Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publisher：Japanese Proteomics Society (Japan Human Proteome Organisation)  

DOI： 10.14889/jhupo.2012.0.56.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The chromodomain helicase DNA-binding (CHD) family of enzymes is thought to regulate gene expression, but their role in the regulation of specific genes has been unclear. Here we show that CHD8 is expressed at a high level during early embryogenesis and prevents apoptosis mediated by the tumour suppressor protein p53. CHD8 was found to bind to p53 and to suppress its transactivation activity. CHD8 promoted the association of p53 and histone H1, forming a trimeric complex on chromatin that was required for inhibition of p53-dependent transactivation and apoptosis. Depletion of CHD8 or histone H1 resulted in p53 activation and apoptosis. Furthermore, Chd8(-/-) mice died early during embryogenesis, manifesting widespread apoptosis, whereas deletion of p53 ameliorated this developmental arrest. These observations reveal a mode of p53 regulation mediated by CHD8, which may set a threshold for induction of apoptosis during early embryogenesis by counteracting p53 function through recruitment of histone H1.

DOI： 10.1038/ncb1831

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

The authors have investigated gene expression of ST2 in the lung tissue of a bleomycin (BLM)-induced lung fibrosis model in vivo and in a human lung fibroblast cell line, WI38, and a human type II alveolar epithelial cell line, A549, reacting to proinflammatory and type 2 helper T cell (Th2)-type cytokine stimuli in vitro. The lung mRNA expression of interleukin (IL)-4, IL-5, IL-1 beta, and tumor necrosis factor (TNT)-alpha increased significantly at day 7 after instillation of BLM, whereas interferon (IFN)-gamma mRNA expression did not increase. ST2 and transforming growth factor (TGF)-beta 1 mRNA expression of the lung increased significantly between days 7 and 21, and increased to maximal levels at day 14 post-BLM challenge. ST2 mRNA expression statistically correlated with TGF-beta 1 mPLNA expression. In addition, the combination of IL-1 beta, TAT-alpha, and IL-4 had an additive effect on ST2 mRNA expression from A549 cells and WI38 cells. These findings suggest that soluble ST2 gene may increase, possibly reflecting the development of the inflammatory process and the Th2-type immune response in the fibrotic lung tissue, and may modulate a process of pulmonary fibrosis.

DOI： 10.1080/01902140701198583

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CLINICAL INVESTIGATION INC  

Factors that determine the spectrum of target organs involved in autoimmune destruction are poorly understood. Although loss of function of autoimmune regulator (AIRE) in thymic epithelial cells is responsible for autoimmunity, the pathogenic roles of AIRE in regulating target-organ specificity remain elusive. In order to gain insight into this issue, we have established NOD mice, an animal model of type 1 diabetes caused by autoimmune attack against P cell islets, in which Aire has been abrogated. Remarkably, acinar cells rather than P cell islets were the major targets of autoimmune destruction in Aire-deficient NOD mice, and this alteration of intra-pancreatic target-organ specificity was associated with production of autoantibody against pancreas-specific protein disulfide isomerase (PDIp), an antigen expressed predominantly by acinar cells. Consistent with this pathological change, the animals were resistant to the development of diabetes. The results suggest that Aire not only is critical for the control of self-tolerance but is also a strong modifier of target-organ specificity through regulation of T cell repertoire diversification. We also demonstrated that transcriptional expression of PDIp was retained in the Aire-deficient NOD thymus, further supporting the concept that Aire may regulate the survival of autoreactive T cells beyond transcriptional control of self-protein expression in the thymus.

DOI： 10.1172/JCI26971

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

I kappa B kinase (IKK) alpha exhibits diverse biological activities through protein kinase-dependent and -independent functions, the former mediated predominantly through a noncanonical NF-kappa B activation pathway. The in vivo function of IKK alpha, however, still remains elusive. Because a natural strain of mice with mutant NF-kappa B-inducing kinase (NIK) manifests autoimmunity as a result of disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma, we speculated that the NIK-IKK alpha axis might constitute an essential step in the thymic organogenesis that is required for the establishment of self-tolerance. An autoimmune disease phenotype was induced in athymic nude mice by grafting embryonic thymus from IKK alpha-deficient mice. The thymic microenvironment that caused autoimmunity in an IKK alpha-dependent manner was associated with defective processing of NF-kappa B2, resulting in the impaired development of thymic epithelial cells. Thus, our results demonstrate a novel function for IKK alpha in thymic organogenesis for the establishment of central tolerance that depends on its protein kinase activity in cooperation with NIK.

DOI： 10.4049/jimmunol.176.7.3995

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Autoimmune regulator ( AIRE) is responsible for the development of organ-specific autoimmune disease in a monogenic fashion. Rare and low levels of tissue expression together with the lack of AIRE-expressing cell lines have hampered a detailed analysis of the molecular dynamics of AIRE. Here we have established cell lines stably transfected with AIRE and studied the regulatory mechanisms for its subcellular expression. We found that nuclear body ( NB) formation by AIRE was dependent on the cell cycle. Biochemical fractionation revealed that a significant proportion of AIRE is associated with the nuclear matrix, which directs the functional domains of chromatin to provide sites for gene regulation. Upon proteasome inhibition, AIRE NBs were increased with concomitant reduced expression in the cytoplasm, suggesting that subcellular targeting of AIRE is regulated by a ubiquitin-proteasome pathway. We also found that AIRE NBs compete for cAMP-response element-binding protein-binding protein/p300, a common coactivator of transcription, with the promyelocytic leukemia gene product. These results suggest that the transcriptional regulating activities of AIRE within a cell are controlled and organized in a spatiotemporal manner.

DOI： 10.1074/jbc.M400702200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The SCF complex, which consists of the invariable components Skp1, Cul1, and Rbx1 as well as a variable F-box protein, functions as an E3 ubiquitin ligase. The mechanism by which the activity of this complex is regulated, however, has been unclear. The application of tandem affinity purification has now resulted in the identification of a novel Cul1-binding protein: TATA-binding protein-interacting protein 120A (TIP120A, also called CAND1). Immunoprecipitation, immunoblot, and immunofluorescence analyses with mammalian cells revealed that TIP120A physically associates with Cull in the nucleus and that this interaction is mediated by a central region of Cull distinct from its binding sites for Skp1 and Rbx1. Furthermore, TIP120A was shown to interact selectively with Cull that is not modified by NEDD8. The Cul1-TIP120A complex does not include Skp1, raising the possibility that TIP120A competes with Skp1 for binding to Cul1. These observations thus suggest that TIP120A may function as a negative regulator of the SCF complex by binding to nonneddylated Cul1 and thereby preventing assembly of this ubiquitin ligase. (C) 2003 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(03)00501-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Targeting of the cyclin-dependent kinase inhibitor p27(Kip1) for proteolysis has been thought to be mediated by Skp2, the F-box protein component of an SCF ubiquitin ligase complex. Degradation of p27(Kip1) at the Go-G, transition of the cell cycle has now been shown to proceed normally in Shp2(-/-) lymphocytes, whereas p27(Kip1) proteolysis during S-G(2) phases is impaired in these Skp2-deficient cells. Degradation of p27(Kip1) at the G,-G(1), transition was blocked by lactacystin, a specific proteasome inhibitor, suggesting that it is mediated by the ubiquitin-proteasome pathway. The first cell cycle of stimulated Shp2(-/-) lymphocytes appeared normal, but the second cycle was markedly inhibited, presumably as a result of P27(Kip1) accumulation during S-G, phases of the first cell cycle. Polyubiquitination of p27(Kip1) in the nucleus is dependent on Skp2 and phosphorylation of P27(Kip1) on threonine 187. However, polyubiquitination activity was also detected in the cytoplasm of Shp2(-/-) cells, even with a threonine 187 --> alanine mutant of P27(Kip1) as substrate. These results suggest that a polyubiquitination activity in the cytoplasm contributes to the early phase of p27(Kip1) degradation in a Skp2-independent manner, thereby promoting cell cycle progression from Go to G,.

DOI： 10.1074/jbc.M107274200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

A peptidase was purified from seeds of Canavalia ensiformis by extraction with water, ammonium sulfate precipitation, and successive chromatographies on DEAE-Toyopearl 650M, butyl-Toyopearl 650M, and G-3000 SW columns. The enzyme has an apparent molecular weight of 41,000. Activity is maximal at pH 9 and 60 degreesC. The enzyme hydrolyzed synthetic substrates at Arg-X and Lys-X bonds more rapidly than bovine trypsin did, and did not cleave protein or ester substrates. The enzyme was inhibited by alkylamines and several serine protease inhibitors such as diisopropylfluorophosphate, chymostatin, leupeptin, and benzamidine. Cysteine protease-, metalloprotease-, and proteinous trypsin inhibitors were ineffective. Inhibition by alkylamines was dependent on length of the alkyl chains. From the substrate specificity and susceptibility to chemicals, the enzyme is a unique peptidase with trypsin-like specificity.

DOI： 10.1271/bbb.64.2186

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

The complete amino acid sequence of brevilysin H6 (H6), a zinc-protease isolated from Gloydius halys brevicaudus venom, was determined by a manual Edman degradation method. HG has an amino-terminal pyroglutamic acid and consists of a total of 419 residues, An N-linked sugar chain is attached at Asn-181, The molecule is composed of three domains (metalloprotease, disintegrin-like and cysteine-rich domains), as commonly found in other high molecular mass: metalloproteases from snake venoms. In the absence of calcium ions, H6 is autocatalytically degraded with a half-life of 47 min to give 29 and 45 kDa fragments, which correspond to residues 208-419 and 99-419 of H6, respectively. Thus, the autoproteolysis seemed to start from the cleavage of either the Leu(98)-Leu(99) or Asp(207)-Ile(208) bond. Calcium ions suppressed both the formation of the 45 kDa fragment and the rate of autoproteolysis, Calcium ions also contributed to the stability of BG against pH, heating, urea and cysteine. More than twenty-five peptide bonds adjacent to hydrophobic residues in the metalloprotease domain were progressively cleaved during the autoproteolysis.

DOI： 10.1093/oxfordjournals.jbchem.a022737

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Two platelet aggregation inhibitors, ussuristatin 1 (US-l) and 2 (US-2), were newly isolated from the venom of Chinese viper (Agkistrodon ussuriensis) by SP-Toyopearl 650M column chromatography and reverse-phase HPLC. The Ms of these polypeptides were estimated to be about 8,000 by SDS-PAGE. Analytical gel filtration revealed that US-2 exists as a dimer, Both polypeptides comprised 71 amino acids, whose sequences showed high similarities to those of other disintegrins. US-1 had a typical Arg-Gly-Asp (RGD) sequence, which is responsible for blocking the binding of fibrinogen to the receptor. In US-2, the corresponding sequence was Lys-G;ly-Asp (KGD). US-1 strongly suppressed platelet aggregation induced by ADP, collagen, thrombin, and epinephrine with IC50=17-33 nM. US-2 also inhibited the platelet aggregation, but the IC(50)s were about ten times higher. US-1 also dose-dependently inhibited the adhesion of human melanoma cells to fibrinogen and fibronectin, while US-2 did not inhibit the cell adhesion to fibronectin. This indicates that the KGD-bearing disintegrin is a specific inhibitor for the fibrinogen receptor.

DOI： 10.1093/oxfordjournals.jbchem.a022264

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