Identification of the gene for vitamin K epoxide reductase

Vitamin K epoxide reductase (VKOR) is the target of warfarin, the most widely prescribed anticoagulant for thromboembolic disorders. Although estimated to prevent twenty strokes per induced bleeding episode, warfarin is under-used because of the difficulty of controlling dosage and the fear of inducing bleeding. Although identified in 1974 (ref. 2), the enzyme has yet to be purified or its gene identified. A positional cloning approach has become possible after the mapping of warfarin resistance to rat chromosome 1 (ref. 3) and of vitamin K-dependent protein deficiencies to the syntenic region of human chromosome 16 (ref. 4). Localization of VKOR to 190 genes within human chromosome 16p12-q21 narrowed the search to 13 genes encoding candidate transmembrane proteins, and we used short interfering RNA (siRNA) pools against individual genes to test their ability to inhibit VKOR activity in human cells. Here, we report the identification of the gene for VKOR based on specific inhibition of VKOR activity by a single siRNA pool. We confirmed that MGC11276 messenger RNA encodes VKOR through its expression in insect cells and sensitivity to warfarin. The expressed enzyme is 163 amino acids long, with at least one transmembrane domain. Identification of the VKOR gene extends our understanding of blood clotting, and should facilitate development of new anticoagulant drugs.     

Identification of UBIAD1 as a novel human menaquinone-4 biosynthetic enzyme

Vitamin?K occurs in the natural world in several forms, including a plant form, phylloquinone (PK), and a bacterial form, menaquinones (MKs). In many species, including humans, PK is a minor constituent of hepatic vitamin?K content, with most hepatic vitamin?K content comprising long-chain MKs. Menaquinone-4 (MK-4) is ubiquitously present in extrahepatic tissues, with particularly high concentrations in the brain, kidney and pancreas of humans and rats. It has consistently been shown that PK is endogenously converted to MK-4 (refs 4-8). This occurs either directly within certain tissues or by interconversion to menadione (K(3)), followed by prenylation to MK-4 (refs 9-12). No previous study has sought to identify the human enzyme responsible for MK-4 biosynthesis. Previously we provided evidence for the conversion of PK and K(3) into MK-4 in mouse cerebra. However, the molecular mechanisms for these conversion reactions are unclear. Here we identify a human MK-4 biosynthetic enzyme. We screened the human genome database for prenylation enzymes and found UbiA prenyltransferase containing 1 (UBIAD1), a human homologue of Escherichia coli prenyltransferase menA. We found that short interfering RNA against the UBIAD1 gene inhibited the conversion of deuterium-labelled vitamin?K derivatives into deuterium-labelled-MK-4 (MK-4-d(7)) in human cells. We confirmed that the UBIAD1 gene encodes an MK-4 biosynthetic enzyme through its expression and conversion of deuterium-labelled vitamin?K derivatives into MK-4-d(7) in insect cells infected with UBIAD1 baculovirus. Converted MK-4-d(7) was chemically identified by (2)H-NMR analysis. MK-4 biosynthesis by UBIAD1 was not affected by the vitamin?K antagonist warfarin. UBIAD1 was localized in endoplasmic reticulum and ubiquitously expressed in several tissues of mice. Our results show that UBIAD1 is a human MK-4 biosynthetic enzyme; this identification will permit more effective decisions to be made about vitamin?K intake and bone health.     

Active gamma-carboxylated human factor IX expressed using recombinant DNA techniques

Factor IX (Christmas factor), a vitamin K-dependent plasma protein made in the liver, functions in the middle phase of the intrinsic pathway of blood coagulation. A functional deficiency of factor IX underlies haemophilia B, a chromosome X-linked recessive disease for which the major therapeutic approach is replacement treatment using factor IX concentrates. The cloning and characterization of the gene for human factor IX would mean that human factor IX could be produced in greater yield and purity through using recombinant DNA techniques. We have now used a human factor IX cDNA clone, inserted into a vaccinia virus-derived vector, to infect human hepatoma cells which normally produce no factor IX, and mouse fibroblasts. Fully active factor IX was produced by the hepatoma cells, whereas the fibroblasts produced a protein less active than natural factor IX, even in the presence of high levels of vitamin K. Human factor IX is extensively post-translationally modified, and thus represents probably the most complex protein produced in active form by recombinant DNA techniques to date. Our study also illustrates the potential of vaccinia virus-based vectors for expressing significant amounts of complex, clinically useful proteins in eukaryotic cells, in addition to its already demonstrated usefulness for producing live recombinant vaccines.     

Vitamin K-dependent blood coagulation proteins form hetero-dimers

The later stages of the blood coagulation cascade are characterized by the presence of vitamin K-dependent proteins and their involvement in membrane-bound, multi-protein converting complexes with an essential requirement for calcium ions. Specific interactions between zymogens and activating enzymes have not yet been identified. Here we describe a crystallographic study of prothrombin fragment 1 (residues 1-156 of prothrombin) which indicates that vitamin K-dependent coagulation proteins have specific association sites that allow them to form hetero-dimers. The calcium-induced formation of a hetero-dimer between fragment 1 and factor X is demonstrated by cross-linking. Such hetero-dimers of vitamin K-dependent proteins could be significant in the coagulation system.     

Crystal structure of human cytochrome P450 2C9 with bound warfarin

Cytochrome P450 proteins (CYP450s) are membrane-associated haem proteins that metabolize physiologically important compounds in many species of microorganisms, plants and animals. Mammalian CYP450s recognize and metabolize diverse xenobiotics such as drug molecules, environmental compounds and pollutants. Human CYP450 proteins CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are the major drug-metabolizing isoforms, and contribute to the oxidative metabolism of more than 90% of the drugs in current clinical use. Polymorphic variants have also been reported for some CYP450 isoforms, which has implications for the efficacy of drugs in individuals, and for the co-administration of drugs. The molecular basis of drug recognition by human CYP450s, however, has remained elusive. Here we describe the crystal structure of a human CYP450, CYP2C9, both unliganded and in complex with the anti-coagulant drug warfarin. The structure defines unanticipated interactions between CYP2C9 and warfarin, and reveals a new binding pocket. The binding mode of warfarin suggests that CYP2C9 may undergo an allosteric mechanism during its function. The newly discovered binding pocket also suggests that CYP2C9 may simultaneously accommodate multiple ligands during its biological function, and provides a possible molecular basis for understanding complex drug-drug interactions.     

抗凝血药物华法灵钠与人血清白蛋白的相互作用研究

用荧光光谱、紫外光谱和圆二色谱法研究了抗凝血药物华发灵钠与人血清白蛋白(HSA)的相互作用.初步确定华法灵钠对人血清白蛋白的荧光产生猝灭现象,猝灭方式为静态,并计算了二者形成化合物的结合常数K和结合位点数n.根据不同温度下的热力学函数,确定了华发灵钠与人血清白蛋白的相互作用力类型为氢键和范德华力.发现华法灵钠的存在明显改变人血清白蛋白的构象,并讨论了华发灵钠使人血清白蛋白构象发生变化的可能原因.     

Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation

Many bacterial pathogens secrete proteins that activate host trypsinogen-like enzyme precursors, most notably the proenzymes of the blood coagulation and fibrinolysis systems. Staphylococcus aureus, an important human pathogen implicated in sepsis and endocarditis, secretes the cofactor staphylocoagulase, which activates prothrombin, without the usual proteolytic cleavages, to directly initiate blood clotting. Here we present the 2.2 A crystal structures of human alpha-thrombin and prethrombin-2 bound to a fully active staphylocoagulase variant. The cofactor consists of two domains, each with three-helix bundles; this is a novel fold that is distinct from known serine proteinase activators, particularly the streptococcal plasminogen activator streptokinase. The staphylocoagulase fold is conserved in other bacterial plasma-protein-binding factors and extracellular-matrix-binding factors. Kinetic studies confirm the importance of isoleucine 1 and valine 2 at the amino terminus of staphylocoagulase for zymogen activation. In addition to making contacts with the 148 loop and (pro)exosite I of prethrombin-2, staphylocoagulase inserts its N-terminal peptide into the activation pocket of bound prethrombin-2, allosterically inducing functional catalytic machinery. These investigations demonstrate unambiguously the validity of the zymogen-activation mechanism known as 'molecular sexuality'.     

采用相对饱和深度评价潜艇脱险减压病危险性的动物模型研究

摘要: 目的观察相同减压负荷的模拟潜艇脱险( simulated submarine escape,sSE) 暴露和相对饱和深度( equal air depth,EAD) 饱和减压暴露减压反应程度的差异,评估采用EAD 饱和减压动物模型对潜艇脱险减压负荷的评价效果。方法山羊12 只,分为4 组,分别给予7 m 饱和后模拟150 m 潜艇脱险( 1 组,EAD = 12. 4 m) 、12. 4 m 饱和后直接减压( 2 组) 、12 m 饱和后模拟150 m 潜艇脱险( 3 组,EAD = 17. 1 m) 和17. 1 m 饱和后直接减压暴露( 4组) ,暴露前后采血检测血常规、血凝和NO、iNOS,暴露后15 min 观察行为表现并进行血管内气泡检测,观察动物存活情况。结果相同EAD 不同处理组间血管内气泡Spencer 分级、血常规和血凝指标、NO 和NOS 无显著性差异( P ＞ 0. 05) ; EAD 大的处理组( 3 组、4 组) 动物死亡率高( 50% ) ; 4 组较1 组PT 显著增加( P ＜ 0. 01) ,较2组APTT、PT 显著增加( P ＜ 0. 05) ; EAD 为17. 1 m 的实验组NO 含量显著高于EAD 为12. 4 m 的实验组( P ＜0. 01) 。结论采用EDA 计算不同压力预饱和暴露后的模拟潜艇脱险的减压负荷是可行的。红细胞的流变学变化和血管活性因子可能在机体的减压应激反应中起到了一定的作用,这为开展减压应激标志物的研究提供了参考。     

Specific inhibition of herpesvirus ribonucleotide reductase by synthetic peptides

Ribonucleotide reductase is an essential enzyme for DNA synthesis in all prokaryotic and eukaryotic cells; it catalyses the reductive conversion of ribonucleotides to deoxyribonucleotides. Several herpesviruses including herpes simplex virus type 1 (HSV-1), HSV-2, pseudorabies virus (PRV), equine herpesvirus type 1 (EHV-1) and Epstein-Barr virus (EBV) have been found to induce novel ribonucleotide reductase activities. There is evidence that the HSV-1 ribonucleotide reductase activity is virus-encoded and essential for virus replication. This makes herpesvirus ribonucleotide reductases potential targets for antiviral chemotherapy. The HSV-1-encoded enzyme consists of two subunits: V136, the large subunit of relative molecular mass (Mr) 136,000 (136K) (RR1), which has been shown to be essential for enzyme activity, and V38, the small subunit (RR2) which forms a complex with the large subunit and is also likely to be essential for enzyme activity. Two particular features of the enzyme make it an attractive antiviral target. First, there is evidence for a common, highly conserved herpesvirus ribonucleotide reductase and second, the interaction between the large and small subunits may itself be exploitable. Here we identify a synthetic peptide which specifically inhibits the activity of virus-induced enzyme. We deduce that the mechanism of inhibition involves interference with the normal interaction between the two types of subunit.     

Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex

The serine proteinase alpha-thrombin causes blood clotting through proteolytic cleavage of fibrinogen and protease-activated receptors and amplifies its own generation by activating the essential clotting factors V and VIII. Thrombomodulin, a transmembrane thrombin receptor with six contiguous epidermal growth factor-like domains (TME1-6), profoundly alters the substrate specificity of thrombin from pro- to anticoagulant by activating protein C. Activated protein C then deactivates the coagulation cascade by degrading activated factors V and VIII. The thrombin-thrombomodulin complex inhibits fibrinolysis by activating the procarboxypeptidase thrombin-activatable fibrinolysis inhibitor. Here we present the 2.3 A crystal structure of human alpha-thrombin bound to the smallest thrombomodulin fragment required for full protein-C co-factor activity, TME456. The Y-shaped thrombomodulin fragment binds to thrombin's anion-binding exosite-I, preventing binding of procoagulant substrates. Thrombomodulin binding does not seem to induce marked allosteric structural rearrangements at the thrombin active site. Rather, docking of a protein C model to thrombin-TME456 indicates that TME45 may bind substrates in such a manner that their zymogen-activation cleavage sites are presented optimally to the unaltered thrombin active site.     

Specific inhibition of herpesvirus ribonucleotide reductase by a nonapeptide derived from the carboxy terminus of subunit 2

Ribonucleotide reductase, an essential enzyme for the synthesis of deoxyribonucleotides, is formed by the association of two nonidentical subunits in almost all prokaryotic and eukaryotic cells. The same model probably holds for the herpes simplex virus (HSV)-encoded ribonucleotide reductase; two polypeptides of relative molecular mass 136,000 (136K; H1) and 40K (H2) (referred to elsewhere as RR1 and RR2; see for example, Dutia et al.) have been associated with the viral enzyme by both genetic and immunological studies. Furthermore, DNA sequence analyses have shown significant stretches of amino-acid homology between these viral polypeptides and those of, respectively, subunit 1 (ref. 12) and subunit 2 (ref. 13) of the Escherichia coli and mammalian enzymes. To assess the involvement of the 40K polypeptide in reductase activity, we synthesized a nonapeptide corresponding to the sequence of its carboxy terminus with the intention of raising neutralizing antibodies specific for the viral activity (E.A.C. et al., in preparation). We report here the unexpected finding that the nonapeptide itself specifically inhibits the HSV ribonucleotide reductase activity in a reversible, non-competitive manner, and we suggest that it does this by impairment of the correct association of the two subunits. This phenomenon emphasizes the potential usefulness of synthetic peptides in probing critical sites involved in macromolecular interactions.     

Is ribonucleotide reductase the transforming function of herpes simplex virus 2?

Transformation of cells by herpes simplex virus 2 (HSV-2) can be induced by the BglII C (0.43-0.58 map units) or N (0.58-0.625) fragments of the viral genome. Sequences partially overlapping both fragments (0.566-0.602) encode two 3' coterminal mRNAs; these in turn direct the synthesis of two related polypeptides of molecular weight 140,000 (140K) and 35K (refs 4, 7), which may be involved in transformation. Recently, a temperature-sensitive (ts) mutation affecting HSV-induced ribonucleotide reductase has been mapped within this common region (B.M. Dutia, personal communication). We have partially purified the induced reductase and raised a rabbit antiserum to it which inhibits the enzyme activity and immunoprecipitates from infected cells a 144K polypeptide and minor species including a 38K polypeptide. Here we show that a monoclonal antibody to the putative transforming proteins competes with the rabbit serum for the 144K and 38K antigens and also immunoprecipitates specifically the induced reductase activity. These results suggest a possible role for ribonucleotide reductase in HSV-2-induced transformation.     

人工饲养树鼩和猕猴的部分凝血因子凝集时间的测定及分析

目的目前有关人工饲养树鼩与猕猴部分凝血因子凝集时间的资料甚少,拟初步建立这两种动物部分凝血因子凝集时间的参考值范围。方法采用全自动血凝仪测定树鼩、猕猴的血浆凝血酶时间（TT）、纤维蛋白原（Fib）、活化部分凝血活酶时间（APTT）、凝血酶原时间（PT）。结果树鼩的TT、PT、Fib和APTT值分别为19.27、17.34、30.51和27.88 s。猕猴的TT、PT、Fib和APTT值分别为20.81、9.82、18.73和33.56 s。人工饲养树鼩与猕猴的PT值、Fib值存在显著性差异（P〈0.01）,APTT值、TT值存在差异（P〈0.05）。结论人工饲养树鼩和猕猴部分凝血因子的凝集时间存在一定差异,同一物种雌雄个体间部分凝血因子的凝集时间没有明显差异。     

维生素K3对安定血药浓度及药理作用的影响

用动物实验研究了维生素Ｋ３对安定血药浓度及药理作用的影响。结果表明，维生素Ｋ３能明显缩短安定血药浓度分布半衰期和消除半衰期，提高安定体内清除率。增加家兔呼吸频率，但不影响麻醉后家兔的心电图、血压和心率。能缩短安定所致小鼠睡眠时间，具有催醒作用。     

Structure of the C2 domain of human factor VIII at 1.5 A resolution

Human factor VIII is a plasma glycoprotein that has a critical role in blood coagulation. Factor VIII circulates as a complex with von Willebrand factor. After cleavage by thrombin, factor VIIIa associates with factor IXa at the surface of activated platelets or endothelial cells. This complex activates factor X (refs 6, 7), which in turn converts prothrombin to thrombin in the presence of factor Va (refs 8, 9). The carboxyl-terminal C2 domain of factor VIII contains sites that are essential for its binding to von Willebrand factor and to negatively charged phospholipid surfaces. Here we report the structure of human factor VIII C2 domain at 1.5 A resolution. The structure reveals a beta-sandwich core, from which two beta-turns and a loop display a group of solvent-exposed hydrophobic residues. Behind the hydrophobic surface lies a ring of positively charged residues. This motif suggests a mechanism for membrane binding involving both hydrophobic and electrostatic interactions. The structure explains, in part, mutations in the C2 region of factor VIII that lead to bleeding disorders in haemophilia A.     

Homogeneous interferon-inducing 22K factor is related to endogenous pyrogen and interleukin-1

In vitro stimulation of mononuclear cells from human peripheral blood with mitogens causes the release of factors (monokines and lymphokines) which possess distinct biological activities. One such factor, termed 22K, can induce production of human beta-interferon (HuIFN-beta) in cultured human fibroblasts, thereby rendering these cells resistant to virus infection. Here we report the complete purification and partial sequencing (39 N-terminal amino acids) of this factor, whose relative molecular mass was estimated by SDS-polyacrylamide gel electrophoresis to be 17,000 (17K). In addition to an antiviral effect, the pure protein exhibits several other biological activities. Most significantly, intravenous (i.v.) injection of the factor in rabbits caused fever and granulopenia at doses of 0.1-1 microgram per kg, effects which we attribute to a monokine called endogenous pyrogen (EP). In vitro, the protein was scored as positive in a LAF (lymphocyte-activating factor) assay at 0.1-1 ng ml-1. LAF and EP are considered to be members of one family of monokines, called interleukin-1 (IL-1). For this reason, and also because the amino-acid sequence of the 22K factor is at least partially homologous to a complementary DNA-derived IL-1 sequence, we postulate that the 22K factor also belongs to the IL-1 family.     

Plasma protease inhibitors in mouse and man: divergence within the reactive centre regions

The plasma protease inhibitors control a wide variety of physiological functions including blood coagulation, complement activation and aspects of the inflammatory response. The inhibitors function by forming a 1:1 complex with a specific protease within the reactive centre region of the inhibitor. Little is known about the evolutionary relationships of these inhibitors. We report here the sequences of cDNAs which represent the C-terminal halves of the two major murine plasma protease inhibitors. One of these, murine alpha 1-antitrypsin, more appropriately called alpha 1-proteinase inhibitor (alpha 1-PI), has diverged from its human counterpart at a vital position in the reactive centre but this has not led to a physiologically significant change in function. Also, we have determined the partial sequence of a recently characterized protein termed contrapsin, which inhibits trypsin-like proteases. We show, surprisingly, that contrapsin is highly homologous to human alpha 1-antichymotrypsin, an inhibitor of chymotrypsin-like proteases. The reactive centre regions of these two inhibitors have diverged considerably, which may account for the differences in specificity. We propose that the genes for contrapsin and human alpha 1-antichymotrypsin are the descendents of a single gene that have evolved since rodent and primate divergence to encode proteins with different functions.     

甲醇-甲乙酮-1-辛基-3-甲基咪唑六氟磷酸盐物系等压汽液平衡数据的测定

在101.3kPa下，用改进的Othmer釜测定了含摩尔分数分别为5%、8%、10%和20%离子液体1-辛基-3-甲基咪唑六氟磷酸盐（[OMIM]PF₆）的甲醇-甲乙酮-[OMIM]PF₆汽液平衡数据，并采用NRTL方程进行关联。实验结果表明，当离子液体[OMIM]PF₆的摩尔分数为5%时，甲醇-甲乙酮二组分物系汽液平衡线发生偏离，当离子液体[OMIM]PF₆摩尔分数达到8%时，就可以消除甲醇-甲乙酮物系的共沸点；离子液体[OMIM]PF₆摩尔分数越大，盐效应越明显，离子液体[OMIM]PF₆可以作为甲醇-甲乙酮的萃取剂。     

TAK1 is a ubiquitin-dependent kinase of MKK and IKK.

TRAF6 is a signal transducer that activates IkappaB kinase (IKK) and Jun amino-terminal kinase (JNK) in response to pro-inflammatory mediators such as interleukin-1 (IL-1) and lipopolysaccharides (LPS). IKK activation by TRAF6 requires two intermediary factors, TRAF6-regulated IKK activator 1 (TRIKA1) and TRIKA2 (ref. 5). TRIKA1 is a dimeric ubiquitin-conjugating enzyme complex composed of Ubc13 and Uev1A (or the functionally equivalent Mms2). This Ubc complex, together with TRAF6, catalyses the formation of a Lys 63 (K63)-linked polyubiquitin chain that mediates IKK activation through a unique proteasome-independent mechanism. Here we report the purification and identification of TRIKA2, which is composed of TAK1, TAB1 and TAB2, a protein kinase complex previously implicated in IKK activation through an unknown mechanism. We find that the TAK1 kinase complex phosphorylates and activates IKK in a manner that depends on TRAF6 and Ubc13-Uev1A. Moreover, the activity of TAK1 to phosphorylate MKK6, which activates the JNK-p38 kinase pathway, is directly regulated by K63-linked polyubiquitination. We also provide evidence that TRAF6 is conjugated by the K63 polyubiquitin chains. These results indicate that ubiquitination has an important regulatory role in stress response pathways, including those of IKK and JNK.