Stabilization Versus Inhibition of TAFIa by Competitive Inhibitors in Vitro

Walker, John B.; Hughes, Bernadette; James, Ian; Haddock, Peter; Kluft, C.; Bajzar, Laszlo

doi:10.1074/jbc.m205006200

Cited by 55 publications

(62 citation statements)

References 29 publications

(40 reference statements)

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“…TAFI constitutes a risk factor for thrombosis and coronary artery disease, and its inhibition leads to an enhancement of clot lysis (40,41). The doubleheaded architecture provides TCI with a 5-fold lower K i toward TAFI compared with potato carboxypeptidase inhibitor and a 10-fold acceleration of fibrinolysis with respect to this singleheaded inhibitor used as a model in thrombolytic assays (42,43). In this way, and as previously discussed, the Nt domain could also contribute to improve the affinity/modulate the specificity of TCI toward CPBs, constituting a good target for the design of new TCI molecules to be used as thrombolytic drugs.…”

Section: Discussionmentioning

confidence: 99%

Characterizing the Tick Carboxypeptidase Inhibitor

Arolas

Bronsoms

Ventura

et al. 2006

Journal of Biological Chemistry

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Tick carboxypeptidase inhibitor (TCI) is a small, disulfiderich protein that selectively inhibits metallocarboxypeptidases and strongly accelerates the fibrinolysis of blood clots. TCI consists of two domains that are structurally very similar, each containing three disulfide bonds arranged in an almost identical fashion. The oxidative folding and reductive unfolding pathways of TCI and its separated domains have been characterized by kinetic and structural analysis of the acid-trapped folding intermediates. TCI folding proceeds through a sequential formation of 1-, 2-, 3-, 4-, 5-, and 6-disulfide species to reach the native form. Folding intermediates of TCI comprise two predominant 3-disulfide species (named IIIa and IIIb) and a major 6-disulfide scrambled isomer (Xa) that consecutively accumulate along the reaction and are strongly prevented by the presence of protein disulfide isomerase. This study demonstrates that IIIa and IIIb are 3-disulfide species containing the native disulfide pairings of the N-and C-terminal domains of TCI, respectively, and explains why the two domains of TCI fold sequentially and independently. Also, we show that the reductive unfolding of TCI undergoes two main independent unfolding events through the formation of IIIa and IIIb intermediates. Together, the comparison of the folding, stability, and inhibitory activity of TCI with those of the isolated domains reveals the reasons behind the two-domain nature of this protein: both domains contribute to the specificity and high affinity of its double-headed binding to carboxypeptidases. The results obtained herein provide valuable information for the design of more potent and selective TCI molecules.The mechanism by which an unfolded protein achieves its native state is one of the most complex problems in structural biology. Understanding the sequence of folding events in proteins may not only help to predict protein structures from amino acid sequences but also provide invaluable information to a variety of related fields, such as protein design or protein misfolding associated with pathological diseases (1, 2). A number of studies concerning folding have been focused on small, disulfide-rich proteins, given that the chemistry of disulfide bond formation allows the trapping and subsequent characterization of the folding intermediates that accumulate, something difficult to attain with disulfide-free proteins (3, 4). In oxidative folding, reduced and denatured proteins are allowed to recover both its native disulfide bonds and native structures in the absence or presence of redox agents. Bovine pancreatic trypsin inhibitor, ribonuclease A, lysozyme, ␣-lactalbumin, and hirudin have been extensively investigated using this methodology (5-9). The heterogeneity and structures of the intermediates that occur during the process define their respective folding landscapes (10).Although most of the analyzed proteins comprise a small, single-domain fold containing three or four disulfide bonds, a great diversity of folding mechanisms is observ...

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Section: Discussionmentioning

confidence: 99%

Characterizing the Tick Carboxypeptidase Inhibitor

Arolas

Bronsoms

Ventura

et al. 2006

Journal of Biological Chemistry

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“…Plasma in Vitro Clot Lysis Assays-Clot lysis assays were performed essentially as described previously (29,30). In short, human plasma, diluted 1:3 in 20 mM Hepes, 150 mM NaCl, pH 7.4, was added to 96-well microtiter plates.…”

Section: Matrix-assisted Laser Desorption Ionization Mass Spectrometrmentioning

confidence: 99%

Thrombin-activable Fibrinolysis Inhibitor (TAFI) Zymogen Is an Active Carboxypeptidase

Valnickova

Thøgersen

Potempa

et al. 2007

Journal of Biological Chemistry

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Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase found in human plasma, presumably as an inactive zymogen. The current dogma is that proteolytic activation by thrombin/thrombomodulin generates the active enzyme (TAFIa), which down-regulates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin. In this study, we have shown that the zymogen exhibits continuous and stable carboxypeptidase activity against large peptide substrates, and we suggest that the activity downregulates fibrinolysis in vivo.Human thrombin-activable fibrinolysis inhibitor (TAFI) 3 (EC 3.4.17.20; MEROPS, M14.009; UniProt, Q96IY4) is a single chain 60-kDa protein secreted by the liver and found in plasma at an average concentration of 13.4 g/ml (220 nM) (1). TAFI is N-glycosylated, and the attached glycans account for ϳ20% of the overall size of the protein. All five potential N-linked glycosylation sites are occupied, and four of these are located on the activation peptide and one on the enzyme moiety (2). The protein belongs to the metallocarboxypeptidase family and is also known as plasma pro-carboxypeptidases B, R, and U (3-5). TAFI was discovered by several groups as a plasminogen-binding protein (3, 5-7) leading investigators to explore a role in fibrinolysis. Indeed, the protein appears to be an important regulator of fibrinolysis by removing surface-exposed C-terminal Lys from partially degraded fibrin clots, thus reducing the number of plasminogen-binding sites (8). Activated TAFI (TAFIa) has also been implicated in the regulation of the inflammatory response by inactivating complement-derived inflammatory peptides (9).Several different proteases are able to activate TAFI in vitro, but thrombin/thrombomodulin has been suggested as the most likely physiologically relevant activator (10). At the present, the proteolytic activity is exclusively assigned to TAFIa. This form is generated by cleavage of the Arg 92 -Ala 93 peptide bond releasing the heavily glycosylated activation peptide. The dissociation of the activation peptide from the bulk of the molecule causes the isoelectric point of the enzyme moiety to shift from a pH value of ϳ5 to 8, rendering the protein significantly more basic and less soluble (2). In addition, the enzymatic activity of TAFIa is unstable, with a half-life ranging from 1.4 to 15 min at 37°C, depending on the isoform (11, 12). This inactivation is highly temperature-dependent and is most likely caused by a change in the structure of TAFIa (11, 12). The structural change appears to expose regions with affinity for ␣ 2 -macroglobulin and pregnancy zone protein, as these interactions are properties of TAFIa only (13) and may function as a clearance mechanism to remove TAFIa that escapes covalent crosslinking to the clot catalyzed by transglutaminase (FXIII) during coagulation (14).TAFI is homologous to the pancreatic carboxypeptidases human procarboxypeptidase B (pro-CPB) and human procarboxypeptidases A1 and A2 (pro-CPA1 and pro-CPA2). These are characterized by ...

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“…13,14 Thermal stability of TAFIa is increased by binding of small peptide substrates, like hippuryl-arginine, 15 or carboxypeptidase inhibitors such as 2-guanidino-ethyl-mercaptosuccinic acid (GEMSA). 16,17 In addition, extensive mutagenesis studies have identified 5 mutations (S305C, T325I, T329I, H333Y, and H335Q) which, in combination, stabilize TAFIa activity 180-fold. [18][19][20] In this paper we present crystal structures of wild-type TAFI, a TAFI-inhibitor complex, and a TAFI mutant with a 70-fold more stable active enzyme.…”

Section: Introductionmentioning

confidence: 99%

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

Marx

Brondijk²,

Plug

et al. 2008

Blood

115

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Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombincleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors. (Blood. 2008;112: 2803-2809) Introduction TAFI 1,2 is a pro-metallocarboxypeptidase that links the coagulation and fibrinolytic systems. TAFI is activated by thrombin, the thrombin-thrombomodulin complex or plasmin. 3 Activated TAFI (TAFIa) inhibits plasmin-mediated blood clot lysis by removing C-terminal lysine residues from partially degraded fibrin that are required for positive feedback in tissue plasminogen-activator dependent plasmin generation. In addition, TAFIa has been implicated in modulation of the inflammatory response by inactivating bradykinin and the anaphylatoxins C3a and C5a. 4,5 Although it is a powerful antifibrinolytic agent, there are no known physiologic inhibitors of TAFIa. Instead, the half-life of TAFIa activity is regulated by its intrinsic instability. The inactivation rate, 5 to 10 minutes at 37°C, is highly temperature-dependent, suggesting that inactivation involves a large conformational change. 6 This is also suggested by the susceptibility of the inactive enzyme, TAFIai to proteolytic cleavage by thrombin at Arg302, a site that is cryptic in TAFI and TAFIa. 6,7 The stability of TAFIa is an important determinant for its antifibrinolytic potential because TAFIa inhibits fibrinolysis through a threshold-dependent mechanism. [8][9][10] Full-length TAFI consists of 401 amino acids divided into 2 domains: the first 92 amino acids form the activation peptide; the next 309 amino acids form the catalytic domain. The activation peptide restricts substrate access to the catalytic cleft in the zymogen. TAFI is activated through cleavage at Arg92, which releases the activation peptide.TAFI is highly homologous to the pancreatic procarboxypeptidases with 42% sequence identity to human procarboxypeptidase B (pro...

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Stabilization Versus Inhibition of TAFIa by Competitive Inhibitors in Vitro

Cited by 55 publications

References 29 publications

Characterizing the Tick Carboxypeptidase Inhibitor

Characterizing the Tick Carboxypeptidase Inhibitor

Thrombin-activable Fibrinolysis Inhibitor (TAFI) Zymogen Is an Active Carboxypeptidase

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

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