Non-canonical proteolytic activation of human prothrombin by subtilisin from Bacillus subtilis may shift the procoagulant-anticoagulant equilibrium toward thrombosis.

Blood coagulation is a finely regulated physiological process culminating with the factor Xa (FXa)-mediated conversion of the prothrombin (ProT) zymogen to active α-thrombin (αT). In the prothrombinase complex on the platelet surface, FXa cleaves ProT at Arg-271, generating the inactive precursor prethrombin-2 (Pre2), which is further attacked at Arg-320-Ile-321 to yield mature αT. Whereas the mechanism of physiological ProT activation has been elucidated in great detail, little is known about the role of bacterial proteases, possibly released in the bloodstream during infection, in inducing blood coagulation by direct proteolytic ProT activation. This knowledge gap is particularly concerning, as bacterial infections are frequently complicated by severe coagulopathies. Here, we show that addition of subtilisin (50 nm to 2 µm), a serine protease secreted by the non-pathogenic bacterium Bacillus subtilis, induces plasma clotting by proteolytically converting ProT into active σPre2, a nicked Pre2 derivative with a single cleaved Ala-470-Asn-471 bond. Notably, we found that this non-canonical cleavage at Ala-470-Asn-471 is instrumental for the onset of catalysis in σPre2, which was, however, reduced about 100-200-fold compared with αT. Of note, σPre2 could generate fibrin clots from fibrinogen, either in solution or in blood plasma, and could aggregate human platelets, either isolated or in whole blood. Our findings demonstrate that alternative cleavage of ProT by proteases, even by those secreted by non-virulent bacteria such as B. subtilis, can shift the delicate procoagulant-anticoagulant equilibrium toward thrombosis.

Thrombin inhibits the anti-myeloperoxidase and ferroxidase functions of ceruloplasmin: relevance in rheumatoid arthritis.

Human ceruloplasmin (CP) is a multifunctional copper-binding protein produced in the liver. CP oxidizes Fe(2+) to Fe(3+), decreasing the concentration of Fe(2+) available for generating harmful oxidant species. CP is also a potent inhibitor of leukocyte myeloperoxidase (MPO) (Kd=130nM), a major source of oxidants in vivo. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting flexible joints and characterized by activation of both inflammatory and coagulation processes. Indeed, the levels of CP, MPO, and thrombin are markedly increased in the synovial fluid of RA patients. Here we show that thrombin cleaves CP in vitro at (481)Arg-Ser(482) and (887)Lys-Val(888) bonds, generating a nicked species that retains the native-like fold and the ferroxidase activity of the intact protein, whereas the MPO inhibitory function of CP is abrogated. Analysis of the synovial fluid of 24 RA patients reveals that CP is proteolytically degraded to a variable extent, with a fragmentation pattern similar to that observed with thrombin in vitro, and that proteolysis is blocked by hirudin, a highly potent and specific thrombin inhibitor. Using independent biophysical techniques, we show that thrombin has intrinsic affinity for CP (Kd=60-270nM), independent of proteolysis, and inhibits CP ferroxidase activity (KI=220±20nM). Mapping of thrombin binding sites with specific exosite-directed ligands (i.e., hirugen, fibrinogen γ'-peptide) and thrombin analogues having the exosites variably compromised (i.e., prothrombin, prethrombin-2, ßT-thrombin) reveals that the positively charged exosite-II of thrombin binds to the negatively charged upper region of CP, while the protease active site and exosite-I remain accessible. These results suggest that thrombin can exacerbate inflammation in RA by impairing the MPO inhibitory function of CP via proteolysis and by competitively inhibiting CP ferroxidase activity. Notably, local administration of hirudin, a highly potent and specifc thrombin inhibitor, reduces the concentration of active MPO in the synovial fluid of RA patients and has a beneficial effect on the clinical symptoms of the disease.

A novel protein from the serum of Python sebae, structurally homologous with type-γ phospholipase A(2) inhibitor, displays antitumour activity.

Cytotoxic and antitumour factors have been documented in the venom of snakes, although little information is available on the identification of cytotoxic products in snake serum. In the present study, we purified and characterized a new cytotoxic factor from serum of the non-venomous African rock python (Python sebae), endowed with antitumour activity. PSS (P. sebae serum) exerted a cytotoxic activity and reduced dose-dependently the viability of several different tumour cell lines. In a model of human squamous cell carcinoma xenograft (A431), subcutaneous injection of PSS in proximity of the tumour mass reduced the tumour volume by 20%. Fractionation of PSS by ion-exchange chromatography yielded an active protein fraction, F5, which significantly reduced tumour cell viability in vitro and, strikingly, tumour growth in vivo. F5 is composed of P1 (peak 1) and P2 subunits interacting in a 1:1 stoichiometric ratio to form a heterotetramer in equilibrium with a hexameric form, which retained biological activity only when assembled. The two peptides share sequence similarity with PIP {PLI-γ [type-γ PLA(2) (phospholipase A(2)) inhibitor] from Python reticulatus}, existing as a homohexamer. More importantly, although PIP inhibits the hydrolytic activity of PLA(2), the anti-PLA(2) function of F5 is negligible. Using high-resolution MS, we covered 87 and 97% of the sequences of P1 and P2 respectively. In conclusion, in the present study we have identified and thoroughly characterized a novel protein displaying high sequence similarity to PLI-γ and possessing remarkable cytotoxic and antitumour effects that can be exploited for potential pharmacological applications.

RNase A oligomerization through 3D domain swapping is favoured by a residue located far from the swapping domains.

Bovine pancreatic ribonuclease A forms 3D domain-swapped oligomers by lyophilization from 40% acetic acid solutions or if subjected to various thermally-induced denaturation procedures. Considering that the intrinsic swapping propensity of bovine seminal RNase, the only member of the pancreatic-type RNase super-family that is dimeric in nature, is decreased from 70 to 30% if Arg80 is substituted by Ser (the corresponding residue in native RNase A), we introduced the opposite mutation in position 80 of the pancreatic enzyme. Our aim was to detect if the RNase A tendency to aggregate through domain swapping could increase. Aggregation of the S80R-RNase A mutant was induced either through the 'classic' acetic acid lyophilization, or through a thermally-induced method. The results indicate that the S80R mutant aggregates to a higher extent than the native protein, and that the increase occurs especially through N-terminal swapping. Additional investigations on the dimeric and multimeric species formed indicate that the S80R mutation increases their stability against regression to monomer, and does not significantly change their structural and functional features.

o-Nitrotyrosine and p-iodophenylalanine as spectroscopic probes for structural characterization of SH3 complexes.

High-throughput screening of protein-protein and protein-peptide interactions is of high interest both for biotechnological and pharmacological applications. Here, we propose the use of the noncoded amino acids o-nitrotyrosine and p-iodophenylalanine as spectroscopic probes in combination with circular dichroism and fluorescence quenching techniques (i.e., collisional quenching and resonance energy transfer) as a means to determine the peptide orientation in complexes with SH3 domains. Proline-rich peptides bind SH3 modules in two alternative orientations, according to their sequence motifs, classified as class I and class II. The method was tested on an SH3 domain from a yeast myosin that is known to recognize specifically class I peptides. We exploited the fluorescence quenching effects induced by o-nitrotyrosine and p-iodophenylalanine on the fluorescence signal of a highly conserved Trp residue, which is the signature of SH3 domains and sits directly in the binding pocket. In particular, we studied how the introduction of the two probes at different positions of the peptide sequence (i.e., N-terminally or C-terminally) influences the spectroscopic properties of the complex. This approach provides clear-cut evidence of the orientation of the binding peptide in the SH3 pocket. The chemical strategy outlined here can be easily extended to other protein modules, known to bind linear sequence motifs in a highly directional manner.

3-Nitrotyrosine as a spectroscopic probe for investigating protein protein interactions.

3-Nitrotyrosine (NT) is approximately 10(3)-fold more acidic than Tyr, and its absorption properties are strongly pH-dependent. NT absorbs radiation in the wavelength range where Tyr and Trp emit fluorescence (300-450 nm), and it is essentially nonfluorescent. Therefore, NT may function as an energy acceptor in resonance energy transfer (FRET) studies for investigating ligand protein interactions. Here, the potentialities of NT were tested on the hirudin thrombin system, a well-characterized protease inhibitor pair of key pharmacological importance. We synthesized two analogs of the N-terminal domain (residues 1-47) of hirudin: Y3NT, in which Tyr3 was replaced by NT, and S2R/Y3NT, containing the substitutions Ser2 --> Arg and Tyr3 --> NT. The binding of these analogs to thrombin was investigated at pH 8 by FRET and UV/Vis-absorption spectroscopy. Upon hirudin binding, the fluorescence of thrombin was reduced by approximately 50%, due to the energy transfer occurring between the Trp residues of the enzyme (i.e., the donors) and the single NT of the inhibitor (i.e., the acceptor). The changes in the absorption spectra of the enzyme inhibitor complex indicate that the phenate moiety of NT in the free state becomes protonated to phenol in the thrombin-bound form. Our results indicate that the incorporation of NT can be effectively used to detect protein protein interactions with sensitivity in the low nanomolar range, to uncover subtle structural features at the ligand protein interface, and to obtain reliable Kd values for structure activity relationship studies. Furthermore, advances in chemical and genetic methods, useful for incorporating noncoded amino acids into proteins, highlight the broad applicability of NT in biotechnology and pharmacological screening.

Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin.

In the present work, the effect of Na+ binding on the conformational, stability and molecular recognition properties of thrombin was investigated. The binding of Na+ reduces the CD signal in the far-UV region, while increasing the intensity of the near-UV CD and fluorescence spectra. These spectroscopic changes have been assigned to perturbations in the environment of aromatic residues at the level of the S2 and S3 sites, as a result of global rigidification of the thrombin molecule. Indeed, the Na+-bound form is more stable to urea denaturation than the Na+-free form by approximately 2 kcal/mol (1 cal identical with 4.184 J). Notably, the effects of cation binding on thrombin conformation and stability are specific to Na+ and parallel the affinity order of univalent cations for the enzyme. The Na+-bound form is even more resistant to limited proteolysis by subtilisin, at the level of the 148-loop, which is suggestive of the more rigid conformation this segment assumes in the 'fast' form. Finally, we have used hirudin fragment 1-47 as a molecular probe of the conformation of thrombin recognition sites in the fast and 'slow' form. From the effects of amino acid substitutions on the affinity of fragment 1-47 for the enzyme allosteric forms, we concluded that the specificity sites of thrombin in the Na+-bound form are in a more open and permissible conformation, compared with the more closed structure they assume in the slow form. Taken together, our results indicate that the binding of Na+ to thrombin serves to stabilize the enzyme into a more open and rigid conformation.