A new cryptic host defense peptide identified in human 11-hydroxysteroid dehydrogenase-1 ß-like: from in silico identification to experimental evidence.

BACKGROUND: Host defence peptides (HDPs) are evolutionarily conserved components of innate immunity. Human HDPs, produced by a variety of immune cells of hematopoietic and epithelial origin, are generally grouped into two families: beta structured defensins and variably-structured cathelicidins. We report the characterization of a very promising cryptic human HDP, here called GVF27, identified in 11-hydroxysteroid dehydrogenase-1 ß-like protein. METHODS: Conformational analysis of GVF27 and its propensity to bind endotoxins were performed by NMR, Circular Dichroism, Fluorescence and Dynamic Light Scattering experiments. Crystal violet and WST-1 assays, ATP leakage measurement and colony counting procedures were used to investigate antimicrobial, anti-biofilm, cytotoxicity and hemolytic activities. Anti-inflammatory properties were evaluated by ELISA. RESULTS: GVF27 possesses significant antibacterial properties on planktonic cells and sessile bacteria forming biofilm, as well as promising dose dependent abilities to inhibit attachment or eradicate existing mature biofilm. It is unstructured in aqueous buffer, whereas it tends to assume a helical conformation in mimic membrane environments as well as it is able to bind lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Notably it is not toxic towards human and murine cell lines and triggers a significant innate immune response by attenuating expression levels of pro-inflammatory interleukins and release of nitric oxide in LPS induced macrophages. CONCLUSION: Human GVF27 may offer significant advantages as leads for the design of human-specific therapeutics. GENERAL SIGNIFICANCE: Human cryptic host defence peptides are naturally no immunogenic and for this they are a real alternative for solving the lack of effective antibiotics to control bacterial infections.

Combinatorial experimental protocols for Erbicin-derived immunoagents and Herceptin.

Erbicin is a human anti-ErbB2 single-chain antibody fragment with high affinity and selectivity for ErbB2-positive cancer cells. Two anti-ErbB2 immunoconjugates, called Erb-hRNase and Erb-hcAb, have been prepared and found to be selectively cytotoxic on ErbB2-positive cancer cells in vitro and vivo. In Erb-hRNase, Erbicin is linked to a human RNase and in Erb-hcAb it is linked to the key structural and functional regions of a human IgG. Herceptin is an anti-ErbB2 humanised antibody successfully used in the immunotherapy of breast cancer. We report here that the Erbicin-derived immunoagents target on breast cancer cells an ErbB2 epitope different than that of Herceptin. This finding led us to verify the effects of Herceptin on breast cancer cells when it was used in combination with the Erbicin-derived immunoagents. The results indicated that in combination experiments the antitumour action of Herceptin and that of the novel agents were significantly increased in an additive fashion. An inspection of the mechanism of action of Erb-hRNase or Erb-hcAb combined with Herceptin provided evidence that the antibody combinations engendered an increased downregulation of the ErbB2 receptor, and led to an enhanced apoptotic cell death.

Effective expression and purification of recombinant onconase, an antitumor protein.

Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward several tumor cell lines. One of these is onconase, the smallest member of the RNase A superfamily, which is at present undergoing phase III clinical trials. We report here the expression of recombinant onconase in Escherichia coli inclusion bodies, the correct processing of the protein, followed by its purification in high yields. The recombinant protein has biological and catalytic properties identical to those of the natural enzyme.

Circular dichroism study of ribonuclease A mutants containing the minimal structural requirements for dimerization and swapping.

Four residues Pro19. Leu28, Cys31 and Cys32 proved to be the minimal structural requirements in determining the dimeric structure and the N-terminal segment swapping of bovine seminal ribonuclease, BS-RNase. We analyzed the content of secondary and tertiary structures in RNase A, P-RNase A, PL-RNase A, MCAM-PLCC-RNase A and MCAM-BS-RNase, performing near and far-UV CD spectra. It results that the five proteins have very similar native conformations. Thermal denaturation at pH 5.0 of the proteins. studied by means of CD measurements. proved reversible and well represented by the two-state N<==>D transition model. Thermodynamic data are discussed in the light of the structural information available for RNase A and BS-RNase.

New muteins of RNase A with enhanced antitumor action.

Monomeric bovine pancreatic RNase A has been transformed into a dimeric ribonuclease with antitumor activity (Di Donato, A., Cafaro, V. and D'Alessio, G. (1994) J. Biol. Chem. 269, 17394-17396). This was accomplished by replacing the residues located in the RNase chain at positions 19, 28, 31, and 32, with proline, leucine, and two cysteine residues, respectively, i.e. those present at identical positions in the subunit of bovine seminal RNase, a dimeric RNase of the pancreatic-type superfamily, endowed with a powerful antitumor action. However, as an antitumor agent this mutant dimeric RNase A is not as powerful as seminal RNase. We report here site-directed mutagenesis experiments which have led to the identification of two other amino acid residues, glycine 38 and 111, whose substitution in the polypeptide chain of the first generation dimeric mutant of RNase A, is capable of conferring to the mutein the full cytotoxic activity characteristic of native seminal RNase.

A recombinant ribosome-inactivating protein from the plant Phytolacca dioica L. produced from a synthetic gene.

Phytolacca dioica L. leaves produce at least two type-I ribosome-inactivating proteins. Each polypeptide chain is subjected to different post-translational modifications giving rise to PD-L1 and PD-L2, and PD-L3 and PD-L4, each polypeptide pair having the same primary structure. With the aim of exploiting the cytotoxic properties of these proteins as potential biological phytodrugs, a gene encoding PD-L4 was designed based on criteria expected to maximize the translation efficiency in tomato. The gene was constructed from 18 oligonucleotides and preliminarily expressed in Escherichia coli, using the T7 promoter system. The protein produced was insoluble and accumulated in inclusion bodies to about 300 mg/l of culture. Ribosome-inactivating activity was generated by controlled oxidation of the reduced and denatured protein. The recombinant protein was indistinguishable from natural PD-L4 as isolated from leaves of Phytolacca dioica, in both catalytic activity and primary structure.

Protein engineering of ribonucleases.

Natural bovine seminal RNase possesses a potent antitumor action. We have mutagenized monomeric bovine pancreatic RNase A, devoid of any cytotoxic action, to insert residues present at corresponding positions in the subunit of dimeric, antitumor, seminal RNase. Like naturally dimeric seminal RNase, the mutant dimeric RNases display selective toxicity for malignant cells, which is absent in the monomeric mutants.

From ribonuclease A toward bovine seminal ribonuclease: a step by step thermodynamic analysis.

A proline, a leucine, and two cysteine residues, introduced at positions 19, 28, 31, and 32 of bovine pancreatic RNase A, i.e. the positions occupied by these residues in the subunit of bovine seminal RNase, the only dimeric RNase of the pancreatic-type superfamily, transform monomeric RNase A into a dimeric RNase, endowed with the same ability of BS-RNase of swapping its N-terminal segments. The thermodynamic consequences of the progressive introduction of these four residues into RNase A polypeptide chain have been studied by comparing the temperature- and urea-induced denaturation of three mutants of RNase A with that of a stable monomeric derivative of BS-RNase. The denaturation processes proved reversible for all proteins, and well represented by the two-state N<-->D transition model. The progressive introduction of the four residues into RNase A led to a gradual shift of the protein stability toward that characteristic of monomeric BS-RNase, which, in turn, is markedly less stable than RNase A with respect to both temperature- and urea-induced denaturation. On the other hand, the thermal stability of a dimeric active mutant of RNase A is found to approach that of wild-type seminal RNase.

Hints on the evolutionary design of a dimeric RNase with special bioactions.

Residues P19, L28, C31, and C32 have been implicated (Di Donato A, Cafaro V, D'Alessio G, 1994, J Biol Chem 269:17394-17396; Mazzarella L, Vitagliano L, Zagari A, 1995, Proc Natl Acad Sci USA: forthcoming) with key roles in determining the dimeric structure and the N-terminal domain swapping of seminal RNase. In an attempt to have a clearer understanding of the structural and functional significance of these residues in seminal RNase, a series of mutants of pancreatic RNase A was constructed in which one or more of the four residues were introduced into RNase A. The RNase mutants were examined for: (1) the ability to form dimers; (2) the capacity to exchange their N-terminal domains; (3) resistance to selective cleavage by subtilisin; and (4) antitumor activity. The experiments demonstrated that: (1) the presence of intersubunit disulfides is both necessary and sufficient for engendering a stably dimeric RNase; (2) all four residues play a role in determining the exchange of N-terminal domains; (3) the exchange is the molecular basis for the RNase antitumor action; and (4) this exchange is not a prerequisite in an evolutionary mechanism for the generation of dimeric RNases.

Full antitumor action of recombinant seminal ribonuclease depends on the removal of its N-terminal methionine.

Bovine seminal RNase (BS-RNase) is a dimeric member of the pancreatic-like ribonuclease superfamily, with antitumor activity. We report here that recombinant Met(-1) BS-RNase is a less potent cytotoxic factor, while structurally and catalytically indistinguishable from BS-RNase isolated from natural sources. Mature recombinant BS-RNase instead displays full antitumor action. This suggests that the conformation of the N-terminal region of BS-RNase is among the structural determinants of its antitumor action, in addition to its catalytic activity and its quaternary structure.

The antitumor action of seminal ribonuclease and its quaternary conformations.

It has been previously shown that the antitumor action of bovine seminal ribonuclease (BS-RNase) is dependent on its dimeric structure. However, two distinct quaternary structures, each in equilibrium with the other, have been described for the enzyme: one in which the two subunits exchange their N-terminal ends, the other with no exchange. Antitumor activity assays, carried out on homogeneous quaternary forms of the enzyme, as well as on dimeric mutants of bovine pancreatic RNase A, reveal that another structural determinant of the antitumor activity of BS-RNase is the exchange of N-terminal ends between subunits.

Ribonuclease A can be transformed into a dimeric ribonuclease with antitumor activity.

A cDNA coding for bovine pancreatic RNase A was mutagenized to insert a proline, a leucine, and 2 cysteine residues, i.e. the residues present at corresponding positions in the subunit of seminal RNase, the only dimeric RNase of the pancreatic-type superfamily. The mutant, expressed in Escherichia coli, eventually aggregated into catalytically active dimers. Like naturally dimeric seminal RNase, at equilibrium the mutant dimeric RNase A adopted two quaternary structures (one with an exchange of the N-terminal segments between partner subunits, the other with no exchange) and displayed a selective toxicity for malignant cells, absent in the monomeric, parent protein.

The determinants of the dimeric structure of seminal ribonuclease are located in its N-terminal region.

A chimeric cDNA was constructed, coding for the N-terminal region of BS-RNase (residues 1-49) and the C-terminal region of RNase A (residues 50-124). The resulting chimeric DNA was expressed in Escherichia coli and found to code for RNase chains that spontaneously assembled into a covalently dimeric ribonuclease.

Abruptio placentae.

Analysis of blood samples from pregnant women has shown that the mean histamine level starts to rise when the plasma ascorbic acid level falls below 1.0 mg/100 ml; it is doubled when the ascorbate level falls to 0.5 mg/100 ml and quadrupled when it falls below 0.2 mg/100 ml. The incidence of abruptio placentae was found to be seven out of 355 (or 2.0%) in women who had plasma ascorbic acid levels above 0.4 mg/100 ml and six out of 31 (19.4%) in women with plasma ascorbate levels below 0.4 mg/100 ml. This difference is highly significant. It is suggested that ascorbic acid deficiency and histamine excess play leading roles in the etiology of abruptio placentae.