Human Pancreatic Ribonuclease Research Articles

Engineering Human Pancreatic RNase 1 as an Immunotherapeutic Agent for Cancer Therapy Through Computational and Experimental Studies.

Most plant and bacterial toxins are highly immunogenic with non-specific toxic effects. Human ribonucleases are thought to provide a promising basis for reducing the toxic agent's immunogenic properties, which are candidates for cancer therapy. In the cell, the ribonuclease inhibitor (RI) protein binds to the ribonuclease enzyme and forms a tight complex. This study aimed to engineer and provide a gene construct encoding an improved version of Human Pancreatic RNase 1 (HP-RNase 1) to reduce connection to RI and modulate the immunogenic effects of immunotoxins. To further characterize the interaction complex of HP-RNase 1 and RI, we established various in silico and in vitro approaches. These methods allowed us to specifically monitor interactions within native and engineered HP-RNase 1/RI complexes. In silico research involved molecular dynamics (MD) simulations of native and mutant HP-RNase 1 in their free form and when bound to RI. For HP-RNase 1 engineering, we designed five mutations (K8A/N72A/N89A/R92D/E112/A) based on literature studies, as this combination proved effective for the intended investigation. Then, the cDNA encoding HP-RNase 1 was generated by RT-PCR from blood and cloned into the pSYN2 expression vector. Consequently, wild-type and the engineered HP-RNase 1 were over-expressed in E. coli TG1 and purified using an IMAC column directed against a poly-his tag. The protein products were detected by SDS-PAGE and Western blot analysis. HP-RNase 1 catalytic activity, in the presence of various concentrations of RI, demonstrated that the mutated version of the protein is able to escape the ribonuclease inhibitor and target the RNA substrate 2.5 folds more than that of the wild type. From these data, we tend to suggest the engineered recombinant HP-RNase 1 potentially as a new immunotherapeutic agent for application in human cancer therapy.

A Nuclear-Directed Ribonuclease Variant Targets Cancer Stem Cells and Inhibits Migration and Invasion of Breast Cancer Cells.

Simple SummaryDuring the past decades the achievements made in treating cancers have significantly improved the survival of patients. However, cancer is still one of the leading causes of mortality. It is suggested that treatment failure is mediated by a subpopulation of tumor cells named cancer stem cells that can survive after treatment and promote cancer relapse. Targeting these cells is important to improve cancer therapy. The aim of our study is to determine the effect of a human ribonuclease variant on breast cancer cells grown in 3D and on cancer stem cells. Moreover, we study its effect on the ability of breast cancer cells to migrate and produce metastasis, responsible for about 90% of cancer deaths. We show that this ribonuclease arrests tumor cells grown in 3D without affecting normal breast cells, and this significantly inhibits cancer stem cell development. Additionally, it reduces the migratory and invasive capacities of tumor cells.Despite the significant advances in cancer research made in recent years, this disease remains one of the leading causes of death worldwide. In part, this is due to the fact that after therapy, a subpopulation of self-renewing tumor cells can survive and promote cancer relapse, resistance to therapies and metastasis. Targeting these cancer stem cells (CSCs) is therefore essential to improve the clinical outcome of cancer patients. In this sense, multi-targeted drugs may be promising agents targeting CSC-associated multifocal effects. We have previously constructed different human pancreatic ribonuclease (RNase) variants that are cytotoxic for tumor cells due to a non-classical nuclear localization signal introduced in their sequence. These cytotoxic RNases affect the expression of multiple genes involved in deregulated metabolic and signaling pathways in cancer cells and are highly cytotoxic for multidrug-resistant tumor cell lines. Here, we show that these cytotoxic nuclear-directed RNases are highly selective for tumor cell lines grown in 3D, inhibit CSCs’ development and diminish the self-renewal capacity of the CSCs population. Moreover, these human RNase variants reduce the migration and invasiveness of highly invasive breast cancer cells and downregulate N-cadherin expression.

Specific Targeting of Recombinant Human Pancreatic Ribonuclease 1 using Gonadotropin-Releasing Hormone Targeting Peptide toward Gonadotropin-Releasing Hormone Receptor-Positive Cancer Cells.

Background: Targeted drug delivery is a novel method to specifically deliver anticancer therapeutics to tumor sites. Gonadotropin-releasing hormone (GnRH) is a decapeptide, and its target binding property has attracted attention as a means of targeted drug delivery. Human pancreatic ribonuclease 1 (hpRNase1) has been shown to exert anticancer properties, when fused to a targeting moiety. The goal of the present study was to add a GnRH targeting peptide to the N-terminus of hpRNase1 to specifically target GnRH receptor (GnRH-R) expressing cells.Methods: This in vitro study was conducted at Shiraz Institute for Cancer Research (Shiraz, Iran) in 2019. The coding sequence of GnRH and hpRNase1 were fused, and the chimeric protein together with non-fused hpRNase1 were produced in E. coli (BL21). The recombinant proteins were purified, and their biological activity was evaluated using MTT and apoptosis assays. Non-parametric Kruskal–Wallis tests with Dunn’s post hoc tests were performed to determine the significant differences between the study groups.Results: GnRH-hpRNase1 chimeric protein specifically inhibited the proliferation of PC-3 (P=0.021), LNCaP (P=0.034), and AD-Gn (P=0.041) cells, while the growth of negative cells (AD-293) was not significantly affected (P=0.081). GnRH-hpRNase1 decreased the IC50 values more than non-fused hpRNase1, by approximately 26.5-fold (P=0.036) for PC-3 cells, and exerted its growth inhibitory effects through apoptosis induction.Conclusion: Fusion of GnRH to hpRNase1 structure produced an enzyme, which could specifically target tumor cells. This approach can be used to eliminate tumors that harbor GnRH-R.

The Selectivity for Tumor Cells of Nuclear-Directed Cytotoxic RNases Is Mediated by the Nuclear/Cytoplasmic Distribution of p27KIP1.

Although single targeted anti-cancer drugs are envisaged as safer treatments because they do not affect normal cells, cancer is a very complex disease to be eradicated with a single targeted drug. Alternatively, multi-targeted drugs may be more effective and the tumor cells may be less prone to develop drug resistance although these drugs may be less specific for cancer cells. We have previously developed a new strategy to endow human pancreatic ribonuclease with antitumor action by introducing in its sequence a non-classical nuclear localization signal. These engineered proteins cleave multiple species of nuclear RNA promoting apoptosis of tumor cells. Interestingly, these enzymes, on ovarian cancer cells, affect the expression of multiple genes implicated in metabolic and signaling pathways that are critic for the development of cancer. Since most of these targeted pathways are not highly relevant for non-proliferating cells, we envisioned the possibility that nuclear directed-ribonucleases were specific for tumor cells. Here, we show that these enzymes are much more cytotoxic for tumor cells in vitro. Although the mechanism of selectivity of NLSPE5 is not fully understood, herein we show that p27KIP1 displays an important role on the higher resistance of non-tumor cells to these ribonucleases.

Semisynthesis of Human Ribonuclease-S.

Since its conception, the ribonuclease S complex (RNase S) has led to historic discoveries in protein chemistry, enzymology, and related fields. Derived by the proteolytic cleavage of a single peptide bond in bovine pancreatic ribonuclease (RNase A), RNase S serves as a convenient and reliable model system for incorporating unlimited functionality into an enzyme. Applications of the RNase S system in biomedicine and biotechnology have, however, been hindered by two shortcomings: (1) the bovine-derived enzyme could elicit an immune response in humans, and (2) the complex is susceptible to dissociation. Here, we have addressed both limitations in the first semisynthesis of an RNase S conjugate derived from human pancreatic ribonuclease and stabilized by a covalent interfragment cross-link. We anticipate that this strategy will enable unprecedented applications of the "RNase-S" system.

Biological Activities of Secretory RNases: Focus on Their Oligomerization to Design Antitumor Drugs.

Ribonucleases (RNases) are a large number of enzymes gathered into different bacterial or eukaryotic superfamilies. Bovine pancreatic RNase A, bovine seminal BS-RNase, human pancreatic RNase 1, angiogenin (RNase 5), and amphibian onconase belong to the pancreatic type superfamily, while binase and barnase are in the bacterial RNase N1/T1 family. In physiological conditions, most RNases secreted in the extracellular space counteract the undesired effects of extracellular RNAs and become protective against infections. Instead, if they enter the cell, RNases can digest intracellular RNAs, becoming cytotoxic and having advantageous effects against malignant cells. Their biological activities have been investigated either in vitro, toward a number of different cancer cell lines, or in some cases in vivo to test their potential therapeutic use. However, immunogenicity or other undesired effects have sometimes been associated with their action. Nevertheless, the use of RNases in therapy remains an appealing strategy against some still incurable tumors, such as mesothelioma, melanoma, or pancreatic cancer. The RNase inhibitor (RI) present inside almost all cells is the most efficacious sentry to counteract the ribonucleolytic action against intracellular RNAs because it forms a tight, irreversible and enzymatically inactive complex with many monomeric RNases. Therefore, dimerization or multimerization could represent a useful strategy for RNases to exert a remarkable cytotoxic activity by evading the interaction with RI by steric hindrance. Indeed, the majority of the mentioned RNases can hetero-dimerize with antibody derivatives, or even homo-dimerize or multimerize, spontaneously or artificially. This can occur through weak interactions or upon introducing covalent bonds. Immuno-RNases, in particular, are fusion proteins representing promising drugs by combining high target specificity with easy delivery in tumors. The results concerning the biological features of many RNases reported in the literature are described and discussed in this review. Furthermore, the activities displayed by some RNases forming oligomeric complexes, the mechanisms driving toward these supramolecular structures, and the biological rebounds connected are analyzed. These aspects are offered with the perspective to suggest possible efficacious therapeutic applications for RNases oligomeric derivatives that could contemporarily lack, or strongly reduce, immunogenicity and other undesired side-effects.

Construction of Highly Stable Cytotoxic Nuclear-Directed Ribonucleases.

Ribonucleases are proteins whose use is promising in anticancer therapy. We have previously constructed different human pancreatic ribonuclease variants that are selectively cytotoxic for tumor cells by introducing a nuclear localization signal into their sequence. However, these modifications produced an important decrease in their stability compromising their behavior in vivo. Here, we show that we can significantly increase the thermal stability of these cytotoxic proteins by introducing additional disulfide bonds by site-directed mutagenesis. One of these variants increases its thermal stability by around 17 °C, without affecting its catalytic activity while maintaining the cytotoxic activity against tumor cells. We also show that the most stable variant is significantly more resistant to proteolysis when incubated with proteinase K or with human sera, suggesting that its half-live could be increased in vivo once administered.

PO-427 A nuclear-directed human pancreatic ribonuclease variant is cytotoxic for breast cancer cells cultured in 3D and kills cancer stem cells

IntroductionRibonucleases are a new class of antitumor RNA-degrading drugs that control the malignant phenotype at different levels. We produced nuclear-directed human pancreatic ribonucleases (ND-RNases) that showed selective cytotoxicity for tumour cells using two-dimensional cultures in vitro.Material and methodsNow we have studied the effect of the most effective ND-RNase in 2D cultures on different breast cancer cell lines representative of different molecular subtypes in three-dimensional assays.Results and discussionsThe results show that the ND-RNase cytotoxicity is selective for these cancer cell lines grown in 3D in a laminin-rich extracellular matrix, a better model to predict drug sensitivity in vivo than cells cultured on a 2D substrate. On the other hand, we have studied the effect of this ND-RNase on the oncogenic cell signalling cascades through phospho-kinase array profiling assays. The results show that the ND-RNase strongly enhance phosphorylation of multiple signalling cascades in breast carcinoma cells. Moreover, we show that the ND-RNase have a marked ability to decrease the capacity of formation of mammospheres and their self-renewal in all breast cancer cell lines assayed.ConclusionIn conclusion, mammosphere formation assays are indicative that this ND-RNase is able to kill cancer stem cells, which are thought responsible for tumour progression, metastasis, resistance to therapy, and subsequent tumour recurrence. The pleitropic effects of this ND-RNase can be explained through its effect on the phosphorylation of multiple oncogenic cell signalling cascades.

A ribonuclease inhibitor resistant dimer of human pancreatic ribonuclease displays specific antitumor activity

Human pancreatic ribonuclease (HPR) and bovine seminal ribonuclease (BS-RNase) are members of the RNase A superfamily. HPR is monomeric, whereas BS-RNase is dimeric. BS-RNase has strong antitumor and cytotoxic activities. However, HPR lacks cytotoxic activity as it is inactivated by intracellular cytosolic ribonuclease inhibitor (RI). Earlier, an RI-resistant cytotoxic variant of HPR, termed HPR-KNE was generated which contained three residues Lys7, Asn71 and Glu111 of HPR, known to interact with RI, mutated to alanine. In this study, we have engineered HPR to develop two dimeric RI-resistant molecules having anti-tumor activity. By incorporating two cysteines in HPR and HPR-KNE, we generated disulfide linked dimeric HPR, and a dimer of HPR-KNE, termed as HPR-D and HPR-KNE-D respectively. HPR-KNE-D was resistant towards inhibition by RI, and was found to be highly toxic to a variety of cells. On J774A.1 cells HPR-KNE-D was >375-fold more cytotoxic than HPR, and 15-fold more toxic than HPR-D. Further, on U373 cells HPR-KNE-D was >65-fold more cytotoxic than HPR, and 9-fold more toxic than HPR-D. The study demonstrates that combining dimerization and RI-resistance results in providing potent anti-tumor activity to HPR. The cytotoxic variants of HPR will be useful in designing protein therapeutics with low immunogenicity.

An interactomics overview of the human and bovine milk proteome over lactation.

BackgroundMilk is the most important food for growth and development of the neonate, because of its nutrient composition and presence of many bioactive proteins. Differences between human and bovine milk in low abundant proteins have not been extensively studied. To better understand the differences between human and bovine milk, the qualitative and quantitative differences in the milk proteome as well as their changes over lactation were compared using both label-free and labelled proteomics techniques. These datasets were analysed and compared, to better understand the role of milk proteins in development of the newborn.MethodsHuman and bovine milk samples were prepared by using filter-aided sample preparation (FASP) combined with dimethyl labelling and analysed by nano LC LTQ-Orbitrap XL mass spectrometry.ResultsThe human and bovine milk proteome show similarities with regard to the distribution over biological functions, especially the dominant presence of enzymes, transport and immune-related proteins. At a quantitative level, the human and bovine milk proteome differed not only between species but also over lactation within species. Dominant enzymes that differed between species were those assisting in nutrient digestion, with bile salt-activated lipase being abundant in human milk and pancreatic ribonuclease being abundant in bovine milk. As lactation advances, immune-related proteins decreased slower in human milk compared to bovine milk. Notwithstanding these quantitative differences, analysis of human and bovine co-expression networks and protein-protein interaction networks indicated that a subset of milk proteins displayed highly similar interactions in each of the different networks, which may be related to the general importance of milk in nutrition and healthy development of the newborn.ConclusionsOur findings promote a better understanding of the differences and similarities in dynamics of human and bovine milk proteins, thereby also providing guidance for further improvement of infant formula.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-016-0110-0) contains supplementary material, which is available to authorized users.

A novel fully human anti-NCL immunoRNase for triple-negative breast cancer therapy.

Breast cancer is the most common cancer in women worldwide. A new promising anti-cancer therapy involves the use of monoclonal antibodies specific for target tumor-associated antigens (TAAs). A TAA of interest for immunotherapy of Triple Negative Breast Cancer (TNBC) is nucleolin (NCL), a multifunctional protein, selectively expressed on the surface of cancer cells, which regulates the biogenesis of specific microRNAs (miRNAs) involved in tumor development and drug-resistance. We previously isolated a novel human anti-NCL scFv, called 4LB5, that is endowed with selective anti-tumor effects. Here we report the construction and characterization of a novel immunoRNase constituted by 4LB5 and a human pancreatic RNase (HP-RNase) called “4LB5-HP-RNase”. This immunoRNase retains both the enzymatic activity of human pancreatic RNase and the specific binding of the parental scFv to a panel of surface NCL-positive breast cancer cells. Notably, 4LB5-HP-RNase dramatically and selectively reduced the viability and proliferation of NCL-positive tumor cells in vitro and in vivo. Specifically, it induced apoptosis and reduced the levels of the tumorigenic miRNAs miR-21, -221 and -222. Thus, this novel immunoagent could be a valuable tool for the treatment of TNBC patients ineligible for currently available targeted treatments.

Ribonuclease (RNase) Prolongs Survival of Grafts in Experimental Heart Transplantation.

BackgroundCell damage, tissue and vascular injury are associated with the exposure and release of intracellular components such as RNA, which promote inflammatory reactions and thrombosis. Based on the counteracting anti‐inflammatory and cardioprotective functions of ribonuclease A (RNase A) in this context, its role in an experimental model of heart transplantation in rats was studied.Methods and ResultsInbred BN/OrlRj rat cardiac allografts were heterotopically transplanted into inbred LEW/OrlRj rats. Recipients were intravenously treated every other day with saline or bovine pancreatic RNase A (50 μg/kg). Toxic side effects were not found (macroscopically and histologically). Heart tissue flow cytometry and quantitative morphological analyses of explanted hearts at postoperative day 1 or postoperative day 4 showed reduced leukocyte infiltration, edema, and thrombus formation in RNase A‐treated rats. In allogeneic mixed lymphocyte reactions, RNase A decreased the proliferation of effector T cells. RNase A treatment of rats resulted in prolonged median graft survival up to 10.5 days (interquartile range 1.8) compared to 6.5 days (interquartile range 1.0) in saline treatment (P=0.001). Treatment of rats with a new generated (recombinant) human pancreatic RNase 1 prolonged median graft survival similarly, unlike treatment with (recombinant) inactive human RNase 1 (each 50 μg/kg IV every other day, 11.0 days, interquartile range 0.3, versus 8.0 days, interquartile range 0.5, P=0.007).ConclusionsUpon heart transplantation, RNase administration appears to present a promising and safe drug to counteract ischemia/reperfusion injury and graft rejection. Furthermore, RNase treatment may be considered in situations of critical reperfusion after percutaneous coronary interventions or in cardiac surgery using the heart–lung machine.

A nuclear-directed human pancreatic ribonuclease (PE5) targets the metabolic phenotype of cancer cells.

Ribonucleases represent a new class of antitumor RNA-damaging drugs. However, many wild-type members of the vertebrate secreted ribonuclease family are not cytotoxic because they are not able to evade the cytosolic ribonuclease inhibitor. We previously engineered the human pancreatic ribonuclease to direct it to the cell nucleus where the inhibitor is not present. The best characterized variant is PE5 that kills cancer cells through apoptosis mediated by the p21WAF1/CIP1 induction and the inactivation of JNK. Here, we have used microarray-derived transcriptional profiling to identify PE5 regulated genes on the NCI/ADR-RES ovarian cancer cell line. RT-qPCR analyses have confirmed the expression microarray findings. The results show that PE5 cause pleiotropic effects. Among them, it is remarkable the down-regulation of multiple genes that code for enzymes involved in deregulated metabolic pathways in cancer cells.

Increased N-glycosylation of Asn⁸⁸ in serum pancreatic ribonuclease 1 is a novel diagnostic marker for pancreatic cancer.

Alterations of carbohydrate structures in cancer cells are the most promising targets for developing clinical diagnostic reagents. Pancreatic cancer is one of the most difficult cancers to diagnose because it lacks definitive symptoms. Two antibodies were raised against human pancreatic ribonuclease 1 that bind to the enzyme containing unglycosylated Asn88, but not when its Asn88 is N-glycosylated. Differential studies using these antibodies in immunoassays and Western blot analyses showed a significant increase in the serum levels of pancreatic ribonuclease 1 containing N-glycosylated Asn88 in pancreatic cancer patients compared with normal human subjects. Focusing on the increase in an N-glycosylated Asn residue of serum pancreatic ribonuclease 1, specifically Asn88, affords a new diagnostic marker for pancreatic cancer. This is the first report of a diagnostic cancer marker that takes advantage of the presence or absence of N-glycosylation at a specific Asn residue of a glycoprotein.

Bovine Brain Ribonuclease Is the Functional Homolog of Human Ribonuclease 1

Mounting evidence suggests that human pancreatic ribonuclease (RNase 1) plays important roles in vivo, ranging from regulating blood clotting and inflammation to directly counteracting tumorigenic cells. Understanding these putative roles has been pursued with continual comparisons of human RNase 1 to bovine RNase A, an enzyme that appears to function primarily in the ruminant gut. Our results imply a different physiology for human RNase 1. We demonstrate distinct functional differences between human RNase 1 and bovine RNase A. Moreover, we characterize another RNase 1 homolog, bovine brain ribonuclease, and find pronounced similarities between that enzyme and human RNase 1. We report that human RNase 1 and bovine brain ribonuclease share high catalytic activity against double-stranded RNA substrates, a rare quality among ribonucleases. Both human RNase 1 and bovine brain RNase are readily endocytosed by mammalian cells, aided by tight interactions with cell surface glycans. Finally, we show that both human RNase 1 and bovine brain RNase are secreted from endothelial cells in a regulated manner, implying a potential role in vascular homeostasis. Our results suggest that brain ribonuclease, not RNase A, is the true bovine homolog of human RNase 1, and provide fundamental insight into the ancestral roles and functional adaptations of RNase 1 in mammals.

Evaluation of human pancreatic RNase as effector molecule in a therapeutic antibody platform

Human antibody-ribonuclease (RNase) fusion proteins, referred to as immunoRNases, have been proposed as an alternative to heterologous immunotoxins, without their immunogenicity and unspecific toxicity issues. In this study, we investigated if human pancreatic RNase will be suitable as effector component in a therapeutic antibody development platform. We generated several fusion proteins consisting of tumor-specific human immunoglobulins (IgGs) and human pancreatic RNase. Transient mammalian cell production was efficient and IgG-RNases were purified to homogeneity. Antigen binding was comparable to the parental antibodies and RNase catalytic activity was retained even in the presence of 50-fold molar excess of human cytosolic RNase inhibitor (RI). Serum stability, cell binding and internalization of IgG-RNases were comparable to the parental IgGs. Despite these promising properties, none of the IgG-RNases revealed significant inhibition of tumor cell growth in vitro even when targeting different antigens putatively employing different endocytotic pathways. The introduction of different linkers containing endosomal protease cleavage sites into the IgG-RNase did not enhance cytotoxicity. Similarly, RI evasive human pancreatic RNase variants mediated only small inhibiting effects on tumor cell growth at high concentrations, potentially reflecting inefficient cytosolic translocation. Taken together, human pancreatic RNase and variants did not prove to be generally suitable as effector component for a therapeutic antibody drug development platform.

Three-dimensional domain swapping and supramolecular protein assembly: insights from the X-ray structure of a dimeric swapped variant of human pancreatic RNase

The deletion of five residues in the loop connecting the N-terminal helix to the core of monomeric human pancreatic ribonuclease leads to the formation of an enzymatically active domain-swapped dimer (desHP). The crystal structure of desHP reveals the generation of an intriguing fibril-like aggregate of desHP molecules that extends along the c crystallographic axis. Dimers are formed by three-dimensional domain swapping. Tetramers are formed by the aggregation of swapped dimers with slightly different quaternary structures. The tetramers interact in such a way as to form an infinite rod-like structure that propagates throughout the crystal. The observed supramolecular assembly captured in the crystal predicts that desHP fibrils could form in solution; this has been confirmed by atomic force microscopy. These results provide new evidence that three-dimensional domain swapping can be a mechanism for the formation of elaborate large assemblies in which the protein, apart from the swapping, retains its original fold.

Human Ribonuclease with a Pendant Poly(Ethylene Glycol) Inhibits Tumor Growth in Mice

Human pancreatic ribonuclease (RNase 1) is a small secretory protein that catalyzes the cleavage of RNA. This highly cationic enzyme can enter human cells spontaneously but is removed rapidly from circulation by glomerular filtration. Here, this shortcoming is addressed by attaching a poly(ethylene glycol) (PEG) moiety to RNase 1. The pendant has no effect on ribonucleolytic activity but does increase persistence in circulation. The RNase 1-CPEG conjugates inhibit the growth of tumors in a xenograft mouse model of human lung cancer. Both retention in circulation and tumor growth inhibition correlate with the size of the pendant PEG. A weekly dose of the 60-kDa conjugate at 1 μmol/kg inhibited nearly all tumor growth without affecting body weight. Its molecular efficacy is ~5000-fold greater than that of erlotinib, which is a small molecule in clinical use for the treatment of lung cancer. These data demonstrate that the addition of a PEG moiety can enhance the in vivo efficacy of human proteins that act within cells and highlight a simple means of converting an endogenous human enzyme into a cytotoxin with potential clinical utility.

A hydrophobic segment of some cytotoxic ribonucleases

The exact mechanism by which cytotoxic ribonucleases reach the cytosol of tumor cells remains unclear. The interaction of ribonucleases with a lipid bilayer is involved in the translocation of ribonucleases across the endosomal membrane. Here, we aimed to study the hydropathy character of toxic antitumor ribonucleases (bovine seminal ribonuclease and binase) and two non-toxic ribonucleases (bovine pancreatic ribonuclease and human pancreatic ribonuclease) by sliding-window hydrophobicity analysis. Comparative hydropathy plot analysis of the non-toxic pancreatic ribonucleases and their toxic variants was also performed. The data obtained indicate that some cytotoxic ribonucleases have a hydrophobic segment, which is sterically available for the hydrophobic interaction with a tumor cell membrane and endosomal membrane. After dissociation, subunits of dimeric ribonucleases are probably capable of thermodynamically favorable interaction with the interfacial region of a lipid bilayer. Remarkably the hydrophobic segment is not identified in the amino acid sequences of non-toxic ribonucleases. The paper describes the hydrophobic properties of toxic RNases that are essential for both the model of a lipid–protein interaction and the cytotoxicity mechanism unraveling.

A novel fully human antitumor ImmunoRNase resistant to the RNase inhibitor

The ErbB2 tyrosine kinase receptor is an attractive target for immunotherapy, as it is overexpressed in many carcinomas. ImmunoRNases, made up of a human anti-ErbB2 scFv (single chain antibody fragment) and human RNases, have been engineered to overcome the limits of immunotoxins, made up of mouse antibodies and plant or bacterial toxins, such as immunogenicity and non-specific toxicity. Here we describe the construction and characterization of a second-generation anti-ErbB2 immunoRNase, called ERB-HP-DDADD-RNase, obtained by fusing Erbicin, a human ErbB2-directed scFv, with an inhibitor-resistant variant of human pancreatic RNase (HP-DDADD-RNase). This novel immunoRNase retains both the enzymatic activity of human pancreatic RNase and the specific binding of the parental scFv to ErbB2-positive cells, showing an affinity comparable with that of the previously reported parental immunoRNase (ERB-HP-RNase). Moreover, the novel immunoRNase is endowed with an effective and selective in vitro antiproliferative action for ErbB2-positive tumor cells, which is more potent than that of the parental immunoRNase on tumor cells expressing low levels of ErbB2, due to its resistance to the RNase inhibitor. Thus, the novel immunoRNase could represent a valuable tool for ErbB2-positive cancer therapy.