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(A) Sequence alignment of RNase 3 and RNase 7. Primary sequences (UniProt codes: P12724 and Q9H1E1) were used, respectively. RNase 3 three-dimensional structure is indicated (PDB ID: 4OXF). Cationic residues are shown in both proteins in green and fuchsia boxes, respectively. The alignment was performed using the ESPript program (http://espript.ibcp.fr/EsPript/). (B) Three dimensional representation of crystal structures of wildtype RNase 3 (yellow; PDB ID: 4OXF) and active site mutant RNase 3-H15A (purple; PDB ID: 4OWZ). Mutated residues (His 15 and Trp 35) are depicted in baton sticks. (C) Detail of active centre in both proteins. Picture was drawn with PyMOL Molecular Graphics System (Schrödinger, LLC).
Source publication
Human antimicrobial RNases, which belong to the vertebrate RNase A superfamily and are secreted upon infection, display a wide spectrum of antipathogen activities. In this work, we examined the antifungal activity of the eosinophil RNase 3 and the skin-derived RNase 7, two proteins expressed by innate cell types that are directly involved in the ho...
Contexts in source publication
Context 1
... RNase 7 is a member of the RNase A superfamily (Fig. 1). This family, which includes other se- cretory RNases with antimicrobial properties (Boix and Nogués 2007), is a protein family that is suggested to have emerged with an ancestral host defense role (Pizzo and D'Alessio 2007;Rosenberg 2008). Antimicrobial RNases are expressed by epithelial tissues and blood cell types, and their ...
Context 2
... its turn, RNase 3, another of the main antimicrobial RNases within the RNase A superfamily (Fig. 1), also called the Eosinophil Cationic Protein (ECP), is involved in inflammatory processes mediated by eosinophils and is released by the secondary granules upon infection (Acharya and Ackerman 2014). RNase 3 has also been reported to display high antimicrobial activity against both gram-negative bacteria, such as E. coli, Acinetobacter ...
Context 3
... media impaired considerably the RNases fungicidal activity (Table S2), as previously reported for the bactericidal action of RNase 3 ( Lehrer et al. 1989) and other AMPs ( Krishnakumari et al. 2009). Binding to Candida cells was assessed by monitoring the free unbound protein after incubation at sublethal concentrations in PBS for 1 h at 37°C (Fig. S1). No binding was achieved in equivalent assay conditions by the homologous RNase A, used here as a non anti- microbial reference protein. 50 , given as mean ± SD, were determined using the Bactiter-Glo™ kit as detailed in the Experimental Procedures. Values are averaged from three replicates of two independent experiments. For the ...
Context 4
... of the catalytic activity of the RNases on their antimicrobial action, we prepared mutants of both RNases with defective active sites. The active site mutants were designed by making substitutions at His 15 within the active site catalytic triad ( Boix et al. 1999;Huang et al. 2007), where His 15 is the counterpart of His 12 in RNase A (Fig. 1) ( Cuchillo et al. 2011). The histidine to alanine substitution almost abolished all of the protein enzymatic activity for both RNases (Table 4) as previously reported by Raines and coworkers for the RNase A-H12A mutant counterpart (Park et al. 2001). Additionally, the overall three-dimensional structure and the active site architecture ...
Context 5
... for both RNases (Table 4) as previously reported by Raines and coworkers for the RNase A-H12A mutant counterpart (Park et al. 2001). Additionally, the overall three-dimensional structure and the active site architecture of the mutant protein were maintained, as confirmed by solving the RNase 3-H15A mutant X-ray crystal structure (PDB ID: 4OWZ) ( Fig. 1 and Table S1). The functional characterization of both active site mutants confirmed that these mutant proteins conserved their membrane lytic activity, showing an equivalent leakage activity on lipid vesicles (Table 4). Furthermore, the po- tential contribution of the H15 residue on the proteins' affinity for the cell membrane was ...
Context 6
... the po- tential contribution of the H15 residue on the proteins' affinity for the cell membrane was discarded, being a residue poorly exposed to the protein surface (solvent accessible surface area, SASA, for His 15 ~ 14 Å; 4A2Y. pdb (Boix et al. 2012a)). ...
Context 7
... we assayed an RNase 3 mutant ver- sion (W35A), previously reported as defective in its protein-membrane interaction without affecting its RNase activity ( Carreras et al. 2003;Nikolovski et al. 2006;Torrent et al. 2007). The present results confirm the key role of the surface exposed Trp (Fig. 1B) in the toxicity of the protein to yeast cells. The W35A mutant displays a two to threefold reduction in its fungicidal (Table 1) and membrane destabilizing activities (Table 2). Mostly, the abilities of RNase 3 to cause membrane depolarization and disruption were severely impaired (Table 2). Indeed, by confocal microscopy, we ...
Citations
... While studies focusing on the N-terminal antibacterial activity of HsR3 peptides have refuted the involvement of the enzymatic activity in this function (25,26,45), Salazar et al. (46) highlighted the importance of ribonucleolytic activity in the anti-yeast function of HsR3. Besides causing membrane destabilization, HsR3 can also enter Candida albicans cells and target cellular RNA (46). Further sequence minimization and structure-activity relationship assays should provide additional details on their respective mechanism of action against bacterial and mammalian cells. ...
Evolutionarily conserved structural folds can give rise to diverse biological functions, yet predicting atomic-scale interactions that contribute to the emergence of novel activities within such folds remains challenging. Pancreatic-type ribonucleases illustrate this complexity, sharing a core structure that has evolved to accommodate varied functions. In this study, we used ancestral sequence reconstruction to probe evolutionary and molecular determinants that distinguish biological activities within eosinophil members of the RNase 2/3 subfamily. Our investigation unveils functional, structural, and dynamical behaviors that differentiate the evolved ancestral ribonuclease (AncRNase) from its contemporary eosinophil RNase orthologs. Leveraging the potential of ancestral reconstruction for protein engineering, we used AncRNase predictions to design a minimal 4-residue variant that transforms human RNase 2 into a chimeric enzyme endowed with the antimicrobial and cytotoxic activities of RNase 3 members. This work provides unique insights into mutational and evolutionary pathways governing structure, function, and conformational states within the eosinophil RNase subfamily, offering potential for targeted modulation of RNase-associated functions.
... Nucleases hydrolyzing the RNA and DNA of fungi attract particular attention among enzymes that have antifungal activity [93,94,99]. The use of several nucleases at once [96][97][98] or nuclease in combination with glucanase [99] leads to the fact that not only is the growth of fungal cells stopped, but membrane destruction (permeabilization and depolymerization) is observed. ...
Active research of metal-containing compounds and enzymes as effective antifungal agents is currently being conducted due to the growing antifungal resistance problem. Metals are attracting special attention due to the wide variety of ligands that can be used for them, including chemically synthesized and naturally obtained variants as a result of the so-called “green synthesis”. The main mechanism of the antifungal action of metals is the triggering of the generation and accumulation of reactive oxygen species (ROS). Further action of ROS on various biomolecules is nonspecific. Various hydrolytic enzymes (glucanases and proteases), in turn, exhibit antifungal properties by affecting the structural elements of fungal cells (cell walls, membranes), fungal quorum sensing molecules, fungal own protective agents (mycotoxins and antibiotics), and proteins responsible for the adhesion and formation of stable, highly concentrated populations in the form of biofilms. A wide substrate range of enzymes allows the use of various mechanisms of their antifungal actions. In this review, we discuss the prospects of combining two different types of antifungal agents (metals and enzymes) against mycelial fungi and yeast cells. Special attention is paid to the possible influence of metals on the activity of the enzymes and the possible effects of proteins on the antifungal activity of metal-containing compounds.
... Nucleases hydrolyzing RNA and DNA of fungi attract particular attention among enzymes that have antifungal activity [96,97,102]. The use of several nucleases at once [96][97][98] or nuclease in combination with glucanase [102] leads to the fact that not only the growth of fungal cells is stopped, but membrane destruction (permeabilization and depolymerization) is observed, lowering of mitochondrial membrane potential, degradation of target cellular nucleic acids and the death of microbial cells. ...
... Nucleases hydrolyzing RNA and DNA of fungi attract particular attention among enzymes that have antifungal activity [96,97,102]. The use of several nucleases at once [96][97][98] or nuclease in combination with glucanase [102] leads to the fact that not only the growth of fungal cells is stopped, but membrane destruction (permeabilization and depolymerization) is observed, lowering of mitochondrial membrane potential, degradation of target cellular nucleic acids and the death of microbial cells. ...
Active research of metal-containing compounds and enzymes as effective antifungal agents is currently noted. The interest in metals is due to the wide variety of ligands that can be used for metals, including chemically synthesized and naturally obtained variants as a result of the so-called "green synthesis". The main mechanism of antifungal action of metals is the triggering of generation and accumulation of reactive oxygen species (ROS). Further action of ROS on various biomolecules is nonspecific. This review highlights various hydrolytic enzymes (glucanases and proteases) that affect the structural elements of fungal cells (cell walls, membranes), fungal quorum sensing molecules, fungal own protective agents (mycotoxins and antibiotics), proteins responsible for the adhesion and formation of stable highly concentrated populations in the form of biofilms. A wide range of the substrates for enzymes allows the use of various mechanisms of their antifungal actions. The prospects of combining two different types of antifungal agents (metals and enzymes) for mycelial fungi and yeast cells are discussed in this review. Special attention is paid to the possible influence of metals on activity of the enzymes and the possible effects of proteins on antifungal activity of metal-containing compounds.
... Another antimicrobial protein is RNase7, the dominant RNase in human skin, which is constitutively expressed but can also be induced by pro-inflammatory cytokines or microorganisms. In C. albicans it causes RNA cleavage and cell lysis [52,53]. Similar to the other AMPs, infection of the skin models in this study resulted in a significant increase in RNASE7 gene expression. ...
Cutaneous candidiasis is characterized by an overgrowth of Candida leading to skin inflammation and infection. Similar to bacteria, Candida can develop tolerance to common antifungal drugs. Cold atmospheric plasma (CAP), with its proven antimicrobial properties, offers a promising alternative to the prevailing methods. Because of plasma heterogeneity each new device must be tested individually for its effectiveness. Antimicrobial activity is usually studied using planktonic microorganisms or animal models, making it difficult to extrapolate the results to the human system. Therefore, a 3D skin model of cutaneous candidiasis for the antimicrobial testing of CAP was established. First, the reaction of the 3D-skin model to Candida infection was examined using various histological and molecular–biological methods. Infection with C. albicans resulted in increased expression and secretion of pro-inflammatory cytokines and augmented expression of antimicrobial peptides. Within 48 h, hyphal growth spread throughout the model and caused tissue damage. Second, the CAP treatment was employed. It was shown that CAP significantly reduced the spread of the yeast in the infected skin models as well as decreased the expression and secretion of the infection markers. The plasma device exhibited a high antifungal activity by completely inhibiting hyphal growth and reducing inflammation at the highest treatment duration.
... However, recent work is showing that catalytic activity has a role in several aspects of the immune system properties of RNase proteins [15][16][17]. For example, the antiviral and fungicidal activities of RNases seem to be related to the RNase activity [18,19]. ...
Bacterial resistance to antibiotics urges the development of alternative therapies. Based on the structure-function of antimicrobial members of the RNase A superfamily, we have developed a hybrid enzyme. Within this family, RNase 1 exhibits the highest catalytic activity and the lowest cytotoxicity; in contrast, RNase 3 shows the highest bactericidal action, alas with a reduced catalytic activity. Starting from both parental proteins, we designed a first RNase 3/1-v1 chimera. The construct had a catalytic activity much higher than RNase 3, unfortunately without reaching an equivalent antimicrobial activity. Thus, two new versions were created with improved antimicrobial properties. Both of these versions (RNase 3/1-v2 and -v3) incorporated an antimicrobial loop characteristic of RNase 3, while a flexible RNase 1-specific loop was removed in the latest construct. RNase 3/1-v3 acquired both higher antimicrobial and catalytic activities than previous versions, while retaining the structural determinants for interaction with the RNase inhibitor and displaying non-significant cytotoxicity. Following, we tested the constructs’ ability to eradicate macrophage intracellular infection and observed an enhanced ability in both RNase 3/1-v2 and v3. Interestingly, the inhibition of intracellular infection correlates with the variants’ capacity to induce autophagy. We propose RNase 3/1-v3 chimera as a promising lead for applied therapeutics.
... While the role and mechanisms of AMPs in controlling the growth of bacteria on the skin surface have been intensely studied, 4-6 their involvement in fungal infections of the skin just started. [7][8][9][10][11][12] Superficial tinea was reported as the most frequent cutaneous fungal infection in Germany, caused by dermatophytes in up 90% of all cases. 13 In more than 50% 14,15 dermatomycosis affects the skin of the feet and is known as tinea pedis. ...
... A recent study demonstrated that RNase 7 effectively inhibits the growth of the yeast Candida (C.) albicans by lysis of the cell membrane and subsequent enzymatic RNA degradation. 11 Additionally, the authors described that the minimal fungicidal concentration of RNase 7 is approximately 2-4 µmol/l (30-60 µg/ml). 11 Based on these results, it can be hypothesised that the inhibition of dermatophytes follows similar mechanisms and minimal fungicidal concentrations. ...
... 11 Additionally, the authors described that the minimal fungicidal concentration of RNase 7 is approximately 2-4 µmol/l (30-60 µg/ml). 11 Based on these results, it can be hypothesised that the inhibition of dermatophytes follows similar mechanisms and minimal fungicidal concentrations. ...
Background
Tinea pedis is often chronic or recurrent, but not all individuals are equally susceptible to this infection. Dermatophytes are able to induce the expression of antimicrobial peptides and proteins (AMPs) in human keratinocytes and certain AMPs can inhibit the growth of dermatophytes.
Objective
The focus of this study was to analyze the secretion of relevant AMPs, especially RNase 7, human beta‐defensin‐2 (hBD‐2) and the S‐100 protein psoriasin (S100A7), in patients with confirmed tinea pedis.
Methods
To verify the diagnosis, skin scales were obtained from all patients (n=13) and the dermatophytes were identified by potassium hydroxide mount, culture and molecular analysis. To determine the AMP concentrations, the lesional skin area of the foot was rinsed with a buffer that was subsequently analyzed by ELISA. The corresponding area of the other unaffected foot as well as defined healthy skin areas of the forearm and forehead and samples from age and gender‐matched healthy volunteers served as controls.
Results
In tinea pedis patients the AMP concentrations were higher in lesional skin than in non‐lesional skin and in healthy skin of controls. In particular, concentrations of hBD‐2 and psoriasin were significantly elevated.
Conclusions
The induction of AMPs in tinea pedis might be triggered directly by the dermatophytes; furthermore, attendant inflammation and/or differentiation processes may play a role. Our results indicate that there is no defect in the constitutive expression and induction of the analyzed AMPs by dermatophytes in the epidermis of affected patients. However, it is not known why the elevated AMP concentrations fail to efficiently combat dermatophyte growth.
... In addition, RNase3 expression is induced by infection and might contribute to the protection of biological fluids. The protein exhibits antimicrobial activity against a wide range of microorganisms, such as bacteria, yeast, viruses, and parasites [3,[6][7][8][9][10]. Abundance of surface-exposed cationic and hydrophobic residues can mediate the protein binding and subsequent destabilization of bacterial membranes through a carpet-like mechanism characteristic of many host defence antimicrobial peptides (AMPs) [10][11][12]. ...
... Although the protein triggers the pathogen death mainly through a direct mechanical action at the cell envelope, the targeting of intracellular components, such as nucleic acids cannot be disregarded. Indeed, RNase3 internalization is observed in treated yeast cells and protozoa [8,13]. Besides, RNase3 can enter the macrophage and eradicate the intracellular dwelling bacteria [14]. ...
... RNase3 and RNase3-H15A recombinant proteins were produced as previously reported [8]. Briefly, E. coli BL21(DE3)/pET11c cells were induced by 1 mM Isopropyl β-d-1-thiogalactopyranoside (IPTG, St. Louis, MO, USA) and the inclusion bodies enriched pellet was resuspended in 80 mL of 10 mM Tris-HCl pH 8.5, 2 mM EDTA and left incubating 30 min with 40 µg/mL of lysozyme prior to sonication. ...
The human RNase3 is a member of the RNaseA superfamily involved in host immunity. RNase3 is expressed by leukocytes and shows broad-spectrum antimicrobial activity. Together with a direct antimicrobial action, RNase3 exhibits immunomodulatory properties. Here, we have analysed the transcriptome of macrophages exposed to the wild-type protein and a catalytic-defective mutant (RNase3-H15A). The analysis of differently expressed genes (DEGs) in treated THP1-derived macrophages highlighted a common pro-inflammatory “core-response” independent of the protein ribonucleolytic activity. Network analysis identified the epidermal growth factor receptor (EGFR) as the main central regulatory protein. Expression of selected DEGs and MAPK phosphorylation were inhibited by an anti-EGFR antibody. Structural analysis suggested that RNase3 activates the EGFR pathway by direct interaction with the receptor. Besides, we identified a subset of DEGs related to the protein ribonucleolytic activity, characteristic of virus infection response. Transcriptome analysis revealed an early pro-inflammatory response, not associated to the protein catalytic activity, followed by a late activation in a ribonucleolytic-dependent manner. Next, we demonstrated that overexpression of macrophage endogenous RNase3 protects the cells against infection by Mycobacterium aurum and the human respiratory syncytial virus. Comparison of cell infection profiles in the presence of Erlotinib, an EGFR inhibitor, revealed that the receptor activation is required for the antibacterial but not for the antiviral protein action. Moreover, the DEGs related and unrelated to the protein catalytic activity are associated to the immune response to bacterial and viral infection, respectively. We conclude that RNase3 modulates the macrophage defence against infection in both catalytic-dependent and independent manners.
... A number of other human AMP/HDP possess documented antifungal activity, including RNases, psoriasin, dermcidin, lactoferricin, antileukprotease/secretory leukocyte protease inhibitor (SLPI), calprotectin, trappin-2/pre-elafin, granulysin, thrombocidins, hepcidins, α-melanocyte stimulating hormone, the chemokine CCL20, substance P, calcitonin generelated peptide, neuropeptide Y, amyloid β-peptide and vasostatin-1 (136)(137)(138)(139)(140)(141)(142)(143)(144)(145)(146)(147)(148)(149)(150)(151)(152). ...
... RNase 3 and RNase 7 demonstrated activity against C. albicans (MFC 2.5-5.0 µmol/L) (151), whereas dermcidin demonstrated pH-dependent activity against C. albicans with optimal activity at pH 5.5-6.5 (143). SLPI was active against A. fumigatus, including spores (137) and C. albicans (153). ...
The purpose of this review is to describe antifungal therapeutic candidates in preclinical and clinical development derived from, or directly influenced by, the immune system, with a specific focus on antimicrobial peptides (AMP). Although the focus of this review is AMP with direct antimicrobial effects on fungi, we will also discuss compounds with direct antifungal activity, including monoclonal antibodies (mAb), as well as immunomodulatory molecules that can enhance the immune response to fungal infection, including immunomodulatory AMP, vaccines, checkpoint inhibitors, interferon and colony stimulating factors as well as immune cell therapies. The focus of this manuscript will be a non-exhaustive review of antifungal compounds in preclinical and clinical development that are based on the principles of immunology and the authors acknowledge the incredible amount of in vitro and in vivo work that has been conducted to develop such therapeutic candidates.
... RNase3 shows activity, in vitro, against both Gramnegative and Gram-positive bacteria as well as against fungi such as C. albicans. Its mechanism of toxicity involves targeting both the bacterial cell wall and the cytoplasmic membrane [336,364]. Moreover, its high cationic charge and aggregation region promotes RNase3 self-aggregation and mediates agglutination of bacterial cells [323,365]. ...
... RNase7 is expressed by several tissues (epithelial cells, liver, kidney, muscles) and contributes mainly to the protection of the epidermis and urinary tract sterility (Figs. 4 and 5) [323,[393][394][395][396]. Potent antimicrobial activity against several infective microorganisms [364,390,393,397,398] and some immune-modulatory properties have been reported [323]. Inflammatory cytokines such as IL-1β and IFNγ [397] and activation of the PI3K/AKT pathway [399] also enhance RNase7 expression. ...
Chronic obstructive pulmonary disease (COPD) represents a heightened inflammatory
response in the lung generally resulting from tobacco smoking-induced recruitment and
activation of inflammatory cells and/or activation of lower airway structural cells. Several
mediators can modulate activation and recruitment of these cells, particularly those belonging
to the chemokines (conventional and atypical) family. There is emerging evidence for complex
roles of atypical chemokines and their receptors [such as high mobility group box 1
(HMGB1), antimicrobial peptides, receptor for advanced glycosylation end products (RAGE)
or toll-like receptors (TLRs)] in the pathogenesis of COPD, both in the stable disease and during
exacerbations. Modulators of these pathways represent potential novel therapies for
COPD and many are now in preclinical development. Inhibition of only a single atypical
chemokine or receptor may not block inflammatory processes because there is redundancy in
this network. However, there are many animal studies that encourage studies for modulating
the atypical chemokine network in COPD. Thus, few pharmaceutical companies maintain a
significant interest in developing agents that target these molecules as potential antiinflammatory
drugs. Antibody-based (biological) and small molecule drug (SMD)-based
therapies targeting atypical chemokines and/or their receptors are mostly at the preclinical
stage and their progression to clinical trials is eagerly awaited. These agents will most likely
enhance our knowledge about the role of atypical chemokines in COPD pathophysiology and
thereby improve COPD management.
... Although the catalytic activity of the protein was reduced, the antibacterial activity was maintained and increased After immobilization on MNP. Recent studies have demonstrated that indeed, the antimicrobial and catalytic activities appear unrelated [16,86]. Finally, it should be noted that the obtained nanobioconjugates were prepared at a single RNases:MNPs ratio, as detailed in the Materials and Methods section. ...
Current treatments against bacterial infections have severe limitations, mainly due to the emergence of resistance to conventional antibiotics. In the specific case of Pseudomonas aeruginosa strains, they have shown a number of resistance mechanisms to counter most antibiotics. Human secretory RNases from the RNase A superfamily are proteins involved in a wide variety of biological functions, including antimicrobial activity. The objective of this work was to explore the intracellular antimicrobial action of an RNase 3/1 hybrid protein that combines RNase 1 high catalytic and RNase 3 bactericidal activities. To achieve this, we immobilized the RNase 3/1 hybrid on Polyetheramine (PEA)-modified magnetite nanoparticles (MNPs). The obtained nanobioconjugates were tested in macrophage-derived THP-1 cells infected with Pseudomonas aeruginosa PAO1. The obtained results show high antimicrobial activity of the functionalized hybrid protein (MNP-RNase 3/1) against the intracellular growth of P. aeruginosa of the functionalized hybrid protein. Moreover, the immobilization of RNase 3/1 enhances its antimicrobial and cell-penetrating activities without generating any significant cell damage. Considering the observed antibacterial activity, the immobilization of the RNase A superfamily and derived proteins represents an innovative approach for the development of new strategies using nanoparticles to deliver antimicrobials that counteract P. aeruginosa intracellular infection.
