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The ribonuclease A superfamily is a vertebrate-specific family of proteins that encompasses eight functional members in humans. The proteins are secreted by diverse innate immune cells, from blood cells to epithelial cells and their levels in our body fluids correlate with infection and inflammation processes. Recent studies ascribe a prominent role to secretory RNases in the extracellular space. Extracellular RNases endowed with immuno-modulatory and antimicrobial properties can participate in a wide variety of host defense tasks, from performing cellular housekeeping to maintaining body fluid sterility. Their expression and secretion are induced in response to a variety of injury stimuli. The secreted proteins can target damaged cells and facilitate their removal from the focus of infection or inflammation. Following tissue damage, RNases can participate in clearing RNA from cellular debris or work as signaling molecules to regulate the host response and contribute to tissue remodeling and repair. We provide here an overall perspective on the current knowledge of human RNases’ biological properties and their role in health and disease. The review also includes a brief description of other vertebrate family members and unrelated extracellular RNases that share common mechanisms of action. A better knowledge of RNase mechanism of actions and an understanding of their physiological roles should facilitate the development of novel therapeutics.

The increasing rate in antibiotic-resistant bacterial strains has become an imperative health issue. Thus, pharmaceutical industries have focussed their efforts to find new potent, non-toxic compounds to treat bacterial infections. Antimicrobial peptides (AMPs) are promising candidates in the fight against antibiotic-resistant pathogens due to their low toxicity, broad range of activity and unspecific mechanism of action. In this context, bioinformatics' strategies can inspire the design of new peptide leads with enhanced activity. Here, we describe an artificial neural network approach, based on the AMP's physicochemical characteristics, that is able not only to identify active peptides but also to assess its antimicrobial potency. The physicochemical properties considered are directly derived from the peptide sequence and comprise a complete set of parameters that accurately describe AMPs. Most interesting, the results obtained dovetail with a model for the AMP's mechanism of action that takes into account new concepts such as peptide aggregation. Moreover, this classification system displays high accuracy and is well correlated with the experimentally reported data. All together, these results suggest that the physicochemical properties of AMPs determine its action. In addition, we conclude that sequence derived parameters are enough to characterize antimicrobial peptides.

The eosinophil cationic protein (ECP) is an eosinophil-secreted RNase involved in the immune host defense, with a cytotoxic activity against a wide range of pathogens. The protein displays antimicrobial activity against both Gram-negative and Gram-positive strains. The protein can destabilize lipid bilayers, although the action at the membrane level can only partially account for its bactericidal activity. We have now shown that ECP can bind with high affinity to the bacteria-wall components. We have analyzed its specific association to lipopolysaccharides (LPSs), its lipid A component, and peptidoglycans (PGNs). ECP high-affinity binding capacity to LPSs and lipid A has been analyzed by a fluorescent displacement assay, and the corresponding dissociation constants were calculated using the protein labeled with a fluorophor. The protein also binds in vivo to bacteria cells. Ultrastructural analysis of cell bacteria wall and morphology have been visualized by scanning and transmission electron microscopy in both Escherichia coli and Staphylococcus aureus strains. The protein damages the bacteria surface and induces the cell population aggregation on E. coli cultures. Although both bacteria strain cells retain their shape and no cell lysis is patent, the protein can induce in E. coli the outer membrane detachment. ECP also activates the cytoplasmic membrane depolarization in both strains. Moreover, the depolarization activity on E. coli does not require any pretreatment to overcome the outer membrane barrier. The protein binding to the bacteria-wall surface would represent a first encounter step key in its antimicrobial mechanism of action.

Eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are proteins of the ribonuclease A (RNase A) superfamily that have developed biological properties related to the function of eosinophils. ECP is a potent cytotoxic molecule, and although the mechanism is still unknown this cytotoxic activity has been associated with its highly cationic character. Using liposome vesicles as a model, we have demonstrated that ECP tends to disrupt preferentially acidic membranes. On the basis of structure analysis, ECP variants modified at basic and hydrophobic residues have been constructed. Changes in the leakage of liposome vesicles by these ECP variants have indicated the role of both aromatic and basic specific amino acids in cellular membrane disruption. This is the case with the two tryptophans at positions 10 and 35, but not phenylalanine 76, and the two arginines 101 and 104. The bactericidal activity of both native ECP and point-mutated variants, tested against Escherichia coli and Staphylococcus aureus, suggests that basic amino acids play, in addition to the effect on the disruption of the cellular membrane, other roles such as specific binding on the surface of the bacteria cell.

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Antimicrobial proteins and peptides (AMPs) are important effectors of the innate immune system that play a vital role in the prevention of infections. Recent advances have highlighted the similarity between AMPs and amyloid proteins. Using the Eosinophil Cationic Protein as a model, we have rationalized the structure-activity relationships between amyloid aggregation and antimicrobial activity. Our results show how protein aggregation can induce bacteria agglutination and cell death. Using confocal and total internal reflection fluorescence microscopy we have tracked the formation in situ of protein amyloid-like aggregates at the bacteria surface and on membrane models. In both cases, fibrillar aggregates able to bind to amyloid diagnostic dyes were detected. Additionally, a single point mutation (Ile13 to Ala) can suppress the protein amyloid behavior, abolishing the agglutinating activity and impairing the antimicrobial action. The mutant is also defective in triggering both leakage and lipid vesicle aggregation. We conclude that ECP aggregation at the bacterial surface is essential for its cytotoxicity. Hence, we propose here a new prospective biological function for amyloid-like aggregates with potential biological relevance.

Supplementary data are available at Bioinformatics online.