Acute phase mediators modulate thrombin-activable fibrinolysis inhibitor (TAFI) gene expression in HepG2 cells

Thrombin-activable fibrinolysis inhibitor (TAFI) has recently been identified as a positive acute phase protein in mice, an observation that may have important implications for the interaction of the coagulation, fibrinolytic, and inflammatory systems. Activated TAFI (TAFIa) inhibits fibrinolysis by removing the carboxyl-terminal lysines from partially degraded fibrin that are important for maximally efficient plasminogen activation. In addition, TAFIa has been shown to be capable of removing the carboxyl-terminal arginine residues from the anaphylatoxins and bradykinin, thus implying a role for the TAFI pathway in the vascular responses to inflammation. In the current study, we investigated the ability of acute phase mediators to modulate human TAFI gene expression in cultured human hepatoma (HepG2) cells. Surprisingly, we found that treatment of HepG2 cells with a combination of interleukin (IL)-1 and IL-6 suppressed endogenous TAFI mRNA abundance in HepG2 cells (~60% decrease), while treatment with IL-1 or IL-6 alone had no effect. Treatment with IL-1 and/or IL-6 had no effect on TAFI promoter activity as measured using a luciferase reporter plasmid containing the human TAFI 5'-flanking region, whereas treatment with IL-1 and IL-6 in combination, but not alone, decreased the stability of the endogenous TAFI mRNA. Treatment with the synthetic glucocorticoid dexamethasone resulted in a 2-fold increase of both TAFI mRNA levels and promoter activity. We identified a functional glucocorticoid response element (GRE) in the human TAFI promoter between nucleotides 92 and 78. The GRE was capable of binding the glucocorticoid receptor, as assessed by gel mobility shift assays, and mutation of this element markedly decreased the ability of the TAFI promoter to be activated by dexamethasone.     

Amino acid residues in the P6-P'3 region of thrombin-activable fibrinolysis inhibitor (TAFI) do not determine the thrombomodulin dependence of TAFI activation.

Thrombin bound to thrombomodulin activates thrombin-activable fibrinolysis inhibitor (TAFI) and protein C much more efficiently than thrombin alone. Although thrombomodulin has been proposed to alter the thrombin active site, the recently determined structure of the thrombin-thrombomodulin complex does not support this proposal. In this study, the contribution of amino acids near the activation site of TAFI toward thrombomodulin dependence was determined, utilizing four variants of TAFI with specific substitutions in the P6-P'3 region surrounding the Arg-92 cleavage site. Two point mutants had either the Ser-90 or Asp-87 of TAFI replaced with Ala, a third mutant had the thrombin activation site of the fibrinogen Bbeta-chain substituted into positions 91-95 of TAFI, and a fourth mutant had the thrombin activation site of protein C substituted into positions 90-95 of TAFI. Each of these mutants was expressed, purified, and characterized with respect to activation kinetics and functional properties of the enzyme. Even though fibrinogen is poorly cleaved by thrombin-thrombomodulin, the fibrinogen activation site does not significantly alter the thrombomodulin dependence of TAFI activation. The TAFI variant with the protein C activation sequence is only slowly activated by thrombin-thrombomodulin, and not at all by free thrombin. Mutating Asp-87 to Ala increases the catalytic efficiency of activation 3-fold both in the presence and absence of thrombomodulin, whereas mutating Ser-90 to Ala effects only minor kinetic differences compared with wild type TAFI. The thermal stabilities and antifibrinolytic properties of the enzymes were not substantially altered by any of the mutations that allowed for efficient activation of the enzyme. We conclude that residues in the P6-P'3 region of TAFI do not determine the thrombomodulin dependence of activation, which lends support to the argument that the role of thrombomodulin is to optimally orient thrombin and its substrate, rather than to allosterically alter the specificity of the thrombin active site.     

Human procarboxypeptidase B: three-dimensional structure and implications for thrombin-activatable fibrinolysis inhibitor (TAFI)

Besides their classical role in alimentary protein degradation, zinc-dependant carboxypeptidases also participate in more selective regulatory processes like prohormone and neuropeptide processing or fibrinolysis inhibition in blood plasma. Human pancreatic procarboxypeptidase B (PCPB) is the prototype for those human exopeptidases that cleave off basic C-terminal residues and are secreted as inactive zymogens. One such protein is thrombin-activatable fibrinolysis inhibitor (TAFI), also known as plasma PCPB, which circulates in human plasma as a zymogen bound to plasminogen. The structure of human pancreatic PCPB displays a 95-residue pro-segment consisting of a globular region with an open-sandwich antiparallel-alpha antiparallel-beta topology and a C-terminal alpha-helix, which connects to the enzyme moiety. The latter is a 309-amino acid residue catalytic domain with alpha/beta hydrolase topology and a preformed active site, which is shielded by the globular domain of the pro-segment. The fold of the proenzyme is similar to previously reported procarboxypeptidase structures, also in that the most variable region is the connecting segment that links both globular moieties. However, the empty active site of human procarboxypeptidase B has two alternate conformations in one of the zinc-binding residues, which account for subtle differences in some of the key residues for substrate binding. The reported crystal structure, refined with data to 1.6A resolution, permits in the absence of an experimental structure, accurate homology modelling of TAFI, which may help to explain its properties.     

Elements of the primary structure of thrombomodulin required for efficient thrombin-activable fibrinolysis inhibitor activation

Deletion and point mutants of soluble thrombomodulin were used to compare and contrast elements of primary structure required for the activation of thrombin-activable fibrinolysis inhibitor (TAFI) and protein C. The smallest mutant capable of efficiently promoting TAFI activation contained residues including the c-loop of epidermal growth factor-3 (EGF3) through EGF6. This mutant is 13 residues longer than the smallest mutant that functioned well with protein C; the latter consisted of residues from the interdomain loop connecting EGF3 and EGF4 through EGF6. Alanine point mutants showed no loss of function in protein C activation for mutations within the c-loop of EGF3. In TAFI activation, however, alanine mutations cause a 50% reduction at Tyr-337, 67% reductions at Asp-338 and Leu-339, and 90% or greater reductions at Val-340, Asp-341, and Glu-343. A mutation at Asp-349 in the peptide connecting EGF3 to EGF4 eliminated activity against both TAFI and protein C. Oxidation of Met-388 in the peptide connecting EGF5 to EGF6 reduced the rate of protein C activation by 80% but marginally, if at all, affected the rate of TAFI activation. Mutation at Phe-376 severely reduced protein C activation but only marginally influenced that of TAFI. A Q387P mutation, however, severely reduced both activities. TAFI activation was shown to be Ca(2+)-dependent. The response, unlike that of protein C, was monotonic and was half-maximal at 0.25 mm Ca(2+). Like protein C activation, TAFI activation was eliminated by a monoclonal antibody directed at the thrombin-binding domain (EGF5) but was not affected by one directed at EGF2. Thus, elements of structure in the thrombin-binding domain are needed for the activation of both protein C and TAFI, but more of the primary structure is needed for TAFI activation. In addition, some residues are needed for one of the reactions but not the other.     

Structural basis for the selective inhibition of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) by a selenium-containing inhibitor with chloro-aminopyridine as a basic group

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) is a target molecule for treating thromboembolic disorders. We previously reported that design and synthesis of compound 1 containing a selenol group and chloloaminopyridine. Compound 1 showed high inhibitory activity towards TAFIa, with a high degree of selectivity for TAFIa over carboxypeptidase N (CPN). Here we report investigation of this selectivity. To obtain co-crystal of 1/pp-CPB (a surrogate of TAFIa), we synthesized protected compound 5 as a stabilized precursor of 1. The X-ray crystal structure and docking study indicated that the Cl substituent is accommodated in the pp-CPB specific pocket whereas CPN has no identical pocket. This is important information for the design of drugs targeting TAFIa with high selectivity.     

Mutations in the substrate binding site of thrombin-activatable fibrinolysis inhibitor (TAFI) alter its substrate specificity

Thrombin-activable fibrinolysis inhibitor (TAFI) is a zymogen that inhibits the amplification of plasmin production when converted to its active form (TAFIa). TAFI is structurally very similar to pancreatic procarboxypeptidase B. TAFI also shares high homology in zinc binding and catalytic sites with the second basic carboxypeptidase present in plasma, carboxypeptidase N. We investigated the effects of altering residues involved in substrate specificity to understand how they contribute to the enzymatic differences between TAFI and carboxypeptidase N. We expressed wild type TAFI and binding site mutants in 293 cells. Recombinant proteins were purified and characterized for their activation and enzymatic activity as well as functional activity. Although the thrombin/thrombomodulin complex activated all the mutants, carboxypeptidase B activity of the activated mutants against hippuryl-arginine was reduced. Potato carboxypeptidase inhibitor inhibited the residual activity of the mutants. The functional activity of the mutants in a plasma clot lysis assay correlated with their chromogenic activity. The effect of the mutations on other substrates depended on the particular mutation, with some of the mutants possessing more activity against hippuryl-His-leucine than wild type TAFIa. Thus mutations in residues around the substrate binding site of TAFI resulted in altered C-terminal substrate specificity.     

Combined segregation-linkage analysis of plasma thrombin activatable fibrinolysis inhibitor (TAFI) antigen levels with TAFI gene polymorphisms

By decreasing plasminogen binding to fibrin surface, the thrombin activatable fibrinolysis inhibitor (TAFI) has been hypothesized to constitute an early marker for atherothrombotic diseases. Previous studies have shown that plasma TAFI levels exhibit a high interindividual variability that is only poorly explained by lifestyle factors. Several polymorphisms of the TAFI gene have been described, and a combination of a C+1542G substitution in the 3' untranslated region and an Ala147Thr amino acid change has been shown to explain 60% of TAFI variability in a sample of unrelated individuals. A segregation-linkage analysis was performed to determine whether these polymorphisms are directly involved in the genetic regulation of TAFI levels, or whether they are only markers in linkage disequilibrium (LD) with unmeasured TAFI-linked quantitative trait loci (QTLs). The sample consisted of 97 healthy nuclear families from the Stanislas Cohort. The C+1542G and Ala147Thr polymorphisms were in complete negative LD, with minor allele frequencies of 0.27 and 0.28, respectively. Results of the segregation-linkage analysis provided evidence of two TAFI-linked QTLs in LD with the two measured polymorphisms, which would explain 78% of the TAFI variance, as compared with 55% explained by the C+1542G and the Ala147Thr polymorphisms combined. The two putative QTLs would have minor allele frequencies of 0.45 and 0.32, respectively. The hypothesis that one of the measured polymorphisms is one of the QTLs was rejected. The putative QTLs also did not seem compatible with the other TAFI gene polymorphisms that we have previously described. More extensive sequencing of the TAFI gene is necessary to identify the functional variants.     

Design and synthesis of conformationally restricted inhibitors of active thrombin activatable fibrinolysis inhibitor (TAFIa)

A series of 4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid and 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid derivatives designed as inhibitors of TAFIa has been prepared via a common hydrogenation–alkylation sequence starting from the appropriate benzimidazole and imidazopyridine system. We present a successful design strategy using a conformational restriction approach resulting in potent and selective inhibitors of TAFIa. The X-ray structure of compound 5 in complex with a H333Y/H335Q double mutant TAFI indicate that the conformational restriction is responsible for the observed potency increase.     

3-Mercaptopropionic acids as efficacious inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa)

A novel series of cyclic potent, selective, small molecule, thiol-based inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) and the crystal structures of TAFIa inhibitors bound to porcine pancreatic carboxypeptidase B are described. Three series of cyclic arginine and lysine mimetic inhibitors vary significantly in their selectivity against other human basic carboxypeptidases, carboxypeptidase N and carboxypeptidase B. (-)2a displays TAFIa IC50 = 3 nM and 600-fold selectivity against CPN. Inhibition of TAFIa with (rac)2a resulted in dose dependent acceleration of human plasma clot lysis in vitro and was efficacious as an adjunct to tPA in an in vivo rabbit jugular vein thrombolysis model.     

The crystal structure of human GlnRS provides basis for the development of neurological disorders

Cytosolic glutaminyl-tRNA synthetase (GlnRS) is the singular enzyme responsible for translation of glutamine codons. Compound heterozygous mutations in GlnRS cause severe brain disorders by a poorly understood mechanism. Herein, we present crystal structures of the wild type and two pathological mutants of human GlnRS, which reveal, for the first time, the domain organization of the intact enzyme and the structure of the functionally important N-terminal domain (NTD). Pathological mutations mapping in the NTD alter the domain structure, and decrease catalytic activity and stability of GlnRS, whereas missense mutations in the catalytic domain induce misfolding of the enzyme. Our results suggest that the reduced catalytic efficiency and a propensity of GlnRS mutants to misfold trigger the disease development. This report broadens the spectrum of brain pathologies elicited by protein misfolding and provides a paradigm for understanding the role of mutations in aminoacyl-tRNA synthetases in neurological diseases.     

The crystal structure of seabream antiquitin reveals the structural basis of its substrate specificity

The crystal structure of seabream antiquitin in complex with the cofactor NAD(+) was solved at 2.8A resolution. The mouth of the substrate-binding pocket is guarded by two conserved residues, Glu120 and Arg300. To test the role of these two residues, we have prepared the two mutants E120A and R300A. Our model and kinetics data suggest that antiquitin's specificity towards the substrate alpha-aminoadipic semialdehyde is contributed mainly by Glu120 which interacts with the alpha-amino group of the substrate. On the other hand, Arg300 does not have any specific interaction with the alpha-carboxylate group of the substrate, but is important in maintaining the active site conformation.     

Platelet factor 4 inhibits thrombomodulin-dependent activation of thrombin-activatable fibrinolysis inhibitor (TAFI) by thrombin

Thrombomodulin (TM) is a cofactor for thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI) and thereby helps coordinate coagulation, anticoagulation, fibrinolysis, and inflammation. Platelet factor 4 (PF4), a platelet α-granule protein and a soluble cofactor for TM-dependent protein C activation, stimulates protein C activation in vitro and in vivo. In contrast to stimulation of protein C activation, PF4 is shown here to inhibit activation of TAFI by thrombin-TM. Consequences of inhibition of TAFI activation by PF4 included loss of TM-dependent prolongation of clot lysis times in hemophilia A plasma and loss of TM-stimulated conversion of bradykinin (BK) to des-Arg(9)-BK by TAFIa in normal plasma. Thus, PF4 modulates the substrate specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation, thereby preventing the generation of the antifibrinolytic and anti-inflammatory activities of TAFIa. To block the inhibitory effects of PF4 on TAFI activation, heparin derivatives were tested for their ability to retain high affinity binding to PF4 despite having greatly diminished anticoagulant activity. N-acetylated heparin (NAc-Hep) lacked detectable anticoagulant activity in activated partial thromboplastin time clotting assays but retained high affinity binding to PF4 and effectively reversed PF4 binding to immobilized TM. NAc-Hep permitted BK conversion to des-Arg(9)-BK by TAFIa in the presence of PF4. In a clot lysis assay on TM-expressing cells using hemophilia A plasma, NAc-Hep prevented PF4-mediated inhibition of TAFI activation and the antifibrinolytic functions of TAFIa. Accordingly, NAc-Hep or similar heparin derivatives might provide therapeutic benefits by diminishing bleeding complications in hemophilia A via restoration of TAFIa-mediated protection of clots against premature lysis.     

The intrinsic threshold of the fibrinolytic system is modulated by basic carboxypeptidases, but the magnitude of the antifibrinolytic effect of activated thrombin-activable fibrinolysis inhibitor is masked by its instability

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) is intrinsically unstable, a property that complicates the study of its role in regulating fibrinolysis. To investigate the effect of basic carboxypeptidases on fibrinolysis under conditions of constant carboxypeptidase activity, we employed pancreatic carboxypeptidase B (CPB), a homologous, stable basic carboxypeptidase, as a surrogate for TAFIa. Clots formed from TAFI-depleted plasma or from purified components were supplemented with tissue-type plasminogen activator and either CPB or TAFIa. The clot lysis data indicate that the down-regulation of fibrinolysis mediated by basic carboxypeptidases involves a threshold mechanism. At carboxypeptidase concentrations above the threshold, plasminogen activation is maintained in a fully down-regulated state; experiments in plasma showed that fibrinolysis is essentially halted by saturating concentrations of TAFIa and that fibrinolysis can be prolonged more than 45-fold by a stable carboxypeptidase. The threshold carboxypeptidase concentration was dependent on tissue-type plasminogen activator and antiplasmin concentrations, indicating that the threshold is determined by the steady-state plasmin concentration. Although obvious with CPB, the threshold was masked by the intrinsic instability of TAFIa and became apparent only when the effect of TAFIa was investigated over the picomolar concentration range. Because of the threshold effect and the instability of TAFIa, exponential increases in TAFIa concentration generate linear increases in lysis time. A model relating lysis time to TAFIa concentration, TAFIa half-life, and the threshold concentration of TAFIa is provided. The threshold effect has potentially important implications regarding the role of TAFIa and the regulation of clot lysis in vivo.     

The 1.5 A crystal structure of a highly selected antiviral T cell receptor provides evidence for a structural basis of immunodominance

Despite a potential repertoire of >10(15) alphabeta T cell receptors (TcR), the HLA B8-restricted cytolytic T cell response to a latent antigen of Epstein-Barr virus (EBV) is strikingly limited in the TcR sequences that are selected. Even in unrelated individuals this response is dominated by a single highly restricted TcR clonotype that selects identical combinations of hypervariable Valpha, Vbeta, D, J, and N region genes. We have determined the 1.5 A crystal structure of this "public" TcR, revealing that five of the six hypervariable loops adopt novel conformations providing a unique combining site that contains a deep pocket predicted to overlay the HLA B8-peptide complex. The findings suggest a structural basis for the immunodominance of this clonotype in the immune response to EBV.     

Crystal structure of the N domain of human somatic angiotensin I-converting enzyme provides a structural basis for domain-specific inhibitor design

Human somatic angiotensin I-converting enzyme (sACE) is a key regulator of blood pressure and an important drug target for combating cardiovascular and renal disease. sACE comprises two homologous metallopeptidase domains, N and C, joined by an inter-domain linker. Both domains are capable of cleaving the two hemoregulatory peptides angiotensin I and bradykinin, but differ in their affinities for a range of other substrates and inhibitors. Previously we determined the structure of testis ACE (C domain); here we present the crystal structure of the N domain of sACE (both in the presence and absence of the antihypertensive drug lisinopril) in order to aid the understanding of how these two domains differ in specificity and function. In addition, the structure of most of the inter-domain linker allows us to propose relative domain positions for sACE that may contribute to the domain cooperativity. The structure now provides a platform for the design of "domain-specific" second-generation ACE inhibitors.     

The crystal structure of the globular head of complement protein C1q provides a basis for its versatile recognition properties

C1q is a versatile recognition protein that binds to an amazing variety of immune and non-immune ligands and triggers activation of the classical pathway of complement. The crystal structure of the C1q globular domain responsible for its recognition properties has now been solved and refined to 1.9 A of resolution. The structure reveals a compact, almost spherical heterotrimeric assembly held together mainly by non-polar interactions, with a Ca2+ ion bound at the top. The heterotrimeric assembly of the C1q globular domain appears to be a key factor of the versatile recognition properties of this protein. Plausible three-dimensional models of the C1q globular domain in complex with two of its physiological ligands, C-reactive protein and IgG, are proposed, highlighting two of the possible recognition modes of C1q. The C1q/human IgG1 model suggests a critical role for the hinge region of IgG and for the relative orientation of its Fab domain in C1q binding.     

The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity.

From the lysosomal cysteine proteinase cathepsin B, isolated from human liver in its two-chain form, monoclinic crystals were obtained which contain two molecules per asymmetric unit. The molecular structure was solved by a combination of Patterson search and heavy atom replacement methods (simultaneously with rat cathepsin B) and refined to a crystallographic R value of 0.164 using X-ray data to 2.15 A resolution. The overall folding pattern of cathepsin B and the arrangement of the active site residues are similar to the related cysteine proteinases papain, actinidin and calotropin DI. 166 alpha-carbon atoms out of 248 defined cathepsin B residues are topologically equivalent (with an r.m.s. deviation of 1.04 A) with alpha-carbon atoms of papain. However, several large insertion loops are accommodated on the molecular surface and modify its properties. The disulphide connectivities recently determined for bovine cathepsin B by chemical means were shown to be correct. Some of the primed subsites are occluded by a novel insertion loop, which seems to favour binding of peptide substrates with two residues carboxy-terminal to the scissile peptide bond; two histidine residues (His110 and His111) in this "occluding loop' provide positively charged anchors for the C-terminal carboxylate group of such polypeptide substrates. These structural features explain the well-known dipeptidyl carboxypeptidase activity of cathepsin B. The other subsites adjacent to the reactive site Cys29 are relatively similar to papain; Glu245 in the S2 subsite favours basic P2-side chains. The above mentioned histidine residues, but also the buried Glu171 might represent the group with a pKa of approximately 5.5 near the active site, which governs endo- and exopeptidase activity. The "occluding loop' does not allow cystatin-like protein inhibitors to bind to cathepsin B as they do to papain, consistent with the reduced affinity of these protein inhibitors for cathepsin B compared with the related plant enzymes.     

The crystal structure of R-specific alcohol dehydrogenase from Lactobacillus brevis suggests the structural basis of its metal dependency

The crystal structure of the apo-form of an R-specific alcohol dehydrogenase from Lactobacillus brevis (LB-RADH) was solved and refined to 1.8A resolution. LB-RADH is a member of the short-chain dehydrogenase/reductase (SDR) enyzme superfamily. It is a homotetramer with 251 amino acid residues per subunit and uses NADP(H) as co-enzyme. NADPH and the substrate acetophenone were modelled into the active site. The enantiospecificity of the enzyme can be explained on the basis of the resulting hypothetical ternary complex. In contrast to most other SDR enzymes, the catalytic activity of LB-RADH depends strongly on the binding of Mg(2+). Mg(2+) removal by EDTA inactivates the enzyme completely. In the crystal structure, the Mg(2+)-binding site is well defined. The ion has a perfect octahedral coordination sphere and occupies a special position concerning crystallographic and molecular point symmetry, meaning that each RADH tetramer contains two magnesium ions. The magnesium ion is no direct catalytic cofactor. However, it is structurally coupled to the putative C-terminal hinge of the substrate-binding loop and, via an extended hydrogen bonding network, to some side-chains forming the substrate binding region. Therefore, the presented structure of apo-RADH provides plausible explanations for the metal dependence of the enzyme.     

Leishmania pyruvate kinase: the crystal structure reveals the structural basis of its unique regulatory properties

Glycolysis occupies a central role in cellular metabolism, and is of particular importance for the catabolic production of ATP in protozoan parasites such as Leishmania and Trypanosoma. In these organisms pyruvate kinase plays a key regulatory role, and is unique in responding to fructose 2,6-bisphosphate as allosteric activator. The determination of the crystal structure of the first eukaryotic pyruvate kinase in the T-state (the inactive or 'tense' conformation of allosteric enzymes) is described. A comparison of the effector sites of the Leishmania and yeast enzymes reveals the structural basis for the different effector specificity. Two loops, comprising residues 443-453 and 480-489, adopt very different conformations in the two enzymes, and Lys-453 and His-480 that are a feature of trypanosomatid enzymes provide probable ligands for the 2-phospho group of the effector molecule. These and other differences offer an opportunity for the design of drugs that would exploit regulatory differences between parasite and host.