Journal of Biochemistry - Aktuelle Forschungsartikel
Aktuelle Forschungsartikel: Biochemie
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Journal of Biochemistry - Verlag: Oxford Journals - Herausgeber: The Japanese Biochemical Society
JB veröffentlicht die Ergebnisse aktueller Forschung auf den Gebieten der Biochemie, Molekularbiologie, Zellbiologie und Biotechnologie.
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.
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-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]–L-Ala–L-Arg–NH2. 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.
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.
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 Ca2+ ion levels ([Ca2+]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 A1 (PS-PLA1) that deacylates fatty acid at the sn-1 position of PS and produces 2-acyl-LysoPS, but not catalytically inactive mutant PS-PLA1, 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-PLA1 but not with the mutant PS-PLA1. PS treated with PLA1 was much more effective at stimulating AP-TGFα release than PS treated with PLA2. 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.
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.
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.
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.
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.
Human matrix metalloproteinase 7 (MMP-7) activity exhibits broad bell-shaped pH profile with the acidic and alkaline pKa (pKe1 and pKe2) values of about 4 and 10. The ionizable group for pKe2 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 pKe2 [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-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2, 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. pKe2 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 pKe2.
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, Km 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.
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.
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.
Posted on 26 April 2012 | 6:43 am
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