PDT

The steroids synthesized in the central nervous system (CNS) are the neurosteroids. Since little information is currently available concerning the roles of the retinoic acids (RAs) during steroidogenesis in the CNS, we investigated the effects of RAs upon their synthesis in our current study. Specifically, we analyzed the effects of all-trans-retinoic acid (ATRA) upon the expression of neurosteroid biosynthesis genes in the human glial cell line GI-1, in which the major steroidogenic genes are expressed. Treatment with ATRA (10 µM) induced a 4.9-fold increase in the expression of the cytochrome P450scc (CYP11A1) gene, the product of which cleaves the cholesterol side chain, a rate-limiting step during steroidogenesis. ATRA also strongly induced the expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) (an increase of 5- and 50-fold, respectively). A retinoic acid receptor (RAR)-specific agonist, TTNPB, was unable to mimic this induction whereas a retinoid X receptor (RXR)-specific agonist, methoprene acid, in addition to 9-cis-RA, could do so. These data indicate that ATRA is isomerized to 9-cis-RA in the culture medium, as reported previously, and that 9-cis-RA activates the RXR. In addition, ATRA also induced the de novo synthesis of neurosteroids such as pregnenolone and progesterone. These results suggest that ATRA might induce the de novo neurosteroid synthesis via the induction of steroidogenic genes in human glial cells. The multiple effects of vitamin A upon CNS functions might therefore be partly explained by the induction of neurosteroidogenesis by RAs, since neurosteroids have also been reported to have multiple effects in the CNS.

Expression and functional analysis of a predicted AtsG arylsulfatase identified from Mycobacterium tuberculosis genomic data

Hossain, Md. M., Kawarabayasi, Y., Kimura, M., Kakuta, Y. — Fri, 04 Sep 2009 02:49:46 PDT

Sulfatase family enzymes hydrolyze the sulfate ester, found on the pathogens cell surface and playing an important role for host-pathogen interaction. The AtsG, homologue of arylsulfatase, predicted in the Mycobacterium tuberculosis genomic data, was successfully expressed in Escherichia coli. The recombinant AtsG protein exhibited hydrolysis of para-nitrophenyl sulfate and para-nitrocatechol sulfate, and binding affinity to the Heparin-Sepharose resin. This is the first report of molecular evidence for an arylsulfatase activity of the AtsG protein. The maximum activity was detected at pH 8.0 and 37°C. As EDTA completely inhibited this activity, a divalent cation was required for the activity.

Correlation between Proton Translocation and Growth: Genetic Analysis of the Respiratory Chain of Corynebacterium glutamicum

Kabashima, Y., Kishikawa, J.-i., Kurokawa, T., Sakamoto, J. — Fri, 04 Sep 2009 02:49:45 PDT

Corynebacterium glutamicum contains at least two terminal oxidases in the respiratory chain; cytochrome aa₃–type cytochrome c oxidase and bd–type menaquinol oxidase. Thus, the chain has two branches of electron flow. The bcc–aa₃ branch translocates three protons per electron transferred, while the bd branch translocates only one. In this study, we constructed two mutant strains, lacking of either subunit I of the cytochrome c oxidase (cta D) or subunits I and II of the quinol oxidase (cydAB), and also plasmids to complement the deficient genes to investigate their effects on energy conservation and cell growth. We measured H⁺/O ratios of C. glutamicum wild-type and mutant cells grown aerobically. The H⁺/O ratio of the wild-type cells grown in the semi-synthetic medium was 3.94 ± 0.30, while the value was 2.76 ± 0.25 for the cta D mutant. In contrast, the value was 5.23 ± 0.36 for the cydAB mutant. The cells grown in the LB medium showed higher value compared to that of cells grown in the semi-synthetic medium. The cta D mutant grew less than the wild-type in LB medium, while they grew about equally in semi-synthetic medium. Correlation between bioenergetics and growth of C. glutamicum was significantly affected by the growth nutrients.

The Syntaxin 5 isoforms Syx5 and Syx5L have distinct effects on the processing of {beta}-amyloid precursor protein

Suga, K., Saito, A., Tomiyama, T., Mori, H., Akagawa, K. — Sun, 30 Aug 2009 21:59:41 PDT

In this study, we examined the interaction of Syntaxin 5L (Syx5L), a Syx5 isoform that has an N-terminal extension containing a di-arginine ER-retrieval motif, with presenilin (PS) and its effects on the processing of β-amyloid precursor protein (βAPP). Similar to Syx5, Syx5L bound to PS1 holoprotein but not to its N- or C-terminal fragments. Unlike Syx5, Syx5L overexpression did not cause marked accumulation of intracellular βAPP holoprotein, and did not inhibit amyloid β peptide (Aβ) secretion. Analyses using deletion mutants of Syx5L revealed that, in addition to the difference in the intracellular localization between the isoforms, the presence of the N-terminal extension in Syx5L was critical for suppressing its inhibition of βAPP processing. Treatment of cells that overexpressed Syx5L with brefeldin A, an inhibitor of transport from the ER to the Golgi compartments, resulted in substantial accumulation of intracellular βAPP holoprotein and reduction in the secretion of Aβ. Although Syx5 and Syx5L share lengthy regions of amino acid identity, they appear to play distinct roles in modulating the metabolism and trafficking of βAPP in the early secretory compartment.

Induction of Mitochondrial Permeability Transition by the DNA-intercalating Cationic Dye Ethidium Bromide

Fri, 28 Aug 2009 21:42:52 PDT

This work shows that the DNA cationic probe, ethidium bromide, induces the transition from selective to non-selective mitochondrial permeability. This statement is based on the findings, indicating: i) ethidium bromide induced the release of accumulated Ca²⁺ through a mechanism sensitive to cyclosporin A and octylguanidine; ii) ethidium bromide induced the release of cytochrome c, and iii) ethidium bromide induced mitochondrial swelling. Interestingly, mersalyl inhibited, in a non-competitive fashion, ethidium bromide uptake, which would indicate that the uptake may be carried out through a protein membrane system. This work also shows that the effect of the dye on permeability transition was stimulated by carboxyatractyloside. Taking into account the facts that ethidium bromide inhibited the ADP exchange reaction and increased the binding of the fluorescent probe eosin-5-maleimide to adenine nucleotide translocase, it is tempting to assume a possible interaction between ethidium bromide and the ADP/ATP carrier.

Succinylation - Induced Conformational Destabilization of Lysozyme as Studied by Guanidine Hydrochloride Denaturation

Ong, H. N., Arumugam, B., Tayyab, S. — Fri, 28 Aug 2009 21:42:52 PDT

Using 100 molar excess of succinic anhydride, about 99 % of lysine residues of hen egg white lysozyme (HEWL) were modified. Succinylated (S₉₉) HEWL showed both charge and size homogeneity as judged by polyacrylamide gel electrophoresis and gel filtration, respectively. Hydrodynamic parameters such as Stokes radius and frictional ratio (f/f_o) showed more expanded conformation of S99 HEWL compared to native HEWL as evident from the increase in Stokes radius (from 1.36 to 1.86 nm) and f/f_o (from 0.86 to 1.15) values. Guanidine hydrochloride (GdnHCl) denaturation studies using fluorescence spectroscopy connoted a marked decrease in conformational stability of HEWL upon succinylation. Complete denaturation of S₉₉ HEWL was achieved at lower GdnHCl concentration (~3.8 M) compared to native HEWL (~5 M). Furthermore, free energy of stabilization (G_D^H2O) value also showed a notable decrease from 8559 and 7956 cal/mol (for native HEWL) to 4404 and 4669 cal/mol (for succinylated HEWL) using excitation at 280 and 295 nm, respectively. Both expanded conformation and decreased G_D^H2O can be attributed to the increase in the net negative charge on the protein upon succinylation. All these results manifested the importance of positively charged lysine residues in maintaining the conformational stability of HEWL through electrostatic interactions.

FGF-1-Induced Reactions for Biogenesis of apoE-HDL are Mediated by Src in Rat Astrocytes

Nishida, T., Ito, J.-i., Nagayasu, Y., Yokoyama, S. — Thu, 27 Aug 2009 09:14:17 PDT

Fibroblast growth factor-1 (FGF-1) is released from astrocytes in stress and stimulates MEK/ERK and PI3K/Akt pathways in autocrine fashion to increase synthesis of cholesterol and 25-OH cholesterol, and to induce transport and secretion of apoE, respectively. FGF-1 induced phosphorylation of Src, and phosphorylation of MEK, ERK and Ark was inhibited by Src inhibitors in rat astrocytes. Src inhibitors also suppressed FGF-1-induced increase of biosynthesis and release of cholesterol and increase of apolipoprotein E (apoE) secretion. The results were reproduced in rat astrocytoma cells transfected by rat apoE and in 3T3 cells. Down-regulation of Src expression reduced FGF-1-induced phosphorylation of the signaling protein and subsequent reactions. Increase by FGF-1 of messages of apoE and HMG-CoA reductase was not influenced by Src inhibitors or by its down-regulation. We conclude that FGF-1 activates Src for activation of MEK/ERK and PI3K/Akt pathways, while Src may not be involved in enhancement of transcription of the cholesterol-related genes.

Biotinylated bionanocapsules for displaying diverse ligands toward cell specific delivery

Tue, 25 Aug 2009 22:35:53 PDT

Bionanocapsule (BNC) is hollow nanoparticle composed of the L protein of the hepatitis B virus surface antigen. BNC allows targeted delivery of either genes or drugs only to hepatocytes, but not to other cell types. In this study, we attempted to alter the specificity of BNC by insertion of biotin-acceptor peptide (BAP), which is efficiently biotinylated using biotin ligase BirA from E. coli. Using streptavidin as a linker, biotinylated BNC could be display various biotinylated ligands that are otherwise difficult to fuse with BNC, such as antibodies, synthetic peptides, and functional molecules. BAP-fused BNC was efficiently biotinylated and effectively displayed streptavidin. Furthermore, we demonstrated that biotinylated BNC was internalized into targeted cells via biotinylated Nanobody displayed on the BNC surface. Biotinylated BNC permit display of diverse ligands, and thus have potential as a versatile carrier for drug delivery to a variety of target cells.

GDP-GTP exchange processes of G{alpha}i1 protein are accelerated/decelerated depending on the type and the concentration of added detergents

Kubota, M., Tanaka, T., Kohno, T., Wakamatsu, K. — Mon, 24 Aug 2009 07:49:48 PDT

Although detergents have been widely used in G protein studies to increase solubility and stability of the protein, we noticed that detergents modulate the nucleotide-binding properties of G proteins. Hence, we analyzed the effects of detergents on guanine nucleotide exchange reactions of G_i1. Lubrol PX, a non-ionic detergent which has been widely used in nucleotide dissociation/binding assays, was found to accelerate both GDP dissociation and GTPS binding from/to G in parallel at above its critical micelle concentration (cmc). Sodium cholate, an anionic detergent, which have been used to extract G proteins from animal tissues, decelerated and accelerated GDP dissociation below and above its cmc, respectively. Surprisingly, micellar cholate decelerated GTPS binding, and the binding rate constant was decreased by three orders of magnitude in the presence of 2% cholate. These results demonstrate that the guanine nucleotide exchange reactions of G_i1 are drastically modulated by detergents differently depending on the type and the state (monomeric or micellar) of the detergents and that dissociation of GDP from G_i1 does not necessarily lead to immediate binding of GTP to G_i1 in some cases. These effects of detergents on G proteins must be taken into account in G protein experiments.

Tyrosine sulfation of sphingosine 1-phosphate 1 (S1P1) is required for S1P-mediated cell migration in primary cultures of human umbilical vein endothelial cells

Huang, Y.-L., Lin, H.-S., Chen, S.-U., Lee, H. — Thu, 20 Aug 2009 07:03:24 PDT

Sphingosine 1-phosphate (S1P), a lysophospholipid mediator, regulates diverse functions of many types of cells by binding to specific G protein-coupled receptors termed S1P₁~S1P₅. In T cells, tyrosine sulfation of S1P₁ is required for high-affinity binding of S1P and fully functional signaling. In this study, we showed that tyrosine sulfation of S1P₁ is necessary for S1P-induced Src phosphorylation and migration in human umbilical vein endothelial cells (HUVECs). Both substitution of phenylalanine (F) for tyrosine (Y) in S1P₁ and inhibition of tyrosine sulfation blocked c-Src phosphorylation and migration in HUVECs. In addition, overexpression of mutant (F19, 22F) S1P₁, lacking tyrosine sulfation sites, suppressed native S1P₁ effects on migration, actin rearrangement, and lamellipodia formation. Therefore, tyrosine sulfation of S1P₁ thus is required for its optimal transduction of signals from S1P in HUVECs.

Amyloid Formation by the Model Protein Muscle Acylphosphatase is Accelerated by Heparin and Heparan Sulfate Through a Scaffolding-Based Mechanism

Motamedi-Shad, N., Monsellier, E., Chiti, F. — Wed, 12 Aug 2009 06:42:05 PDT

Amyloid formation is the hallmark of many diseases. The propensity of a protein to aggregate depends on a number of biological factors like the presence of sulfated polysaccharides termed glycosaminoglycans (GAGs). Here we assessed whether the polymeric nature of GAGs is responsible for their protein aggregation promoting effect. We studied the effect of different monosaccharide-derivatives, featuring the main characteristics of heparin and heparan sulfate (HS) building blocks, on the aggregation kinetics of human muscle acylphosphatase (mAcP), a useful model protein for these studies. We observed that while heparin and HS changed the mAcP aggregation kinetic profile, the monosaccharide derivatives had no effect, whatever their concentration and both when they are studied separately or in combination. By contrast, heparin fragments with 6 or more monosaccharides reproduced the effects of HS and in part those of heparin. We conclude that the effect of heparin and HS on protein aggregation arises from the clustering and regular distribution of their composing units on a polymeric structure. We propose a model in which heparin and HS promote mAcP aggregation through a scaffolding-based mechanism, in which the regularly spaced sulfate moieties of the polymer interact with mAcP molecules increasing their local concentration and facilitating their orientation.

Ro52-mediated monoubiquitination of IKK{beta} downregulates NF-{kappa}B signaling

Wada, K., Niida, M., Tanaka, M., Kamitani, T. — Wed, 12 Aug 2009 06:42:04 PDT

Upon activation, NF-B translocates into the nucleus and initiates biological events. This NF-B signaling is mainly regulated by the protein kinese IKKβ. Early in this signaling pathway, IKKβ is phosphorylated for activation by several factors, such as proinflammatory cytokines and the Tax oncoprotein of HTLV-1. In cells infected by HTLV-1, IKKβ is persistently phosphorylated and conjugated with monoubiquitin due to Tax expression. Although this Tax-induced monoubiquitination appears to be an important regulation system for IKKβ, how the monoubiquitination occurs is unknown, and its role in NF-B signaling is still unclear. Here, we show that an E3-ubiquitin ligase Ro52 interacts weakly with wild-type IKKβ but strongly with a phosphomimetic mutant IKKβ to conjugate monoubiquitin in cooperation with an E2-ubiquitin-conjugating enzyme UbcH5B. These results suggest that the Tax-induced phosphorylation of IKKβ causes an interaction with Ro52 for the subsequent monoubiquitination. NF-B reporter assays have shown that the IKKβ activity is suppressed by wild-type Ro52, but not by its inactive mutant. In addition, monoubiquitin fusion of IKKβ reduced its activity for NF-B signaling. We also found that Ro52 dramatically reduces the level of Tax. These results suggest that Ro52 downregulates Tax-induced NF-B signaling by monoubiquitinating IKKβ and by reducing the level of Tax.

PERIOD1 (PER1) is an anti-apoptotic factor in human pancreatic and hepatic cancer cells.

Wed, 12 Aug 2009 06:42:03 PDT

PERIOD1 (PER1) is a clock gene. We examined the effect of knockdown of PER1 on apoptosis in pancreatic cancer (MIA PaCa-2 and PANC-1) and hepatocellular carcinoma (HepG2) cells. Transfection of siRNA against PER1 into these cells increased the cleaved forms of caspases and poly-ADP-ribose-polymerase (PARP) and induced apoptosis in all three cell lines. In the two pancreatic cancer cell lines, PER1 knockdown resulted in upregulation of Bax and downregulation of Bcl-2. Expression of p53 was not altered in the two pancreatic cancer cell lines containing mutated p53, but was upregulated in the HepG2 cells containing wild type p53. Cell proliferation of MIA PaCa-2 and HepG2 was inhibited by PER1 knockdown. We also examined, by immunohistochemical staining, the expression of PER1 in pancreatic cancer tissue and found that PER1 was strongly expressed in pancreatic cancer cells. These results indicate that PER1 acts as an anti-apoptotic factor in pancreatic cancer cells.

The Genomic Structure of Thermus Bacteriophage {varphi}IN93

Matsushita, I., Yanase, H. — Wed, 12 Aug 2009 06:42:03 PDT

We have determined the complete nucleotide sequence of the phage IN93 is 19,604-bp long and contains 39 putative open reading frames (ORFs). The functions for 20% of IN93 gene products are similar to those expressed by other known phages and bacteria, and include peptidase, lytic enzymes, integrase, repressor protein, and replication protein. The structural proteins of the IN93 virion were identified through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and found to have no similarity to those of other phages. We also determined the transcription initiation sites and classified four transcription units using the primer extension method. Three transcription units were transcribed in the same direction as part of the lytic cycle, while the remaining unit was transcribed in the opposite direction as part of the lysogenic cycle.

Cyclic AMP represses the hypoxic induction of hypoxia-inducible factors in PC12 cells

Torii, S., Okamura, N., Suzuki, Y., Ishizawa, T., Yasumoto, K.-i., Sogawa, K. — Tue, 11 Aug 2009 04:16:18 PDT

Hypoxia-inducible factor 1 (HIF-1) is a master regulator for hypoxic activation of genes for angiogenesis, hormone synthesis, glycolysis and cell survival. In addition to hypoxic stimulus, various effectors and reagents were reported to affect HIF-1 activity. Here we show that cyclic AMP (cAMP) downregulates the HIF-1 activity in pheochromocytoma PC12 cells but not in Hep3B and HeLa cells. Hypoxia response element-dependent reporter activity was decreased by the addition of dibutyryl cAMP. Expression of protein kinase A (PKA) catalytic subunits repressed the HIF-1 activity. HIF-1 and HLF (HIF-2 or EPAS1) protein levels were decreased by the treatment with dibutyryl cAMP. Although CREB was also served as a negative factor for the HIF-1 activity, it may not be a major PKA target in the cAMP-dependent HIF- repression pathway. Induction of hypoxia responsive genes was suppressed by dibutyryl cAMP. Our results provide additional insight into a regulatory mechanism of hypoxic response.

Possible role of mitochondrial remodeling on cellular triacylglycerol accumulation

Kita, T., Nishida, H., Shibata, H., Niimi, S., Higuti, T., Arakaki, N. — Mon, 10 Aug 2009 22:09:11 PDT

Mitochondrial fusion and fission processes play a role in a variety of cell functions, including energy metabolism, cell differentiation, and programmed cell death. Still, it is not clear how these processes contribute to the cell functions. Here we investigated the role of mitochondrial remodeling on lipid metabolism in adipocytes. In 3T3-L1 preadipocytes, the morphology of mitochondria is organized as a continuous reticulum. Upon differentiation of adipocytes manifested by cellular triacylglycerol accumulation, mitochondrial morphology altered from filamentous to fragmented and/or punctate structures. When the mitochondrial fusion was induced in adipocytes by silencing of mitochondrial fission proteins including Fis1 and Drp1, the cellular triacylglycerol content was decreased. In contrast, the silencing of mitochondrial fusion proteins including mitofusin 2 and Opa1 increased the cellular triacylglycerol content followed by fragmentation of mitochondria. It also appears that polyphenolic phytochemicals, negative regulators of lipid accumulation, have mitochondrial fusion activity and that there is a good correlation between mitochondrial fusion activity and the cellular TG accumulation-reducing activity of the phytochemicals. These results suggest that cellular triacylglycerol accumulation is regulated, at least in part, via mitochondrial fusion and fission processes.

A Novel Insertion Sequence Transposed to Thermophilic Bacteriophage {varphi}IN93

Matsushita, I., Yanase, H. — Mon, 10 Aug 2009 22:09:11 PDT

A novel insertion sequence (IStaqTZ2) was transposed from the genome of Thermus thermophilus TZ2 to that of the thermophilic bacteriophage IN93. The complete nucleotide sequence of IStaqTZ2 was determined and was found to be 1,258 bps in length and to contain an open reading frame (ORF1179), which is predicted to encode a transposase. IStaqTZ2 was also found to contain two terminal inverted repeats with 48 and 52 bps, respectively. Based on homology analysis, IStaqTZ2 was classified as a member of the IS256 family.

Molecular Pathogenesis of Protein Misfolding Diseases: Pathological Molecular Environments versus Quality Control Systems against Misfolded Proteins

Naiki, H., Nagai, Y. — Thu, 30 Jul 2009 02:31:09 PDT

Diverse human diseases, including various neurodegenerative disorders and amyloidoses, are thought to result from the misfolding and aggregation of disease-causative proteins, and thus are collectively called protein misfolding diseases. Natively folded disease-causative proteins generally undergo a β-sheet conformational transition through an energetically unfavorable process, and further polymerize into amyloid fibrils. In the case of β₂-microglobulin-related amyloidosis, an extracellular protein misfolding disease, many kinds of biological molecules including glycosaminoglycans, proteoglycans and lipids partially unfold β₂-microglobulin and catalyze its subsequent nucleus formation. After amyloid fibrils are formed, these biological molecules stabilize the β₂-microglobulin fibrils. In the polyglutamine neurodegenerative diseases, an intracellular protein misfolding disease, molecular chaperones as well as the ubiquitin-proteasome and autophagy-lysosome protein degradation systems, which are called the protein quality control systems, strictly regulate protein misfolding, aggregation and disease progression. A family of extracellular chaperones also binds to misfolded proteins and inhibit amyloid fibril formation in the extracellular space. Protein misfolding and aggregation may be an ideal therapeutic target for protein misfolding diseases in general.

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. — Fri, 26 Jun 2009 08:19:05 PDT

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

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

Fri, 13 Jun 2008 04:49:51 PDT

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

Helmy, M, El-Adl, N — Mon, 26 Mar 2007 10:03:34 PDT

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.