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  • Articles  (975)
  • Oxford University Press  (975)
  • PANGAEA
  • 2010-2014  (975)
  • Journal of Biochemistry  (309)
  • 740
  • 1
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    Affinity maturation of a CDR3-grafted VHH using in silico analysis and surface plasmon resonance (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: A requirement for advancing antibody-based medicine is the development of proteins that can bind with high affinity to a specific epitope related to a critical protein activity site. As a part of generating such proteins, we have succeeded in creating a binding protein without changing epitope by complementarity-determining region 3 (CDR3) grafting (Inoue et al. , Affinity transfer to a human protein by CDR3 grafting of camelid VHH. Protein Sci. 20, 1971–1981). However, the affinity of the target-binding protein was low. In this manuscript, the affinity maturation of a target-binding protein was examined using CDR3-grafted camelid single domain antibody (VHH) as a model protein. Several amino acids in the CDR1 and CDR2 regions of VHH were mutated to tyrosines and/or serines and screened for affinity-matured proteins by using in silico analysis. The mutation of two amino acids in the CDR2 region to arginine and/or aspartic acid increased the affinity by decreasing the dissociation rate. The affinity of designed mutant increased by ~20-fold over that of the original protein. In the present study, candidate mutants were narrowed down using in silico screening and computational modelling, thus avoiding much in vitro analytical effort. Therefore, the method used in this study is expected to be one of the useful for promoting affinity maturation of antibodies.
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  • 2
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    Characterization of deamidation at Asn138 in L-chain of recombinant humanized Fab expressed from Pichia pastoris (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ~30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.
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  • 3
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    TRIM proteins as trim tabs for the homoeostasis (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: The tripartite motif (TRIM) or RBCC proteins are characterized by the TRIM composed of a RING finger, B-box and coiled-coil domains. TRIM proteins often play roles in the post-translational protein modification, including ubiquitylation and other ubiquitin-like modifications. Evidence has accumulated in regard to the contribution of TRIM proteins to diverse cellular processes, including such as cell cycle progression, apoptosis, immunity and transcriptional regulation. In particular, some of the TRIM proteins have been characterized to exert oncogenic or tumour suppressor-like functions depending on the context. A recent report by Inoue and his colleagues has revealed that Terf/TRIM17 stimulates the degradation of a kinetochore protein ZWINT and regulates the proliferation of breast cancer cells. Terf has also been paid attention as a factor promoting neuronal apoptosis, by degrading a Bcl2-like anti-apoptotic protein Mcl-1. Like aircraft trim tabs, TRIM proteins trim the balance of homoeostasis by modulating various biological pathways through protein–protein interactions.
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  • 4
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    Inhibition of DNA binding of MCM2-7 complex by phosphorylation with cyclin-dependent kinases (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: Cyclin-dependent kinase (CDK) that plays a central role in preventing re-replication of DNA phosphorylates several replication proteins to inactivate them. MCM4 in MCM2-7 and RPA2 in RPA are phosphorylated with CDK in vivo . There are inversed correlations between the phosphorylation of these proteins and their chromatin binding. Here, we examined in vitro phosphorylation of human replication proteins of MCM2-7, RPA, TRESLIN, CDC45 and RECQL4 with CDK2/cyclinE, CDK2/cyclinA, CDK1/cyclinB, CHK1, CHK2 and CDC7/DBF4 kinases. MCM4, RPA2, TRESLIN and RECQL4 were phosphorylated with CDKs. Effect of the phosphorylation by CDK2/cyclinA on DNA-binding abilities of MCM2-7 and RPA was examined by gel-shift analysis. The phosphorylation of RPA did not affect its DNA-binding ability but that of MCM4 inhibited the ability of MCM2-7. Change of six amino acids of serine and threonine to alanines in the amino-terminal region of MCM4 rendered the mutant MCM2-7 insensitive to the inhibition with CDK. These biochemical data suggest that phosphorylation of MCM4 at these sites by CDK plays a direct role in dislodging MCM2-7 from chromatin and/or preventing re-loading of the complex to chromatin.
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  • 5
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    Effects of alcohols on the stability and low-frequency local motions that control the slow changes in structural dynamics of ferrocytochrome c (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: To determine the effects of alcohols on the low-frequency local motions that control slow changes in structural dynamics of native-like compact states of proteins, we have studied the effects of alcohols on structural fluctuation of M80-containing -loop by measuring the rate of thermally driven CO dissociation from a natively folded carbonmonoxycytochrome c under varying concentrations of alcohols (methanol, ethanol, 1-propanol, 2-propanol, 3°-butanol, 2,2,2-trifluoroethanol). As alcohol is increased, the rate coefficient of CO dissociation ( k diss ) first decreases in subdenaturing region and then increases on going from subdenaturing to denaturing milieu. This decrease in k diss is more for 2,2,2-trifluroethanol and 1-propanol and least for methanol, indicating that the first phase of motional constraint is due to the hydrophobicity of alcohols and intramolecular protein cross-linking effect of alcohols, which results in conformational entropy loss of protein. The thermal denaturation midpoint for ferrocytochrome c decreases with increase in alcohol, indicating that alcohol decrease the global stability of protein. The stabilization free energy ( G ) in alcohols’ solution was calculated from the slope of the Wyman–Tanford plot and water activity. The m -values obtained from the slope of G versus alcohols plot were found to be more negative for longer and linear chain alcohols, indicating destabilization of proteins by alcohols through disturbance of hydrophobic interactions and hydrogen bonding.
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  • 6
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    Linear ubiquitination-mediated NF-{kappa}B regulation and its related disorders (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: Ubiquitination is a post-translational modification involved in the regulation of a broad variety of cellular functions, such as protein degradation and signal transduction, including nuclear factor-B (NF-B) signalling. NF-B is crucial for inflammatory and immune responses, and aberrant NF-B signalling is implicated in multiple disorders. We found that linear ubiquitin chain assembly complex (LUBAC), composed of HOIL-1L, HOIP and SHARPIN, generates a novel type of Met1 (M1)-linked linear polyubiquitin chain and specifically regulates the canonical NF-B pathway. Moreover, specific deubiquitinases, such as CYLD, A20 (TNFAIP3) and OTULIN/gumby, inhibit LUBAC-induced NF-B activation by different molecular mechanisms, and several M1-linked ubiquitin-specific binding domains have been structurally defined. LUBAC and these linear ubiquitination-regulating factors contribute to immune and inflammatory processes and apoptosis. Functional impairments of these factors are correlated with multiple disorders, including autoinflammation, immunodeficiencies, dermatitis, B-cell lymphomas and Parkinson’s disease. This review summarizes the molecular basis and the pathophysiological implications of the linear ubiquitination-mediated NF-B activation pathway regulation by LUBAC.
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  • 7
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    Different circadian expression of major matrix-related genes in various types of cartilage: modulation by light-dark conditions (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: We screened circadian-regulated genes in rat cartilage by using a DNA microarray analysis. In rib growth-plate cartilage, numerous genes showed statistically significant circadian mRNA expression under both 12:12 h light–dark and constant darkness conditions. Type II collagen and aggrecan genes—along with several genes essential for post-translational modifications of collagen and aggrecan, including prolyl 4-hydroxylase 1, lysyl oxidase, lysyl oxidase-like 2 and 3'-phosphoadenosine 5'-phosphosulphate synthase 2—showed the same circadian phase. In addition, the mRNA level of SOX9, a master transcription factor for the synthesis of type II collagen and aggrecan, has a similar phase of circadian rhythms. The circadian expression of the matrix-related genes may be critical in the development and the growth of various cartilages, because similar circadian expression of the matrix-related genes was observed in hip joint cartilage. However, the circadian phase of the major matrix-related genes in the rib permanent cartilage was almost the converse of that in the rib growth-plate cartilage under light–dark conditions. We also found that half of the oscillating genes had conserved clock-regulatory elements, indicating contribution of the elements to the clock outputs. These findings suggest that the synthesis of the cartilage matrix macromolecules is controlled by cell-autonomous clocks depending upon the in vivo location of cartilage.
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  • 8
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    Association of epigenetic alterations in the human C7orf24 gene with the aberrant gene expression in malignant cells (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: Human chromosome 7 open reading frame 24 (C7orf24)/-glutamyl cyclotransferase has been suggested to be a potential diagnostic marker for several cancers, including carcinomas in the bladder urothelium, breast and endometrial epithelium. We here investigated the epigenetic regulation of the human C7orf24 promoter in normal diploid ARPE-19 and IMR-90 cells and in the MCF-7 and HeLa cancer cell lines to understand the transcriptional basis for the malignant-associated high expression of C7orf24. Chromatin immunoprecipitation analysis revealed that histone modifications associated with active chromatin were enriched in the proximal region but not in the distal region of the C7orf24 promoter in HeLa and MCF-7 cells. In contrast, elevated levels of histone modifications leading to transcriptional repression and accumulation of heterochromatin proteins in the C7orf24 promoter were observed in the ARPE-19 and IMR-90 cells, compared to the levels in HeLa and MCF-7 cancer cells. In parallel, the CpG island of the C7orf24 promoter was methylated to a greater extent in the normal cells than in the cancer cells. These results suggest that the transcriptional silencing of the C7orf24 gene in the non-malignant cells is elicited through heterochromatin formation in its promoter region; aberrant expression of C7orf24 associated with malignant alterations results from changes in chromatin dynamics.
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  • 9
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    Identification and characterization of interactions between abscisic acid and human heat shock protein 70 family members (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: Abscisic acid (ABA) is a stress-inducible plant hormone comprising an inevitable component of the human diet. Recently, stress-induced accumulation of autocrine ABA was shown in humans, as well as ABA-mediated modulation of a number of disease-associated systems. Now, the application of a chemical proteomics approach to gain further insight into ABA mechanisms of action in mammalian cells is reported. An ABA mimetic photoaffinity probe was applied to intact mammalian insulinoma and embryonic cells, leading to the identification of heat shock protein 70 (HSP70) family members, (including GRP78 and HSP70-2) as putative human ABA-binding proteins. In vitro characterization of the ABA–HSP70 interactions yielded K d s in the 20–60 µM range, which decreased several fold in the presence of co-chaperone. However, ABA was found to have only variable- and co-chaperone-independent effects on the ATPase activity of these proteins. The potential implications of these ABA–HSP70 interactions are discussed with respect to the intracellular protein folding and extracellular receptor-like activities of these stress-inducible proteins. While mechanistic and functional relevance remain enigmatic, we conclude that ABA can bind to human HSP70 family members with physiologically relevant affinities and in a co-chaperone-dependent manner.
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  • 10
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    RETRACTION of: Down-regulation of protein tyrosine phosphatase gene expression in lactating mouse mammary gland (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
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  • 11
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    The vasohibin family: a novel family for angiogenesis regulation (2012)
    Oxford University Press
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    Publication Date: 2012-12-22
    Description: Angiogenesis, a formation of neovessels, is regulated by the local balance between angiogenesis stimulators and inhibitors. A number of such endogenous regulators of angiogenesis have been found in the body. Recently, vasohibin-1 (VASH1) was isolated as a negative feedback regulator of angiogenesis produced by endothelial cells (ECs) and subsequently vasohibin-2 (VASH2) as a homologue of VASH1. It was then explored that VASH1 is expressed in ECs to terminate angiogenesis, whereas VASH2 is expressed in cells other than ECs to promote angiogenesis in the mouse model of angiogenesis. This review will focus on the vasohibin family members, which are novel regulators of angiogenesis.
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  • 12
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    Activation of the Wnt/{beta}-catenin pathway and tissue inhibitor of metalloprotease 1 during tertiary dentinogenesis (2012)
    Oxford University Press
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    Publication Date: 2012-12-22
    Description: Tertiary dentin is deposited inside teeth after various stimuli and serves as a major defensive wall to preserve pulp cells. However, the molecular mechanisms of the activation of quiescent odontoblasts, immature pulp cells and tertiary dentin formation are still unclear. Therefore, we performed a comprehensive gene expression analysis of pulp cells after cavity preparation of 9-week-old rat molars to clarify the critical molecules in tertiary dentinogenesis. As a result, mRNA expression of various molecules was up- or down-regulated. Notably, several members of the matrix metalloprotease family and their endogenous inhibitors were up-regulated after cavity preparation. In situ hybridization showed that tissue inhibitor of metalloprotease 1 ( Timp1 ) was widely and continuously distributed in the pulp beneath the cavity in vivo . We also observed accumulation of β-catenin in the pulp cells beneath the cavity by fluorescence immunohistochemistry. Furthermore, Timp1 transcription was repressed by a dominant-negative TCF4 in immature undifferentiated mesenchymal cells, but not altered in mature odontoblast-like cells. These results indicate that cavity preparation may activate the Wnt/β-catenin pathway and the Wnt/β-catenin pathway and Timp1 may be correlatively involved in pulp repair. Timp1 might play crucial roles in reactivation of immature pulp cells for tertiary dentinogenesis.
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  • 13
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    Phosphoinositide 5-phosphatases: how do they affect tumourigenesis? (2012)
    Oxford University Press
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    Publication Date: 2012-12-22
    Description: The activity of biological molecules is often affected by their phosphorylation state. Regulatory phosphorylation operates as a binary switch and is usually controlled by counteracting kinases and phosphatases. However, phosphatidylinositol (PtdIns) has three phosphorylation sites on its inositol ring. The phosphorylation status of PtdIns is controlled by multiple kinases and phosphatases with distinct substrate specificities, serving as a ‘lipid code’ or ‘phosphoinositide code’. Class I phosphoinositide 3-kinase (PI3K) converts PtdIns(4,5)P 2 to PtdIns(3,4,5)P 3 , which plays a pivotal role in signals controlling glucose uptake, cytoskeletal reorganization, cell proliferation and apoptosis. PI3K is pro-oncogenic, whereas phosphoinositide phosphatases that degrade PtdIns(3,4,5)P 3 are not always anti-oncogenic. Recent studies have revealed the unique characteristics of phosphoinositide 5-phosphatases.
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  • 14
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    Vascular endothelial growth factor and its receptor system: physiological functions in angiogenesis and pathological roles in various diseases (2012)
    Oxford University Press
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    Publication Date: 2012-12-22
    Description: Vascular endothelial growth factors (VEGFs) belong to the platelet-derived growth factor supergene family, and they play central roles in the regulation of angiogenesis and lymphangiogenesis. VEGF-A, the major factor for angiogenesis, binds to two tyrosine kinase (TK) receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), and regulates endothelial cell proliferation, migration, vascular permeability, secretion and other endothelial functions. VEGFR-2 exhibits a strong TK activity towards pro-angiogenic signals, whereas the soluble VEGFR-1 (sFlt-1) functions as an endogenous VEGF inhibitor. sFlt-1 is abnormally overexpressed in the placenta of preeclampsia patients, resulting in the major symptoms of the disease due to abnormal trapping of VEGFs. The VEGF-VEGFR system is crucial for tumour angiogenesis, and anti-VEGF-VEGFR molecules are now widely used in the clinical field to treat cancer patients. The efficacy of these molecules in prolonging the overall survival of patients has been established; however, some cancers do not respond well and reduced tumour sensitivity to anti-VEGF signals may occur after long-term treatment. The molecular basis of tumour refractoriness should be determined to improve anti-angiogenic therapy.
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  • 15
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    Accurate determination of tissue steroid hormones, precursors and conjugates in adult male rat (2012)
    Oxford University Press
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    Publication Date: 2012-12-22
    Description: The actual levels of steroid hormones in organs are vital for endocrine, reproductive and neuronal health and disorders. We developed an accurate method to determine the levels of steroid hormones and steroid conjugates in various organs by an efficient preparation using a solid-phase-extraction cartridge. Each steroid was identified by the precursor ion spectra using liquid chromatography–electrospray ionization time-of-flight mass spectrometry, and the respective steroids were quantitatively analysed in the selected reaction monitoring mode by liquid chromatograph-mass spectrometry/mass spectrometry (LC-MS/MS). The data showed that significant levels of testosterone, corticosterone and precursors of both hormones were detected in all organs except liver. The glucuronide conjugates of steroid hormones and the precursors were detected in all organs except liver, but sulfate conjugates of these steroids were observed only in the target organs of the hormones and kidney. Interestingly, these steroids and the conjugates were not observed in the liver except pregnenolone. In conclusion, an accurate determination of tissue steroids was developed using LC-MS analysis. Biosynthesis of steroid hormones from the precursors was estimated even in the target organs, and the delivery of these steroid conjugates was also suggested via the circulation without any significant hepatic participation.
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  • 16
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    Enzymatic characterization of germination-specific cysteine protease-1 expressed transiently in cotyledons during the early phase of germination (2012)
    Oxford University Press
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    Publication Date: 2012-12-22
    Description: Papain-like cysteine protease activity that shows a unique transient expression profile in cotyledons of daikon radish during germination was detected. The enzyme showed a distinct elution pattern on DEAE-cellulose compared with cathepsin B-like and Responsive to dessication-21 cysteine protease. Although this activity was not detected in seed prior to imbibition, the activity increased markedly and reached a maximum at 2 days after imbibition and then decreased rapidly and completely disappeared after 5 days. Using cystatin-Sepharose, the 26 kDa cysteine protease (DRCP26) was isolated from cotyledons at 2 days after imbibition. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that DRCP26 is an orthologue of Arabidopsis unidentified protein, germination-specific cysteine protease-1, belonging to the C1 family of cysteine protease predicted from genetic information. In an effort to characterize the enzymatic properties of DRCP26, the enzyme was purified to homogeneity from cotyledons at 48 h after imbibition. The best synthetic substrate for the enzyme was carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide. All model peptides were digested to small peptides by the enzyme, suggesting that DRCP26 possesses broad cleavage specificity. These results indicated that DRCP26 plays a role in the mobilization of storage proteins in the early phase of seed germination.
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  • 17
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    Single-molecule imaging with a tagged ribosome to explore trans-translation (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Single-molecule imaging is a powerful technique to visualize molecular interactions and movements. Translation is one of the most interesting targets for researchers with the molecular-imaging skills, since mRNA, tRNA and translation factors interact with or move inside or on the ribosome in an ordered manner. Trans -translation is a bacterial quality control system to rescue the ribosomes stalled at the 3' end of the mRNA, and this phenomenon is recapitulated in vitro with defined factors including two trans -translation-specific entities tmRNA and SmpB. Zhou et al. (Single molecule imaging of the trans -translation entry process via anchoring of the tagged ribosome. J Biochem 2011;149:609-618.) successfully visualized the interaction of the tmRNA–SmpB complex with the ribosome by immobilizing the ribosome on the quartz surface with the HaloTag technology. This ribosome-anchoring system may be useful for the imaging analysis of other processes of translation.
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  • 18
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    The functional roles of S1P in immunity (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: The lipid mediator sphingosine-1-phosphate (S1P) is generated within cells from sphingosine by two sphingosine kinases (SPHK1 and SPHK2). Intracellularly synthesized S1P is released into the extracellular fluid by S1P transporters, including SPNS2. Released S1P binds specifically to the G protein-coupled S1P receptors (S1PR1/S1P 1 –S1PR5/S1P 5 ), which activate a diverse range of downstream signalling pathways. Recent studies have proposed that one of the central physiological functions of intercellular S1P signalling is in lymphocyte trafficking in vivo because genetic disruption of SPHK1/2, SPNS2 or S1PR1/S1P 1 in mice induces a lymphopenia phenotype. In this review, we discuss the current understanding of intercellular S1P signalling in the context of immunity.
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  • 19
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    Cdc6: a trifunctional AAA+ ATPase that plays a central role in controlling the G1-S transition and cell survival (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Cdc6 is the AAA+ ATPase that assembles prereplicative complexes on replication origins in eukaryotic chromosomes. Recently, the same Cdc6 protein was found to exert two more functions in mammalian cells to promote cell proliferation and survival: ATP-dependent activation of p21 CIP1 - or p27 KIP1 -bound Cdk2-cyclin A/E complexes and obstruction of apoptosome assembly and consequent cell death by forming stable complexes with activated Apaf-1 molecules. These findings not only redefined the biological role of mammalian Cdc6 but also led the discovery of entirely new mechanisms controlling Cdk2 activity and apoptosis. This review focuses on this multi-functional AAA+ ATPase and the newly discovered mechanisms by which it controls the G 1 –S transition and cell survival during proliferation.
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  • 20
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    Pseudomonas putida PydR, a RutR-like transcriptional regulator, represses the dihydropyrimidine dehydrogenase gene in the pyrimidine reductive catabolic pathway (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: The pyrimidine reductive catabolic pathway is important for the utilization of uracil and thymine as sources of nitrogen and carbon. The pathway is controlled by three enzymes: dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase and β-alanine synthase. The putative DPD genes, pydX and pydA , are tandemly arranged in the Pseudomonas putida genome. Intriguingly, a putative transcriptional regulator, PydR, homologous to Escherichia coli RutR, a repressor of the Rut-dependent pyrimidine degradation pathway, is located downstream of pydX and pydA . In this study, we show that a pydA strain of P. putida fails to grow on a minimal media containing uracil or thymine as a sole nitrogen source, demonstrating the physiological importance of DPD in the reductive pathway. The expression of pydA and DPD activity in the absence of uracil were significantly higher in a pydR strain than in the wild-type strain, indicating that PydR acts as a repressor of the pyrimidine reductive pathway in P. putida . Phylogenetic analysis of RutR and PydR suggests that these homologous repressors may have evolved from a common ancestral protein that regulates pyrimidine degradation.
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  • 21
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    Mediator lipidomics in acute inflammation and resolution (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Acute inflammation is an indispensable host response to foreign challenges or tissue injury. In healthy conditions, inflammatory processes are self-limiting and self-resolving, suggesting the existence of endogenous mechanisms for the control of inflammation and resolution. A comprehensive understanding of the cellular and molecular events of a well-orchestrated inflammatory response is required, and recent studies have uncovered the roles of endogenous lipid mediators derived from polyunsaturated fatty acids (i.e. lipoxins, resolvins, protectins) in controlling the resolution of inflammation. This review presents recent advances in understanding the formation and action of these mediators, especially focusing on the LC-MS/MS-based lipidomics approach and the emerging roles of eosinophils and eosinophil-derived lipid mediators in controlling acute inflammation and resolution.
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    Matrix control of transforming growth factor-{beta} function (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: The cytokine transforming growth factor-beta (TGF-β) has multiple effects in both physiological and pathological conditions. TGF-β is secreted as part of a tripartite complex from which it must be released in order to bind to its receptor. Sequestration of latent TGF-β in the extracellular matrix (ECM) is crucial for proper mobilization of the latent cytokine and its activation. However, contrary to expectation, loss-of-function mutations in genes encoding certain matrix proteins that bind TGF-β yield elevated, rather than decreased, TGF-β levels, posing a ‘TGF-β paradox.’ In this review, we discuss recent findings concerning the relationship of TGF-β, ECM molecules, and latent TGF-β activation and propose a model to resolve the ‘TGF-β paradox.’
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    Identification of the catalytic residues of sequence-specific and histidine-free ribonuclease colicin E5 (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Colicin E5 cleaves tRNAs for Tyr, His, Asn and Asp in their anticodons to abolish protein synthesis in Escherichia coli . We previously showed how its C-terminal RNase domain, E5-CRD, recognizes the anticodon bases but the catalytic mechanism remained to be elucidated. Although the reaction products with 5'-OH and 2',3'-cyclic phosphate ends suggested a similar mechanism to those of RNases A and T1, E5-CRD does not have the His residues necessary as a catalyst in usual RNases. To identify residues important for the catalytic reaction, mutants as to all residues within 5 Å from the central phosphorus of the scissile phosphodiester bond were prepared. Evaluation of the killing activities of the mutant colicins and the RNase activities of the mutant E5-CRDs suggested direct involvement of Arg33, Lys25, Gln29 and Lys60 in the reaction. Particularly, Arg33 plays a critical role and Ile94 provides a structural support of Arg33. Crystal structure of the complex of E5-CRD(R33Q)/dGpdUp showed structural and binding functional integrity of this mutant protein, suggesting involvement of Arg33 in the catalytic reaction. The structure of the free E5–CRD, we also determined, showed great flexibility of a flap region, which facilitates the access of Lys60 to the substrate in an induced-fit manner.
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    Sulfatide negatively regulates the fusion process of human parainfluenza virus type 3 (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Sulfatide (HSO 3 -3-galactosylceramide), which enriched in lipid rafts of plasma membranes in various epithelial cell lines, is a critical component of host cells for effective production of influenza A virus. However, the function of sulfatide in other virus infections targeting epithelial cells remains unknown. In this study, the effect of sulfatide on infection of human parainfluenza virus type 3 (hPIV3) was demonstrated by using genetically produced sulfatide-enriched cells and by treatment of hPIV3-infected cells with anti-sulfatide monoclonal antibody (GS-5) as well as by addition of sulfatide to the cells. hPIV3 was found to bind to sulfatide in a virus overlay assay and a solid-phase binding assay. Genetic expression of sulfatide in COS-7 cells defective in sulfatide suppressed initial hPIV3 infection and formation of multinucleate virus-infected cells. Treatment of virus-infected LLC-MK2 cells with GS-5 promoted formation of multinucleate cells. In contrast, exogenous addition of sulfatide to hPIV3-infected COS-7 cells and cells expressing the hPIV3- hemagglutinin-neuraminidase ( HN ) gene and fusion ( F ) gene conspicuously reduced the formation of multinucleate cells. The results suggest that sulfatide negatively regulates the fusion process of hPIV3, possibly through interaction with HN or F glycoprotein on the cell surface.
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    Annexin A3 as a negative regulator of adipocyte differentiation (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Annexin A3 is a protein belonging to the annexin family, and it is mainly present in cellular membranes as a phospholipid-binding protein that binds via the calcium ion. However, its physiological function remains to be clarified. We examined the expression of annexin A3 in mouse tissues and found for the first time that annexin A3 mRNA and its protein were expressed more strongly in adipose tissues than in other tissues. In adipose tissues, annexin A3-expressing cells were present in the stromal vascular fraction, and precisely identical to Pref-1-positive preadipocytes, Pref-1 being an epidermal growth factor repeat-containing transmembrane protein that inhibits adipogenesis. In 3T3-L1 cells, used as a model of adipogenesis, annexin A3 was down-regulated at an early phase of adipocyte differentiation, and this pattern paralleled that of Pref-1. Suppression of annexin A3 in these cells with siRNA caused elevation of the PPAR2 mRNA level and lipid droplet accumulation. In conclusion, our data suggest that annexin A3 is a negative regulator of adipocyte differentiation.
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    PKL01, an Ndr kinase homologue in plant, shows tyrosine kinase activity (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Protein phosphorylation by protein tyrosine (Tyr) kinases plays important roles in a variety of signalling pathways in cell growth, differentiation and oncogenesis in animals. Despite the absence of classical Tyr kinases in plants, a similar ratio of phosphotyrosine residues in phosphorylated proteins was found in Arabidopsis thaliana as in human. However, protein kinases responsible for tyrosine phosphorylation in plants except some dedicated dual-specificity kinases still remain unclear. In this study, we found that PKL01, a nuclear Dbf2-related (Ndr) kinase homologue in Lotus japonicus , was autophosphorylated at a tyrosine residue when it was expressed in Escherichia coli , but kinase-dead mutant of PKL01 was not. Tyrosine phophorylation site in PKL01 was identified as Tyr-56 by LC-MS/MS analysis. Recombinant PKL01, which had been dephosphorylated by an alkaline phosphatase, could be phosphorylated again at the Tyr residue when it was incubated with ATP. Furthermore, other Ndr kinases in plants and PKL01 phosphorylated on Tyr residues in the exogenous substrates such as poly(Glu, Tyr) 4:1 and casein. Therefore, the Ndr kinases in plants, which had been assumed as protein serine (Ser)/threonine (Thr) kinases, turned out to be dual-specificity kinases responsible for phosphorylation of Tyr residues and Ser/Thr residues in plant proteins.
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    The critical role of amino acid residue at position 117 of mouse UDP-glucuronosyltransfererase 1a6a and 1a6b in resveratrol glucuronidation (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Mouse UDP-glucuronosyltransferase 1a6 (Ugt1a6) contains two functional copies of 1a6a and 1a6b that share high sequence homology (98%). Only 10 amino acids located around the substrate recognition region are different out of 531 total residues. Although Ugt1a6 plays important roles in conjugating phenolic compounds, the functional characteristics of these isozymes are unclear. We performed functional analyses of mouse Ugt1a6a and Ugt1a6b using two isomeric polyphenols ( trans - and cis -resveratrol). The cDNAs of mouse Ugt1a6a and Ugt1a6b were cloned and constructed as recombinant proteins using a yeast expression system, and kinetic parameters were evaluated. The wild-type Ugt1a6a and Ugt1a6b proteins catalysed trans - and cis -resveratrol 3- O -glucuronidation. Although the K m value for trans -resveratrol was significantly lower for Ugt1a6a compared with Ugt1a6b, the K m values for cis -resveratrol were comparable for the isozymes. Despite high sequence homology, significant kinetic differences were observed between the isozymes. To identify the critical residues for resveratrol glucuronidation, we constructed 10 variants of Ugt1a6a (T81P, N96R, H98Q, L100V, S104P, N115S, I117L, V118T, V119L and D120E). The I117L variant had Ugt1a6b-like enzymatic properties of K m in trans -resveratrol, and V max and K si in cis -form, suggesting that the residues located at position 117 of Ugt1a6a and Ugt1a6b play an important role in resveratrol glucuronidation.
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    Role of PKC-{alpha} in NF-{kappa}B-MT1-MMP-mediated activation of proMMP-2 by TNF-{alpha} in pulmonary artery smooth muscle cells (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: We sought to evaluate the mechanism(s) associated with pro matrix metalloprotease 2 (proMMP-2) activation in bovine pulmonary artery smooth muscle cells. Preincubation of cells with anti-TNFR1 antibody prevented tumour necrosis factor-α (TNF-α)-induced proMMP-2 activation and increase in membrane type 1 matrix metalloprotease (MT1-MMP) expression as well as inhibition of tissue inhibitor of metalloproteinase 2 (TIMP-2) expression, indicating the role of TNFR1 receptor during TNF-α stimulation. Anti-MT1-MMP antibody abrogated proMMP-2 activation by TNF-α-stimulated cell membrane, suggesting the involvement of MT1-MMP in proMMP-2 activation. Induction of MT1-MMP expression in response to TNF-α occurs via activation of nuclear factor (NF)-B on inhibitory B kinase (IKK) activation and subsequently phosphorylation/degradation of IB-α. Inhibition of protein kinase C (PKC)-α activity by Go6976 and PKC-α siRNA prevented TNF-α-induced IKK activity, IB-α phosphorylation/degradation and NF-B activation. Inhibition of PKC-α activity also prevented TNF-α-induced MT1-MMP expression and proMMP-2 activation as well as down regulation of TIMP-2 expression. Inhibition of IB-α phosphorylation by PS-1145, an IKK selective inhibitor, prevented TNF-α-induced increase in MT1-MMP expression and proMMP-2 activation, which although did not alter inhibition of TIMP-2 expression. Overall, we unravelled a hitherto unknown mechanism of the involvement of PKC-α in proMMP-2 activation and inhibition of TIMP-2 expression by NF-B–MT1-MMP–dependent and –independent pathway, respectively, during TNF-α stimulation in pulmonary artery smooth muscle cells.
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    Tumour endothelial cells acquire drug resistance in a tumour microenvironment (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: Tumour growth is dependent on angiogenesis, and tumour blood vessels are recognized as an important target for cancer therapy. Tumour endothelial cells (TECs) are the main targets of anti-angiogenic therapy. Unlike the traditionally held view, some TECs may be genetically abnormal and might acquire drug resistance. Therefore, we investigated the drug resistance of TECs and the mechanism by which it is acquired. TECs show resistance to paclitaxel through greater mRNA expression of multidrug resistance 1, which encodes P-glycoprotein, as compared with normal endothelial cells. We found that high levels of vascular endothelial growth factor in tumour-conditioned medium may be responsible for upregulated P-glycoprotein expression. This is a novel mechanism for the acquisition of drug resistance by TECs in a tumour microenvironment. This review focuses on the possibility that TECs can acquire drug resistance.
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    Studies of inositol 1-phosphate analogues as inhibitors of the phosphatidylinositol phosphate synthase in mycobacteria (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: We previously reported a novel pathway for the biosynthesis of phosphatidylinositol in mycobacteria via phosphatidylinositol phosphate (PIP) [Morii H., Ogawa, M., Fukuda, K., Taniguchi, H., and Koga, Y (2010) J. Biochem . 148, 593–602]. PIP synthase in the pathway is a promising target for the development of new anti-mycobacterium drugs. In the present study, we evaluated the characteristics of the PIP synthase of Mycobacterium tuberculosis . Four types of compounds were chemically synthesized based on the assumption that structural homologues of inositol 1-phosphate, a PIP synthase substrate, would act as PIP synthase inhibitors, and the results confirmed that all synthesized compounds inhibited PIP synthase activity. The phosphonate analogue of inositol 1-phosphate (Ino-C-P) had the greatest inhibitory effect among the synthesized compounds examined. Kinetic analysis indicated that Ino-C-P acted as a competitive inhibitor of inositol 1-phosphate. The IC 50 value for Ino-C-P inhibition of the PIP synthase activity was estimated to be 2.0 mM. Interestingly, Ino-C-P was utilized in the same manner as the normal PIP synthase substrate, leading to the synthesis of a phosphonate analogue of PIP (PI-C-P), which had a structure similar to that of the natural product, PIP. In addition, PI-C-P had high inhibitory activity against PIP synthase.
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    Transforming growth factor-{beta}1 induces epithelial-mesenchymal transition and integrin {alpha}3{beta}1-mediated cell migration of HSC-4 human squamous cell carcinoma cells through Slug (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: We investigated whether transforming growth factor (TGF)-β1 promoted epithelial–mesenchymal transition (EMT) and migration of human oral squamous cell carcinoma (hOSCC) cells. Among 6 hOSCC cell lines investigated, Smad2 phosphorylation and TGF-β target genes expression were most clearly upregulated following TGF-β1 stimulation in HSC-4 cells, indicating that HSC-4 cells were the most responsive to TGF-β1. In addition, the expression levels of the mesenchymal markers N-cadherin and vimentin were most clearly induced in HSC-4 cells among the hOSCC cell lines by TGF-β1 stimulation. Interestingly, E-cadherin and β-catenin at the cell surface were internalized in HSC-4 cells stimulated with TGF-β1. In addition, the expression levels of the EMT-related transcription factor Slug was significantly upregulated on TGF-β1 stimulation. Moreover, the downregulation of Slug by RNA interference clearly inhibited the TGF-β1-induced expression of mesenchymal marker and the migration of HSC-4 cells. Proteomics analysis also revealed that the expression levels of integrin α3β1-targeted proteins were upregulated in TGF-β1-stimulated HSC-4 cells. Neutral antibodies against integrin α3 and β1, as well as a focal adhesion kinase (FAK) inhibitor, clearly suppressed TGF-β1-induced cell migration. These results suggest that the EMT and integrin α3β1/FAK pathway–mediated migration of TGF-β1-stimulated HSC-4 hOSCC cells is positively controlled by Slug.
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    All members of the EPI64 subfamily of TBC/RabGAPs also have GAP activities towards Ras (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: The importance of interconnective signalling networks between distinct GTPases and their regulators is being recognized. EPI64C/TBC1D10C/carabin, a haematopoietically enriched GTPase-activating protein (GAP) for Rab35, has been shown to exhibit RasGAP activity. Owing to the diverged Rab specificities among the EPI64 members (EPI64A–C) and the relatively weak sequence conservation between EPI64A/B and EPI64C in their catalytic TBC domains, it is difficult to predict whether EPI64A and B will also have RasGAP activities. Therefore, in this study, we examined the RasGAP activities of all three EPI64 subfamily members. We found that EPI64A–C exhibited in vivo GAP activities towards Ras using three independent methods, spectrofluorometry with Förster resonance energy transfer (FRET) sensors, the Bos' pull-down assay and time-lapse FRET imaging. EPI64A and B were predominantly localized at the periphery of COS-7 cells. In COS-7 cells, confocal FRET imaging showed that H-Ras activity was higher at the Golgi than at the plasma membrane. Thus, we propose that EPI64A and B, which are ubiquitously expressed members of the EPI64 subfamily, inactivate Ras and certain Rabs at the periphery of cells.
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    Granting specificity for breast cancer cells using a hepatitis B core particle with a HER2-targeted affibody molecule (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: Capsid-like particles consisting of a hepatitis B core (HBc) protein have been studied for their potential as carriers for drug delivery systems (DDS). The hollow HBc particle, which is formed by the self-assembly of core proteins comprising 183 aa residues, has the ability to bind to various cells non-specifically via the action of an arginine-rich domain. In this study, we developed an engineered HBc particle that specifically recognizes and targets human epidermal growth factor receptor-related 2 (HER2)-expressing breast cancer cells. To despoil the non-specific binding property of an HBc particle, we genetically deleted the C-terminal 150–183 aa part of the core protein that encodes the arginine-rich domain (HBc). Then, we genetically inserted a Z HER2 affibody molecule into the 78–81 aa position of the core protein to confer the ability of target-cell-specific recognition. The constructed Z HER2 -displaying HBc (Z HER2 -HBc) particle specifically recognized HER2-expressing SKBR3 and MCF-7 breast cancer cells. In addition, the Z HER2 -HBc particle exhibited different binding amounts in accordance with the HER2 expression levels of cancer cells. These results show that the display of other types of affibody molecules on HBc particles would be an expandable strategy for targeting several kinds of cancer cells that would help enable a pinpoint DDS.
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    Preparation and characterization of a monoclonal antibody against the refolded and functional extracellular domain of rat P2X4 receptor (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: The P2X4 purinergic receptor is a key molecule in neuropathic pain, particularly in the allodynia after peripheral nerve injury. We therefore sought to establish an anti-P2X4 receptor monoclonal antibody that would be useful for detection and characterization of the P2X4 receptor. We first prepared the refolded extracellular domain of the rat P2X4 receptor expressed in Escherichia coli . Then, we established a B-cell hybridoma producing the monoclonal antibody for the head domain of the rat P2X4 receptor with strict recognition, including S-S bond formation. In addition, we succeeded in the detection and immune precipitation of rat P2X4 receptor molecules on cultured cells. As the antibody specifically binds to the rat P2X4 receptor molecule, it is expected that the established monoclonal antibody will be applicable as a tool for detecting increasing expression levels of the P2X4 receptor molecule accompanied with increasing intensity of neuropathic pain.
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    A simple system for expression of proteins containing 3-azidotyrosine at a pre-determined site in Escherichia coli (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: We developed an efficient method for introduction of 3-azidotyrosine (N 3 -Y) into proteins in Escherichia coli cells. We constructed a plasmid that is adaptable for the constitutive expression of both Methanosarcina acetivorans tyrosyl-tRNA synthetase (TyrRS) and tRNA ( CUA) , and made an orthogonal tRNA (CUA) that is recognized as a substrate only by the archaeal TyrRS. Random mutations were introduced into M. acetivorans TyrRS around the tyrosine binding pocket, and a TyrRS mutant recognizing N 3 -Y was selected. We then expressed rat calmodulin (CaM) containing N 3 -Y, using the CaM gene with an amber codon at position 80. Mass analyses confirmed production of CaM containing N 3 -Y, but a significant amount of CaM containing 3-aminotyrosine was also detected. To more efficiently express CaM containing N 3 -Y, we added an arabinose-inducible gene for the mutant TyrRS to the plasmid carrying the mutant TyrRS/tRNA ( CUA) gene. Although the yields of full-length CaM increased ~ 3-fold, the ratio of N 3 -Y introduction was not significantly improved. Following screening for a suitable host cell, we found that CaM expressed in E. coli SHuffle (K-12) had 97% N 3 -Y at the pre-determined site. Finally, we obtained up to 2 mg of CaM containing N 3 -Y per 100 ml of culture media, sufficient for use in various proteomics experiments, including photo-crosslinking.
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    Pharmacophore identification of ascofuranone, potent inhibitor of cyanide-insensitive alternative oxidase of Trypanosoma brucei (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: Trypanosoma brucei is a parasite that causes human African trypanosomiasis (HAT). The parasites depend on the cyanide-insensitive trypanosome alternative oxidase (TAO) for their vital aerobic respiration. Ascofuranone (AF), a potent and specific sub-nanomolar inhibitor of the TAO quinol oxidase, is a potential novel drug with selectivity for HAT, because mammalian hosts lack the enzyme. To elucidate not only the inhibition mechanism but also the inhibitor–enzyme interaction, AF derivatives were designed and synthesized, and the structure–activity relationship was evaluated. Here we identified the pharmacophore of AF that interacts with TAO. The detailed inhibitory profiles indicated that the 1-formyl and 6-hydroxyl groups, which might contribute to intramolecular hydrogen bonding and/or serve as hydrogen-bonding donors, were responsible for direct interaction with the enzyme.
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    Sulfation of ractopamine and salbutamol by the human cytosolic sulfotransferases (2012)
    Oxford University Press
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    Publication Date: 2012-08-29
    Description: Feed additives such as ractopamine and salbutamol are pharmacologically active compounds, acting primarily as β-adrenergic agonists. This study was designed to investigate whether the sulfation of ractopamine and salbutamol may occur under the metabolic conditions and to identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating two major feed additive compounds, ractopamine and salbutamol. A metabolic labelling study showed the generation and release of [ 35 S]sulfated ractopamine and salbutamol by HepG2 human hepatoma cells labelled with [ 35 S]sulfate in the presence of these two compounds. A systematic analysis using 11 purified human SULTs revealed SULT1A3 as the major SULT responsible for the sulfation of ractopamine and salbutamol. The pH dependence and kinetic parameters were analyzed. Moreover, the inhibitory effects of ractopamine and salbutamol on SULT1A3-mediated dopamine sulfation were investigated. Cytosol or S9 fractions of human lung, liver, kidney and small intestine were examined to verify the presence of ractopamine-/salbutamol-sulfating activity in vivo . Of the four human organs, the small intestine displayed the highest activity towards both compounds. Collectively, these results imply that the sulfation mediated by SULT1A3 may play an important role in the metabolism and detoxification of ractopamine and salbutamol.
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    C-H ... {pi} interplay between Ile308 and Tyr310 residues in the third repeat of microtubule binding domain is indispensable for self-assembly of three- and four-repeat tau (2012)
    Oxford University Press
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    Publication Date: 2012-08-29
    Description: Information on the structural scaffold for tau aggregation is important in developing a method of preventing Alzheimer’s disease (AD). Tau contains a microtubule binding domain (MBD) consisting of three or four repeats of 31 and 32 similar residues in its C-terminal half. Although the key event in tau aggregation has been considered to be the formation of β-sheet structures from a short hexapeptide 306 VQIVYK 311 in the third repeat of MBD, its aggregation pathway to filament formation differs between the three- and four-repeated MBDs, owing to the intermolecular and intramolecular disulphide bond formations, respectively. Therefore, the elucidation of a common structural element necessary for the self-assembly of three-/four-repeated full-length tau is an important research subject. Expanding the previous results on the aggregation mechanism of MBD, in this paper, we report that the C–H ... interaction between the Ile308 and Tyr310 side chains in the third repeat of MBD is indispensable for the self-assembly of three-/four-repeated full-length tau, where the interaction provides a conformational seed for triggering the molecular association. On the basis of the aggregation behaviours of a series of MBD and full-length tau mutants, a possible self-association model of tau is proposed and the relationship between the aggregation form (filament or granule) and the association pathway is discussed.
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    Roles of transcriptional network during the formation of lymphatic vessels (2012)
    Oxford University Press
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    Publication Date: 2012-08-29
    Description: The lymphatic vascular system, also known as the second vascular system in vertebrates, plays crucial roles in various physiological and pathological processes. It participates in the maintenance of normal tissue fluid balance, trafficking of the immune cells and absorption of fatty acids in the gut. Furthermore, lymphatic system is associated with the pathogenesis of a number of diseases, including lymphedema, inflammatory diseases and tumour metastasis. Lymphatic vessels are comprised of lymphatic endothelial cells (LECs), which are differentiated from blood vascular endothelial cells. This review highlights recent advances in our understanding of the transcriptional control of LEC fate determination and reflects on efforts to understand the roles of transcriptional networks during this discrete developmental process.
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    Mediator CDK subunits are platforms for interactions with various chromatin regulatory complexes (2012)
    Oxford University Press
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    Publication Date: 2012-08-29
    Description: The Mediator complex consists of more than 20 subunits. This is composed of four modules: head, middle, tail and CDK/Cyclin. Importantly, Mediator complex is known to play pivotal roles in transcriptional regulation, but its molecular mechanisms are still elusive. Many studies, including our own, have revealed that CDK8, a kinase subunit of the CDK/Cyclin module, is one of the key subunits involved in these roles. Additionally, we previously demonstrated that a novel CDK component, CDK19, played similar roles. It is assumed that various factors that directly affect transcriptional regulation target these two CDKs; thus, we conducted yeast two-hybrid screenings to isolate the CDK19-interacting proteins. From a screening of 40 million colonies, we obtained 287 clones that provided positive results encoded mRNAs, and it turned out that 59 clones of them encoded nuclear proteins. We checked the reading frames of the candidate clones and obtained three positive clones, all of which encoded the transcriptional cofactors, Brahma-related gene 1, B-cell CLL/lymphoma 6 and suppressor of zeste 12 homolog. Intriguingly, these three cofactors are also related to chromatin regulation. Further studies demonstrated that those could bind not only to CDK19 but also to CDK8. These results help elucidate the functional mechanism for the mutual regulations between transcription and chromatin.
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    Binding of von Willebrand factor cleaving protease ADAMTS13 to Lys-plasmin(ogen) (2012)
    Oxford University Press
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    Publication Date: 2012-08-29
    Description: The metalloprotease ADAMTS13 affects platelet adhesion and aggregation through depolymerization of von Willebrand factor (VWF) multimers. Identification of ADAMTS13-binding proteins would reveal the hitherto unrecognized mechanisms underlying microvascular thrombus. To identify ADAMTS13-binding proteins, we performed a yeast two-hybrid screen using the Cys-rich and spacer domains of ADAMTS13, the critical regions for the binding and cleavage of VWF, as a bait region. We identified Lys-plasminogen, an amino-terminal truncated form of plasminogen, as the binding protein to ADAMTS13. Intact Glu-plasminogen did not bind to ADAMTS13. Active-site blocked Lys-plasmin bound to ADAMTS13. Domain truncation of ADAMTS13 and elastase digest of plasminogen indicated that the Cys-rich and spacer domains of ADAMTS13 and the kringle 5 and protease domains of plasminogen served as the main binding sites. Biacore measurements revealed that Lys-plasminogen bound to ADAMTS13 with a K d of 1.9 ± 0.1 x 10 –7 M and Glu-plasminogen exhibited a significantly lower affinity to ADAMTS13. Specific activity measurements revealed that ADAMTS13 and Lys-plasmin were still active even after the binary complex was formed. The binding of ADAMTS13 to Lys-plasminogen may play an important role to localize these two proteases at sites of thrombus formation or vascular injury where the fibrinolytic system is activated.
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    Critical roles of the mitochondrial complex II in oocyst formation of rodent malaria parasite Plasmodium berghei (2012)
    Oxford University Press
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    Publication Date: 2012-08-29
    Description: It is generally accepted that the mitochondria play central roles in energy production of most eukaryotes. In contrast, it has been thought that Plasmodium spp., the causative agent of malaria, rely mainly on cytosolic glycolysis but not mitochondrial oxidative phosphorylation for energy production during blood stages. However, Plasmodium spp. possesses all genes necessary for the tricarboxylic acid (TCA) cycle and most of the genes for electron transport chain (ETC) enzymes. Therefore, it remains elusive whether oxidative phosphorylation is essential for the parasite survival. To elucidate the role of TCA metabolism and ETC in malaria parasites, we deleted the gene for flavoprotein (Fp) subunit, Pbsdha , one of four components of complex II, a catalytic subunit for succinate dehydrogenase activity. The Pbsdha(-) parasite grew normally at blood stages in mouse. In contrast, ookinete formation of Pbsdha(-) parasites in the mosquito stage was severely impaired. Finally, Pbsdha(-) ookinetes failed in oocyst formation, leading to complete malaria transmission blockade. These results suggest that malaria parasite may switch the energy metabolism from glycolysis to oxidative phosphorylation to adapt to the insect vector where glucose is not readily available for ATP production.
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    Exploration of a new drug that targets YAP (2012)
    Oxford University Press
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    Publication Date: 2012-08-29
    Description: Yes-associated protein (YAP) has been shown to play a critical role in the growth of various tumours. Phosphorylation of Ser127 of YAP leads to the inhibition of YAP translocation into nucleus and subsequent failure to regulate the expression of target genes that induce cell proliferation. Chemical manipulation of YAP localization or expression may provide an efficient method for cancer treatment. In a recent work published by Bao et al. ( J. Biochem . 2011;150:199–208), various compounds were screened in human osteosarcoma cells that stably express Green Fluorescent Protein-labeled YAP by monitoring subcellular localization of GFP-YAP. Using this cell-based assay, they found that dobutamine, a β-adrenergic receptor agonist, attenuated YAP-dependent transcription by inhibiting its nuclear translocation. The authors suggest dobutamine as a possible drug for cancer treatment.
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    Effects of site-directed mutagenesis of Asn116 in the {beta}-hairpin of the N-terminal domain of thermolysin on its activity and stability (2012)
    Oxford University Press
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    Publication Date: 2012-08-29
    Description: In the N-terminal domain of thermolysin, two anti-parallel β-strands, Asn112–Ala113–Phe114–Trp115 and Ser118–Gln119–Met120–Val121–Tyr122 are connected by an Asn116–Gly117 turn to form a β-hairpin structure. In this study, we examined the role of Asn116 in the activity and stability of thermolysin by site-directed mutagenesis. Of the 19 Asn116 variants, four (N116A, N116D, N116T and N116Q) were produced in Escherichia coli , by co-expressing the mature and pro domains separately, while the other 15 were not. In the hydrolysis of N -[3-(2-furyl)acryloyl]-glycyl- l -leucine amide (FAGLA) at 25°C, the intrinsic k cat / K m value of N116D was 320% of that of the wild-type thermolysin (WT), and in the hydrolysis of N -carbobenzoxy- l -aspartyl- l -phenylalanine methyl ester (ZDFM) at pH 7.5 at 25°C, the k cat / K m value of N116D was 140% of that of WT, indicating that N116D exhibited higher activity than WT. N116Q exhibited similar activity as WT, and N116A and N116T exhibited reduced activities. The first-order rate constants, k obs , of the thermal inactivation at 80°C were in the order N116A, N116D, N116T 〉 N116Q 〉 WT at all CaCl 2 concentrations examined (1–100 mM), indicating that all variants exhibited reduced stabilities. These results suggest that Asn116 plays an important role in the activity and stability of thermolysin presumably by stabilizing this β-hairpin structure.
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    Function analysis of conserved amino acid residues in a Mn2+-dependent protein phosphatase, Pph3, from Myxococcus xanthus (2012)
    Oxford University Press
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    Publication Date: 2012-08-29
    Description: The Myxococcus xanthus protein phosphatase Pph3 belongs to the Mg 2+ - or Mn 2+ -dependent protein phosphatase (PPM) family. Bacterial PPMs contain three divalent metal ions and a flap subdomain. Putative metal- or phosphate-ion binding site-specific mutations drastically reduced enzymatic activity. Pph3 contains a cyclic nucleotide monophosphate (cNMP)-binding domain in the C-terminal region, and it requires 2-mercaptoethanol for phosphatase activity; however, the C-terminal deletion mutant showed high activity in the absence of 2-mercaptoethanol. The phosphatase activity of the wild-type enzyme was higher in the presence of cAMP than in the absence of cAMP, whereas a triple mutant of the cNMP-binding domain showed slightly lower activities than those of wild-type, without addition of cAMP. In addition, mutational disruption of a disulphide bond in the wild-type enzyme increased the phosphatase activity in the absence of 2-mercaptoethanol, but not in the C-terminal deletion mutant. These results suggested that the presence of the C-terminal region may lead to the formation of the disulphide bond in the catalytic domain, and that disulphide bond cleavage of Pph3 by 2-mercaptoethanol may occur more easily with cAMP bound than with no cAMP bound.
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    Vitamin D3 enhances ATRA-mediated neurosteroid biosynthesis in human glioma GI-1 cells (2012)
    Oxford University Press
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    Publication Date: 2012-08-29
    Description: Emerging evidence indicates that vitamin D (VD) is an important modulator of brain development and function. To investigate whether VD modulates neurosteroid biosynthesis in neural cells, we investigated the effect of VD 3 on steroidogenic gene expression in human glioma GI-1 cells. We found that VD 3 enhanced CYP11A1 and 3β-hydroxysteroid dehydrogenase gene expression. The induction of CYP11A1 gene expression by VD 3 was dose- and incubation time-dependent. Calcipotriol, a VD 3 receptor (VDR) agonist, also induced CYP11A1 gene expression in GI-1 cells, indicating that VDR is involved in this induction. The induction of progesterone (PROG) de novo synthesis was observed along with the induction of steroidogenic genes by VD 3 . Furthermore, VD 3 enhanced all- trans retinoic acid (ATRA)-induced CYP11A1 gene expression and PROG production. This suggests cooperative regulation of steroidogenic gene expression by the two fat-soluble vitamins, A and D. In addition, a mixed culture of neuronal IMR-32 cells and GI-1 cells treated with ATRA and VD 3 resulted in the induction of PROG-responsive gene expression in the IMR-32 cells. This result shows a paracrine action of PROG that is induced in and released by the GI-1 cells. The relationship between neurological dysfunction associated with VD deficiency and neurosteroid induction by VD is discussed.
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    Reactive oxygen species production and activation mechanism of the rice NADPH oxidase OsRbohB (2012)
    Oxford University Press
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    Publication Date: 2012-07-05
    Description: Reactive oxygen species (ROS) produced by plant NADPH oxidases (NOXes) are important in plant innate immunity. The Oryza sativa respiratory burst oxidase homologue B ( OsRbohB ) gene encodes a NOX the regulatory mechanisms of which are largely unknown. Here, we used a heterologous expression system to demonstrate that OsRbohB shows ROS-producing activity. Treatment with ionomycin, a Ca 2+ ionophore, and calyculin A, a protein phosphatase inhibitor, activated ROS-producing activity; it was thus OsRbohB activated by both Ca 2+ and protein phosphorylation. Mutation analyses revealed that not only the first EF-hand motif but also the upstream amino-terminal region were necessary for Ca 2+ -dependent activation, while these regions are not required for phosphorylation-induced ROS production.
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    Expression of RASSF6 in kidney and the implication of RASSF6 and the Hippo pathway in the sorbitol-induced apoptosis in renal proximal tubular epithelial cells (2012)
    Oxford University Press
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    Publication Date: 2012-07-05
    Description: RASSF6, a member of RASSF tumour suppressor proteins, binds to mammalian Ste20-like kinases (MST1/2), core kinases of the proapoptotic Hippo pathway and cooperates with the Hippo pathway to induce apoptosis. We originally identified RASSF6 as a putative interactor of membrane-associated guanylate kinase inverted (MAGI)-1 by the yeast two-hybrid screening. We used human kidney cDNA library for the screening. MAGI-1 is abundantly expressed in kidney and is a core component of the slit diaphragm. These findings suggest that RASSF6 is expressed in kidney. However, the function of RASSF6 in kidney is not yet studied. We performed this study to confirm the interaction of RASSF6 with MAGI-1, to analyse the expression of RASSF6 in kidney and to gain insight into the function of RASSF6 in kidney. RASSF6 binds to PDZ domains of MAGI-1 through its C-terminal PDZ-binding motif and is coimmunoprecipitated with MAGI-1 from rat liver. RASSF6 is localized in normal kidney glomerulus but disappears when the slit diaphragm is disrupted in nephrotic kidney. RASSF6 is also localized on apical membranes in renal proximal tubular epithelial cells. We demonstrated that RASSF6 as well as the Hippo pathway are involved in the sorbitol-induced apoptosis in immortalized human proximal renal tubular epithelial HK-2 cells.
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    A half-site of the p53-binding site on the keratin 14 promoter is specifically activated by p63 (2012)
    Oxford University Press
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    Publication Date: 2012-07-05
    Description: Three members of p53 family, p53, p63 and p73, can transactivate their specific target genes through a p53 consensus sequence-binding motif which consists with direct repeats of PuPuPuC(T/A)(T/A)GPyPyPy as a whole-site of p53-binding site. p63, an epidermal stem cells marker, can regulate epidermal development and differentiation, but p53 has no similar biological activity. One isoform of p63, TAp63α, can active an epidermal basal cell marker, keratin 14. However, the p53-binding site does not exist as a whole-site in the K14 promoter region, although it contains three putative p53 half-binding sites at –269 to –1 of the K14 promoter. Two of three putative half-sites of the p53-binding site can be bound by p63α by electrophoresis mobility shift assay and DNA affinity purification assay. Only mutation of the p53 half-binding site at –140 to –131, the TAp63α induced K14 promoter activity can be abolished. This half-site was specifically activated by p63, but not by p53. Once we extend this p53 half-site to a whole p53-binding site in K14 promoter, both p53 and p63 expression vectors can activate its activity. Therefore, we propose that the different length of p53-binding site would determinate the gene regulated by different p53 family proteins.
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    Liver autophagy: physiology and pathology (2012)
    Oxford University Press
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    Publication Date: 2012-07-05
    Description: Autophagy has long been thought of as a bulk degradation system in which cytoplasmic components are sequestered by double-membrane structures called autophagosomes, and the contents are then degraded after autophagosomes fuse with lysosomes. Genetic experiments in yeast identified a set of Autophagy-related (ATG) genes that are essential for autophagy. We have since elucidated many of the molecular underpinnings of autophagy and the physiologic roles of these processes in various systems. This review summarizes the physiologic roles of autophagy with a particular focus on liver autophagy based on analyses of knockout mice lacking Atg genes.
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    Roles of Dppa2 in the regulation of the present status and future of pluripotent stem cells (2012)
    Oxford University Press
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    Publication Date: 2012-07-05
    Description: Maintenance of undifferentiated states of pluripotent embryonic stem cells is regulated by a complex network of transcription factors and signaling pathways. Recent reports revealed that developmental pluripotency-associated 2 (Dppa2), which regulates chromatin structures, plays important roles in the maintenance of pluripotency and proliferation of embryonic stem cells. Interestingly, developmental pluripotency-associated 2 is involved not only in the normal development of lung but also in the pathogenesis of non-small cell lung cancers. These results suggest that an epigenetic regulator of pluripotent stem cells plays important roles in normal development and tumorigenesis.
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    2'-O,4'-C-ethylene bridged nucleic acid modification enhances pyrimidine motif triplex-forming ability under physiological condition (2012)
    Oxford University Press
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    Publication Date: 2012-07-05
    Description: Since pyrimidine motif triplex DNA is unstable at physiological neutral pH, triplex stabilization at physiological neutral pH is important for improvement of its potential to be applied to various methods in vivo , such as repression of gene expression, mapping of genomic DNA and gene-targeted mutagenesis. For this purpose, we studied the thermodynamic and kinetic effects of a chemical modification, 2'- O ,4'- C -ethylene bridged nucleic acid (ENA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological neutral pH. Thermodynamic investigations indicated that the modification achieved more than 10-fold increase in the binding constant of the triplex formation. The increased number of the modification in TFO enhanced the increased magnitude of the binding constant. On the basis of the obtained thermodynamic parameters, we suggested that the remarkably increased binding constant by the modification may result from the increased stiffness of TFO in the unbound state. Kinetic studies showed that the considerably decreased dissociation rate constant resulted in the observed increased binding constant by the modification. We conclude that ENA modification of TFO could be a useful chemical modification to promote the triplex formation under physiological neutral condition, and may advance various triplex formation-based methods in vivo.
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    Proinsulin C-peptide activates {alpha}-enolase: implications for C-peptide-cell membrane interaction (2012)
    Oxford University Press
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    Publication Date: 2012-07-05
    Description: Proinsulin C-peptide shows beneficial effects on microvascular complications of Type 1 diabetes. However, the possible occurrence of membrane C-peptide receptor(s) has not been elucidated. The aim of this study was to identify and characterize membrane proteins to which C-peptide binds. The enzyme α-enolase was co-immunoprecipitated with C-peptide after chemical cross-linking to HL-60 cell surface proteins and identified by mass spectrometry. Recombinant α-enolase activity was modulated by C-peptide, with a significant decrease in K m for 2-phosphoglycerate without affecting V max . The enzyme modulation by C-peptide was abolished when C-terminal basic lysine residue (K434) of the enzyme was replaced by neutral alanine or acidic glutamate, but not with basic arginine. The enzyme modulation by C-peptide was reproduced with the C-peptide fragments containing glutamate corresponding to position 27 (E27) of the full-length C-peptide. Addition of a lysine analogue to the assay and A31 cell culture abrogated the enzyme modulation and MAP kinase activation by C-peptide, respectively. The results indicate that C-peptide has the capacity to activate α-enolase through a specific interaction between E27 of the peptide and K434 of the enzyme. Since α-enolase plays a role as a cell surface receptor for plasminogen, it may conceivably also serve as a receptor for C-peptide in vivo .
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    Specific conformation and Ca2+-binding mode of yeast calmodulin: insight into evolutionary development (2012)
    Oxford University Press
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    Publication Date: 2012-07-05
    Description: The vertebrate calmodulin is configured with two structurally independent globular lobes in N- and C-terminus, and a flexible central linker. Distinctly, two lobes of calmodulin from Saccharomyces cerevisiae (yCaM) interact and influence the Ca 2+ -binding profile of each other. We explored this further using the mutant proteins with eliminated Ca 2+ -binding ability in one of the lobes and found that the Ca 2+ -bound N-lobe associates with the Ca 2+ -free C-lobe to gain the Ca 2+ affinity of a wild-type level. Next, analysing series of C-terminal residue truncation mutant, we found that the truncation of C-terminal three residues induce the hyper Ca 2+ affinity. These residues are also important for the general structural behaviour of calmodulin, such as Ca 2+ -induced slow mobility shift in polyacrylamide gel electrophoresis and for the ability to activate Cmk1p (yeast calmodulin kinase). These suggest: (i) when Ca 2+ occupies only N-lobe, two lobes interact and form the stable intermediate leading to a proper level of Ca 2+ affinity; (ii) the C-terminal three residues are required to prohibit abnormal stabilization of the intermediate promoting abnormally high Ca 2+ affinity and for recognition of target enzymes. A model for Ca 2+ and target bindings of yCaM is proposed. Evolutional aspect concerning the biological significance of this model was discussed.
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    Gangliosides stimulate bradykinin B2 receptors to promote calmodulin kinase II-mediated neuronal differentiation (2012)
    Oxford University Press
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    Publication Date: 2012-07-05
    Description: Gangliosides mediate neuronal differentiation and maturation and are indispensable for the maintenance of brain function and survival. As part of our ongoing efforts to understand signaling pathways related to ganglioside function, we recently demonstrated that neuronal cells react to exogenous gangliosides GT1b and GD1b. Both of these gangliosides are enriched in the synapse-forming area of the brain and induce Ca 2+ release from intracellular stores, activation of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and activation of cdc42 to promote reorganization of cytoskeletal actin and dendritic differentiation. Here, we show that bradykinin B2 receptors transduce these reactions as a mediator for ganglioside glycan signals. The B2 antagonist Hoe140 inhibited ganglioside-induced CaMKII activation, actin reorganization and early development of axon- and dendrite-like processes of primary cultured hippocampal neurons. Furthermore, we confirmed by yeast reporter assay that major b-series gangliosides, GT1b, GD1b and GD3, stimulated B2 bradykinin receptors. We hypothesize that this B2 receptor-mediated ganglioside signal transduction pathway is one mechanism that modulates neuronal differentiation and maturation.
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    The crystal structure of an intermediate dimer of aspergilloglutamic peptidase that mimics the enzyme-activation product complex produced upon autoproteolysis (2012)
    Oxford University Press
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    Publication Date: 2012-07-05
    Description: Aspergilloglutamic peptidase from Aspergillus niger var. macrosporus (AGP) is one of the so-called pepstatin-insensitive acid endopeptidases, which are distinct from the well-studied aspartic peptidases. Among the known homologues of the glutamic peptidases, AGP is a unique two-chain enzyme with a light chain and a heavy chain bound non-covalently with each other, and thus is an interesting target for protein structure–function relationship studies. In this article, we report the crystal structure of a dimeric form of the enzyme at a resolution of 1.6 Å. This form has a unique structure in which the C-terminal region of the light chain of one of the molecules binds to the active site cleft of the other molecule like a part of a substrate. This form mimics the enzyme-activation product complex produced upon autoproteolysis, and provides a structural clue that could help to clarify the activation mechanism. This type of dimeric structure of a peptidase is here reported for the first time.
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    Crystal structure of the lectin of Camptosema pedicellatum: implications of a conservative substitution at the hydrophobic subsite (2012)
    Oxford University Press
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    Publication Date: 2012-07-05
    Description: Lectins have been used as models for studies of the molecular basis of protein–carbohydrate interaction and specificity by deciphering codes present in the glycan structures. The purpose of the present study was to purify and solve the complete primary and crystal structure of the lectin of Camptosema pedicellatum (CPL) complexed with 5-bromo-4-chloro-3-indolyl-α- d -mannose (X-Man) using tandem mass spectrometry. CPL was purified by single-step affinity chromatography. Mass spectrometry findings revealed that purified CPL features a combination of chains weighing 25,298 ± 2 (α-chain), 12,835 ± 2 (β-chain) and 12,481 ± 2 Da (-chain). The solved crystal structure of CPL features a conservative mutation in the hydrophobic subsite, a constituent of the carbohydrate recognition domain (CRD), indicating the relevance of hydrophobic interactions in the establishment of interactions with carbohydrates. The substitution and the analysis of the interactions with X-Man also revealed that the hydrophobic effect caused by a minor change in the hydrophobic subsite interferes in the formation of H-bonds due to the reorientation of the indolyl group in the CRD.
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    Mitochondrial P5, a member of protein disulphide isomerase family, suppresses oxidative stress-induced cell death (2012)
    Oxford University Press
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    Publication Date: 2012-07-05
    Description: P5, one of the protein disulphide isomerase (PDI) family members, catalyses disulphide bond formation in proteins and exhibits molecular chaperone and calcium binding activities in vitro , whereas its physiological significance remains controversial. Recently, we have reported that P5 localizes not only in the ER but also in mitochondria, although it remains unclear so far about its physiological significance(s) of its dual localization. Here we report that H 2 O 2 – or rotenone-induced cell death is suppressed in MTS-P5 cells, which stably express P5 in mitochondria. H 2 O 2 -induced cell death in Saos-2 cells occurred, in large part, through caspase-independent and poly(ADP-ribose) polymerase (PARP)-dependent manner. In MTS-P5 cells challenged with H 2 O 2 treatment, PARP was still activated, whereas release of cytochrome c or apoptosis-inducing factor and intramitochondrial superoxide generation were suppressed. We also found that mitochondrial P5 was in close contact with citrate synthase and maintained large parts of its activity under H 2 O 2 exposure. These results suggest that mitochondrial P5 may upregulate tricarboxylic acid cycle and possibly, other intramitochondrial metabolism.
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    Deuterium kinetic isotope effects in heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96: the anionic form of the substrate in the enzyme-substrate complex is a reactive species (2012)
    Oxford University Press
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    Publication Date: 2012-06-06
    Description: Heterotetrameric sarcosine oxidase is a flavoprotein that catalyses the oxidative demethylation of sarcosine. It is thought that the dehydrogenated substrate is the anionic form of sarcosine. To verify this assumption, the rate of flavin-adenine dinucleotide (FAD) reduction ( k red ) was analysed using protiated and deuterated sarcosine ( N -methyl- d 3 -Gly) at various pH values using stopped-flow method. By increasing the pH from 6.2 to 9.8, k red increased for both substrates and reached a plateau, but the p K a value (reflecting the ionization of the enzyme–substrate complex) was 6.8 and 7.1 for protiated and deuterated sarcosine, respectively, and the kinetic isotope effect of k red decreased from approximately 19 to 8, indicating deprotonation of the bound sarcosine. The k red / K d ( K d , sarcosine dissociation constant) increased with increasing pH and reached a plateau. The p K (reflecting the ionization of free enzyme or free sarcosine) was 7.0 for both substrates, suggesting deprotonation of the βLys358 residue, which has a p K a of 6.7, as the p K a of the free sarcosine amine proton was determined to be approximately 10.1. These results indicate that the amine proton of sarcosine is transferred to the unprotonated Lys residue in the enzyme–substrate complex.
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    SwrA regulates assembly of Bacillus subtilis DegU via its interaction with N-terminal domain of DegU (2012)
    Oxford University Press
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    Publication Date: 2012-06-06
    Description: The Bacillus subtilis response regulator DegU controls many physiological events including swarming motility and exoprotease production. Swarming motility is a multicellular movement of hyper-flagellated cells on a surface. The swarming motility regulator SwrA and DegU cooperatively drive transcription of fla/che encoding flagella components, chemotaxis constituents and motility-specific sigma factor, which is regarded as the primary event in the development of motility. We have identified ycdA involved in swarming motility, encoding a putative lipoprotein. We showed that the ycdA gene is positively regulated by DegU and SwrA. Mutational analysis of ycdA–lacZ revealed that SwrA changes the use of cis -acting sites for DegU. This suggested that SwrA operates the DegU-regulation mode through changes in the DegU assembly state. DegU binding to the ycdA –promoter region carrying an unusual arrangement of DegU-recognition sequences with low affinity was found to be stimulated by SwrA in electrophoretic mobility shift assay and DNase I footprinting. Yeast two- and three-hybrid analyses revealed that the N-terminal domain of DegU interacts with whole DegU, which is facilitated by SwrA. Together, these results demonstrate that SwrA can stabilize the binding of DegU to the ycdA promoter with low affinity. Thus, SwrA is a novel type of bacterial transcription factor in this regard.
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    Implication of TGF-{beta} as a survival factor during tumour development (2012)
    Oxford University Press
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    Publication Date: 2012-06-06
    Description: Transforming growth factor (TGF)-β is a pleiotropic secretory protein which inhibits and potentiates tumour progression during early and late stage of tumourigenicity, respectively. However, it still remains veiled how TGF-β signalling reveals its two faces. Hoshino et al. (Autocrine TGF-β protects breast cancer cells from apoptosis through reduction of BH3-only protein, Bim, J. Biochem. 2011;149:55–65) demonstrated a new aspect of TGF-β as a survival factor in highly metastatic breast cancer cells from which TGF-β1 and TGF-β3 are abundantly expressed. They found that TGF-β suppressed the expression of BH3-only protein Bim which promotes programmed death signalling via release of cytochrome c from mitochondria. Further interestingly, forkhead box C1 (Foxc1) whose expression is suppressed upon TGF-β stimulation is involved in the expression of Bim. Based on their results, autocrine TGF-β signalling in certain breast cancers promotes cell survival via inhibition of apoptotic signalling. Thus, the inhibitors for activin receptor-like kinase (ALK)5 kinase might exert a curative influence on certain types of metastatic breast cancers.
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    Transcriptional and Post-transcriptional Regulation in TGF-{beta}-mediated epithelial-mesenchymal transition (2012)
    Oxford University Press
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    Publication Date: 2012-06-06
    Description: Epithelial-mesenchymal transition (EMT) is a crucial event in appropriate embryonic development as well as in wound healing, tissue repair and cancer progression in adult tissues. EMT endows cells with migratory and invasive properties, inhibits apoptosis and senescence, contributes to immunosuppression and induces stress resistance and stem cell properties. Many secreted polypeptide factors act in a sequential or cooperative manner to elicit EMT. Transforming growth factor (TGF)-β can initiate and maintain EMT by activating intracellular signalling pathways. Recent studies have provided new insights into molecular mechanisms by which TGF-β mediates changes in transcription of EMT regulators and EMT marker proteins, as well as changes in alternative splicing controlled by epithelial splicing regulatory proteins 1 and 2. Here, we present some of the emerging molecular mechanisms that mediate EMT upon exposure to TGF-β.
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    Identification of the P-body component PATL1 as a novel ALG-2-interacting protein by in silico and far-Western screening of proline-rich proteins (2012)
    Oxford University Press
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    Publication Date: 2012-06-06
    Description: ALG-2 (also named PDCD6) is a 22-kDa Ca 2+ -binding protein that belongs to the penta-EF-hand family including calpain small subunit and interacts with various proteins such as ALIX and Sec31A at their specific sites containing an ALG-2-binding motif (ABM) present in their respective Pro-rich region (PRR). In this study, to search for novel ALG-2-interacting proteins, we first performed in silico screening of ABM-containing PRRs in a human protein database. After selecting 17 sequences, we expressed the PRR or full-length proteins fused with green fluorescent protein (GFP) in HEK293T cells and analysed their abilities to bind to ALG-2 by Far-Western blotting using biotinylated ALG-2 as a probe. As a result, we found 10 positive new ALG-2-binding candidates with different degrees of binding ability. For further investigation, we selected PATL1 (alternatively designated Pat1b), a component of the P-body, which is a cytoplasmic non-membranous granule composed of translation-inactive mRNAs and proteins involved in mRNA decay. Interactions between endogenous PATL1 and ALG-2 proteins were demonstrated by a co-immunoprecipitation assay using their specific antibodies. Furthermore, in immunofluorescence microscopic analyses, PATL1 as well as DCP1A, a well-known P-body marker, co-localized with a subset of ALG-2. This is the first report showing interaction of ALG-2 with a P-body component.
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    Smad7-deficient mice show growth retardation with reduced viability (2012)
    Oxford University Press
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    Publication Date: 2012-06-06
    Description: Smad7 is an inhibitory molecule induced by members of the transforming growth factor-β (TGF-β) family, including TGF-β, activin, nodal and bone morphogenetic proteins (BMPs). To elucidate the in vivo functions of Smad7, we generated conditional Smad7 -knockout mice in which the Mad homology 2 (MH2) domain and the poly (A) signal sequence were flanked with loxP sites (floxed). The Smad7 -floxed mice exhibited no obvious phenotype. Smad7 total-null mice on a C57BL/6 background died within a few days of birth, whereas mice with an ICR background developed to adulthood but were significantly smaller than wild-type mice. Unexpectedly, phospho-Smad2 and phospho-Smad3 were decreased in Smad7 -deficient mouse embryonic fibroblast (MEF) cells, whereas phospho-Smad1/5/8 was similarly expressed in wild-type and Smad7 -deficient MEF cells. Moreover, expression levels of TGF-β type I receptor (ALK5) were higher in Smad7 -deficient MEF cells than in wild-type MEF cells. Plasminogen activator inhibitor-1 (PAI-1) and inhibitor of differentiation-1 (Id-1) mRNA were similarly expressed in wild-type and Smad7 -deficient MEF cells. Some differences were observed in mitogen-activated protein kinase (MAPK)-signalling between wild-type and Smad7 -deficient MEF cells. We demonstrated that Smad7 plays an important role in normal mouse growth and provide a useful tool for analysing Smad7 functions in vivo .
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    Fine tuning of cell signals by glycosylation (2012)
    Oxford University Press
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    Publication Date: 2012-06-06
    Description: Carbohydrates on the glycoproteins and glycosphingolipids expressed on the cell surface membrane play crucial roles in the determination of cell fates by being involved in the fine tuning of cell signalling as reaction molecules in the front line to various extrinsic stimulants. In glycoproteins, modification of proteins is performed by substitution of sugar chains to one or multiple sites of individual proteins, leading to quantitative and qualitative changes of receptor functions in the cell membrane. As for glycosphingolipids, majority of them consist of two moieties, i.e. carbohydrates and ceramides, and are localized in the microdomains such as lipid rafts or detergent-resistant microdomains. They generate and/or modulate cell signals to determine the cell fates by interacting with various carbohydrate-recognizing proteins. Modes of glycosylation and mechanisms by which glycosylation is involved in the regulation of cell signals are now hot subjects in glycobiology.
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    Structural mechanism of disulphide bond-mediated redox switches (2012)
    Oxford University Press
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    Publication Date: 2012-06-06
    Description: The oxidation of cysteine sulphydryl in proteins produces sulphenic acid that can form a reversible disulphide bond with another cysteine. The disulphide bond formation often triggers switches in protein structure and activity, especially when the distance between the two cysteine sulphur atoms is longer than the resulting disulphide bond distance. As an early example for the reversible disulphide bond-mediated functional switches, the reduced and oxidized forms of the bacterial transcription factor OxyR were characterized by X-ray crystallography. Recently, the Drosophila vision signalling protein, the association of inactivation-no-afterpotential D (INAD) was analysed by structural and functional methods. The two conserved cysteines of INAD were found to cycle between reduced and oxidized states during the light signal processing in Drosophila eyes, which was achieved by conformation dependent modulation of the disulphide bond redox potential. The production of the hypertension control peptide angiotensins was also shown to be controlled by the reversible disulphide bond in the precursor protein angiotensinogen. The crystal structure of the complex of angiotensiongen with its processing enzyme renin elucidated the role of the disulphide bond in stabilizing the precursor–enzyme complex facilitating the production of angiotensins. The increasing importance of the disulphide bond-mediated redox switches in normal and diseased states has implications in the development of novel antioxidant-based therapeutic approaches.
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    Crystal structure of mitochondrial quinol-fumarate reductase from the parasitic nematode Ascaris suum (2012)
    Oxford University Press
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    Publication Date: 2012-06-06
    Description: In the anaerobic respiratory chain of the parasitic nematode Ascaris suum , complex II couples the reduction of fumarate to the oxidation of rhodoquinol, a reverse reaction catalyzed by mammalian complex II. In this study, the first structure of anaerobic complex II of mitochondria was determined. The structure, composed of four subunits and five co-factors, is similar to that of aerobic complex II, except for an extra peptide found in the smallest anchor subunit of the A. suum enzyme. We discuss herein the structure–function relationship of the enzyme and the critical role of the low redox potential of rhodoquinol in the fumarate reduction of A. suum complex II.
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    Alzheimer's disease-related protein hGas7b interferes with kinesin motility (2012)
    Oxford University Press
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    Publication Date: 2012-06-06
    Description: In the previous study, we reported the important properties of hGas7b (i) that binds to phospho-tau and facilitates microtubule polymerization and (ii) the level of hGas7b is very low in the brains of patients with Alzheimer’s disease. These results led us to study the function of hGas7b in detail. We focused on the effect of hGas7b on microtubule dynamics in the absence of tau, on the assumption of healthy tau decrease in the brains of Alzheimer’s disease. hGas7b binds to microtubule directly without tau, although this binding does not enhance microtubule polymerization. Excess hGas7b interferes with kinesin motility on microtubules. These results suggest that regulation to maintain an appropriate concentration of hGas7b is required for healthy neurotransmission.
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    Transcriptional regulation of neutral sphingomyelinase 2 in all-trans retinoic acid-treated human breast cancer cell line, MCF-7 (2012)
    Oxford University Press
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    Publication Date: 2012-06-06
    Description: Effects of all- trans retinoic acid (ATRA) on sphingomyelinase expression were examined using MCF-7 (ATRA-sensitive) and MDA-MB-231 (ATRA-resistant) breast cancer cells. Increased NSMase activity, NSMase2 mRNA and protein were observed in ATRA-treated MCF-7 but not in ATRA-treated MDA-MB-231. Increased NSMase2 mRNA of ATRA-treated MCF-7 was mostly due to enhanced transcription. Promoter analysis revealed the important 5'-promoter region of NSMase2 between –148 and –42 bp containing three Sp1 sites but no retinoic acid responsive elements. Experiments using mutated Sp1 sites of the NSMase2 promoter, Mithramycin A (a Sp inhibitor) and Sp family over-expression demonstrated the importance of Sp family protein and the three Sp1 sites for ATRA-induced NSMase2 transcription of MCF-7 cells. Although no quantitative change of bound Sp1 on NSMase2 promoter region after ATRA treatment was detected, Sp1 phosphorylation (activation) by ATRA was observed. Interestingly, PKC was involved in ATRA-induced increased NSMase2 transcription. ATRA-induced PKC phosphorylation and then activated PKC phosphorylated Sp1. Chromatin immunoprecipitation (ChIP) assay showed Sp1, RARα and RXRα complex formation in MCF-7 cells regardless of ATRA treatment and ATRA-induced acetylated histone H3 of the 5'-promoter. Thus, NSMase2 mRNA expression enhanced by ATRA was due to increased transcription via phosphorylated Sp1 caused by PKC activation, followed by chromatin remodelling with histone H3 acetylation.
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    Role of down-regulated neutral ceramidase during all-trans retinoic acid-induced neuronal differentiation in SH-SY5Y neuroblastoma cells (2012)
    Oxford University Press
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    Publication Date: 2012-06-06
    Description: Neutral ceramidase (NCDase) is considered to be a critical enzyme for controlling the turnover of ceramide, an important bioactive lipid, which determines cell's fate. All- trans retinoic acid (ATRA) has been reported to induce neuronal differentiation and cell-cycle arrest [Lopez-Carballo, Moreno, Masia, Perez, and Barettino (Activation of the phosphatidylinositol 3-kinase/Akt signalling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells. J Biol Chem 2002:277:25297–304.)]. In this study, we observed that ATRA-induced cellular ceramide accumulation, cell-growth arrest and differentiation accompanied with down-regulation of NCDase in SH-SY5Y cells, without a decrease in sphingosine or sphingosine 1-phosphate. We examined whether the down-regulation of NCDase was involved in the increase in ceramide and cell differentiation. ATRA was found to down-regulate mRNA, protein and the enzyme activity of NCDase. Interestingly, GATA-2 was also decreased with ATRA treatment, and experiments using its expression vector and siRNA and chromatin immunoprecipitation assay demonstrated GATA-2 acted as transcription-factor of NCDase gene expression. By establishing stable transfectants with decreased NCDase expression and activity, we clarified the significance of NCDase down-regulation for ATRA-induced neuronal differentiation. Those sub-clones showed both increased cellular ceramide and reduced cell growth as well as neuronal differentiation phenotypes. These results demonstrate that down-regulation of NCDase through ATRA-induced GATA-2 decrease plays an important role in induction of ceramide accumulation and neuronal differentiation in SH-SY5Y cells.
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    PUGNAc treatment leads to an unusual accumulation of free oligosaccharides in CHO cells (2012)
    Oxford University Press
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    Publication Date: 2012-04-25
    Description: Free oligosaccharides (fOS) are generated as the result of N- glycoproteins catabolism that occurs in two distinct principal pathways: the endoplasmic reticulum-associated degradation (ERAD) of misfolded newly synthesized N- glycoproteins and the mature N- glycoproteins turnover pathway. The O- (2-acetamid O- 2-deoxy- d -glucopyranosylidene) amino- N -phenylcarbamate (PUGNAc) is a potent inhibitor of the O- GlcNAcase (OGA) catalysing the cleavage of β- O- linked 2-acetamido-2-deoxy-β- d -glucopyranoside ( O- GlcNAc) from serine and threonine residues of post-translationaly O- GlcNAc modified proteins. In order to estimate the impact of O- GlcNAc modification on N- glycoproteins catabolism, fOS were analysed by mass spectrometry (MS). MS analysis revealed the appearance of an unusual population of fOS after PUGNAc treatment. The structures representing this population have been identified as containing non-reducing end GlcNAc residues resulting from incomplete lysosomal fOS degradation. Only observed after PUGNAc treatment, the NButGt, another OGA inhibitor, did not lead to the appearance of this population. These abnormal fOS structures have clearly been shown to accumulate in membrane fractions as the consequence of lysosomal β-hexosaminidases inhibition by PUGNAc. As lysosomal storage disorders (LSD) are characterized by the accumulation of storage material as fOS in lysosomes, our study evokes that the use of PUGNAc could mimic a LSD. This study clearly points out another off target effects of PUGNAc that need to be taken into account in the use of this drug.
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    Matrix metalloproteinase-9 activity detected in body fluids is the result of two different enzyme forms (2012)
    Oxford University Press
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    Publication Date: 2012-04-27
    Description: In vitro activation of matrix metalloproteinase-9 (MMP-9) (Gelatinase B) with MMP-3 shows the presence of two different forms: an 82 kDa, N-terminal truncated form, and a 65 kDa, N- and C-terminal truncated form. So far the presence of the 65 kDa form has not been reported in vivo . Affinity chromatography was performed to separate MMP-9 from MMP-2 and immunoprecipitation to isolate ~65 kDa MMP-9 from 82 kDa MMP-9 in sera of healthy donors. The presence of ~65 kDa active MMP-9 was demonstrated both with gelatin zymography and western blot analysis. The ~65 kDa MMP-9 lacks the haemopexin domain required for the high-affinity binding of the tissue inhibitor TIMP-1, and can be evaluated by activity assay in the presence of TIMP-1. This opens the possibility to investigate the role of this form of MMP-9 that escapes physiological regulation.
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    Crystal structure of non-phosphorylated MAP2K6 in a putative auto-inhibition state (2012)
    Oxford University Press
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    Publication Date: 2012-04-27
    Description: Mitogen-activated protein kinase kinase 6 (MAP2K6) plays a crucial role in the p38 MAP kinase signal cascade that regulates various stress-induced responses and is associated with pathological conditions. The crystal structure of human non-phosphorylated MAP2K6 (npMAP2K6) complexed with an ATP analogue was determined at 2.6 Å resolution and represents an auto-inhibition state of MAP2K6. Three characteristics of short α-helices configured in the activation loop region, termed activation helices (AH1, AH2 and AH3), are important in controlling the auto-inhibition mechanism. AH1 displaces the αC-helix, a component essential for forming the active configuration, away from the active site. AH1 and AH2 were found to enclose the -phosphate, the leaving group of ATP. A comparison with the related enzymes, MAP2K1 and MAP2K4 reveals that MAP2K6 has the unique auto-inhibition mechanism mediated by the three activation helices.
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    Interaction of 8-anilinonaphthalene 1-sulphonate (ANS) and human matrix metalloproteinase 7 (MMP-7) as examined by MMP-7 activity and ANS fluorescence (2012)
    Oxford University Press
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    Publication Date: 2012-04-27
    Description: Human matrix metalloproteinase 7 (MMP-7) is the smallest matrix metalloproteinase. It plays important roles in tumour invasion and metastasis. 8-Anilinonaphthalene 1-sulphonate (ANS) is a fluorescent probe widely used for the analysis of proteins. It emits large fluorescence energy when its anilinonaphthalene group binds with hydrophobic regions of protein. In this study, we analysed the interaction of ANS and MMP-7. At pH 4.5–9.5, ANS inhibited MMP-7 activity in the hydrolysis of (7-methoxycoumarin-4-yl)acetyl– L -Pro– L -Leu–Gly– L -Leu-[ N 3 -(2,4-dinitrophenyl)- L -2,3-diaminopropionyl]– L -Ala– L -Arg–NH 2 . The inhibition was a non-competitive manner and depended on the time for pre-incubation of ANS and MMP-7. At pH 4.5–9.5, the fluorescence of ANS was not changed by the addition of MMP-7. At pH 3.5, MMP-7 lacked activity, and the fluorescence of ANS was increased by the addition of MMP-7. These results suggest that at pH 4.5–9.5, the sulphonic group of ANS binds with MMP-7 through electrostatic interaction, whereas at pH 3.5, the anilinonaphthalene group of ANS binds with MMP-7 through hydrophobic interaction.
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    Ikuo Yamashina: a pioneer who established the basis of current glycobiology (2012)
    Oxford University Press
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    Publication Date: 2012-04-27
    Description: Ikuo Yamashina determined the two notable structures of N -glycans, N -acetylglucosaminylasparagine and β-mannosidic linkages, which are generally present in sugar–amino acid and innermost mannose residue of the N -glycans, respectively. He detected mucins with unusual O -glycans and proteoheparan sulphate in the plasma membranes of AH66 ascites hepatoma cells. Unusual O -glycans were identified as tumour-associated carbohydrate antigens after the development of monoclonal antibodies against these O -glycans. Epitopic structures of some antigens were determined to comprise clusters of short O -glycans aligned on the core peptide, which may be not only antigenic but also functional in relation to tumour behaviour. With respect to proteoheparan sulphate, this finding led to study on membrane-bound proteoglycans.
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    Phosphorylation in the activation loop as the finishing touch in c-Kit activation (2012)
    Oxford University Press
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    Publication Date: 2012-04-27
    Description: Receptor tyrosine kinases are a group of transmembrane proteins that transmit signals in response to stimulation by ligands including growth factors and cytokines. They share a common mechanism of activation through receptor dimerization or oligomerization, but subsequent routes to their full activation appear to be various. A recent paper published by DiNitto et al. (Function of activation loop tyrosine phosphorylation in the mechanism of c-Kit autoactivation and its implication in sunitinib resistance. J. Biochem. 2010;147:601–609) analysed a process of c-Kit autoactivation in detail. They revealed that phosphorylation in the activation loop, which is crucial for activation of many types of tyrosine kinases, is dispensable for c-Kit activation. However, the phosphorylation affects the sensitivity of c-Kit to kinase inhibitors, thus representing the finishing touch in c-Kit activation.
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    A new structural insight into differential interaction of cyanobacterial and plant ferredoxins with nitrite reductase as revealed by NMR and X-ray crystallographic studies (2012)
    Oxford University Press
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    Publication Date: 2012-04-27
    Description: Ferredoxin (Fd), which plays a pivotal role in photosynthesis as an electron carrier, forms a transient complex with various Fd-dependent enzymes, such as nitrite reductase (NiR), to achieve efficient intermolecular electron transfer. We studied the protein–protein interaction of Fd and NiR by NMR spectroscopy and determined three acidic regions of Fd to be major sites for the interaction with NiR, indicating that the complex is stabilized through electrostatic interaction. During this study, we found Fds from higher plant and cyanobacterium, in spite of their high structural similarities including the above acidic regions, differ remarkably in the interaction with cyanobacterial NiR. In activity assay of NiR, K m value for maize Fd (74.6 µM) was 9.6 times larger than that for Leptolyngbya boryana Fd (7.8 µM). The two Fds also showed a similar difference in binding assay to NiR-immobilized resin. Comparative site-specific mutagenesis of two Fds revealed that their discriminative ability for the interaction with NiR is attributed mainly to non-charged residues in the peripheral region of [2Fe–2S] cluster. These non-charged residues are conserved separately between Fds of plant and cyanobacterial origins. Our data highlight that intermolecular force(s) other than electrostatic attraction is(are) also crucial for the molecular interaction between Fd and partner enzyme.
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    GPR34 is a receptor for lysophosphatidylserine with a fatty acid at the sn-2 position (2012)
    Oxford University Press
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    Publication Date: 2012-04-27
    Description: GPR34 is a G protein-coupled receptor belonging to the P2Y family. Here, we attempted to resolve conflicting reports about whether it is a functional lysophosphatidylserine (LysoPS) receptor. In HEK293 cells expressing human, mouse or rat GPR34 and Gα chimera between Gαq and Gαi1(Gq/i1), LysoPS quickly elevated intracellular Ca 2+ ion levels ([Ca 2+ ] i ). LysoPS also stimulated alkaline phosphatase (AP)-tagged TGFα (AP-TGFα) release in GPR34-expressing HEK293 cells and induced the migration of CHO-K1 cells expressing GPR34. Other lysophospholipids did not induce these actions. Replacement of the serine residue of LysoPS abolished the reactivity of LysoPS with GPR34, indicating that GPR34 strictly recognizes the serine head group of LysoPS. Recombinant phosphatidylserine-specific phospholipase A 1 (PS-PLA 1 ) that deacylates fatty acid at the sn -1 position of PS and produces 2-acyl-LysoPS, but not catalytically inactive mutant PS-PLA 1 , stimulated the release of AP-TGFα from GPR34-expressing cells. Consistent with the result, LysoPS was detected in the cells treated with wild-type PS-PLA 1 but not with the mutant PS-PLA 1 . PS treated with PLA 1 was much more effective at stimulating AP-TGFα release than PS treated with PLA 2 . In addition, migration-resistant 2-acyl-1-deoxy-LysoPS, a 2-acyl-LysoPS analogue, was much more potent than 1-acyl-2-deoxy-LysoPS. The present studies confirm that GPR34 is a cellular receptor for LysoPS, especially with a fatty acid at the sn -2 position.
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    Translational regulation by the 5'-UTR of thyroid hormone receptor {alpha} mRNA (2012)
    Oxford University Press
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    Publication Date: 2012-04-27
    Description: Thyroid hormone (TH) regulates gene transcription by binding to the thyroid hormone receptor (TR) and plays a critical role in the regulation of development, growth and metabolism. The ligated TR activates many effector genes, which contributes to the orchestrated remodelling of the amphibian metamorphosis. However, the mechanisms regulating TRα protein level remain unknown. We determined the nucleotide sequences of the 5'-untranslated regions (5'-UTRs) in amphibian TRα mRNAs. The TRα 5'-UTR contains evolutionarily conserved regions. We demonstrated that a 161-nt stretch of the Xenopus TRα 5'-UTR strongly represses the translation of the downstream open reading frame in both frog and human cell lines, as well as in a cell-free translation system. An analysis using successive deletions of the TRα 5'-UTR revealed five elements possessing translational repressive activity. We analysed two elements, the 14-nt GC-rich region and the 15-nt upstream open reading frame (uORF), by introducing point mutations. This analysis showed that the GC-rich region, which shares its nucleotide sequence with the Sp1-binding site, requires stringent sequence specificity at a nucleotide level for translational repression to take place, whereas under our study conditions, the uORF does not. This study provides an example of complex translational regulation by multiple elements.
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    Role of Rab family GTPases and their effectors in melanosomal logistics (2012)
    Oxford University Press
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    Publication Date: 2012-04-25
    Description: Rab GTPases constitute a family of small GTPases that regulate a variety of membrane trafficking events in all eukaryotic cells by recruiting their specific effector molecules. Recent accumulating evidence indicates that members of the mammalian Rab small GTPase family are involved in certain physiological and pathological processes. In particular, functional impairments of specific Rab proteins, e.g . Rab38 and Rab27A, their regulators or their effectors cause pigmentation disorders in humans and coat colour variations in mice because such impairments cause defects in melanosomal logistics, i.e . defects in melanosome biogenesis and transport. Genetic and biochemical analyses of the gene products responsible for mammalian pigmentation disorders in the past decade have revealed that Rab-mediated endosomal transport systems and melanosome transport systems play crucial roles in the efficient darkening of mammalian hair and skin. In this article, we review current knowledge regarding melanosomal logistics, with particular focus on the roles of Rab small GTPases and their effectors.
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    Effects of phosphatase and proteasome inhibitors on Borealin phosphorylation and degradation (2012)
    Oxford University Press
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    Publication Date: 2012-04-25
    Description: The chromosomal passenger complex (CPC) senses tension defects at the kinetochore to activate the spindle assembly checkpoint, and helps to position the cleavage furrow. The CPC, consisting of INCENP, Survivin, Borealin and Aurora B localizes to the inner centromere at metaphase and re-localizes to the spindle midzone at anaphase; several CPC functions are regulated by post-translational modification. Borealin is phosphorylated at multiple sites and phosphorylation at S219 causes Borealin to migrate more slowly upon electrophoresis. Here we find that Cdk1 can induce a mobility shift of Borealin, suggesting that S219 phosphorylation is under Cdk1 control. However, Cdk1 is inefficient at phosphorylating purified Borealin in vitro . A yeast orthologue of Borealin, Npl1, is dephosphorylated by the phosphatase Cdc14. We find no difference in the mobility shift of Borealin in human cells lacking either Cdc14A or Cdc14B. In contrast, the phosphatase inhibitor okadaic acid does delay the dephosphorylation of Borealin as cells exit mitosis. The proteasome inhibitor MG132 reduces Borealin phosphorylation in mitosis and increases the steady-state level of Borealin, especially in mutants lacking the C-terminus. However, a second, structurally unrelated proteasome inhibitor, lactacystin did not up-regulate Borealin. These results suggest that the effect of MG132 on Borealin is due to the inhibition of an intracellular protease other than the proteasome.
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    Varied effects of Pyrococcus furiosus prefoldin and P. furiosus chaperonin on the refolding reactions of substrate proteins (2012)
    Oxford University Press
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    Publication Date: 2012-04-25
    Description: Prefoldin is a molecular chaperone found in the archaeal and eukaryotic cytosol. Prefoldin can stabilize tentatively nascent polypeptide chains or non-native forms of mainly cytoskeletal proteins, which are subsequently delivered to group II chaperonin to accomplish their precise folding. However, the detailed mechanism is not well known, especially with regard to endogenous substrate proteins. Here, we report the effects of Pyrococcus furiosus prefoldin (PfuPFD) on the refolding reactions of Pyrococcus furiosus citrate synthase (PfuCS) and Aequorea enhanced green fluorescence protein (GFPuv) in the presence or absence of Pyrococcus furiosus chaperonin (PfuCPN). We confirmed that both PfuPFD and PfuCPN interacted with PfuCS and GFPuv refolding intermediates. However, the interactions between chaperone and substrate were different for each case, as was the final effect on the refolding reaction. Effects on the refolding reaction varied from passive effects such as ATP-dependent binding and release (PfuCPN towards GFPuv) and binding which leads to folding arrest (PfuPFD towards GFPuv), to active effects such as net increase in thermal stability (PfuCPN towards PfuCS) to an active improvement in refolding yield (PfuPFD towards PfuCS). We postulate that differences in molecular interactions between substrate and chaperone lead to these differences in chaperoning effects.
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    Pleiotropic functions of the CXC-type chemokine CXCL14 in mammals (2012)
    Oxford University Press
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    Publication Date: 2012-04-27
    Description: CXCL14 is a member of the CXC chemokine family. CXCL14 possesses chemoattractive activity for activated macrophages, immature dendritic cells and natural killer cells. CXCL14-deficient mice do not exhibit clear immune system abnormalities, suggesting that the function of CXCL14 can be compensated for by other chemokines. However, CXCL14 does appear to have unique biological roles. It suppresses the in vivo growth of lung and head-and-neck carcinoma cells, whereas the invasiveness of breast and prostate cancer cells appears to be promoted by CXCL14. Moreover, recent evidence revealed that CXCL14 participates in glucose metabolism, feeding behaviour-associated neuronal circuits, and anti-microbial defense. Based on the expression patterns of CXCL14 and CXCL12 during embryonic development and in the perinatal brain in mice, the functions of these two chemokines may be opposite or interactive. Although CXCL14 receptors have not yet been identified, the intracellular activity of CXCL14 in breast cancer cells suggests that the CXCL14 receptor(s) and signal transduction pathway(s) may be different from those of conventional CXC-type chemokines.
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    Roles of stromal microenvironment in colon cancer progression{dagger} (2012)
    Oxford University Press
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    Publication Date: 2012-04-27
    Description: Although our understanding of epithelial cancer cells has advanced significantly, our understanding of the cancer microenvironment is still fragmentary. In contrast to our intuitive impression that our body always suppresses cancer growth, recent pieces of evidence show that cancer often exploits our body reactions to expand, invade local tissues and metastasize to distant organs. Accordingly, investigations of such body reactions in the tumour microenvironment should help us to design novel therapeutic strategies that can be combined with the traditional therapeutics targeted at the cancer cells themselves. In this article, I am going to review our recent efforts in search of novel therapeutic strategies against colon cancer using mouse models.
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    Fine spatial assembly for construction of the phenol-binding pocket to capture bisphenol A in the human nuclear receptor estrogen-related receptor {gamma} (2012)
    Oxford University Press
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    Publication Date: 2012-04-25
    Description: Various lines of evidence have shown that bisphenol A (BPA) acts as an endocrine disruptor that affects various hormones even at merely physiological levels. We demonstrated recently that BPA binds strongly to human nuclear receptor estrogen-related receptor (ERR), one of 48 nuclear receptors. Based on X-ray crystal analysis of the ERR ligand-binding domain (LBD)/BPA complex, we demonstrated that ERR receptor residues, Glu275 and Arg316, function as the intrinsic-binding site of the phenol–hydroxyl group of BPA. If these phenol–hydroxylGlu275 and Arg316 hydrogen bonds anchor the A-benzene ring of BPA, the benzene–phenyl group of BPA would be in a pocket constructed by specific amino acid side chain structures. In the present study, by evaluating the Ala-replaced mutant receptors, we identified such a ligand-binding pocket. Leu268, Leu271, Leu309 and Tyr326, in addition to the previously reported participants Glu275 and Arg316, were found to make a receptacle pocket for the A-ring, whereas Ile279, Ile310 and Val313 were found to assist or structurally support these residues. The results revealed that each amino acid residue is an essential structural element for the strong binding of BPA to ERR.
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    Construction and expression of anti-Tn-antigen-specific single-chain antibody genes from hybridoma producing MLS128 monoclonal antibody (2012)
    Oxford University Press
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    Publication Date: 2012-04-25
    Description: Anti-Tn-antigen monoclonal antibody MLS128 has affinity for three consecutive Tn-antigens (Tn3) more than Tn2. The major aim of this study was to isolate genes encoding MLS128 variable domains to produce a large quantity of recombinant MLS128 antibodies, in turn, allowing the conduct of studies on precise interactions between Tn3- or Tn2-epitopes and MLS128. This study describes cloning of the variable region genes of MLS128, construction of the variable region genes in single-chain variable fragments (scFv) and two scFvs conjugated with human IgG 1 hinge and Fc regions (scFv–Fc) types, and their respective expression in bacterial and mammalian cell. MLS128 scFv protein with the expected specificity and affinity was successfully prepared from inclusion bodies accumulating in Escherichia coli . Construction, expression and purification of two types of MLS128-scFv–Fc proteins with differing linker lengths in Chinese hamster ovary cells demonstrated that the purified scFv–Fc proteins had binding activity specific to the glycoprotein-expressing Tn-antigen clusters. These results revealed that VL and VH genes cloned from the hybridoma represent those of MLS128 and that recombinant antibodies produced from these genes should provide sufficient amounts of binding domains for use in 3D structural studies such as NMR and X-ray analysis.
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    Efficient expression and purification of recombinant human m-calpain using an Escherichia coli expression system at low temperature (2012)
    Oxford University Press
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    Publication Date: 2012-04-25
    Description: Calpain belongs to the superfamily of Ca 2+ -regulated cysteine proteases, which are indispensable to the regulation of various cellular functions. Of the 15 mammalian calpain isoforms, µ- and m-calpains are the best characterized. Both µ- and m-calpain are ubiquitously expressed and exist as heterodimers, containing a distinct 80-kDa catalytic subunit (CAPN1 and CAPN2, respectively) and the common, 30-kDa regulatory subunit (CAPNS1). To date, various expression systems have been developed for producing recombinant calpains for use in structural and physiological studies, however Escherichia coli systems have proven incompatible with large-scale preparation of calpain, with the exception of rat m-calpain. Here, we have established a highly efficient method to purify active recombinant human m-calpain using an E. coli expression system at low temperature (22°C). This was achieved by co-expressing CAPN2 with a C-terminal histidine-tag, and CAPNS1, lacking the first Gly-repeated region at the N-terminal. After three sequential passes through a chromatographic column, ~5 mg of human m-calpain was homogenously purified from 1 l of E. coli culture. Proteins were stable for several months. This is the first report of efficient, large-scale purification of recombinant human m-calpain using an E. coli expression system.
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    Sequence requirements for RNA binding by HuR and AUF1 (2012)
    Oxford University Press
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    Publication Date: 2012-04-25
    Description: The stability of RNAs bearing AU-rich elements in their 3'-UTRs, and thus the level of expression of their protein products, is regulated by interactions with cytoplasmic RNA-binding proteins. Binding by HuR generally leads to mRNA stabilization and increased protein production, whereas binding by AUF1 isoforms generally lead to rapid degradation of the mRNA and reduced protein production. The exact nature of the interplay between these and other RNA-binding proteins remains unclear, although recent studies have shown close interactions between them and even suggested competition between the two for binding to their cognate recognition sequences. Other recent reports have suggested that the sequences recognized by the two proteins are different. We therefore performed a detailed in vitro analysis of the binding site(s) for HuR and AUF1 present in androgen receptor mRNA to define their exact target sequences, and show that the same sequence is contacted by both proteins. Furthermore, we analysed a proposed HuR target within the 3'-UTR of MTA1 mRNA, and show that the contacted bases lie outside of the postulated motif and are a better match to a classical ARE than the postulated motif. The defining features of these HuR binding sites are their U-richness and single strandedness.
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    Activation of receptor protein-tyrosine kinases from the cytoplasmic compartment (2012)
    Oxford University Press
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    Publication Date: 2012-04-25
    Description: It is widely accepted that receptor protein-tyrosine kinases (RTKs) are activated upon dimerization by binding to their extracellular ligands. However, EGF receptor (EGFR) dimerization per se does not require ligand binding. Instead, its cytoplasmic kinase domains have to form characteristic head-to-tail asymmetric dimers to become active, where one ‘activator’ domain activates the other ‘receiver’ domain. The non-catalytic, cytoplasmic regions of RTKs, namely the juxtamembrane and carboxy terminal portions, also regulate kinase activity. For instance, the juxtamembrane region of the RTK MuSK inhibits the kinase domain probably together with a cellular factor(s). These findings suggest that RTKs could be activated by cytoplasmic proteins. Indeed, Dok-7 and cytohesin have recently been identified as such activators of MuSK and EGFR, respectively. Given that failure of Dok-7 signaling causes myasthenia, and inhibition of cytohesin signaling reduces the proliferation of EGFR-dependent cancer cells, cytoplasmic activators of RTKs may provide new therapeutic targets.
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    Human hematopoietic prostaglandin D synthase inhibitor complex structures (2012)
    Oxford University Press
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    Publication Date: 2012-04-25
    Description: In mast and Th2 cells, hematopoietic prostaglandin (PG) D synthase (H-PGDS) catalyses the isomerization of PGH 2 in the presence of glutathione (GSH) to produce the allergic and inflammatory mediator PGD 2 . We determined the X-ray structures of human H-PGDS inhibitor complexes with 1-amino-4-{4-[4-chloro-6-(2-sulpho-phenylamino)-[1,3,5]triazin-2-ylmethyl]-3-sulpho-phenylamino}-9,10-dioxo-9,10-dihydro-anthracene-2-sulphonic acid (Cibacron Blue) and 1-amino-4-(4-aminosulphonyl) phenyl-anthraquinone-2-sulphonic acid (APAS) at 2.0 Å resolution. When complexed with H-PGDS, Cibacron Blue had an IC 50 value of 40 nM and APAS 2.1 μM. The Cibacron Blue molecule was stabilized by four hydrogen bonds and – stacking between the anthraquinone ring and Trp104, the ceiling of the active site H-PGDS pocket. Among the four hydrogen bonds, the Cibacron Blue terminal sulphonic group directly interacted with conserved residues Lys112 and Lys198, which recognize the PGH 2 substrate α-chain. In contrast, the APAS anthraquinone ring was inverted to interact with Trp104, while its benzenesulphonic group penetrated the GSH-bound region at the bottom of the active site. Due to the lack of extended aromatic rings, APAS could not directly hydrogen bond with the two conserved lysine residues, thus decreasing the total number of hydrogen bond from four to one. These factors may contribute to the 50-fold difference in the IC 50 values obtained for the two inhibitors.
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    Membrane-induced alteration of the secondary structure in the SWAP-70 pleckstrin homology domain (2012)
    Oxford University Press
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    Publication Date: 2012-04-25
    Description: Differences in the conformation of the pleckstrin homology (PH) domain of switch-associated protein-70 (SWAP-70) in solution and at the lipid bilayer membrane surface were examined using CD, fluorescence and NMR spectroscopy. Intracellular relocalization of SWAP-70 from the cytoplasm to the plasma membrane and then to the nucleus is associated with its cellular functions. The PH domain of SWAP-70 contains a phosphoinositide-binding site and a nuclear localization signal, which localize SWAP-70 to the plasma membrane and nucleus, respectively. CD and fluorescence spectra showed that a significant conformational alteration involving formation of disordered structure occurs when the PH domain binds to d - myo -phosphatidylinositol 3,4,5-trisphosphate or d - myo -phosphatidylinositol 4,5-bisphosphate embedded in lipid bilayer vesicles. NMR spectra indicate that Ala and Trp residues located in the C-terminal α-helix of the PH domain undergo conformational alterations to form a disordered structure at the vesicle surface. These conformational alterations were not induced by association with inositol 1,3,4,5-tetrakisphosphate in solution or coexistence of phosphatidylcholine vesicles. Interaction with the plane of the lipid bilayer via association with the phosphoinositides is required for the unfolding of the C-terminal α-helix of the PH domain. The unwinding of the C-terminal α-helix could regulate the functions of SWAP-70 at the plasma membrane surface.
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    Membrane-displayed peptide ligand activates the pheromone response pathway in Saccharomyces cerevisiae (2012)
    Oxford University Press
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    Publication Date: 2012-04-27
    Description: The budding yeast, Saccharomyces cerevisiae , is an attractive host for studying G protein-coupled receptors (GPCRs). We developed a system in which a peptide ligand specific for GPCR is displayed on yeast plasma membrane. The model system described here is based on yeast plasma membrane display of an analogue of α-factor, which is a peptide ligand for Ste2p, the GPCR that activates the yeast pheromone response pathway. α-Factor analogues, containing linkers of varying lengths and produced in yeast cells, became attached to the cell plasma membrane by linking to the glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein Yps1p. We were able to demonstrate that an optimized α-factor analogue activated the pheromone response pathway in S. cerevisiae , as assessed by a fluorescent reporter assay. Furthermore, it was shown that linker length strongly influenced signalling pathway activation. To our knowledge, this is the first report documenting functional signalling by a plasma membrane-displayed ligand in S. cerevisiae .
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    A possibility of a protein-bound water molecule as the ionizable group responsible for pKe at the alkaline side in human matrix metalloproteinase 7 activity (2012)
    Oxford University Press
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    Publication Date: 2012-04-27
    Description: Human matrix metalloproteinase 7 (MMP-7) activity exhibits broad bell-shaped pH profile with the acidic and alkaline p K a (p K e1 and p K e2 ) values of about 4 and 10. The ionizable group for p K e2 was assigned to Lys or Arg by thermodynamic analysis; however, no such residues are present in the active site. Hence, based on the crystal structure, we hypothesized that a water molecule bound to the main-chain nitrogen of Ala162 (W1) or the main-chain carbonyl oxygen of Pro217 (W2) is a candidate for the ionizable group for p K e2 [Takeharu, H. et al. (2011) Biochim. Biophys. Acta 1814, 1940–1946]. In this study, we inspected this hypothesis. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl- L -Pro- L -Leu–Gly- L -Leu-[ N 3 -(2,4-dinitrophenyl)- L -2,3-diaminopropionyl]- L -Ala- L -Arg-NH 2 , all 19 variants, in which one of all Lys and Arg residues was replaced by Ala, retained activity, indicating that neither Lys nor Arg is the ionizable group. p K e2 values of A162S, A162V and A162G were 9.6 ± 0.1, 9.5 ± 0.1 and 10.4 ± 0.2, respectively, different from that of wild-type MMP-7 (WT) (9.9 ± 0.1) by 0.3–0.5 pH unit, and those of P217S, P217V and P217G were 10.1 ± 0.1, 9.8 ± 0.1 and 9.7 ± 0.1, respectively, different from that of WT by 0.1–0.2 pH unit. These results suggest a possibility of W1 or W2 as the ionizable group for p K e2 .
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    Nucleocytoplasmic shuttling of IPAS by its unique nuclear import and export signals unshared with other HIF-3{alpha} splice variants (2013)
    Oxford University Press
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    Publication Date: 2013-12-03
    Description: Inhibitory Per/Arnt/Sim (PAS) domain protein (IPAS) is a splice variant of hypoxia-inducible factor (HIF)-3α, and possesses two entirely different functions. One is as a transcriptional repressor against HIF-dependent hypoxic gene activation. The other is as a pro-apoptotic factor by direct binding to the pro-survival protein Bcl-x L and its related proteins on mitochondria. Presently, the regulatory mechanism that determines the intracellular distribution of IPAS to fulfill each of the two functions is unknown. As a first step towards elucidation of the mechanism, nucleocytoplasmic transport signals of IPAS were explored. A bipartite-like nuclear localization signal (NLS) was found in the N-terminal region by the deletion and mutation analysis of EGFP-IPAS. In addition, the helix-loop-helix domain showed weak nuclear import/retention activity. A leptomycin B-sensitive nuclear export signal (NES) was localized in the C-terminal region of the protein. A proline-rich region supported the NES activity. These NLS and NES are not carried by the other variants of HIF-3α due to differential exon usage. These results strongly suggest that IPAS is a nucleocytoplasmic shuttling protein.
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    Enhanced expression of fucosyl GA1 in the digestive tract of immune-deficient scid, nude and pIgR(-/-) mice (2013)
    Oxford University Press
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    Publication Date: 2013-12-03
    Description: Fucosylation of GA1 in murine intestinal epithelia occurs through transcriptional induction of α1,2-fucosyltransferase along with bacterial infection, but the mechanism has not been clearly characterized as to whether it is induced as a result of an immune response to bacteria or of genetic manipulation of the host by bacteria. Accordingly, we analysed the expression of fucosyl GA1 (FGA1) and fucosyltransferase activity in the digestive tracts of immune-deficient scid, nude and pIgR(–/–) mice. In comparison with those in control mice bred under the same SPF circumstances, the amount of FGA1 and the α1,2-fucosyltransferase activity were significantly increased in the immune-deficient mice, indicating that the immune system is not involved in induction of the α1,2-fucosyltransferase gene. Reflecting the enhanced synthesis of FGA1, the total amounts of FGA1 in the intestinal contents of immune-deficient mice were higher than those in control mice. Also, the major faecal bacteria grown on a MRS agar plate were different in immune-deficient and control mice as follows, Lactobacillus murinus for scid and pIgR(–/–) mice, and Lactobacillus johnsonii for their control, and Enterococcus faecalis for nude mice and Lactococcus garvieae for the control, indicating that an alteration in the intestinal lactobacilli is partly involved in the induction of α1,2-fucosyltransferase.
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    Structures and reaction mechanisms of the two related enzymes, PurN and PurU (2013)
    Oxford University Press
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    Publication Date: 2013-12-03
    Description: The crystal structures of glycinamide ribonucleotide transformylases (PurNs) from Aquifex aeolicus ( Aa ), Geobacillus kaustophilus ( Gk ) and Symbiobacterium toebii ( St ), and of formyltetrahydrofolate hydrolase (PurU) from Thermus thermophilus ( Tt ) were determined. The monomer structures of the determined PurN and PurU were very similar to the known structure of PurN, but oligomeric states were different; Aa PurN and St PurN formed dimers, Gk PurN formed monomer and PurU formed tetramer in the crystals. PurU had a regulatory ACT domain in its N-terminal side. So far several structures of PurUs have been determined, yet, the mechanisms of the catalysis and the regulation of PurU have not been elucidated. We, therefore, modelled ligand-bound structures of PurN and PurU, and performed molecular dynamics simulations to elucidate the reaction mechanisms. The evolutionary relationship of the two enzymes is discussed based on the comparisons of the structures and the catalytic mechanisms.
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    Expression and structural characterization of anti-T-antigen single-chain antibodies (scFvs) and analysis of their binding to T-antigen by surface plasmon resonance and NMR spectroscopy (2013)
    Oxford University Press
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    Publication Date: 2013-12-03
    Description: T-antigen (Galβ1-3GalNAcα-1-Ser/Thr), also known as Thomsen–Friedenreich antigen (TF antigen), is an oncofetal antigen commonly found in cancerous tissues. Availability of anti-T-antigen human antibodies could lead to the development of cancer diagnostics and therapeutics. Four groups of single-chain variable fragment (scFv) genes were previously isolated from a phage library (Matsumoto-Takasaki et al. (2009) Isolation and characterization of anti-T-antigen single chain antibodies from a phage library. BioSci Trends 3:87–95.). Here, four anti-T-antigen scFv genes belonging to Group 1–4 were expressed and produced in a Drosophila S2 cell expression system. ELISA and surface plasmon resonance (SPR) analyses confirmed the binding activity of 1E8 scFv protein to various T-antigen presenting conjugates. NMR experiments provided evidence of the folded nature of the 1E8 scFv protein. ScFv-ligand contact was identified by STD NMR, indicating that the galactose unit of T-antigen at the non-reducing end was primarily recognized by 1E8 scFv. This thus provides direct evidence of T-antigen specificity.
    Print ISSN: 0021-924X
    Electronic ISSN: 1756-2651
    Topics: Biology , Chemistry and Pharmacology
    Published by Oxford University Press on behalf of The Japanese Biochemical Society (JBS).
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    Biochemical characterization of endonuclease V from the hyperthermophilic archaeon, Pyrococcus furiosus (2014)
    Oxford University Press
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    Publication Date: 2014-04-29
    Description: Endonuclease V (Endo V) is a DNA repair enzyme that recognizes deoxyinosine and cleaves the second phosphodiester bond on the 3' side of the deaminated base lesion. A database search revealed the presence of homologous genes for Endo V in most archaeal species, but the absence in some methanogenic species. We cloned a gene encoding the sequence homologous to Escherichia coli Endo V from the genome of the hyperthermophilic euryarchaeon, Pyrococcus furiosus and purified gene product (PfuEndoV) to homogeneity. In vitro characterization showed that PfuEndoV possesses specific endonuclease activity for the deoxyinosine-containing DNA strand. The activity of the enzyme was maximal at 90°C. Stable complex formation between PfuEndoV and nicked DNA produced by the cleavage reaction was detected by gel mobility shift assays. The molecular mechanisms of the inosine repair pathway including Endo V in the archaeal cells are discussed. Interestingly, PfuEndoV cleaved inosine-containing RNA strands as well as DNA substrates. PfuEndoV may also be involved in RNA metabolism.
    Print ISSN: 0021-924X
    Electronic ISSN: 1756-2651
    Topics: Biology , Chemistry and Pharmacology
    Published by Oxford University Press on behalf of The Japanese Biochemical Society (JBS).
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    A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid (2014)
    Oxford University Press
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    Publication Date: 2014-04-29
    Description: The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo . The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.
    Print ISSN: 0021-924X
    Electronic ISSN: 1756-2651
    Topics: Biology , Chemistry and Pharmacology
    Published by Oxford University Press on behalf of The Japanese Biochemical Society (JBS).
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    Evaluation of the stability of an SR398/GroES chaperonin complex (2014)
    Oxford University Press
    Details
    Publication Date: 2014-04-29
    Description: The stability of an SR398/GroES chaperonin complex was examined. As was expected, based on the finding of previous studies, the SR398/GroES complex was extremely stable in the presence of an excess amount of free adenosine 5'-[-thio]triphosphate (ATPS) or adenosine 5'-(β,-imido)triphosphate (AMPPNP). However, the complex was not stable in the absence of nucleotides. These results indicate that ATPS and AMPPNP repeatedly associated to and dissociated from the complex in a non-cooperative manner. This nucleotide exchange did not induce the dissociation of GroES and substrate from SR398, suggesting the importance of the cooperative dissociation of nucleotides from the cis -ring to release GroES and substrate proteins in the GroEL/GroES reaction cycle.
    Print ISSN: 0021-924X
    Electronic ISSN: 1756-2651
    Topics: Biology , Chemistry and Pharmacology
    Published by Oxford University Press on behalf of The Japanese Biochemical Society (JBS).
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