Reductive detoxification of acrolein as a potential role for aldehyde reductase (AKR1A) in mammals

Aldehyde reductase (AKR1A), a member of the aldo–keto reductase superfamily, suppresses diabetic complications via a reduction in metabolic intermediates; it also plays a role in ascorbic acid biosynthesis in mice. Because primates cannot synthesize ascorbic acid, a principle role of AKR1A appears to be the reductive detoxification of aldehydes. In this study, we isolated and immortalized mouse embryonic fibroblasts (MEFs) from wild-type (WT) and human Akr1a-transgenic (Tg) mice and used them to investigate the potential roles of AKR1A under culture conditions. Tg MEFs showed higher methylglyoxal- and acrolein-reducing activities than WT MEFs and also were more resistant to cytotoxicity. Enzymatic analyses of purified rat AKR1A showed that the efficiency of the acrolein reduction was about 20% that of glyceraldehyde. Ascorbic acid levels were quite low in the MEFs, and while the administration of ascorbic acid to the cells increased the intracellular levels of ascorbic acid, it had no affect on the resistance to acrolein. Endoplasmic reticulum stress and protein carbonylation induced by acrolein treatment were less evident in Tg MEFs than in WT MEFs. These data collectively indicate that one of the principle roles of AKR1A in primates is the reductive detoxification of aldehydes, notably acrolein, and protection from its detrimental effects.     

Isoform composition of antithrombin in a covalent antithrombin-heparin complex

Antithrombin (AT) circulates in two isoforms, alpha- (90-95%) and beta-AT (5-10%). AT inhibits clotting factors such as thrombin and factor Xa, a reaction catalyzed by heparin. Heparin has been used in many clinical situations but suffers from limitations such as a short intravenous half-life, bleeding risk, and the inability to inhibit thrombin bound to fibrin clots. In order to overcome some of heparin's limitations, we prepared a covalent AT-heparin complex (ATH) that has increased intravenous half-life, reduced bleeding risk, and can directly inhibit clot-bound thrombin. However, structural analysis is required to further develop this promising antithrombotic agent. It was found that the proportion of isoforms in ATH (55% alpha-AT, and 45% beta-AT) was significantly different than that in the commercial AT starting material (80% alpha-AT and 20% beta-AT). Further analysis of the rate of heparin-catalyzed inhibition of thrombin by AT isoforms prepared from ATH revealed that the beta-variant reacted approximately 2-fold faster.     

Separation of latent, prelatent, and native forms of human antithrombin by heparin affinity high-performance liquid chromatography

Latent antithrombin (LAT) is a partially denatured form of human antithrombin (AT). LAT does not inhibit clotting of the blood, but has previously been shown to inhibit angiogenesis and carcinogenesis. Another probably partially denatured form is the so-called prelatent AT (P-LAT), described by Larsson et al. [J. Biol. Chem. 276 (2001) 11996]. In the present work, an analytical heparin affinity chromatography method is described that separates an AT form, which is formed during the pasteurization process and which we believe to be identical to the previously described P-LAT, from native AT and LAT. Non-pasteurized AT was shown to contain no P-LAT, while four, heat-treated commercial AT products all contained P-LAT (1-6%, mean=4%). P-LAT has a slightly lower affinity to heparin than does native AT, but exhibits a much stronger heparin affinity when compared to LAT. P-LAT and native AT were shown to have very similar thrombin inhibiting activity, while LAT lacks such activity.     

Heparin is a major activator of the anticoagulant serpin, protein Z-dependent protease inhibitor

Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa and XIa. Here we provide evidence that, in addition to the established cofactors, protein Z, lipid, and calcium, heparin is an important cofactor of ZPI anticoagulant function. Heparin produced 20-100-fold accelerations of ZPI reactions with factor Xa and factor XIa to yield second order rate constants approaching the physiologically significant diffusion limit (k(a) = 10(6) to 10(7) M(-1) s(-1)). The dependence of heparin accelerating effects on heparin concentration was bell-shaped for ZPI reactions with both factors Xa and XIa, consistent with a template-bridging mechanism of heparin rate enhancement. Maximal accelerations of ZPI-factor Xa reactions required calcium, which augmented the heparin acceleration by relieving Gla domain inhibition as previously shown for heparin bridging of the antithrombin-factor Xa reaction. Heparin acceleration of both ZPI-protease reactions was optimal at heparin concentrations and heparin chain lengths comparable with those that produce physiologically significant rate enhancements of other serpin-protease reactions. Protein Z binding to ZPI minimally affected heparin rate enhancements, indicating that heparin binds to a distinct site on ZPI and activates ZPI in its physiologically relevant complex with protein Z. Taken together, these results suggest that whereas protein Z, lipid, and calcium cofactors promote ZPI inhibition of membrane-associated factor Xa, heparin activates ZPI to inhibit free factor Xa as well as factor XIa and therefore may play a physiologically and pharmacologically important role in ZPI anticoagulant function.     

Control of post-translational modifications in antithrombin during murine post-natal development by miR-200a

Background

Developmental haemostatic studies may help identifying new elements involved in the control of key haemostatic proteins like antithrombin, the most relevant endogenous anticoagulant.

Results

In this study, we showed a significant reduction of sialic acid content in neonatal antithrombin compared with adult antithrombin in mice. mRNA levels of St3gal3 and St3gal4, two sialyltransferases potentially involved in antithrombin sialylation, were 85% lower in neonates in comparison with adults. In silico analysis of miRNAs overexpressed in neonates revealed that mir-200a might target these sialyltransferases. Moreover, in vitro studies in murine primary hepatocytes sustain this potential control.

Conclusions

These data suggest that in addition to the direct protein regulation, microRNAs may also modulate qualitative traits of selected proteins by an indirect control of post-translational processes.     

Tubulin-Tyrosine Ligase,a Long-Lasting Enigma

Tubulins and microtubules are subjected to several post-translational modifications of which the reversible detyrosination/tyrosination of the carboxy-terminal end of most -tubulins has been extensively analysed. This modification cycle involves a specific carboxypeptidase and the activity of the tubulin-tyrosine ligase (TTL). The true physiological function of TTL has so far not been established. This review describes the purification of TTL to homogeneity by biochemical methods, its in vitro properties and the generation of monoclonal antibodies. These mabs not only enabled a very convenient and rapid purification of TTL by immunoaffinity chromatography but also its extensive characterization by protein sequencing, which led to the isolation of the full length cDNA. With this information, gene disruption should be feasible in order to determine the physiological significance of the tyrosination cycle.     

Isolation of a pure octadecasaccharide with antithrombin activity from an ultra-low-molecular-weight heparin

Heparin and low-molecular-weight heparins (LMWHs) are anticoagulant drugs that mainly inhibit the coagulation cascade by indirectly interacting with factor Xa and factor IIa (thrombin). Inhibition of factor Xa by antithrombin (AT) requires the activation of AT by specific pentasaccharide sequences containing 3-O-sulfated glucosamine. Activated AT also inhibits thrombin by forming a stable ternary complex of AT, thrombin, and a polysaccharide (requires at least an 18-mer/octadeca-mer polysaccharide). The full structure of any naturally occurring octadecasaccharide sequence has yet to be determined. In the context of the development of LMWH biosimilars, structural data on such important biological mediators could be helpful for better understanding and regulatory handling of these drugs. Here we present the isolation and identification of an octadecasaccharide with very high anti-factor Xa activity (∼3 times higher than USP [U.S. Pharmacopeia] heparin). The octadecasaccharide was purified using five sequential chromatographic methods with orthogonal specificity, including gel permeation, AT affinity, strong anion exchange, and ion-pair chromatography. The structure of the octadecasaccharide was determined by controlled enzymatic sequencing and nuclear magnetic resonance (NMR). The isolated octadecasaccharide contained three consecutive AT-binding sites and was tested in coagulation assays to determine its biological activity. The isolation of this octadecasaccharide provides new insights into the modulation of thrombin activity.     

Role of P2 glycine in determining the specificity of antithrombin reaction with coagulation proteases

Structural data suggests that bulky hydrophobic residues at the S2-S4 sub-sites of factor Xa (fXa) restrict the preference of this pocket for small and non-polar residues like Gly at the P2 position of substrates and inhibitors. However, kinetic studies monitoring the cleavage specificity of 10-residue peptides by fXa have identified Phe as the most preferred P2 residue and Gln-Phe-Arg-Ser-Leu-Ser as the most preferred P3-P3′ residues for recognition by fXa. To determine whether this mechanism of specificity is also true for fXa reaction with antithrombin (AT), we prepared two AT mutants having either a Phe at the P2 or Gln-Phe-Arg-Ser-Leu-Ser at the P3-P3′ positions of the reactive center loop. Inhibition kinetic studies indicated that the reactivity of P2-Phe with fXa was significantly (∼5-fold) impaired, however, the P3-P3′ mutant exhibited 1.5-fold improved reactivity with the protease, suggesting cooperative effects between P3-P3′ residues influence the P2 specificity of AT. Substitution of Tyr-99 of fXa with a Gly dramatically impaired the reactivity of fXa with wild-type AT, but improved its reactivity with the serpin mutants in the absence, but not in the presence of pentasaccharide. AT with a P2-Phe inhibited thrombin with >150-fold impaired reactivity, however, the defect was restored by either pentasaccharide or by replacing Leu-99 of thrombin with a Gly. The P3-P3′ mutant rapidly inhibited factors VIIa and XIa independent of pentasaccharide. These results indicate that P2-Gly plays a key role in determining the S2 sub-site specificity and target protease selectivity of AT in circulation.     

Influence of ionic strength and pH on the interaction between high-affinity heparin and antithrombin

Binding constants for the binding of high-affinity heparin to antithrombin at different ionic strengths were determined by fluorescence titrations and were also estimated from dissociation curves of the heparin-antithrombin complex. These curves were monitored by near-ultraviolet circular dichroism or fluorescence. The dependence of the binding constant on the activity of NaCl suggested that maximally 5–6 charged groups are directly involved in the interaction between the two macromolecules. Major pH-dependent changes of the interaction, as evident by changes of the spectroscopic properties of the complex between the molecules, were found to occur below pH 5.5 and above pH 8.5. The acid change, which was irreversible, was most likely caused by an irreversible conformational change of antithrombin. At alkaline pH, however, the gross conformation of antithrombin was stable up to pH 12, while the affinity of high-affinity heparin for antithrombin began to decrease markedly at pH 8.5. The dissociation curve, which was reversible, had a midpoint around pH 9.5. This is compatible with the loss of affinity being caused by either a local conformational change, by ionization of tyrosine or by titration of one or more amino groups.     

Arg-129 plays a specific role in the confirmation of antithrombin and in the enhancement of factor Xa inhibition by the pentasaccharode sequence of heparin

Small amounts of a variant antithrombin (AT) bearing an Arg-129 to Gln mutation were purified from plasma by means of affinity chromatography on insolubilized herapin at very low ionic strength. As a control, two variant antithrombins, one bearing on Pro-41 to Leu mutation and the other an Arg-47 to His mutation, were purified in the same way. The biochemical characterization of the variants and the kinetic study of thrombin and activated factor X (F Xa) inhibition in the presence of heparin and heparin derivatives suggest that Arg-129 plays a specific role in AT conformation and F Xa inhibition enhancement. Indeed, the purified variant adopted the locked conformation described ,for AT submitted to mild denaturing conditions (Carrell, R.W., Evans, D.Li and Stein, P.E. (1991) Nature 353, 576–578) and resembling the latent form of plasminogen activator inhibitor (PAI) (Mottonen J., Strand, A., Symersky, J., Sweet, R.M., Danley, D.E., Geohegan, K.F., Gerard, R.D. and Goldsmith, E.J. (1992) Nature 355, 270–273). Moreover, the mutant AT was partially reactivated by heparin for thrombin inhibition, but did not respond to the specific pentasaccharide domain of heparin for F Xa inhibition.     

Characterization of a synthetic peptide from the glycosaminoglycan binding site of heparin cofactor II

Summary We characterized a synthetic peptide based on the glycosaminoglycan (GAG)-binding site of the serine proteinase inhibitor (serpin) heparin cofactor II (HCII): HCII^165–195, K¹⁶⁵DFVNASSKYEITTIHNLFRKLTHRLFRRNF¹⁹⁵. HCII^165–195 negated acceleration of the HCII/thrombin inhibition reaction (IC₅₀ for the peptide shown in parentheses) by heparin (250 nM) and dermatan sulfate (500 nM). Circular dichroism spectra of HCII^165–195 showed that GAGs increase the -helical content of the peptide (percentage -helix of the peptide/GAG complex given in parentheses): no GAG (7%) < low-molecular-weight heparin (32%) < heparin (42%) < dermatan sulfate (55%). A molecular model of HCII predicts that this region is 48% -helix. Our results suggest: (i) HCII^165–195 binds to GAGs; (ii) an -helical conformation is preferable in the presence of GAGs; and (iii) GAGs may help stabilize a specific protein conformation in the HCII GAG-binding site, important for serpin function.     

Modification and inactivation of Cu,Zn-superoxide dismutase by the lipid peroxidation product,acrolein

Acrolein is the most reactive aldehydic product of lipid peroxidation and is found to be elevated in the brain when oxidative stress is high. The effects of acrolein on the structure and function of human Cu,Zn-superoxide dismutase (SOD) were examined. When Cu,Zn-SOD was incubated with acrolein, the covalent crosslinking of the protein was increased, and the loss of enzymatic activity was increased in a dose-dependent manner. Reactive oxygen species (ROS) scavengers and copper chelators inhibited the acrolein-mediated Cu,Zn-SOD modification and the formation of carbonyl compound. The present study shows that ROS may play a critical role in acrolein-induced Cu,Zn-SOD modification and inactivation. When Cu,Zn-SOD that has been exposed to acrolein was subsequently analyzed by amino acid analysis, serine, histidine, arginine, threonine and lysine residues were particularly sensitive. It is suggested that the modification and inactivation of Cu,Zn-SOD by acrolein could be produced by more oxidative cell environments. [BMB Reports 2013; 46(11): 555-560]     

Phosphosite-specific regulation of the oxidative-stress response of Paracoccidioides brasiliensis: a shotgun phosphoproteomic analysis

Paracoccidioides brasiliensis, a thermally dimorphic fungus, is the causative agent of paracoccidioidomycosis, a systemic mycosis that is widespread in Latin America. This fungus is a facultative intracellular pathogen able to survive and replicate inside non-activated macrophages. Therefore, the survival of P. brasiliensis inside the host depends on the ability to adapt to oxidative stress induced by immune cells, especially alveolar macrophages. For several years, reactive oxygen species (ROS) were only associated with pathological processes. Currently, a plethora of roles for ROS in cell signaling have emerged. We have previously reported that low ROS concentrations cause cell proliferation in the human pathogenic fungus P. brasiliensis. In the present report, we investigated the influence of phosphorylation events in that process. Using a mass spectrometry-based approach, we mapped 440 phosphorylation sites in 230 P. brasiliensis proteins and showed that phosphorylation at different sites determines fungal responses to oxidative stress, which are regulated by phosphatases and kinases activities. Furthermore, we present additional evidence for a functional two-component signal transduction system in P. brasiliensis. These findings will help us to understand the phosphorylation events involved in the oxidative stress response.     

Thermal stabilization of antithrombin III by sugars and sugar derivatives and the effects on nonenzymatic glycosylation

A variety of neutral and acidic sugars and related compounds were evaluated in terms of their effect on the midpoint, T_d, of the thermal denaturation curve of antithrombin III. The objectives were to determine which structural features of these molecules are responsible for their stabilizing properties and to identify more efficient stabilizers which combine the effects of lyotropic anions such as citrate with those of the polyols in a single molecule. The presence of one or more carboxylate groups in a sugar molecule invariably increased its stabilizing potency, whereas the number and position of hydroxyl groups appeared to have no influence on the molecules' stabilizing ability. Several compounds were shown to be effective in preserving antithrombin III activity during pasteurization for 10 h at 60°C. However, the presence of reducing sugars invariably resulted in a decrease in activity following pasteurization, in spite of their ablity to increase T_d. In fact, when antithrombin III was pasteurized in the presence of 2 M glucose and 0.5 M citrate, it steadily losts its ability to inhibit thrombin even though T_d under the conditions was 10°C higher than in citrate alone where activity was preserved. This effect was shown to be coincident with the covalent incorporation of glucose into the protein molecule.     

Alpha-1-proteinase inhibitor is a heparin binding serpin: molecular interactions with the Lys rich cluster of helix-F domain

Alpha-1-proteinase (alpha-1-PI) inhibitor is the major circulating serine protease inhibitor in humans. The porcine elastase and trypsin inhibitory activity of human and ovine alpha-1-PI is activated several fold in the presence of anti-coagulant heparin. The activation is allosteric and appears to be characterized by two steps of binding; a weak followed by a strong binding. The Kass for ovine and human alpha-1-PI inhibition of porcine pancreatic elastase was increased approximately 45 fold and 38 fold respectively. Using a combinatorial approach of multiple sequence alignment, surface topology, chemical modification and tryptic peptide mapping to identify the sequence of the heparin bound peptide; we demonstrate that heparin binds to the lysyl rich region of the F-helix of alpha-1-PI, which differs from that of heparin-antithrombin (AT) interactions. Molecular docking prediction using the MEDock algorithm approximates the three positively charged lysines (K154, K155, K174) of human alpha-1-PI in this interaction. This heparin alpha-1-PI interaction has been exploited to develop an affinity purification method, which can be used universally to obtain homogenous preparations of mammalian alpha-1-PIs useful for augmentation therapy. Collectively, all these findings imply that alpha-1-PI has a major role in regulating extra cellular protease activity and the physiological activator is heparin.     

Systematic study on the mechanism of aldehyde oxidation to carboxylic acid by cytochrome P450

The mechanism of aldehyde to carboxylic acid conversion catalyzed by P450 enzymes via a series of reactions was studied systematically for the first time with density functional theory calculations. A two-state reactivity mechanism has been proposed, which can be adopted for many aldehyde oxidation reactions catalyzed by P450 enzymes. The mechanism involves initial hydrogen abstraction as the rate-limiting step and this is followed by steps of oxygen rebound without barriers owing to the quick recombination of the resultant radical species. Meanwhile, in an attempt to explore whether there exist some rules for the hydroxylation of aldehyde substrates by P450, the transition state barriers of the rate-limiting step for a series of aldehyde hydroxylation reactions have been compared. A predictive pattern of extended barrier/bond energy correlation for different hydroxylations of aldehyde substrates by P450 has been established, which was further confirmed to be a reliable reactivity scale by experimental results. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Aldimine to ketoamine isomerization (Amadori rearrangement) potential at the individual nonenzymic glycation sites of hemoglobin a: Preferential inhibition of glycation by nucleophiles at sites of low isomerization potential

The relative roles of the two structural aspects of nonenzymic glycation sites of hemoglobin A, namely the ease with which the amino groups could form the aldimine adducts and the propensity of the microenvironments of the respective aldimines to facilitate the Amadori rearrangement, in dictating the site selectivity of nonenzymic glycation with aldotriose has been investigated. The chemical reactivity of the amino groups of hemoglobin A forin vitro reductive glycation with aldotriose is distinct from that in the nonreductive mode. The reactivity of amino groups of hemoglobin A toward reductive glycation (i.e., propensity for aldimine formation) decreases in the order Val-1(), Val-1(), Lys-66(), Lys-61(), and Lys-16(). The overall reactivity of hemoglobin A toward nonreductive glycation decreased in the order Lys-16(), Val-1(), Lys-66(), Lys-82(), Lys-61(), and Val-1(). Since the aldimine is the common intermediate for both the reductive and nonreductive modification, the differential selectivity of protein for the two modes of glycation is clearly a reflection of the propensity of the microenvironments of nonenzymic glycation sites to facilitate the isomerization reaction (i.e., Amadori rearrangement). A semiquantitative estimate of this propensity of the microenvironment of the nonenzymic glycation sites has been obtained by comparing the nonreductive (nonenzymic) and reductive modification at individual glycation sites. The microenvironment of Lys-16() is very efficient in facilitating the rearrangement and the relative efficiency decreases in the order Lys-16(), Lys-82(), Lys-66(), Lys-61(), Val-1(), and Val-1(). The propensity of the microenvironment of Lys-16() to facilitate the Amadori rearrangement of the aldimine is about three orders of magnitude higher than that of Val-1() and is about 50 times higher than that of Val-1(). The extent of nonenzymic glycation at the individual sites is modulated by various factors, such as thepH, concentration of aldotriose, and the concentration of the protein. The nucleophiles—such as tris, glycine ethyl ester, and amino guanidine—inhibit the glycation by trapping the aldotriose. The nonenzymic glycation inhibitory power of nucleophile is directly related to its propensity to form aldimine. Thus, the extent of inhibition of nonenzymic glycation at a given site by a nucleophile directly reflects the relative role ofpK _a of the site in dictating the glycation at that site. The nonenzymic glycation of an amino group of a protein is an additive/synergestic consequence of the propensity of the site to form aldimine adducts on one hand, and the propensity of its microenvironment to facilitate the isomerization of the aldimines to ketoamines on the other. The isomerization potential of microenvironment plays the dominant role in dictating the site specificity of the nonenzymic glycation of proteins.     

Effects of aldehyde and internal ions on bioluminescence expression of Photobacterium phosphoreum

In a complex medium, cells of Photobacterium phosphoreum (strain 496) grow equally well with 1% and 3% NaCl, but luminescence occurs only with 3% NaCl in the medium. However, the suppression of luminescence is not attributable to the lack of luciferase; log phase cells growing in 1% NaCl will develop luminescence following a shift to 3% NaCl, which is accompanied by an increase of intracellular potassium. Tetradecanal stimulates bioluminescence in a 1% NaCl culture, and also in the presence of nalidixic acid, an inhibitor or gyrase. It is thus suggested that the suppression of luminescence in 1% NaCl or in 3% NaCl with nalidixic acid is due to a deficiency in the synthesis of intracellular aldehyde. The increase in intracellular potassium that occurs upon shifting from 1% to 3% NaCl may also relate to aldehyde synthesis gene expression via activation of gyrase, or via an increase in negative supercoiling of the chromosome. However, since an initial decrease of light intensity is still observed during culture even with the addition of tetradecanal, an additional factor related to cell density must also be involved in bioluminescence expression.Abbreviations nal nalidixic acid - nal-r nalidixic acid resistant strain