Peptide exosite inhibitors of factor VIIa as anticoagulants

Potent anticoagulants have been derived by targeting the tissue factor-factor VIIa complex with naive peptide libraries displayed on M13 phage. The peptides specifically block the activation of factor X with a median inhibitory concentration of 1 nM and selectively inhibit tissue-factor-dependent clotting. The peptides do not bind to the active site of factor VIIa; rather, they work by binding to an exosite on the factor VIIa protease domain, and non-competitively inhibit activation of factor X and amidolytic activity. One such peptide (E-76) has a well defined structure in solution determined by NMR spectroscopy that is similar to the X-ray crystal structure when complexed with factor VIIa. These structural and functional studies indicate an allosteric 'switch' mechanism of inhibition involving an activation loop of factor VIIa and represent a new framework for developing inhibitors of serine proteases.     

Key residues involved in calcium-binding motifs in EGF-like domains

Many extracellular proteins with diverse functions contain domains similar to epidermal growth factor (EGF), a number of which have a consensus Asp/Asn, Asp/Asn, Asp*/Asn*, Tyr/Phe (where the asterisk denotes a beta-hydroxylated residue). These include the coagulation factors IX and X, proteins with two EGF-like domains, the first of which contains the consensus residues. The first EGF-like domain of human factor IX contains a calcium-binding site, which is believed to be responsible for one of the high-affinity sites detected in this protein. Similar results have been obtained for bovine factor X. We have now used protein engineering and 1H-NMR techniques to investigate the importance of individual consensus residues for ligand binding. Measurement of a calcium-dependent Tyr 69 shift in the isolated first EGF-like domain from human factor IX demonstrates that Asp 47, Asp 49, and Asp 64 are directly involved in this binding. Gln 50, whose importance has previously been overlooked, is also involved in this binding. Two mutations in this domain, Asp 47----Glu, and Asp 64----Asn, present in patients with haemophilia B, reduce calcium binding to the domain greater than 4-fold and greater than 1,000-fold, respectively. Furthermore, the defective calcium binding of Asn 64 can be partially rescued by the compensatory mutation Gln 50----Glu. This latter mutation, when introduced singly more than doubles the affinity of the domain for calcium. This study thus defines residues involved in a new type of calcium-binding site and provides strong circumstantial evidence for calcium-binding motifs in many extracellular proteins, including the developmentally important proteins of Drosophila, notch, delta and crumbs.     

The secreted form of the Alzheimer's amyloid precursor protein with the Kunitz domain is protease nexin-II

The A4 protein (or beta-protein) is a 42- or 43-amino-acid peptide present in the extracellular neuritic plaques in Alzheimer's disease and is derived from a membrane-bound amyloid protein precursor (APP). Three forms of APP have been described and are referred to as APP695, APP751 and APP770, reflecting the number of amino acids encoded for by their respective complementary DNAs. The two larger APPs contain a 57-amino-acid insert with striking homology to the Kunitz family of protease inhibitors. Here we report that the deduced amino-terminal sequence of APP is identical to the sequence of a cell-secreted protease inhibitor, protease nexin-II (PN-II). To confirm this finding, APP751 and APP695 cDNAs were over-expressed in the human 293 cell line, and the secreted N-terminal extracellular domains of these APPs were purified to near homogeneity from the tissue-culture medium. The relative molecular mass and high-affinity binding to dextran sulphate of secreted APP751 were consistent with that of PN-II. Functionally, secreted APP751 formed stable, non-covalent, inhibitory complexes with trypsin. Secreted APP695 did not form complexes with trypsin. We conclude that the secreted form of APP with the Kunitz protease inhibitor domain is PN-II.     

A chain initiation factor common to both modular and aromatic polyketide synthases.

Antibiotic-producing polyketide synthases (PKSs) are enzymes responsible for the biosynthesis in Streptomyces and related filamentous bacteria of a remarkably broad range of bioactive metabolites, including antitumour aromatic compounds such as mithramycin and macrolide antibiotics such as erythromycin. The molecular basis for the selection of the starter unit on aromatic PKSs is unknown. Here we show that a component of aromatic PKS, previously named 'chain-length factor', is a factor required for polyketide chain initiation and that this factor has decarboxylase activity towards malonyl-ACP (acyl carrier protein). We have re-examined the mechanism of initiation on modular PKSs and have identified as a specific initiation factor a domain of previously unknown function named KSQ, which operates like chain-length factor. Both KSQ and chain-length factor are similar to the ketosynthase domains that catalyse polyketide chain extension in modular multifunctional PKSs and in aromatic PKSs, respectively, except that the ketosynthase domain active-site cysteine residue is replaced by a highly conserved glutamine in KSQ and in chain-length factor. The glutamine residue is important both for decarboxylase activity and for polyketide synthesis.     

Tissue factor and PAR1 promote microbiota-induced intestinal vascular remodelling

The gut microbiota is a complex ecosystem that has coevolved with host physiology. Colonization of germ-free (GF) mice with a microbiota promotes increased vessel density in the small intestine, but little is known about the mechanisms involved. Tissue factor (TF) is the membrane receptor that initiates the extrinsic coagulation pathway, and it promotes developmental and tumour angiogenesis. Here we show that the gut microbiota promotes TF glycosylation associated with localization of TF on the cell surface, the activation of coagulation proteases, and phosphorylation of the TF cytoplasmic domain in the small intestine. Anti-TF treatment of colonized GF mice decreased microbiota-induced vascular remodelling and expression of the proangiogenic factor angiopoietin-1 (Ang-1) in the small intestine. Mice with a genetic deletion of the TF cytoplasmic domain or with hypomorphic TF (F3) alleles had a decreased intestinal vessel density. Coagulation proteases downstream of TF activate protease-activated receptor (PAR) signalling implicated in angiogenesis. Vessel density and phosphorylation of the cytoplasmic domain of TF were decreased in small intestine from PAR1-deficient (F2r(-/-)) but not PAR2-deficient (F2rl1(-/-)) mice, and inhibition of thrombin showed that thrombin-PAR1 signalling was upstream of TF phosphorylation. Thus, the microbiota-induced extravascular TF-PAR1 signalling loop is a novel pathway that may be modulated to influence vascular remodelling in the small intestine.     

Kunitz抑制剂家族成员抑肽酶对纤溶酶原活化的抑制作用

抑肽酶属Kunitz抑制剂家族成员，能够抑制激肽释放酶、纤溶酶及胰蛋白酶的蛋白水解活性．研究表明，抑肽酶能够抑制尿激酶型-纤溶酶原激活刹（u—PA）和组织型纤溶酶原激活剂（t—PA）对纤溶酶原的激活，但不影响u—PA和t—PA对小分子底物的酰胺水解活性．用u—PA研究了上述作用的机制，发现抑肽酶与u—PA的丝氨酸蛋白酶功能区特异性结合，而与纤溶酶原没有相互作用．抑肽酶与uPA的结合并不阻断u—PA的活性位点，因为u—PA对小分子底物的水解活性仍然保持．上述发现提示抑肽酶可能存在另一种抑制作用模式，该模式不同于以前报道的关于Kunitz抑制剂或纤溶酶原激活酶抑制剂的作用．由于人体内的Kunitz抑制剂与抑肽酶在结构上非常相似，根据研究结果，推测体内纤溶酶原的激活作用并非仅受丝氨酸蛋白酶抑制剂的控制。     

一个在麻疯树根和茎高表达的Kunitz型蛋白酶抑制剂基因JcKTI具有抗虫活性

通过RT-PCR和PCR技术,从麻疯树基因组中克隆得到一个Kunitz型蛋白酶抑制剂基因（JcKTI）的开放阅读框序列。对应开放读码框的基因组序列不含有内含子。该开放阅读框长度为540bp,编码一个含有179个氨基酸残基的成熟多肽,具有典型的Kunitz家族结构特征。组织特异性表达研究显示,JcKTI基因在根和茎中的表达丰度最高,在叶片和种子中表达较低。构建原核表达载体pET32-JcKTI在大肠杆菌BL21中表达,获得纯化的重组蛋白,该蛋白显示出一定的抑制牛胰蛋白酶的活性。将该基因在烟草中过表达,明胶酶法和BAEE法的结果均显示转基因植物的蛋白提取物对胰蛋白酶具有一定抑制作用,进一步的抗虫实验结果表明转基因烟草叶片可使进食后的棉铃虫幼虫生长发育受阻,并减少对叶片的吞食。上述结果暗示JcKTI基因可能在麻疯树根和茎的抗虫应答中扮演着一定的角色。     

Structure of porphobilinogen deaminase reveals a flexible multidomain polymerase with a single catalytic site.

The three-domain structure of porphobilinogen deaminase, a key enzyme in the biosynthetic pathway of tetrapyrroles, has been defined by X-ray analysis at 1.9 A resolution. Two of the domains structurally resemble the transferrins and periplasmic binding proteins. The dipyrromethane cofactor is covalently linked to domain 3 but is bound by extensive salt-bridges and hydrogen-bonds within the cleft between domains 1 and 2, at a position corresponding to the binding sites for small-molecule ligands in the analogous proteins. The X-ray structure and results from site-directed mutagenesis provide evidence for a single catalytic site. Interdomain flexibility may aid elongation of the polypyrrole product in the active-site cleft of the enzyme.     

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.     

Mobile reactive centre of serpins and the control of thrombosis

Two protease inhibitors in human plasma play a key part in the control of thrombosis: antithrombin inhibits coagulation and the plasminogen activator inhibitor PAI-1 inhibits fibrinolysis, the dissolving of clots. Both inhibitors are members of the serpin family and both exist in the plasma in latent or inactive forms. We show here that the reactive centre of the serpins can adopt varying conformations and that mobility of the reactive centre is necessary for the function of antithrombin and its binding and activation by heparin; the identification of a new locked conformation explains the latent inactive state of PAI-1. This ability to vary conformation not only allows the modulation of inhibitory activity but also protects the circulating inhibitor against proteolytic attack. Together these findings explain the retention by the serpins of a large and unconstrained reactive centre as compared to the small fixed peptide loop of other families of serine protease inhibitors.     

Crystal structure of the anthrax lethal factor.

Lethal factor (LF) is a protein (relative molecular mass 90,000) that is critical in the pathogenesis of anthrax. It is a highly specific protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near to their amino termini, leading to the inhibition of one or more signalling pathways. Here we describe the crystal structure of LF and its complex with the N terminus of MAPKK-2. LF comprises four domains: domain I binds the membrane-translocating component of anthrax toxin, the protective antigen (PA); domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of MAPKK-2 before cleavage. Domain II resembles the ADP-ribosylating toxin from Bacillus cereus, but the active site has been mutated and recruited to augment substrate recognition. Domain III is inserted into domain II, and seems to have arisen from a repeated duplication of a structural element of domain II. Domain IV is distantly related to the zinc metalloprotease family, and contains the catalytic centre; it also resembles domain I. The structure thus reveals a protein that has evolved through a process of gene duplication, mutation and fusion, into an enzyme with high and unusual specificity.     

The structures of HsIU and the ATP-dependent protease HsIU-HsIV

The degradation of cytoplasmic proteins is an ATP-dependent process. Substrates are targeted to a single soluble protease, the 26S proteasome, in eukaryotes and to a number of unrelated proteases in prokaryotes. A surprising link emerged with the discovery of the ATP-dependent protease HslVU (heat shock locus VU) in Escherichia coli. Its protease component HslV shares approximately 20% sequence similarity and a conserved fold with 20S proteasome beta-subunits. HslU is a member of the Hsp100 (Clp) family of ATPases. Here we report the crystal structures of free HslU and an 820,000 relative molecular mass complex of HslU and HslV-the first structure of a complete set of components of an ATP-dependent protease. HslV and HslU display sixfold symmetry, ruling out mechanisms of protease activation that require a symmetry mismatch between the two components. Instead, there is conformational flexibility and domain motion in HslU and a localized order-disorder transition in HslV. Individual subunits of HslU contain two globular domains in relative orientations that correlate with nucleotide bound and unbound states. They are surprisingly similar to their counterparts in N-ethylmaleimide-sensitive fusion protein, the prototype of an AAA-ATPase. A third, mostly alpha-helical domain in HslU mediates the contact with HslV and may be the structural equivalent of the amino-terminal domains in proteasomal AAA-ATPases.     

cDNA sequence of human apolipoprotein(a) is homologous to plasminogen

Lipoprotein(a) is an LDL-like lipoprotein whose concentration in plasma is correlated with atherosclerosis. The characteristic protein component of lipoprotein(a) is apolipoprotein(a) which is disulphide-linked to apolipoprotein B-100. Sequencing of cloned human apolipoprotein(a) complementary DNA shows that it is very similar to human plasminogen. It contains a serine protease domain and two types of plasminogen-like kringle domains, one of which is present in 37 copies.     

Formation of beta-amyloid protein deposits in brains of transgenic mice

Deposits of beta-amyloid are one of the main pathological characteristics of Alzheimer's disease. The beta-amyloid peptide constituent (relative molecular mass 4,200) of the deposits is derived from the beta-amyloid precursor protein (beta-APP) which is expressed in several different isoforms. The two most prevalent beta-APP isoforms are distinguished by either the presence (beta-APP751) or absence (beta-APP695) of a Kunitz serine protease inhibitor domain. Changes in the abundance of different beta-APP messenger RNAs in brains of Alzheimer's disease victims have been widely reported. Although these results have been controversial, most evidence favours an increase in the mRNAs encoding protease inhibitor-containing isoforms of beta-APP and it is proposed that this change contributes to beta-amyloid formation. We have now produced an imbalance in the normal neuronal ratio of beta-APP isoforms by preparing transgenic mice expressing additional beta-APP751 under the control of a neural-specific promoter. The cortical and hippocampal brain regions of the transgenic mice display extracellular beta-amyloid immunoreactive deposits varying in size (less than 5-50 microns) and abundance. These results suggest that one mechanism of beta-amyloid formation may involve a disruption of the normal ratio of neuronal beta-APP isoform expression and support a direct relationship between increased expression of Kunitz inhibitor-bearing beta-APP isoforms and beta-amyloid deposition.     

Polymorphisms in the genes for coagulation factor Ⅱ,Ⅴ,Ⅶ in patients undergoing coronary angiography

OBJECTIVE: To determine whether polymorphisms in the genes for coagulation factor II, V, VII could predispose an individual to increase risk for coronary artery disease (CAD) and/or myocardial infarction (MI) in Chinese. METHODS: We screened coagulation factor II(G20210A),V(G1691A),VII (R353Q and HVR4) genotype in 374 patients undergoing coronary angiography by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) assay. RESULTS: The R353Q and HVR4 genotype of the factor VII distribution was in accordance with Hardy-Weinberg equilibrium. The frequencies of FVII genotype or allele did not show statistically significant differences between CAD group and controls or between male and female. The frequencies of the Q allele and (RQ + QQ) genotype were significantly higher among the CAD patients without myocardial infarction (MI) history than among those with MI history (P < 0.05). However, HVR4 polymorphism was not significantly different within groups. We only find one normal control of factor II (G20210A) mutation. No coagulation factor V(G1691A) mutation was found in the CAD patients and controls. CONCLUSION: The factor II(G20210A) ,V(G1691A) mutation is absent and may not be a major genetic factor for CAD and/or MI; the Q allele of the R353Q polymorphism of the factor VII gene may be a protective genetic factor against myocardial infarction in Chinese.     

Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease

Several human bacterial pathogens, including the Gram-negative diplococcus Neisseria gonorrhoeae, produce extracellular proteases that are specific for human immunoglobulin IgA1. Immunoglobulin A (IgA) proteases have been studied extensively and the genes of some species cloned in Escherichia coli, but their role in pathogenesis remains unclear. Recently we derived a DNA fragment of 5 kilobases (kb) from N. gonorrhoeae MS11 directing extracellular active enzyme in E. coli. Although the mature enzyme of strain MS11 was shown to have a relative molecular mass of 106,000 (Mr 106K) in gels, the DNA sequence of this cloned fragment reveals a single gene coding for a 169K precursor of IgA protease. The precursor contains three functional domains, the amino-terminal leader which is assumed to initiate the inner membrane transport of the precursor, the protease, and a carboxyl-terminal 'helper' domain apparently required for extracellular secretion (excretion). Based on the structural features of the precursor, we propose a model in which the helper serves as a pore for excretion of the protease domain through the outer membrane. IgA protease acquires an active conformation as its extracellular transport proceeds and is released as a proform from the membrane-bound helper by autoproteolysis. The soluble proform further matures into the 106 K IgA protease and a small stable alpha-protein.     

A micrococcal nuclease homologue in RNAi effector complexes

RNA interference (RNAi) regulates gene expression by the cleavage of messenger RNA, by mRNA degradation and by preventing protein synthesis. These effects are mediated by a ribonucleoprotein complex known as RISC (RNA-induced silencing complex). We have previously identified four Drosophila components (short interfering RNAs, Argonaute 2 (ref. 2), VIG and FXR) of a RISC enzyme that degrades specific mRNAs in response to a double-stranded-RNA trigger. Here we show that Tudor-SN (tudor staphylococcal nuclease)--a protein containing five staphylococcal/micrococcal nuclease domains and a tudor domain--is a component of the RISC enzyme in Caenorhabditis elegans, Drosophila and mammals. Although Tudor-SN contains non-canonical active-site sequences, we show that purified Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases. Notably, both purified Tudor-SN and RISC are inhibited by a specific competitive inhibitor of micrococcal nuclease. Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi.