Maria Victòria Nogués

Kinetic and Product Distribution Analysis of Human Eosinophil Cationic Protein Indicates a Subsite Arrangement That Favors Exonuclease-type Activity

With the use of a high yield prokaryotic expression system, large amounts of human eosinophil cationic protein (ECP) have been obtained. This has allowed a thorough kinetic study of the ribonuclease activity of this protein. The catalytic efficiencies for oligouridylic acids of the type (Up) n U>p, mononucleotides U>p and C>p, and dinucleoside monophosphates CpA, UpA, and UpG have been interpreted by the specific subsites distribution in ECP. The distribution of products derived from digestion of high molecular mass substrates, such as poly(U) and poly(C), by ECP was compared with that of RNase A. The characteristic cleavage pattern of polynucleotides by ECP suggests that an exonuclease-like mechanism is predominantly favored in comparison to the endonuclease catalytic mechanism of RNase A. Comparative molecular modeling with bovine pancreatic RNase A-substrate analog crystal complexes revealed important differences in the subsite structure, whereas the secondary phosphate-binding site (p2) is lacking, the secondary base subsite (B2) is severely impaired, and there are new interactions at the po, Bo, and p-1sites, located upstream of the P-O-5′ cleavable phosphodiester bond, that are not found in RNase A. The differences in the multisubsites structure could explain the reduced catalytic efficiency of ECP and the shift from an endonuclease to an exonuclease-type mechanism.

The cytotoxicity of eosinophil cationic protein/ribonuclease 3 on eukaryotic cell lines takes place through its aggregation on the cell membrane

Abstract.Human eosinophil cationic protein (ECP)/ ribonuclease 3 (RNase 3) is a protein secreted from the secondary granules of activated eosinophils. Specific properties of ECP contribute to its cytotoxic activities associated with defense mechanisms. In this work the ECP cytotoxic activity on eukaryotic cell lines is analyzed. The ECP effects begin with its binding and aggregation to the cell surface, altering the cell membrane permeability and modifying the cell ionic equilibrium. No internalization of the protein is observed. These signals induce cell-specific morphological and biochemical changes such as chromatin condensation, reversion of membrane asymmetry, reactive oxygen species production and activation of caspase-3-like activity and, eventually, cell death. However, the ribonuclease activity component of ECP is not involved in this process as no RNA degradation is observed. In summary, the cytotoxic effect of ECP is attained through a mechanism different from that of other cytotoxic RNases and may be related with the ECP accumulation associated with the inflammatory processes, in which eosinophils are present.

The antipathogen activities of eosinophil cationic protein.

The eosinophil cationic protein (ECP) is a secretory ribonuclease, which is found in the eosinophilic leukocyte and involved in the innate immune system. Its cytotoxic activity is effective against a wide range of pathogens, suggesting a relatively non-specific mechanism of action. We review here the specific antipathogen activities that have been characterized for ECP. Although eosinophils and ECP are primarily associated with the host defense against nonphagocytosable pathogens, such as helminthic parasites, ECP has also an antibacterial activity, which is not shared by the other, closely-related eosinophil ribonuclease, the eosinophil derived neurotoxin (EDN). Although there is no evidence for direct involvement in vivo of eosinophils in the host response to bacterial infection, ECP is active against both Gram-negative and Gram-positive bacterial strains and its mechanism depends on its action both at the bacterial cell wall and cytoplasmic membrane levels. Other antipathogen activities, including antihelminthic activity, are also discussed. Modulation of the protein activity by posttranslational modifications and the currently identified polymorphisms are reviewed. Antimicrobial RNases, as innate immune proteins with anti-infective and immunomodulatory properties, present substantial therapeutic potential in the drug development industry, both in the search of alternative antibiotics and for the treatment of inflammatory disorders.

The contribution of noncatalytic phosphate-binding subsites to the mechanism of bovine pancreatic ribonuclease A

Abstract. The enzymatic catalysis of polymeric substrates such as proteins, polysaccharides or nucleic acids requires precise alignment between the enzyme and the substrate regions flanking the region occupying the active site. In the case of ribonucleases, enzyme-substrate binding may be directed by electrostatic interactions between the phosphate groups of the RNA molecule and basic amino acid residues on the enzyme. Specific interactions between the nitrogenated bases and particular amino acids in the active site or adjacent positions may also take place. The substrate-binding subsites of ribonuclease A have been characterized by structural and kinetic studies. In addition to the active site (p1 ), the role of other noncatalytic phosphate-binding subsites in the correct alignment of the polymeric substrate has been proposed. p2 and p0 have been described as phosphate-binding subsites that bind the phosphate group adjacent to the 3′ side and 5′ side, respectively, of the phosphate in the active site. In both cases, basic amino acids (Lys-7 and Arg-10 in p2 , and Lys-66 in p0 ) are involved in binding. However, these binding sites play different roles in the catalytic process of ribonuclease A. The electrostatic interactions in p2 are important both in catalysis and in the endonuclease activity of the enzyme, whilst the p0 electrostatic interaction contributes only to binding of the RNA.

The Subsites Structure of Bovine Pancreatic Ribonuclease A Accounts for the Abnormal Kinetic Behavior with Cytidine 2′,3′-Cyclic Phosphate

The kinetics of the hydrolysis of cytidine 2′,3′-cyclic phosphate (C>p) to 3′-CMP by ribonuclease A are multiphasic at high substrate concentrations. We have investigated these kinetics by determining 3′-CMP formation both spectrophotometrically and by a highly specific and quantitative chemical sampling method. With the use of RNase A derivatives that lack a functional p2 binding subsite, evidence is presented that the abnormal kinetics with the native enzyme are caused by the sequential binding of the substrate to the several subsites that make up the active site of ribonuclease. The evidence is based on the following points. 1) Some of the unusual features found with native RNase A and C>p as substrate disappear when the derivatives lacking a functional p2 binding subsite are used. 2) Thek cat/K m values with oligocytidylic acids of increasing lengths (ending in C>p) show a behavior that parallels the specific velocity with C>p at high concentrations: increase in going from the monomer to the trimer, a decrease from tetramer to hexamer, and then an increase in going to poly(C). 3) Adenosine increases the k catobtained with a fixed concentration of C>p as substrate. 4) High concentrations of C>p protect the enzyme from digestion with subtilisin, which results in a more compact molecule, implying large substrate concentration-induced conformational changes. The data for the hydrolysis of C>p by RNase A can be fitted to a fifth order polynomial that has been derived from a kinetic scheme based on the sequential binding of several monomeric substrate molecules.

A human ribonuclease induces apoptosis associated with p21 WAF1/CIP1 induction and JNK inactivation

BackgroundRibonucleases are promising agents for use in anticancer therapy. Among the different ribonucleases described to be cytotoxic, a paradigmatic example is onconase which manifests cytotoxic and cytostatic effects, presents synergism with several kinds of anticancer drugs and is currently in phase II/III of its clinical trial as an anticancer drug against different types of cancer. The mechanism of cytotoxicity of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase.MethodsCytotoxicity was measured by the MTT method and by the tripan blue exclusion assay. Apoptosis was assessed by flow cytometry, caspase enzymatic detection and confocal microscopy. Cell cycle phase analysis was performed by flow cytometry. The expression of different proteins was analyzed by western blot.ResultsWe show that the cytotoxicity of PE5 is produced through apoptosis, that it does not require the proapoptotic activity of p53 and is not prevented by the multiple drug resistance phenotype. We also show that PE5 and onconase induce cell death at the same extent although the latter is also able to arrest the cell growth. We have compared the cytotoxic effects of both ribonucleases in the NCI/ADR-RES cell line by measuring their effects on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins. PE5 increases the number of cells in S and G2/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21WAF1/CIP1 together with the underphosphorylation of p46 forms of JNK. Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAPConclusionsWe conclude that PE5 kills the cells through apoptosis associated with the p21WAF1/CIP1 induction and the inactivation of JNK. This mechanism is significantly different from that found for onconase.

The 1H, 13C, 15N resonance assignment, solution structure, and residue level stability of eosinophil cationic protein/RNase 3 determined by NMR spectroscopy†

Eosinophil cationic protein (ECP)/human RNase 3, a member of the RNase A family, is a remarkably cytotoxic protein implicated in asthma and allergies. These activities are probably due to ECPs ability to interact with and disrupt membranes and depend on two Trp, 19 Arg, and possibly an extremely high conformational stability. Here, we have used NMR spectroscopy to assign essentially all 1H, 15N, and backbone 13C resonances, to solve the 3D structure in aqueous solution and to quantify its residue‐level stability. The NMR solution structure was determined on the basis of 2316 distance constraints and is well‐defined (backbone RMSD = 0.81 Å). The N‐terminus and the loop composed of residues 114–123 are relatively well‐ordered; in contrast, conformational diversity is observed for the loop segments 17–22, 65–68, and 92–95 and most exposed sidechains. The side chain NH groups of the two Trp and 19 Arg showed no significant protection against hydrogen/deuterium exchange. The most protected NH groups belong to the first and last two β‐strands, and curiously, the first α‐helix. Analysis of their exchange rates reveals a strikingly high global stability of 11.8 kcal/mol. This value and other stability measurements are used to better quantify ECPs unfolding thermodynamics.

Protein post-translational modification in host defense: the antimicrobial mechanism of action of human eosinophil cationic protein native forms

Knowledge on the contribution of protein glycosylation in host defense antimicrobial peptides is still scarce. We have studied here how the post‐translational modification pattern modulates the antimicrobial activity of one of the best characterized leukocyte granule proteins. The human eosinophil cationic protein (ECP), an eosinophil specific granule protein secreted during inflammation and infection, can target a wide variety of pathogens. Previous work in human eosinophil extracts identified several ECP native forms and glycosylation heterogeneity was found to contribute to the protein biological properties. In this study we analyze for the first time the antimicrobial activity of the distinct native proteins purified from healthy donor blood. Low and heavy molecular weight forms were tested on Escherichia coli cell cultures and compared with the recombinant non‐glycosylated protein. Further analysis on model membranes provided an insight towards an understanding of the protein behavior at the cytoplasmic membrane level. The results highlight the significant reduction in protein toxicity and bacteria agglutination activity for heavy glycosylated fractions. Notwithstanding, the lower glycosylated fraction mostly retains the lipopolysaccharide binding affinity together with the cytoplasmic membrane depolarization and membrane leakage activities. From structural analysis we propose that heavy glycosylation interferes with the protein self‐aggregation, hindering the cell agglutination and membrane disruption processes. The results suggest the contribution of post‐translational modifications to the antimicrobial role of ECP in host defense.

The first crystal structure of human RNase 6 reveals a novel substrate-binding and cleavage site arrangement

We describe the first human RNase 6 crystal structure in complex with sulfate anions. Kinetic analysis, site-directed mutagenesis and molecular dynamics simulations identified novel substrate recognition and cleavage sites.

Human pancreatic ribonuclease presents higher endonucleolytic activity than ribonuclease A.

Analyzing the pattern of oligonucleotide formation induced by HP-RNase cleavage shows that the enzyme does not act randomly and follows a more endonucleolytic pattern when compared to RNase A. The enzyme prefers the binding and cleavage of longer substrate molecules, especially when the phosphodiester bond that is broken is 8-11 nucleotides away from at least one of the ends of the substrate molecule. This more endonucleolytic pattern is more appropriate for an enzyme with a regulatory role. Deleting two positive charges on the N-terminus (Arg4 and Lys6) modifies this pattern of external/internal phosphodiester bond cleavage preference, and produces a more exonucleolytic enzyme. These residues may reinforce the strength of a non-catalytic secondary phosphate binding (p2) or, alternatively, constitute a new non-catalytic phosphate binding subsite (p3).