Rational Design of Protein C Activators.

In addition to its procoagulant and proinflammatory functions mediated by cleavage of fibrinogen and PAR1, the trypsin-like protease thrombin activates the anticoagulant protein C in a reaction that requires the cofactor thrombomodulin and the endothelial protein C receptor. Once in the circulation, activated protein C functions as an anticoagulant, anti-inflammatory and regenerative factor. Hence, availability of a protein C activator would afford a therapeutic for patients suffering from thrombotic disorders and a diagnostic tool for monitoring the level of protein C in plasma. Here, we present a fusion protein where thrombin and the EGF456 domain of thrombomodulin are connected through a peptide linker. The fusion protein recapitulates the functional and structural properties of the thrombin-thrombomodulin complex, prolongs the clotting time by generating pharmacological quantities of activated protein C and effectively diagnoses protein C deficiency in human plasma. Notably, these functions do not require exogenous thrombomodulin, unlike other anticoagulant thrombin derivatives engineered to date. These features make the fusion protein an innovative step toward the development of protein C activators of clinical and diagnostic relevance.

Dissecting the integrative antioxidant and redox systems in plant mitochondria. Effect of stress and S-nitrosylation.

Mitochondrial respiration provides the energy needed to drive metabolic and transport processes in cells. Mitochondria are a significant site of reactive oxygen species (ROS) production in plant cells, and redox-system components obey fine regulation mechanisms that are essential in protecting the mitochondrial integrity. In addition to ROS, there are compelling indications that nitric oxide can be generated in this organelle by both reductive and oxidative pathways. ROS and reactive nitrogen species play a key role in signaling but they can also be deleterious via oxidation of macromolecules. The high production of ROS obligates mitochondria to be provided with a set of ROS scavenging mechanisms. The first line of mitochondrial antioxidants is composed of superoxide dismutase and the enzymes of the ascorbate-glutathione cycle, which are not only able to scavenge ROS but also to repair cell damage and possibly serve as redox sensors. The dithiol-disulfide exchanges form independent signaling nodes and act as antioxidant defense mechanisms as well as sensor proteins modulating redox signaling during development and stress adaptation. The presence of thioredoxin (Trx), peroxiredoxin (Prx) and sulfiredoxin (Srx) in the mitochondria has been recently reported. Cumulative results obtained from studies in salt stress models have demonstrated that these redox proteins play a significant role in the establishment of salt tolerance. The Trx/Prx/Srx system may be subjected to a fine regulated mechanism involving post-translational modifications, among which S-glutathionylation and S-nitrosylation seem to exhibit a critical role that is just beginning to be understood. This review summarizes our current knowledge in antioxidative systems in plant mitochondria, their interrelationships, mechanisms of compensation and some unresolved questions, with special focus on their response to abiotic stress.

Histone H4 promotes prothrombin autoactivation.

Recent studies have documented the ability of prothrombin to spontaneously convert to the mature protease thrombin when Arg-320 becomes exposed to solvent for proteolytic attack upon mutation of residues in the activation domain. Whether prothrombin autoactivation occurs in the wild-type under conditions relevant to physiology remains unknown. Here, we report that binding of histone H4 to prothrombin under physiological conditions generates thrombin by autoactivation. The effect is abrogated by mutation of the catalytic Ser-525 and requires the presence of the Gla domain. Fluorescence titrations document direct binding of histone H4 to prothrombin with an affinity in the low nm range. Stopped flow data and luminescence resonance energy transfer measurements indicate that the binding mechanism obeys conformational selection. Among the two conformations of prothrombin, collapsed and fully extended, histone H4 binds selectively to the collapsed form and induces a transition toward a new conformation where the distance between Ser-101 in kringle-1 and Ser-210 in kringle-2 increases by 13 Å. These findings confirm the molecular plasticity of prothrombin emerged from recent structural studies and suggest that different conformations of the inter-kringle linker domain determine the functional behavior of prothrombin. The results also broaden our mechanistic understanding of the prothrombotic phenotype observed during cellular damage due to the release of histones in the blood stream. Prothrombin autoactivation induced by histone H4 emerges as a mechanism of pathophysiological relevance through which thrombin is generated independently of activation of the coagulation cascade.

Autoactivation of thrombin precursors.

Trypsin-like proteases are synthesized as inactive zymogens and convert to the mature form upon activation by specific enzymes, often assisted by cofactors. Central to this paradigm is that the zymogen does not convert spontaneously to the mature enzyme, which in turn does not feed back to activate its zymogen form. In the blood, the zymogens prothrombin and prethrombin-2 require the prothrombinase complex to be converted to the mature protease thrombin, which is unable to activate prothrombin or prethrombin-2. Here, we show that replacement of key residues within the activation domain causes these zymogens to spontaneously convert to thrombin. The conversion is started by the zymogen itself, which is capable of binding ligands at the active site, and is abrogated by inactivation of the catalytic residue Ser-195. The product of autoactivation is functionally and structurally equivalent to wild-type thrombin. Zymogen autoactivation is explained by conformational selection, a basic property of the trypsin fold uncovered by structural and rapid kinetics studies. Both the zymogen and protease undergo a pre-existing equilibrium between active and inactive forms. The equilibrium regulates catalytic activity in the protease and has the potential to unleash activity in the zymogen to produce autoactivation. A new strategy emerges for the facile production of enzymes through zymogen autoactivation that is broadly applicable to trypsin-like proteases of biotechnological and clinical interest.

Exposure of R169 controls protein C activation and autoactivation.

Protein C is activated by thrombin with a value of k(cat)/K(m) = 0.11mM(-1)s(-1) that increases 1700-fold in the presence of the cofactor thrombomodulin. The molecular origin of this effect triggering an important feedback loop in the coagulation cascade remains elusive. Acidic residues in the activation domain of protein C are thought to electrostatically clash with the active site of thrombin. However, functional and structural data reported here support an alternative scenario. The thrombin precursor prethrombin-2 has R15 at the site of activation in ionic interaction with E14e, D14l, and E18, instead of being exposed to solvent for proteolytic attack. Residues E160, D167, and D172 around the site of activation at R169 of protein C occupy the same positions as E14e, D14l, and E18 in prethrombin-2. Caging of R169 by E160, D167, and D172 is responsible for much of the poor activity of thrombin toward protein C. The E160A/D167A/D172A mutant is activated by thrombin 63-fold faster than wild-type in the absence of thrombomodulin and, over a slower time scale, spontaneously converts to activated protein C. These findings establish a new paradigm for cofactor-assisted reactions in the coagulation cascade.

Crystal structures of prethrombin-2 reveal alternative conformations under identical solution conditions and the mechanism of zymogen activation.

Prethrombin-2 is the immediate zymogen precursor of the clotting enzyme thrombin, which is generated upon cleavage at R15 and separation of the A chain and catalytic B chain. The X-ray structure of prethrombin-2 determined in the free form at 1.9 Å resolution shows the 215-217 segment collapsed into the active site and occluding 49% of the volume available for substrate binding. Remarkably, some of the crystals harvested from the same crystallization well, under identical solution conditions, diffract to 2.2 Å resolution in the same space group but produce a structure in which the 215-217 segment moves >5 Å and occludes 24% of the volume available for substrate binding. The two alternative conformations of prethrombin-2 have the side chain of W215 relocating >9 Å within the active site and are relevant to the allosteric E*-E equilibrium of the mature enzyme. Another unanticipated feature of prethrombin-2 bears on the mechanism of prothrombin activation. R15 is found buried within the protein in ionic interactions with E14e, D14l, and E18, thereby making its exposure to solvent necessary for proteolytic attack and conversion to thrombin. On the basis of this structural observation, we constructed the E14eA/D14lA/E18A triple mutant to reduce the level of electrostatic coupling with R15 and promote zymogen activation. The mutation causes prethrombin-2 to spontaneously convert to thrombin, without the need for the snake venom ecarin or the physiological prothrombinase complex.

The dual-targeted plant sulfiredoxin retroreduces the sulfinic form of atypical mitochondrial peroxiredoxin.

Sulfiredoxin (Srx) couples the energy of ATP hydrolysis to the energetically unfavorable process of reducing the inactive sulfinic form of 2-cysteine peroxiredoxins (Prxs) to regenerate its active form. In plants, Srx as well as typical 2-cysteine Prx have been considered as enzymes with exclusive chloroplast localization. This work explores the subcellular localization of Srx in pea (Pisum sativum) and Arabidopsis (Arabidopsis thaliana). Immunocytochemistry, analysis of protein extracts from isolated intact organelles, and cell-free posttranslational import assays demonstrated that plant Srx also localizes to the mitochondrion in addition to plastids. The dual localization was in line with the prediction of a signal peptide for dual targeting. Activity tests and microcalorimetric data proved the interaction between Srx and its mitochondrial targets Prx IIF and thioredoxin. Srx catalyzed the retroreduction of the inactive sulfinic form of atypical Prx IIF using thioredoxin as reducing agent. Arabidopsis Srx also reduced overoxidized human Prx V. These results suggest that plant Srx could play a crucial role in the regulation of Prx IIF activity by controlling the regeneration of its overoxidized form in mitochondria, which are sites of efficient reactive oxygen species production in plants.

DNA binding induces dimerization of Saccharomyces cerevisiae Pif1.

In Saccharomyces cerevisiae, Pif1 is involved in a wide range of DNA transactions. It operates both in mitochondria and in the nucleus, where it has telomeric and non-telomeric functions. All of the activities of Pif1 rely on its ability to bind to DNA. We have determined the mode of Pif1 binding to different DNA substrates. While Pif1 is a monomer in solution, we show that binding of ssDNA to Pif1 induces protein dimerization. DNA-induced dimerization of Pif1 is also observed on tailed- and forked-dsDNA substrates, suggesting that on the latter formation of a Pif1 dimer prevents binding of additional Pif1 molecules. A dimer of Pif1 also forms on ssDNA of random composition and in the presence of saturating concentrations of nonhydrolyzable ATP analogues. The observation that a Pif1 dimer is formed on unwinding substrates in the presence of ATP analogues suggests that a dimeric form of the enzyme might constitute the pre-initiation complex leading to its unwinding activity.

Characterization of plant sulfiredoxin and role of sulphinic form of 2-Cys peroxiredoxin.

The antioxidant function of 2-Cys peroxiredoxin (Prx) involves the oxidation of its conserved peroxidatic cysteine to sulphenic acid that is recycled by a reductor agent. In conditions of oxidative stress, the peroxidatic cysteine can be overoxidized to sulphinic acid inactivating the Prx. An enzyme recently discovered, named sulfiredoxin (Srx), reduces the sulphinic 2-Cys Prx (Prx-SO(2)H). To explore the physiological functions of Srx in plants we have cloned, expressed and purified to homogeneity a Srx from Arabidopsis thaliana (AtSrx), as well as five variants by site-directed mutagenesis on amino acids involved in its activity. The activity of sulfiredoxin, determined by a new method, is dependent on the concentration of the sulphinic form of Prx and the conserved Srx is capable of regenerating the functionality of both pea and Arabidopsis Prx-SO(2)H. Molecular modelling of AtSrx and the facts that the R28Q variant shows a partial inactivation, that the activity of the E76A variant is equivalent to that of the native enzyme and that the double mutation R28Q/E76A abolishes the enzymatic activity suggests that the pair His100-Glu76 may be involved in the activation of C72 in the absence of R28. The knock-out mutant plants without Srx or 2-Cys Prx exhibited phenotypical differences under growth conditions of 16 h light, probably due to the signalling role of the sulphinic form of Prx. These mutants showed more susceptibility to oxidative stress than wild-type plants. This work presents the first systematic biochemical characterization of the Srx/Prx system from plants and contributes to a better understanding of its physiological function.

The oligomeric conformation of peroxiredoxins links redox state to function.

Protein-protein associations, i.e. formation of permanent or transient protein complexes, are essential for protein functionality and regulation within the cellular context. Peroxiredoxins (Prx) undergo major redox-dependent conformational changes and the dynamics are linked to functional switches. While a large number of investigations have addressed the principles and functions of Prx oligomerization, understanding of the diverse in vivo roles of this conserved redox-dependent feature of Prx is slowly emerging. The review summarizes studies on Prx oligomerization, its tight connection to the redox state, and the knowledge and hypotheses on its physiological function in the cell as peroxidase, chaperone, binding partner, enzyme activator and/or redox sensor.

Mitochondrial and nuclear localization of a novel pea thioredoxin: identification of its mitochondrial target proteins.

Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in the regulation of the activity of many enzymes through dithiol-disulfide exchange. In addition to chloroplastic and cytoplasmic Trx systems, plant mitochondria contain a reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase and a specific Trx o, and to date, there have been no reports of a gene encoding a plant nuclear Trx. We report here the presence in pea (Pisum sativum) mitochondria and nuclei of a Trx isoform (PsTrxo1) that seems to belong to the Trx o group, although it differs from this Trx type by its absence of introns in the genomic sequence. Western-blot analysis with isolated mitochondria and nuclei, immunogold labeling, and green fluorescent protein fusion constructs all indicated that PsTrxo1 is present in both cell compartments. Moreover, the identification by tandem mass spectrometry of the native mitochondrial Trx after gel filtration using the fast-protein liquid chromatography system of highly purified mitochondria and the in vitro uptake assay into isolated mitochondria also corroborated a mitochondrial location for this protein. The recombinant PsTrxo1 protein has been shown to be reduced more effectively by the Saccharomyces cerevisiae mitochondrial Trx reductase Trr2 than by the wheat (Triticum aestivum) cytoplasmic reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase. PsTrxo1 was able to activate alternative oxidase, and it was shown to interact with a number of mitochondrial proteins, including peroxiredoxin and enzymes mainly involved in the photorespiratory process.

Thermodynamics of 2-Cys peroxiredoxin assembly determined by isothermal titration calorimetry.

Oligomerization is a frequently encountered physical characteristic of biological molecules that occurs for a wide number of transcription factors, ion channels, oxygen-carrying macromolecules such as hemocyanin and enzymes. On the other hand, unwanted protein oligomerization can lead to the formation of pathogenic structures related with Alzheimer and other diseases. Self-assembly is also a well-described phenomenon within peroxiredoxins, a family of thiol peroxidases. Peroxiredoxin hyperaggregate formation is the key mechanism that triggers the switch between Prx activity as peroxidase and chaperone. The oligomerization process is fundamental for understanding the multiple peroxiredoxin function. The chapter gives a detailed description of typical 2-Cys Peroxiredoxin oligomerization using isothermal titration calorimetry (ITC) and provides a recipe for studying the thermodynamic parameters of peroxiredoxin assembly, that is, association and dissociation constant, enthalpy, entropy, and the Gibbs free energy of the process.

Hexameric oligomerization of mitochondrial peroxiredoxin PrxIIF and formation of an ultrahigh affinity complex with its electron donor thioredoxin Trx-o.

Mitochondria from plants, yeast, and animals each contain at least one peroxiredoxin (Prx) that is involved in peroxide detoxification and redox signalling. The supramolecular dynamics of atypical type II Prx targeted to the mitochondrion was addressed in pea. Microcalorimetric (ITC) titrations identified an extremely high-affinity binding between the mitochondrial PsPrxIIF and Trx-o with a K(D) of 126+/-14 pM. Binding was driven by a favourable enthalpy change (DeltaH= -60.6 kcal mol(-1)) which was counterbalanced by unfavourable entropy changes (TDeltaS= -47.1 kcal mol(-1)). This is consistent with the occurrence of large conformational changes during binding which was abolished upon site-directed mutaganesis of the catalytic C59S and C84S. The redox-dependent interaction was confirmed by gel filtration of mitochondrial extracts and co-immunoprecipitation from extracts. The heterocomplex of PsPrxIIF and Trx-o reduced peroxide substrates more efficiently than free PsPrxIIF suggesting that Trx-o serves as an efficient and specific electron donor to PsPrxIIF in vivo. Other Trx-s tested by ITC analysis failed to interact with PsPrxIIF indicating a specific recognition of PsPrxIIF by Trx-o. PsPrxIIF exists primarily as a dimer or a hexamer depending on the redox state. In addition to the well-characterized oligomerization of classical 2-Cys Prx the results also show that atypical Prx undergo large structural reorganization with implications for protein-protein interaction and function.

Thermodynamics of the dimer-decamer transition of reduced human and plant 2-cys peroxiredoxin.

Isothermal titration calorimetry (ITC) is a powerful technique for investigating self-association processes of protein complexes and was expected to reveal quantitative data on peroxiredoxin oligomerization by directly measuring the thermodynamic parameters of dimer-dimer interaction. Recombinant classical 2-cysteine peroxoredoxins from Homo sapiens, Arabidopsis thaliana, and Pisum sativum as well as a carboxy-terminally truncated variant were subjected to ITC analysis by stepwise injection into the reaction vessel under various redox conditions. The direct measurement of the decamer-dimer equilibrium of reduced peroxiredoxin revealed a critical concentration in the very low micromolar range. The data suggest a cooperative assembly above this critical transition concentration where a nucleus facilitates assembly. The rather abrupt transition indicates that assembly processes do not occur below the critical transition concentration while oligomerization is efficiently triggered above it. The magnitude of the measured enthalpy confirmed the endothermic nature of the peroxiredoxin oligomerization. Heterocomplexes between peroxiredoxin polypeptides from different species were not formed. We conclude that a functional constraint conserved the dimer-decamer transition with highly similar critical transition concentrations despite emerging sequence variation during evolution.

Biochemical and molecular characterization of the mitochondrial peroxiredoxin PsPrxII F from Pisum sativum.

The pea peroxiredoxin homologue PsPrxII F of the Arabidopsis thaliana mitochondrial AtPrxII F was isolated as cDNA and genomic DNA, and characterized in respect to its biochemical and molecular properties. The deduced amino acid sequence contains an N-terminal targeting address for mitochondrial import. Mitochondrial location of PsPrxII F was confirmed by immunocytochemistry. The mature enzyme, without the transit peptide, has a molecular mass of 18.75 kDa, and, at positions 59 and 84, carries the two catalytic cysteinyl residues which are characteristic for this particular Prx subgroup. Activity of site-directed mutagenized C84S-variant lacking the so-called resolving Cys dropped to about 12% of WT Prx while C59S lost its peroxidatic activity completely. Likewise, WT PsPrxII F and C84S-variant but not C59S protected plasmid DNA against strand breakage in a mixed function oxidation assay. WT PrxII F and the variant proteins aggregated to high mass oligomers not yet described for type II Prx. Upon oxidation with hydrogen peroxide PsPrxII F focussed in a series of spots of distinct pI but similar molecular masses in two-dimensional gels indicating different oxidation states of the protein. Using this technique, partial oxidation was also detected in leaf extracts and isolated mitochondria. PsPrxII F mRNA and protein accumulated in cold and heavy metals treated pea plants suggesting a particular function under stress.

Cloning, overexpression, purification and preliminary crystallographic studies of a mitochondrial type II peroxiredoxin from Pisum sativum.

A cDNA encoding an open reading frame of 199 amino acids corresponding to a type II peroxiredoxin from Pisum sativum with its transit peptide was isolated by RT-PCR. The 171-amino-acid mature protein (estimated molecular weight 18.6 kDa) was cloned into the pET3d vector and overexpressed in Escherichia coli. The recombinant protein was purified and crystallized by the hanging-drop vapour-diffusion technique. A full data set (98.2% completeness) was collected using a rotating-anode generator to a resolution of 2.8 angstroms from a single crystal flash-cooled at 100 K. X-ray data revealed that the protein crystallizes in space group P1, with unit-cell parameters a = 61.88, b = 66.40, c = 77.23 angstroms, alpha = 102.90, beta = 104.40, gamma = 99.07 degrees, and molecular replacement using a theoretical model predicted from the primary structure as a search model confirmed the presence of six molecules in the unit cell as expected from the Matthews coefficient. Refinement of the structure is in progress.