Developing nucleoside tailoring strategies against SARS-CoV-2 via ribonuclease targeting chimera.

In response to the urgent need for potent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) therapeutics, this study introduces an innovative nucleoside tailoring strategy leveraging ribonuclease targeting chimeras. By seamlessly integrating ribonuclease L recruiters into nucleosides, we address RNA recognition challenges and effectively inhibit severe acute respiratory syndrome coronavirus 2 replication in human cells. Notably, nucleosides tailored at the ribose 2'-position outperform those modified at the nucleobase. Our in vivo validation using hamster models further bolsters the promise of this nucleoside tailoring approach, positioning it as a valuable asset in the development of innovative antiviral drugs.

Treatment with RNase alleviates brain injury but not neuroinflammation in neonatal hypoxia/ischemia.

There is a need for new treatments to reduce brain injuries derived from neonatal hypoxia/ischemia. The only viable option used in the clinic today in infants born at term is therapeutic hypothermia, which has a limited efficacy. Treatments with exogenous RNase have shown great promise in a range of different adult animal models including stroke, ischemia/reperfusion injury, or experimental heart transplantation, often by conferring vascular protective and anti-inflammatory effects. However, any neuroprotective function of RNase treatment in the neonate remains unknown. Using a well-established model of neonatal hypoxic/ischemic brain injury, we evaluated the influence of RNase treatment on RNase activity, gray and white matter tissue loss, blood-brain barrier function, as well as levels and expression of inflammatory cytokines in the brain up to 6 h after the injury using multiplex immunoassay and RT-PCR. Intraperitoneal treatment with RNase increased RNase activity in both plasma and cerebropinal fluids. The RNase treatment resulted in a reduction of brain tissue loss but did not affect the blood-brain barrier function and had only a minor modulatory effect on the inflammatory response. It is concluded that RNase treatment may be promising as a neuroprotective regimen, whereas the mechanistic effects of this treatment appear to be different in the neonate compared to the adult and need further investigation.

The egg ribonuclease SjCP1412 accelerates liver fibrosis caused by Schistosoma japonicum infection involving damage-associated molecular patterns (DAMPs).

Schistosomiasis, a parasite infectious disease caused by Schistosoma japonicum, often leads to egg granuloma and fibrosis due to the inflammatory reaction triggered by egg antigens released in the host liver. This study focuses on the role of the egg antigens CP1412 protein of S. japonicum (SjCP1412) with RNase activity in promoting liver fibrosis. In this study, the recombinant egg ribonuclease SjCP1412, which had RNase activity, was successfully prepared. By analysing the serum of the population, it has been proven that the anti-SjCP1412 IgG in the serum of patients with advanced schistosomiasis was moderately correlated with liver fibrosis, and SjCP1412 may be an important antigen associated with liver fibrosis in schistosomiasis. In vitro, the rSjCP1412 protein induced the human liver cancer cell line Hep G2 and liver sinusoidal endothelial cells apoptosis and necrosis and the release of proinflammatory damage-associated molecular patterns (DAMPs). In mice infected with schistosomes, rSjCP1412 immunization or antibody neutralization of SjCP1412 activity significantly reduced cell apoptosis and necroptosis in liver tissue, thereby reducing inflammation and liver fibrosis. In summary, the SjCP1412 protein plays a crucial role in promoting liver fibrosis during schistosomiasis through mediating the liver cells apoptosis and necroptosis to release DAMPs inducing an inflammatory reaction. Blocking SjCP1412 activity could inhibit its proapoptotic and necrotic effects and alleviate hepatic fibrosis. These findings suggest that SjCP1412 may be served as a promising drug target for managing liver fibrosis in schistosomiasis japonica.

Ribonuclease 1 Enhances Antitumor Immunity against Breast Cancer by Boosting T cell Activation.

The secretory enzyme human ribonuclease 1 (RNase1) is involved in innate immunity and anti-inflammation, achieving host defense and anti-cancer effects; however, whether RNase1 contributes to adaptive immune response in the tumor microenvironment (TME) remains unclear. Here, we established a syngeneic immunocompetent mouse model in breast cancer and demonstrated that ectopic RNase1 expression significantly inhibited tumor progression. Overall changes in immunological profiles in the mouse tumors were analyzed by mass cytometry and showed that the RNase1-expressing tumor cells significantly induced CD4+ Th1 and Th17 cells and natural killer cells and reduced granulocytic myeloid-derived suppressor cells, supporting that RNase1 favors an antitumor TME. Specifically, RNase1 increased expression of T cell activation marker CD69 in a CD4+ T cell subset. Notably, analysis of cancer-killing potential revealed that T cell-mediated antitumor immunity was enhanced by RNase1, which further collaborated with an EGFR-CD3 bispecific antibody to protect against breast cancer cells across molecular subtypes. Our results uncover a tumor-suppressive role of RNase1 through adaptive immune response in breast cancer in vivo and in vitro, providing a potential treatment strategy of combining RNase1 with cancer immunotherapies for immunocompetent patients.

In Silico Identification of Novel Derivatives of Rifampicin Targeting Ribonuclease VapC2 of M. tuberculosis H37Rv: Rifampicin Derivatives Target VapC2 of Mtb H37Rv.

The emergence of multi-drug-resistant Mycobacterium tuberculosis (Mtb) strains has rendered many of the currently available anti-TB drugs ineffective. Hence, there is a pressing need to discover new potential drug targets/candidates. In this study, attempts have been made to identify novel inhibitors of the ribonuclease VapC2 of Mtb H37Rv using various computational techniques. Ribonuclease VapC2 Mtb H37Rv's protein structure was retrieved from the PDB databank, 22 currently used anti-TB drugs were retrieved from the PubChem database, and protein-ligand interactions were analyzed by docking studies. Out of the 22 drugs, rifampicin (RIF), being a first-line drug, showed the best binding energy (-8.8 Kcal/mol) with Mtb H37Rv VapC2; hence, it was selected as a parent molecule for the design of its derivatives. Based on shape score and radial plot criteria, out of 500 derivatives designed through SPARK (Cresset®, Royston, UK) program, the 10 best RIF derivatives were selected for further studies. All the selected derivatives followed the ADME criteria concerning drug-likeness. The docking of ribonuclease VapC2 with RIF derivatives revealed the best binding energy of -8.1 Kcal/mol with derivative 1 (i.e., RIF-155841). A quantitative structure-activity relationship study revealed that derivative 1's activity assists in the inhibition of ribonuclease VapC2. The stability of the VapC2-RIF155841 complex was evaluated using molecular dynamics simulations for 50 ns and the complex was found to be stable after 10 nsec. Further, a chemical synthesis scheme was designed for the newly identified RIF derivative (RIF-155841), which verified that its chemical synthesis is possible for future in vitro/in vivo experimental validation. Overall, this study evaluated the potential of the newly designed RIF derivatives with respect to the Mtb VapC2 protein, which is predicted to be involved in some indispensable processes of the related pathogen. Future experimental studies regarding RIF-155841, including the exploration of the remaining RIF derivatives, are warranted to verify our current findings.

[Recombinant Schistosoma japonicum egg ribonuclease SjCP1412 inhibits the activation of LX-2 hepatic stellate cells in vitro].

OBJECTIVE: To investigate the effect of recombinant Schistosoma japonicum egg ribonuclease SjCP1412 (rSjCP1412) on proliferation, cell cycle, apoptosis and activation of human hepatic stellate cells LX-2 in vitro, and explore the underlying mechanisms. METHODS: The rSjCP1412 protein was expressed in Escherichia coli BL21 by prokaryotic expression, and the highly purified soluble rSjCP1412 protein was prepared by Ni NTA affinity chromatography and urea gradient refolding dialysis. Yeast RNA was digested using 12.5, 25.0, 50.0 µg rSjCP1412 proteins at 37 °C for 2, 3, 4 h, and the enzymatic products were electrophoresed on 1.5% agarose gel to observe the RNAase activity of rSjCP1412 protein. The proliferation of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was measured using CCK-8 assay, and the apoptosis of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was detected using the Annexin V-FITC/PI double staining, while the percentage of LX-2 cells at G0/G1, S and G2/M phases of cell cycle following stimulation with different doses of rSjCP1412 protein for 48 h was detected by DAPI staining. The type I collagen, type III collagen and α-smooth muscle actin (α-SMA) mRNA expression was quantified using quantitative florescent real-time PCR (qPCR) assay and Western blotting at transcriptional and translational levels in LX-2 cells following stimulation with different doses of rSjCP1412 protein for 48 h, while soluble egg antigen (SEA) served a positive control and PBS without rSjCP1412 protein as a normal control in the above experiments. The expression of collagen I, α-SMA and Smad4 protein was determined using Western blotting in LX-2 cells following stimulation with rSjCP1412 protein, transforming growth factor-ß1 (TGF-ß1) alone or in combination, to examine the signaling for the effect of rSjCP1412 protein on LX-2 cells. RESULTS: The rSjCP1412 protein was successfully expressed and the highly purified soluble rSjCP1412 protein was prepared, which had a RNase activity. Compared with the normal group, the survival rates of LX-2 cells significantly decreased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein and SEA for 48 h (F = 22.417 and 20.448, both P values < 0.05). The apoptotic rates of LX-2 cells significantly increased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h (F = 11.350, P < 0.05), and treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h resulted in arrest of LX-2 cells in G0/G1 phase (F = 20.710, P < 0.05). Treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h caused a significant reduction in relative expression levels of collagen I (F = 11.340, P < 0.05), collagen III (F = 456.600, P < 0.05) and α-SMA mRNA (F = 23.100, P < 0.05) in LX-2 cells, and both rSjCP1412 protein and SEA treatment caused a significant reduction in collagen I (F = 1 302.000, P < 0.05), α-SMA (F = 49.750, P < 0.05) and Smad4 protein expression (F = 52.420, P < 0.05) in LX-2 cells. In addition, rSjCP1412 protein treatment inhibited collagen I (F = 66.290, P < 0.05), α-SMA (F = 31.300, P < 0.05) and Smad4 protein expression (F = 27.010, P < 0.05) in LX-2 cells activated by TGF-ß1. CONCLUSIONS: rSjCP1412 protein may induce apoptosis of LX-2 cells and inhibit proliferation, cell cycle and activation of LX-2 cells through down-regulating Smad4 signaling molecules.

Optimization of Anti-SARS-CoV-2 Treatments Based on Curcumin, Used Alone or Employed as a Photosensitizer.

Curcumin, the bioactive compound of the spice Curcuma longa, has already been reported as a potential COVID-19 adjuvant treatment due to its immunomodulatory and anti-inflammatory properties. In this study, SARS-CoV-2 was challenged with curcumin; moreover, curcumin was also coupled with laser light at 445 nm in a photodynamic therapy approach. Curcumin at a concentration of 10 µM, delivered to the virus prior to inoculation on cell culture, inhibited SARS-CoV-2 replication (reduction >99%) in Vero E6 cells, possibly due to disruption of the virion structure, as observed using the RNase protection assay. However, curcumin was not effective as a prophylactic treatment on already-infected Vero E6 cells. Notably, when curcumin was employed as a photosensitizer and blue laser light at 445 nm was delivered to a mix of curcumin/virus prior to the inoculation on the cells, virus inactivation was observed (>99%) using doses of curcumin that were not antiviral by themselves. Photodynamic therapy employing crude curcumin can be suggested as an antiviral option against SARS-CoV-2 infection.

[Combination of RNase Binase and AKT1/2 Kinase Inhibitor Blocks Two Alternative Survival Pathways in Kasumi-1 Cells].

Treatment of malignant neoplasms often requires the use of combinations of chemotherapeutic agents. However, in order to select combinations that are effective against specific tumor cells, it is necessary to understand the mechanisms of action of the drugs that make up the combination. Bacillus pumilus ribonuclease (binase) is considered as an adjuvant antitumor agent, and the sensitivity of malignant cells to the apoptogenic effect of binase depends on the presence of certain oncogenes. In the acute myelogenous leukemia cell line Kasumi-1, binase blocks the proliferation pathway mediated by the mutant tyrosine kinase KIT, which, as shown in our work, activates an alternative proliferation pathway through AKT kinase. In Kasumi-1 cells, binase in combination with an Akt1/2 inhibitor induces apoptosis, and their toxic effects add up: the Akt1/2 inhibitor blocks the binase-induced pathway after suppression of the KIT-dependent pathway. Thus, a combination of binase and AKT kinase inhibitors can effectively block various pathways of tumor cell proliferation and be used for their elimination.

Characterization and cytotoxic activity of ribotoxin-like proteins from the edible mushroom Pleurotus eryngii.

Ribotoxin-like proteins (RL-Ps) represent a novel specific ribonuclease family found in edible mushrooms and are able to inhibit protein synthesis. Here, we report the characterization and cytotoxic effects of four novel RL-Ps, named eryngitins, isolated from fruiting bodies of the king oyster mushroom (Pleurotus eryngii). These proteins induced formation of α-fragment from rabbit ribosomes, characteristic of their enzymatic action. The two 15 kDa eryngitins (3 and 4) are considerably more thermostable than the 21 kDa ones (1 and 2), however their overall structural features, as determined by far-UV CD spectrometry, are similar. Complete in vitro digestibility by pepsin-trypsin, and lack of cytotoxicity towards human HUVEC cells suggest low toxicity of eryngitins, if ingested. However, eryngitins exhibit cytotoxic action against insect Sf9 cells, suggesting their possible use in biotechnological applications as bioinsecticides. This cytotoxicity was not enhanced in the presence of cytolytic protein complexes based on aegerolysin proteins from Pleurotus mushrooms.

Anticancer Activity of Reconstituted Ribonuclease S-Decorated Artificial Viral Capsid.

Ribonuclease S (RNase S) is an enzyme that exhibits anticancer activity by degrading RNAs within cancer cells; however, the cellular uptake efficiency is low due to its small molecular size. Here we generated RNase S-decorated artificial viral capsids with a size of 70-170 nm by self-assembly of the ß-annulus-S-peptide followed by reconstitution with S-protein at neutral pH. The RNase S-decorated artificial viral capsids are efficiently taken up by HepG2 cells and exhibit higher RNA degradation activity in cells compared with RNase S alone. Cell viability assays revealed that RNase S-decorated capsids have high anticancer activity comparable to that of standard anticancer drugs.

Essential Role of Enzymatic Activity in the Leishmanicidal Mechanism of the Eosinophil Cationic Protein (RNase 3).

The recruitment of eosinophils into Leishmania lesions is frequently associated with a favorable evolution. A feasible effector for this process is eosinophil cationic protein (ECP, RNase 3), one of the main human eosinophil granule proteins, endowed with a broad spectrum of antimicrobial activity, including parasites. ECP was active on Leishmania promastigotes and axenic amastigotes (LC50's = 3 and 16 µM, respectively) but, in contrast to the irreversible membrane damage caused on bacteria and reproduced by its N-terminal peptides, it only induced a mild and transient plasma membrane destabilization on Leishmania donovani promastigotes. To assess the contribution of RNase activity to the overall leishmanicidal activity of ECP, parasites were challenged in parallel with a single-mutant version, ECP-H15A, devoid of RNase activity, that fully preserves the conformation and liposome permeabilization ability. ECP-H15A showed a similar uptake to ECP on promastigotes, but with higher LC50's (>25 µM) for both parasite stages. ECP-treated promastigotes showed a degraded RNA pattern, absent in ECP-H15A-treated samples. Moreover ECP, but not ECP-H15A, reduced more than 2-fold the parasite burden of infected macrophages. Altogether, our results suggest that ECP enters the Leishmania cytoplasm by an endocytic pathway, ultimately leading to RNA degradation as a key contribution to the leishmanicidal mechanism. Thus, ECP combines both membrane destabilization and enzymatic activities to effect parasite killing. Taken together, our data highlight the microbicidal versatility of ECP as an innate immunity component and support the development of cell-penetrating RNases as putative leishmanicidal agents.

Ribonuclease-1 treatment after traumatic brain injury preserves blood-brain barrier integrity and delays secondary brain damage in mice.

Traumatic brain injury (TBI) involves primary mechanical damage and delayed secondary damage caused by vascular dysfunction and neuroinflammation. Intracellular components released into the parenchyma and systemic circulation, termed danger-associated molecular patterns (DAMPs), are major drivers of vascular dysfunction and neuroinflammation. These DAMPs include cell-free RNAs (cfRNAs), which damage the blood-brain barrier (BBB), thereby promoting edema, procoagulatory processes, and infiltration of inflammatory cells. We tested the hypothesis that intraperitoneal injection of Ribonuclease-1 (RNase1, two doses of 20, 60, or 180 µg/kg) at 30 min and 12 h after controlled-cortical-impact (CCI) can reduce secondary lesion expansion compared to vehicle treatment 24 h and 120 h post-CCI. The lowest total dose (40 µg/kg) was most effective at reducing lesion volume (- 31% RNase 40 µg/kg vs. vehicle), brain water accumulation (- 5.5%), and loss of BBB integrity (- 21.6%) at 24 h post-CCI. RNase1 also reduced perilesional leukocyte recruitment (- 53.3%) and microglial activation (- 18.3%) at 120 h post-CCI, but there was no difference in lesion volume at this time and no functional benefit. Treatment with RNase1 in the early phase following TBI stabilizes the BBB and impedes leukocyte immigration, thereby suppressing neuroinflammation. RNase1-treatment may be a novel approach to delay brain injury to extend the window for treatment opportunities after TBI.

Modifications of Ribonucleases in Order to Enhance Cytotoxicity in Anticancer Therapy.

Ribonucleases (RNases) are a superfamily of enzymes that have been extensively studied since the 1960s. For a long time, this group of secretory enzymes was studied as an important model for protein chemistry such as folding, stability, and enzymatic catalysis. Since it was discovered that RNases displayed cytotoxic activity against several types of malignant cells, recent investigation has focused mainly on the biological functions and medical applications of engineered RNases. In this review, we describe the structures, functions, and mechanisms of antitumor activity of RNases. They operate at the crossroads of transcription and translation, preferentially degrading tRNA. As a result, this inhibits protein synthesis, induces apoptosis, and causes the death of cancer cells. This effect can be enhanced thousands of times when RNases are conjugated with monoclonal antibodies. Such combinations, called immunoRNases, have demonstrated selective antitumor activity against cancer cells both in vitro and in animal models. This review summarizes the current status of engineered RNases and immunoRNases as promising novel therapeutic agents for different types of cancer. Also, we describe our experimental results from published or previously unpublished research and compare them with other scientific information.

Upregulation of miR-34a-5p, miR-20a-3p and miR-29a-3p by Onconase in A375 Melanoma Cells Correlates with the Downregulation of Specific Onco-Proteins.

Onconase (ONC) is an amphibian secretory ribonuclease displaying cytostatic and cytotoxic activities against many mammalian tumors, including melanoma. ONC principally damages tRNA species, but also other non-coding RNAs, although its precise targets are not known. We investigated the ONC ability to modulate the expression of 16 onco-suppressor microRNAs (miRNAs) in the A375 BRAF-mutated melanoma cell line. RT-PCR and immunoblots were used to measure the expression levels of miRNAs and their regulated proteins, respectively. In silico study was carried out to verify the relations between miRNAs and their mRNA targets. A375 cell transfection with miR-20a-3p and miR-34a-5p mimics or inhibitors was performed. The onco-suppressors miR-20a-3p, miR-29a-3p and miR-34a-5p were highly expressed in 48-h ONC-treated A375 cells. The cytostatic effect of ONC in A375 cells was mechanistically explained by the sharp inhibition of cyclins D1 and A2 expression level, as well as by downregulation of retinoblastoma protein and cyclin-dependent-kinase-2 activities. Remarkably, the expression of kinases ERK1/2 and Akt, as well as of the hypoxia inducible factor-1α, was inhibited by ONC. All these proteins control pro-survival pathways. Finally, many crucial proteins involved in migration, invasion and metastatic potential were downregulated by ONC. Results obtained from transfection of miR-20a-3p and miR-34a-5p inhibitors in the presence of ONC show that these miRNAs may participate in the antitumor effects of ONC in the A375 cell line. In conclusion, we identified many intracellular downregulated proteins involved in melanoma cell proliferation, metabolism and progression. All mRNAs coding these proteins may be targets of miR-20a-3p, miR-29a-3p and/or miR-34a-5p, which are in turn upregulated by ONC. Data suggest that several known ONC anti-proliferative and anti-metastatic activities in A375 melanoma cells might depend on the upregulation of onco-suppressor miRNAs. Notably, miRNAs stability depends on the upstream regulation by long-non-coding-RNAs or circular-RNAs that can, in turn, be damaged by ONC ribonucleolytic activity.

Discovery of antitumor effects of leczymes.

Sialic-acid binding lectin from bullfrog (Rana catesbeiana) eggs, cSBL, is a cytotoxic ribonuclease (RNase) belonging to the RNase A superfamily. cSBL is cytotoxic to tumor cells, such as malignant pleural mesothelioma by inducing apoptotic cell death caused by the degradation of RNA in tumor cells. In addition, we have reported some data that cSBL could be involved in the endoplasmic reticulum stress pathway, and it was also assumed to cause apoptotic cell death. The most significant property of cSBL is its specificity toward malignant cells. Furthermore, since the antitumor activity of cSBL was confirmed by in vivo experiments using mouse xenograft models, it is expected to be a candidate for clinical chemotherapy. Here, we summarize the history of cSBL, alternatively called "leczyme," with its present and future.

Refined immunoRNases for the efficient targeting and selective killing of tumour cells: A novel strategy.

In order to overcome limitations of conventional cancer therapy methods, immunotoxins with the capability of target-specific action have been designed and evaluated pre-clinically, and some of them are in clinical studies. Targeting cancer cells via antibodies specific for tumour-associated surface proteins is a new biomedical approach that could provide the selectivity that is lacking in conventional cancer therapy methods such as radiotherapy and chemotherapy. A successful example of an approved immunotoxin is represented by immunoRNases. ImmunoRNases are fusion proteins in which the toxin has been replaced by a ribonuclease. Conjugation of RNase molecule to monoclonal antibody or antibody fragment was shown to enhance specific cell-killing by several orders of magnitude, both in vitro and in animal models. There are several RNases obtained from different mammalian cells that are expected to be less immunogenic and systemically toxic. In fact, RNases are pro-toxins which become toxic only upon their internalization in target cells mediated by the antibody moiety. The structure and large size of the antibody molecules assembled with the immunoRNases have always been a challenge in the application of immunoRNases as an antitoxin. To overcome this obstacle, we have offered a new strategy for the application of immunoRNases as a promising approach for upgrading immunoRNAses with maximum affinity and high stability in the cell, which can ultimately act as an effective large-scale cancer treatment. In this review, we introduce the optimized antibody-like molecules with small size, approximately 10 kD, which are presumed to significantly enhance RNase activity and be a suitable agent with the potential for anti-cancer functionality. In addition, we also discuss new molecular entities such as monobody, anticalin, nonobody and affilin as refined versions in the development of immunoRNases. These small molecules express their functionality with the suitable small size as well as with low immunogenicity in the cell, as a part of immunoRNases.

Serum circulating proteins from pediatric patients with dilated cardiomyopathy cause pathologic remodeling and cardiomyocyte stiffness.

Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and main indication for heart transplantation in children. Therapies specific to pediatric DCM remain limited due to lack of a disease model. Our previous study showed that treatment of neonatal rat ventricular myocytes (NRVMs) with serum from nonfailing or DCM pediatric patients activates the fetal gene program (FGP). Here we show that serum treatment with proteinase K prevents activation of the FGP, whereas RNase treatment exacerbates it, suggesting that circulating proteins, but not circulating miRNAs, promote these pathological changes. Evaluation of the protein secretome showed that midkine (MDK) is upregulated in DCM serum, and NRVM treatment with MDK activates the FGP. Changes in gene expression in serum-treated NRVMs, evaluated by next-generation RNA-Seq, indicated extracellular matrix remodeling and focal adhesion pathways were upregulated in pediatric DCM serum and in DCM serum-treated NRVMs, suggesting alterations in cellular stiffness. Cellular stiffness was evaluated by Atomic Force Microscopy, which showed an increase in stiffness in DCM serum-treated NRVMs. Of the proteins increased in DCM sera, secreted frizzled-related protein 1 (sFRP1) was a potential candidate for the increase in cellular stiffness, and sFRP1 treatment of NRVMs recapitulated the increase in cellular stiffness observed in response to DCM serum treatment. Our results show that serum circulating proteins promoted pathological changes in gene expression and cellular stiffness, and circulating miRNAs were protective against pathological changes.

Toxicity and membrane perturbation properties of the ribotoxin-like protein Ageritin.

Ageritin is the prototype of a new ribotoxin-like protein family, which has been recently identified also in basidiomycetes. The protein exhibits specific RNase activity through the cleavage of a single phosphodiester bond located at sarcin/ricin loop of the large rRNA, thus inhibiting protein biosynthesis at early stages. Conversely to other ribotoxins, its activity requires the presence of divalent cations. In the present study, we report the activity of Ageritin on both prokaryotic and eukaryotic cells showing that the protein has a prominent effect on cancer cells viability and no effects on eukaryotic and bacterial cells. In order to rationalize these findings, the ability of the protein to interact with various liposomes mimicking normal, cancer and bacterial cell membranes was explored. The collected results indicate that Ageritin can interact with DPPC/DPPS/Chol vesicles, used as a model of cancer cell membranes, and with DPPC/DPPG vesicles, used as a model of bacterial cell membranes, suggesting a selective interaction with anionic lipids. However, a different perturbation of the two model membranes, mediated by cholesterol redistribution, was observed and this might be at the basis of Ageritin selective toxicity towards cancer cells.

Ageritin from Pioppino Mushroom: The Prototype of Ribotoxin-Like Proteins, a Novel Family of Specific Ribonucleases in Edible Mushrooms.

Ageritin is a specific ribonuclease, extracted from the edible mushroom Cyclocybe aegerita (synonym Agrocybe aegerita), which cleaves a single phosphodiester bond located within the universally conserved alpha-sarcin loop (SRL) of 23-28S rRNAs. This cleavage leads to the inhibition of protein biosynthesis, followed by cellular death through apoptosis. The structural and enzymatic properties show that Ageritin is the prototype of a novel specific ribonucleases family named 'ribotoxin-like proteins', recently found in fruiting bodies of other edible basidiomycetes mushrooms (e.g., Ostreatin from Pleurotus ostreatus, Edulitins from Boletus edulis, and Gambositin from Calocybe gambosa). Although the putative role of this toxin, present in high amount in fruiting body (>2.5 mg per 100 g) of C. aegerita, is unknown, its antifungal and insecticidal actions strongly support a role in defense mechanisms. Thus, in this review, we focus on structural, biological, antipathogenic, and enzymatic characteristics of this ribotoxin-like protein. We also highlight its biological relevance and potential biotechnological applications in agriculture as a bio-pesticide and in biomedicine as a therapeutic and diagnostic agent.

A novel strategy for engineering of a smart generation of immune ribonucleases against EGFR+ cells.

The overexpression of epidermal growth factor receptor (EGFR) could result in the development of solid tumors of prostate, breast, gastric, colorectal, ovarian, and head and neck, leading to carcinoma. Antibody therapies are ideal methods to overcome malignant diseases. However, immunoribonucleases are a new generation of antibodies in which an RNase binds to a specific antibody and shows a stronger ability to terminate cancer cells. In this study, we engineered Rana pipiens RNase to bind to the scFv of human antiepidermal growth factor receptor antibody. The molecular dynamic simulations confirmed protein stability and the ability of scFv-ranpirnase (rantoxin) to bind to epidermal growth factor receptor protein. Then, the rantoxin construct was synthesized in a pCDNA 3.1 Neo vector. CHO-K1 cells were used as expression hosts and the construct was transfected. Cells were selected by antibiotic therapies using neomycin, 120 mg/ml, and the high-yield colony was screened by real-time polymerase chain reaction (PCR) methods. Then, the recombinant protein production was confirmed using the sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analyses. The molecular dynamic simulation (MDS) confirmed that the I467, S468, Q408, and H409 amino acids of EGFR bonded well to rantoxin. As revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses, the rantoxin production and PCR analysis showed that the T3 colony can produce rantoxin messenger RNA fourfold higher than the GAPDH gene. The immunotoxin function was assessed in A431 cancer cells and EGFR-negative HEK293 cells, and IC50  values were estimated to be 22.4 ± 3 and >620.4 ± 5 nM, respectively. The results indicated that the immunotoxins produced in this study had the potential for use as anticancer drugs.