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Morpheeins are proteins that reversibly assemble into different oligomers, whose architectures are governed by conformational changes of the subunits. This property could be utilized in bionanotechnology where the building of nanometric and new high-ordered structures is required. By capitalizing on the adaptability of morpheeins to create patterned structures and exploiting their inborn affinity toward inorganic and living matter, "bottom-up" creation of nanostructures could be achieved using a single protein building block, which may be useful as such or as scaffolds for more complex materials. Peroxiredoxins represent the paradigm of a morpheein that can be applied to bionanotechnology. This review describes the structural and functional transitions that peroxiredoxins undergo to form high-order oligomers, e.g., rings, tubes, particles, and catenanes, and reports on the chemical and genetic engineering approaches to employ them in the generation of responsive nanostructures and nanodevices. The usefulness of the morpheeins' behavior is emphasized, supporting their use in future applications.

A novel suicide gene therapy approach was tested in U87 MG glioblastoma multiforme cells. A 26nt G-rich double-stranded DNA aptamer (AS1411) was integrated into a vector at the 5′ of a mammalian codon-optimized saporin gene, under CMV promoter. With this plasmid termed “APTSAP”, the gene encoding ribosome-inactivating protein saporin is driven intracellularly by the glioma-specific aptamer that binds to cell surface-exposed nucleolin and efficiently kills target cells, more effectively as a polyethyleneimine (PEI)-polyplex. Cells that do not expose nucleolin at the cell surface such as 3T3 cells, used as a control, remain unaffected. Suicide gene-induced cell killing was not observed when the inactive saporin mutant SAPKQ DNA was used in the (PEI)-polyplex, indicating that saporin catalytic activity mediates the cytotoxic effect. Rather than apoptosis, cell death has features resembling autophagic or methuosis-like mechanisms. These main findings support the proof-of-concept of using PEI-polyplexed APTSAP for local delivery in rat glioblastoma models.

Le proteine del siero di latte sia nella loro forma nativa che chimicamente modificata e i loro derivati peptidici hanno incontrato già da diversi anni molto interesse frai ricercatori del settore nutraceutico e farmaceutico per le loro numerose proprietà tra cui quelle antimicrobiche, immunomodulanti e antitumorali, tali da rendere il latte e le sue proteine utilizzabili in un ampio spettro di trattamenti sanitari e di prevenzione. Recentemente è stata riscontrata una importante attività antivirale di queste molecole e bisogna sottolineare come lo studio di molecole antivirali con bassa o nulla tossicità, come quelle derivate dagli alimenti, sia attualmente considerato cruciale come trattamento aggiuntivo alle terapie convenzionali per contrastare le infezioni virali già note e quelle più recenti e meno conosciute come il COVID-19. Nella presente rassegna vengono riportate le recenti pubblicazioni riguardanti le proprietà antivirali elle proteine del siero di latte e le loro potenziali applicazioni nel campo della protezione della salute umana.

Novel two-dimensional films and three-dimensional (3D) scaffolds based on chitosan (CHI), apatite (Ap), and graphene oxide (GO) were developed by an in situ synthesis in which self-assembly process was conducted to direct partial reduction of GO by CHI in acidic medium. Physical-chemical characterization was carried out by optical microscopy, scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy. In vitro biological studies using murine fibroblast (MC3T3) and human neuroblastoma (SH-SY5Y) cell lines were also performed. Cell growth and adherence on composites was also checked using SEM. Live and death staining by confocal microscope and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium of the samples were investigated. The results confirmed the incorporation of both Ap and GO sheets, into CHI polymeric matrix. Furthermore, it was confirmed a physical integration between inorganic Ap and organic CHI and strong chemical interaction between CHI and GO in the obtained composites. SH-SY5Y cell line showed preferential adherence on CHI/GO films surface while MC3T3 cell line displayed a good compatibility for all 3D scaffolds. This study confirms the biocompatibility of materials based on CHI, Ap, and GO for future tissues applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1585-1594, 2018.

// Emily Capone 1, * , Francesco Giansanti 2, * , Sara Ponziani 2, 3 , Alessia Lamolinara 4 , Manuela Iezzi 4 , Annamaria Cimini 2, 5, 6 , Francesco Angelucci 2 , Rossana La Sorda 3 , Vincenzo De Laurenzi 1 , Pier Giorgio Natali 3 , Rodolfo Ippoliti 2 , Stefano Iacobelli 1, 3 and Gianluca Sala 1, 3 1 Department of Medical, Oral and Biotechnological Sciences, University of Chieti-Pescara, Chieti, Italy 2 Department of Life, Health and Environmental Sciences, University of L'Aquila, Coppito (AQ) Italy 3 MediaPharma s.r.l., Via della Colonnetta, Chieti, Italy 4 Department of Medicine and Aging Science, Center of Excellence on Aging and Translational Medicine (CeSi-Met), G. D’Annunzio University, Chieti-Pescara, Italy 5 Sbarro Institute for Cancer Research and Molecular Medicine and Center for Biotechnology Temple University, Philadelphia, USA 6 National Institute for Nuclear Physics (INFN), Gran Sasso National Laboratory (LNGS), Assergi, Italy * These authors have contributed equally to this work Correspondence to: Gianluca Sala, email: g.sala@unich.it Stefano Iacobelli, email: iacobell@unich.it Keywords: HER-3, antibody-drug conjugate, melanoma, saporin, target therapy Received: June 22, 2017 Accepted: August 08, 2017 Published: September 08, 2017 ABSTRACT Melanoma is the most biologically aggressive skin cancer of well established constitutive and induced resistance to pharmacological treatment. Despite the recent progresses in immunotherapies, many advanced metastatic melanoma patients still face a significant mortality risk. The aggressive nature of this disease sustains an urgent need for more successful, effective drugs. HER-3 - one of the four member of the tyrosin kinase epidermal growth factor receptors (EGFRs) family- is frequently overexpressed in solid tumors, including melanoma. Moreover, up-regulation of HER-3 and its ligand NRGβ-1 are associated with poor prognosis, thus suggesting this receptor as a suitable target for cancer therapy. Several monoclonal antibodies targeting HER-3 are currently available, but preliminary results from clinical testing of these agents reveal a modest efficacy. Thus, a substantial improvement over this immunotherapeutic approach could be offered by an anti-HER-3 based Antibody-Drug Conjugate (ADC). In the present paper, we describe the generation of an ADC obtained by coupling the HER-3 targeting antibody EV20 linked to the plant toxin Saporin (Sap). In vitro , this ADC displays a powerful, specific and target-dependent cytotoxic activity which correlates with the degree of expression and internalization of HER-3 on tumor cells. Furthermore, in a murine melanoma model, EV20-Sap treatment leads to a significant reduction of the number of pulmonary metastasis.

Background Proteins are efficient supramolecular scaffolds to drive self-assembly of nanomaterials into regular colloidal structures suitable for several purposes, including cell imaging and drug delivery. Proteins, in particular, can bind to gold nanoparticles (AuNPs) through van der Waals and electrostatic forces as well as coordination and hydrogen bonds leading their assembly into responsive nanostructures. Methods Bioconjugation of alkyne Raman tag-labeled 20 nm AuNPs with the ring-shaped protein Peroxiredoxin (Prx), characterized by a symmetric homo-oligomeric circular arrangement, has been investigated by absorption spectroscopy, transmission and scanning electron microscopy. The plasmonic behavior of the resulting hybrid assemblies has been assessed by Surface Enhanced Raman Scattering (SERS). Results The ring-shaped Prx molecules are demonstrated to adsorb onto the gold surface acting as “sticky” bio-linkers between adjacent nanoparticles to drive self-assembly into small colloidal AuNPs arrays. The arrays show nanometric interparticle gaps tailored by the protein ring thickness. The arrays exhibit improved optical activity due to SERS allowing detection of the Raman signals from both the protein and alkyne molecules. Conclusions This method can be used to build up SERS-active nanostructures using Prx as both a bio-linker and platform for attaching dyes, two-dimensional materials, such as graphene, and other biomolecules including DNA and enzymes. General significance The development of colloidal SERS nanostructures is considered a significant step forward in spectroscopic bioanalysis. Though protein-tailored nanofabrication is in a childhood stage, these results demonstrate the versatility of supramolecular proteins as tools to build-up nanostructures which are still impractical to obtain through top-down techniques.

Liver cancer (LC) is an aggressive disease with a markedly poor prognosis. Therapeutic options are limited, and, until recently the only FDA‑approved agent for first‑line treatment of patients with LC was the multi‑kinase inhibitor sorafenib, which exhibits limited activity and an increased overall survival (OS) of only 3 months over placebo. Therefore, the development of alternative therapeutic molecules for the treatment of LC is an urgent medical need. Antibody‑drug conjugates (ADCs) are an emerging class of novel anticancer agents, which have been developed recently for the treatment of malignant conditions, including LC, and are being studied in preclinical and clinical settings. Our group has recently generated an ADC [EV20/monomethyl auristatin F (MMAF)] by coupling the HER3 targeting antibody (EV20) to MMAF via a non‑cleavable maleimidocaproyl linker. This ADC was revealed to possess potent therapeutic activity in melanoma and breast carcinoma. In the present study, using western blot and flow cytometric analysis, it was reported that HER‑3 receptor was highly expressed in LC and activated by its ligand NRG‑1β in a panel of LC cell lines, thus indicating that this receptor may serve as a suitable target for ADC therapy. A novel ADC [EV20‑sss‑valine‑citrulline (vc)/MMAF] was generated, in which the cytotoxic payload MMAF was site‑specifically coupled to an engineered variant of EV20 via a vc cleavable linker. Cytotoxicity assays were performed to investigate in vitro antitumor activity of EV20‑sss‑vc/MMAF and it was compared to EV20/MMAF, which revealed only modest activity in LC.EV20‑sss‑vc/MMAF exhibited a significant cell killing activity in several LC cell lines. Additionally, in vivo xenograft experiments revealed that EV20‑sss‑vc/MMAF inhibited growth of LC tumors. The present data indicated that EV20‑sss‑vc/MMAF is a worthy candidate for the treatment of HER‑3 positive LC.

Simple Summary Several current therapies fail to eradicate tumors due to the ability of cancer stem cells (CSCs) to escape different programmed cell deaths. In fact, apoptosis, autophagy, and necroptosis are deregulated in CSCs. Over the years, it has emerged that CSCs play a key role in tumor formation, progression, and recurrence. Thus, developing CSC-selective and programmed death-inducing therapeutic approaches appears of primary importance. In this review, we focus on signaling pathways involved in CSCs’ escape-death ability and the variety of CSC-targeting agents developed in recent years. Abstract Cancer is one of the primary causes of death worldwide. Tumour malignancy is related to tumor heterogeneity, which has been suggested to be due to a small subpopulation of tumor cells named cancer stem cells (CSCs). CSCs exert a key role in metastasis development, tumor recurrence, and also epithelial–mesenchymal transition, apoptotic resistance, self-renewal, tumorigenesis, differentiation, and drug resistance. Several current therapies fail to eradicate tumors due to the ability of CSCs to escape different programmed cell deaths. Thus, developing CSC-selective and programmed death-inducing therapeutic approaches appears to be of primary importance. In this review, we discuss the main programmed cell death occurring in cancer and the promising CSC-targeting agents developed in recent years. Even if the reported studies are encouraging, further investigations are necessary to establish a combination of agents able to eradicate CSCs or inhibit their growth and proliferation.

Simple Summary Antibody Drug Conjugates are an emerging class of biopharmaceuticals that have seen an impressive increase of attention in the field of cancer therapy. Here, we describe the therapeutic activity of 1959-sss/DM3, a non-internalizing ADC targeting LGALS3BP, a secreted, extracellular vesicles-associated protein expressed by the majority of human cancers, including neuroblastoma. We show that 1959-sss/DM3 treatment can cure mice with established neuroblastoma tumours in pseudometastatic, orthotopic and Patient Derived Xenograft models. Abstract Neuroblastoma is the most common extra-cranial solid tumor in infants and children, which accounts for approximately 15% of all cancer-related deaths in the pediatric population. New therapeutic modalities are urgently needed. Antibody-Drug Conjugates (ADC)s-based therapy has been proposed as potential strategy to treat this pediatric malignancy. LGALS3BP is a highly glycosylated protein involved in tumor growth and progression. Studies have shown that LGALS3BP is enriched in extracellular vesicles (EV)s derived by most neuroblastoma cells, where it plays a critical role in preparing a favorable tumor microenvironment (TME) through direct cross talk between cancer and stroma cells. Here, we describe the development of a non-internalizing LGALS3BP ADC, named 1959-sss/DM3, which selectively targets LGALS3BP expressing neuroblastoma. 1959-sss/DM3 mediated potent therapeutic activity in different types of neuroblastoma models. Notably, we found that treatments were well tolerated at efficacious doses that were fully curative. These results offer preclinical proof-of-concept for an ADC targeting exosomal LGALS3BP approach for neuroblastomas.

Galectin-3-binding protein (Gal-3BP) has been identified as a cancer and metastasis-associated, secreted protein that is expressed by the large majority of cancers. The present study describes a special type of non-internalizing antibody-drug-conjugates that specifically target Gal-3BP. Here, we show that the humanized 1959 antibody, which specifically recognizes secreted Gal-3BP, selectively localized around tumor but not normal cells. A site specific disulfide linkage with thiol-maytansinoids to unpaired cysteine residues of 1959, resulting in a drug-antibody ratio of 2, yielded an ADC product, which cured A375m melanoma bearing mice. ADC products based on the non-internalizing 1959 antibody may be useful for the treatment of several human malignancies, as the cognate antigen is abundantly expressed and secreted by several cancers, while being present at low levels in most normal adult tissues.

The use of nanoscale materials in the design of scaffolds for CNS tissue is increasing, due to their ability to promote cell adhesion, to mimic an extracellular matrix microenvironment and to interact with neuronal membranes. In this framework, one of the major challenges when using undifferentiated neural cells is how to control the differentiation process. Here we report the characterization of a scaffold based on the self-assembled nanotubes of a mutant of the protein peroxiredoxin (from Schistosoma mansoni or Bos taurus), which allows the growth and differentiation of a model neuronal cell line (SHSY5Y). The results obtained demonstrate that SHSY5Y cells grow without any sign of toxicity and develop a neuronal phenotype, as shown by the expression of neuronal differentiation markers, without the use of any differentiation supplement, even in the presence of serum. The prodifferentiation effect is demonstrated to be dependent on the formation of the protein nanotube, since a wild-type (WT) form of the peroxiredoxin from Schistosoma mansoni does not induce any differentiation. The protein scaffold was also able to induce the spread of glioblastoma cancer stem cells growing in neurospheres and allowing the acquisition of a neuron-like morphology, as well as of immature rat cortical neurons. This protein used here as coating agent may be suggested for the development of scaffolds for tissue regeneration or anti-tumour devices. Copyright © 2016 John Wiley & Sons, Ltd.

Ovotransferrin or conalbumin belong to the transferrin protein family and is endowed with both iron-transfer and protective activities. In addition to its well-known antibacterial properties, ovotransferrin displays other protective roles similar to those already ascertained for the homologous mammalian lactoferrin. These additional functions, in many cases not directly related to iron binding, are also displayed by the peptides derived from partial hydrolysis of ovotransferrin, suggesting a direct relationship between egg consumption and human health.

In this paper we have studied a PDZ protein domain as a possible tool for cellular targeting of the ribosome inactivating protein Saporin, exploiting the ability of PDZ domains to recognize and bind short peptide sequences located at the C-terminus of a cognate protein. We have focused our attention on the PDZ domain from hCASK (Human calcium/calmodulin-dependent serine protein kinase) that binds extracellular CD98 in epithelial cells, being this antigen recognized as a marker for several human tumors and particularly considered a negative prognostic marker for human glioblastoma. We produced recombinant fusions of one or two hCASK-PDZ domains with the ribosome inactivating protein Saporin and assayed them on two human glioblastoma cell lines (GL15 and U87). These constructs proved to be toxic, with increasing activity as a function of the number of PDZ domains, and induce cell death by apoptotic mechanisms in a dose-dependent and/or time dependent manner.

Lactoferrin is an iron-binding protein present in large quantities in colostrum and in breast milk, in external secretions and in polymorphonuclear leukocytes. Lactoferrin’s main function is non-immune protection. Among several protective activities shown by lactoferrin, those displayed by orally administered lactoferrin are: (i) antimicrobial activity, which has been presumed due to iron deprivation, but more recently attributed also to a specific interaction with the bacterial cell wall and extended to viruses and parasites; (ii) immunomodulatory activity, with a direct effect on the development of the immune system in the newborn, together with a specific antinflammatory effects; (iii) a more recently discovered anticancer activity. It is worth noting that most of the protective activities of lactoferrin have been found, sometimes to a greater extent, also in peptides derived from limited proteolysis of lactoferrin that could be generated after lactoferrin ingestion. Lactoferrin could therefore be considered an ideal nutraceutic product because of its relatively cheap production from bovine milk and of its widely recognized tolerance after ingestion, along with its well demonstrated protective activities. The most important protective activities shown by orally administered bovine lactoferrin are reviewed in this article.

Lactoferrin belongs to the transferrin protein superfamily and is a major component of the mammalian innate immune system. Proteolytic cleavage of either human or bovine lactoferrin results in the generation of several peptides, most of which manifest the protective activities of Lactoferrin. It is worth noting that these peptides may be naturally produced in the human intestine after milk ingestion. Interestingly, peptides deriving from proteolytic cleavage of Ovotransferrin, a Lactoferrin homologous protein present in reptiles and birds, displays the same protective activities. This mini-review summarizes the most relevant protective activities of peptides deriving from Lactoferrin and Ovotransferrin.

Targeted anticancer therapies demand discovery of new cellular targets to be exploited for the delivery of toxic molecules and drugs. In this perspective, in the last few years, nucleolin has been identified as an interesting surface marker to be used for the therapy of glioblastoma. In this study, we investigated whether a synthetic antagonist of cell-surface nucleolin known as N6L, previously reported to decrease both tumour growth and tumour angiogenesis in several cancer cell lines, including glioblastoma cells, as well as endothelial cells proliferation, could be exploited to deliver a protein toxin (saporin) to glioblastoma cells. The pseudopeptide N6L cross-linked to saporin-S6 induced internalization of the toxin inside glioblastoma cancer cells. Our results in vitro demonstrated the effectiveness of this conjugate in inducing cell death, with an ID50 four orders of magnitude lower than that observed for free N6L. Furthermore, the preliminary "in vivo" study demonstrated efficiency in reducing the tumour mass in an orthotopic mouse model of glioblastoma

Novel two-dimensional films and three-dimensional (3D) scaffolds based on chitosan (CHI), apatite (Ap), and graphene oxide (GO) were developed by an in situ synthesis in which self-assembly process was conducted to direct partial reduction of GO by CHI in acidic medium. Physical-chemical characterization was carried out by optical microscopy, scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy. In vitro biological studies using murine fibroblast (MC3T3) and human neuroblastoma (SH-SY5Y) cell lines were also performed. Cell growth and adherence on composites was also checked using SEM. Live and death staining by confocal microscope and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium of the samples were investigated. The results confirmed the incorporation of both Ap and GO sheets, into CHI polymeric matrix. Furthermore, it was confirmed a physical integration between inorganic Ap and organic CHI and strong chemical interaction between CHI and GO in the obtained composites. SH-SY5Y cell line showed preferential adherence on CHI/GO films surface while MC3T3 cell line displayed a good compatibility for all 3D scaffolds. This study confirms the biocompatibility of materials based on CHI, Ap, and GO for future tissues applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1585-1594, 2018.

The first function attributed to lactoferrin, an iron-binding protein belonging to the non-immune natural de- fences, was antimicrobial activity related to its capability of sequestering iron. Many other antimicrobial and antiviral functions have been ascribed to lactoferrin. In vitro activity towards human pathogenic fungi on the part of both human and bovine lactoferrin has been well documented as well. The antifungal activity appears to be related to lactoferrin inter- ference with the fungal cell surface rather than iron deprivation and some host-mediated mechanisms of action cannot be ruled out. Lactoferrin also displays anti-parasitic activity, although the molecular mechanisms of such activity are even more complex.

Neuroblastoma is a solid tumour affecting the peripheral nervous system accounting for ∼13% of all pediatric cancer mortality. Despite immunotherapy with the GD2 antibody has improved the clinical outcome of a fraction of patients with metastatic neuroblastoma, the majority of patients with relapsing, high risk disease cannot be successfully treated. Recently, it has been shown that non-internalizing, linker-less antibody drug conjugates (ADCs) targeting the tumor extracellular environment can exert a potent therapeutic activity against different tumors. We identified LGALS3BP (aka Mac-2 BP or 90K) as an important target for non-internalizing ADC development, as the protein is largely secreted by the majority of human tumors, while being virtually undetectable in normal adult tissues. In this study, we show that a variant of the humanized 1959 antibody, targeting human LGALS3BP, coupled to the maytansinoid drugs DM3 by means of disulfide linker, bound human neuroblastoma cell lines harboring or not N-myc amplification, although with different affinity. Importantly, normal human fibroblasts were not decorated by the antibody. The ADC potently inhibited metastatic lesions in the liver and lungs of NSG mice injected intravenously with the neuroblastoma cell line SKNAS. Our findings offer a preclinical proof-of-concept for the development of a non-internalizing ADC endowed with potent therapeutic activity for neuroblastoma treatment through an innovative mechanism of action. Citation Format: Emily Capone, Sara Ponziani, Francesco Giansanti, Roberta Gentile, Giulia Di Vittorio, Vincenzo De Laurenzi, Michele Sallese, Sandra Bibbò, Arturo Sala, Rodolfo Ippoliti, Jean Frederic Sauniere, Stefano Iacobelli, Gianluca Sala. Therapeutic activity of the non-internalizing antibody drug conjugate 1959-sss/DM3 targeting galectin3-binding protein in human neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 238.

Mammals posses both serum transferrin and lactoferrin, whose functions are taken over in birds by ovotransferrin, displaying both iron transport and antibacterial activities. Ovotransferrin also exerts antiviral activity towards Marek’s disease virus, an avian member of the herpes family of viruses. This virus infects lymphoid organs and induces the transcription of ovotransferrin in infected chicken embryo fibroblasts. However, it has not yet been established whether ovotransferrin gene transcription is linked to the release of the protein outside the cells or whether ovotransferrin expression and release also occurs in chicken lymphoblastoid cells in which the Marek’s disease viral genome is integrated. Our results indicate that both serum and egg-white isoforms of ovotransferrin are expressed and released in the supernatants of chicken embryo fibroblast and lymphoblastoid cells in the absence of infection. Viral infection of chicken embryo fibroblasts caused a slight increase of ovotransferrin release, whereas viral reinfection of lymphoblastoid cells caused a remarkable ovotransferrin release in a virus concentration-dependent manner. These findings suggest that ovotransferrin release in vivo may play a crucial role in protecting the whole organism from viral infection spreading, and support the hypothesis that the antiviral activity of ovotransferrin is an important part of the innate immune response in birds, resembling the antiviral activity of lactoferrin in mammals.

""Abstract: Lactoferrin is an iron binding protein belonging to the transferrin superfamily. Its main function is the protection. towards infections. In a previous article (Leboffe et al., Anti-Infective Agents in Medicinal Chemistry, 2009), we. have reviewed the antifungal and antiparasitic activity of lactoferrin. In the present updated review we focused the attention. on the newly discovered antifungal properties of lactoferrin and its derived peptides in clinical, diagnostic and biotechnological. applications. In particular, we discuss the new findings on diagnosis of fungal infections utilizing Lf and on. emerging pharmacological treatments exploiting combined effects of Lf and classical antifungal drugs. We also reported. recent studies on the use of Lf antifungal activity for food and beverage preservation. These new properties have found. applications both “in vitro” and “in vivo”, opening new scenarios about the use of this protein as an antinfective agent. both in translational medicine and biotechnology""

Proteins are hetero-polymers made-up of single building blocks (aminoacids) whose composition determines folding and final architecture. Some proteins are able to undergo self-assembly process enabling the formation of ordered molecular aggregates that in some cases assume conformations particularly suitable to nanotechnological applications. In this work we describe the properties of a ring-like decameric protein, Peroxiredoxin (Prx), to build composite materials interacting with or catalyzing the formation of selectively metal nanoparticles that can be trapped over the surface of nanostructured graphene oxide (GO) sheets. We demonstrate furthermore the ability of Prx to guide the formation of 3D layers of GO embedding metal nanoparticles in the composite material. These composites are discussed as possible precursors to electronic and chemical devices.

Graphene oxide (GO) is rapidly emerging worldwide as a breakthrough precursor material for next-generation devices. However, this requires the transition of its two-dimensional layered structure into more accessible three-dimensional (3D) arrays. Peroxiredoxins (Prx) are a family of multitasking redox enzymes, self-assembling into ring-like architectures. Taking advantage of both their symmetric structure and function, 3D reduced GO-based composites are hereby built up. Results reveal that the "double-faced" Prx rings can adhere flat on single GO layers and partially reduce them by their sulfur-containing amino acids, driving their stacking into 3D multi-layer reduced GO-Prx composites. This process occurs in aqueous solution at a very low GO concentration, i.e. 0.2 mg ml(-1). Further, protein engineering allows the Prx ring to be enriched with metal binding sites inside its lumen. This feature is exploited to both capture presynthesized gold nanoparticles and grow in situ palladium nanoparticles paving the way to straightforward and "green" routes to 3D reduced GO-metal composite materials.

Antibody-Drug Conjugates (ADCs) are highly potent biopharmaceuticals that use the targeting ability of monoclonal antibodies to selectively bind to tumor cells where the conjugated cancer drug is released. Traditionally, the anticancer efficacy of ADCs relies on their selective internalization by cancer cells. However, it has recently been shown that non-internalizing, linkerless ADCs targeting the tumor extracellular environment can display a potent therapeutic activity. LGALS3BP (aka Mac-2 BP or 90K) represents an appealing target for non-internalizing ADC development, as the protein is largely secreted by the majority of human tumors, while being virtually undetectable in normal adult tissues except for the prostate and the colorectum. Additionally, once secreted the protein stops from spreading and clusters on the plasma membrane where it binds to some endogenous ligands. Here, we show that an engineered variant of the humanized 1959 antibody to human LGALS3BP selectively localizes around the tumor cells and once site-specifically coupled to the maytansinoid drugs DM3 or DM4 by means of disulfide linker can display a potent therapeutic activity. Both the 1959sss-DM3 and -DM4 conjugates mediated potent antitumor activity in mice bearing human tumor xenografts. Quantitative biodistribution studies confirmed a preferential accumulation of 1959sss-DM3 and -DM4 conjugates around the tumor cells. Notably, we found that both conjugates were well tolerated and cured 100% of tumor-bearing mice. Our findings offer a preclinical proof-of-concept for curative non-internalizing ADC endowed with an innovative mechanism of action. Citation Format: Gianluca Sala, Emily Capone, Enza Piccolo, Sara Ponziani, Roberta Gentile, Rodolfo Ippoliti, Francesco Giansanti, Stefano Iacobelli. Non-internalizing site-specific antibody-drug conjugates based on maytansinoids display curative properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 748.

A critical problem in studying ribosome-inactivating proteins (RIPs) lies in the very limited possibility to produce them in heterologous systems. In fact, their inherent toxicity for the producing organism nearly always prevents their recombinant expression. In this study, we designed, expressed and characterized an engineered form of the RIP saporin (SapVSAV), bearing a C-terminal extra sequence that is recognized and bound by the second PDZ domain from murine PTP-BL protein (PDZ2). The co-expression of SapVSAV and PDZ2 in Escherichia coli BL21 cells greatly enhances the production of the toxin in a soluble form. The increase of production was surprisingly not due to protection from bacterial intoxication, but may arise from a stabilization effect of PDZ2 on the toxin molecule during biosynthesis. We found that once purified, SapVSAV is stable but is not toxic to free ribosomes, while it is fully active against human cancer cells. This strategy of co-expression of a toxin moiety and a soluble PDZ domain may represent a new system to increase the production of recombinant toxic proteins and could allow the selection of new extra sequences to target PDZ domains inside specific mammalian cellular domains.

The use of nanoscale materials in the design of scaffolds for CNS tissue is increasing, due to their ability to promote cell adhesion, to mimic an extracellular matrix microenvironment and to interact with neuronal membranes. In this framework, one of the major challenges when using undifferentiated neural cells is how to control the differentiation process. Here we report the characterization of a scaffold based on the self-assembled nanotubes of a mutant of the protein peroxiredoxin (from Schistosoma mansoni or Bos taurus), which allows the growth and differentiation of a model neuronal cell line (SHSY5Y). The results obtained demonstrate that SHSY5Y cells grow without any sign of toxicity and develop a neuronal phenotype, as shown by the expression of neuronal differentiation markers, without the use of any differentiation supplement, even in the presence of serum. The prodifferentiation effect is demonstrated to be dependent on the formation of the protein nanotube, since a wild-type (WT) form of the peroxiredoxin from Schistosoma mansoni does not induce any differentiation. The protein scaffold was also able to induce the spread of glioblastoma cancer stem cells growing in neurospheres and allowing the acquisition of a neuron-like morphology, as well as of immature rat cortical neurons. This protein used here as coating agent may be suggested for the development of scaffolds for tissue regeneration or anti-tumour devices. Copyright © 2016 John WileySons, Ltd.

In recent years the knowledge of avian cytokines has advanced and new data are continuously added. Nevertheless, some discontinuities persist and the correlations between molecular and functional levels are not completely clear. Most of the studies are focused on chicken, and comparative aspects with other avian groups are limited. The existence of T1 and T3 avian cytokines was assessed long ago and the recent relevant demonstration of the existence of T2 cytokines in birds is a further step in depicting a more complete view on avian immunology. The progressive knowledge of avian cytokines can hopefully help in developing new strategies in prophylaxis and therapy of avian diseases, not always completely controlled due to the emergence of more pathogenic strains.

1.1 Marek’s disease virus (MDV) Marek’s disease virus (MDV) belongs to the alphaherpesvirus family, like Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) and varicella zoster virus (VZV). Marek’s Disease Virus (MDV) is the etiologic agent of Marek’s Disease (MD), a highly contagious malignant lymphoma of chickens. (Morimura, et al. 1998). Marek’s Disease (MD) was first described by Jozsef Marek in 1907 as fowl paralysis caused by mononuclear infiltration into the sciatic nerve plexi (Marek, 1907). Marek’s Disease (MD) is a lymphoproliferative and neuropatic disease of domestic chickens and, less commonly, turkeys and quails (Payne & Venugopal, 2000). Generally, four different clinical forms of the disease are recognized in flocks infected with MDV: A) Classical or neuronal form; B) Acute form; C) Transient paralysis and D) Acute mortality syndrome.

The HER-3 receptor is emerging as an attractive molecule in therapeutics because of its overexpression across many human cancers and because of its role in in several compensatory processes that underlay emergence of resistance to certain cancer drugs. In this study, we present evidence of the preclinical efficacy of a novel Antibody-Drug Conjugate (ADC) targeting HER-3. It consists of a humanized HER-3 monoclonal antibody (mAb EV20), which recognizes the HER-3 extracellular domain, conjugated to different payloads (HER-3-ADC)s. In HER-3 expressing cancer cell lines, these HER-3-ADCs demonstrated a powerful, specific and target-dependent killing activity. High expression levels of HER-3 in tumor cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro. Efficacy studies demonstrated that HER-3-ADCs treatment leads to a long lasting tumor growth inhibition of cell line-based models of human head and neck, breast, pancreatic, prostatic, lung, stomach cancers and melanoma. Overall, these findings validate HER-3 as an attractive therapeutic target in multiple solid tumors and support further clinical development and application of HER-3 targeting ADCs. Citation Format: Gianluca Sala, Manuela Iezzi, Alessia Lamolinara, Emily Capone, Stefano Iacobelli, Jean-Fred Sauniere, Sara Ponziani, Francesco Giansanti, Rodolfo Ippoliti. An Antibody Drug Conjugate targeting HER-3 demonstrates promising antitumor efficacy in a wide range of human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 41. doi:10.1158/1538-7445.AM2017-41

Nanomanipulation of matter to create responsive, ordered materials still remains extremely challenging. Supramolecular chemistry has inspired new strategies by which such nanomaterials can be synthesized step by step by exploiting the self-recognition properties of molecules. In this work, the ring-shaped architecture of the 2-Cys peroxiredoxin I protein from Schistosoma mansoni, engineered to have metal ion-binding sites, is used as a template to build up 1D nanoscopic structures through metal-induced self-assembly. Chromatographic and microscopic analyses demonstrate the ability of the protein rings to stack directionally upon interaction with divalent metal ions and form well-defined nanotubes by exploiting the intrinsic recognition properties of the ring surfaces. Taking advantage of such behavior, the rings are then used to capture colloidal Ni(2+)-functionalized ultrasmall gold nanoparticles and arrange them into 1D arrays through stacking into peapod-like complexes. Finally, as the formation of such nano-peapods strictly depends on nanoparticle dimensions, the peroxiredoxin template is used as a colloidal cut-off device to sort by size the encapsulated nanoparticles. These results open up possibilities in developing Prx-based methods to synthesize new advanced functional materials.

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.

Ovotransferrin and lactoferrin are iron-binding proteins with antiviral and antibacterial activities related to natural immunity, showing marked sequence and structural homologies. The antiviral activity of two hen ovotransferrin fragments DQKDEYELL (hOtrf219–227) and KDLLFK (hOtrf269–301 and hOtrf633–638) towards Marek’s disease virus infection of chicken embryo fibroblasts is reported here. These fragments have sequence homology with two bovine lactoferrin fragments with antiviral activity towards herpes simplex virus, suggesting that these fragments could have a role for the exploitation of the antiviral activity of the intact proteins towards herpes viruses. NMR analysis showed that these peptides, chemically synthetized, did not possess any favourite conformation in solution, indicating that both the aminoacid sequence and the conformation they display in the intact protein are essential for the antiviral activity.

Bovine lactoferrin catalyzes the hydrolysis of synthetic substrates (i.e., Z-aminoacyl-7-amido-4-methylcoumarin). Values of Km and kcat for the bovine lactoferrin catalyzed hydrolysis of Z-Phe-Arg-7-amido-4-methylcoumarin are 50 microM and 0.03 s(-1), respectively, the optimum pH value is 7.5 at 25 degrees C. The bovine lactoferrin substrate specificity is similar to that of trypsin, while the hydrolysis rate is several orders of magnitude lower than that of trypsin. The bovine lactoferrin catalytic activity is irreversibly inhibited by the serine-protease inhibitors PMSF and Pefabloc. Moreover, both iron-saturation of the protein and LPS addition strongly inhibit the bovine lactoferrin activity. Interestingly, bovine lactoferrin undergoes partial auto-proteolytic cleavage at positions Arg415-Lys416 and Lys440-Lys441. pKa shift calculations indicate that several Ser residues of bovine lactoferrin display the high nucleophilicity required to potentially catalyze substrate cleavage. However, a definitive identification of the active site awaits further studies.

Ovotransferrin (formerly conalbumin) is an iron-binding protein present in birds. It belongs to the transferrin family and shows about 50% sequence homology with mammalian serum transferrin and lactoferrin. This protein has been demonstrated to be capable of delivering iron to cells and of inhibiting bacterial multiplication. However, no antiviral activity has been reported for ovotransferrin, although the antiviral activity of human and bovine lactoferrins against several viruses, including human herpes simplex viruses, has been well established. In this report, the antiviral activity of ovotransferrin towards chicken embryo fibroblast infection by Marek's disease virus (MDV), an avian herpesvirus, was clearly demonstrated. Ovotransferrin was more effective than human and bovine lactoferrins in inhibiting MDV infection and no correlation between antiviral efficacy and iron saturation was found. The observations reported here are of interest from an evolutionary point of view since it is likely that the defensive properties of transferrins appeared early in evolution. In birds, the defensive properties of ovotransferrin remained joined to iron transport functions; in mammals, iron transport functions became peculiar to serum transferrin, and the defensive properties towards infections were optimised in lactoferrin.Key words: ovotransferrin, lactoferrin, Marek disease's virus, herpes simplex virus, evolution.

Background Ovotransferrin is an iron-binding glycoprotein, found in avian egg white and in avian serum, belonging to the family of transferrin iron-binding glycoproteins. All transferrins show high sequence homology. In mammals are presents two different soluble glycoproteins with different functions: i) serum transferrin that is present in plasma and committed to iron transport and iron delivery to cells and ii) lactoferrin that is present in extracellular fluids and in specific granules of polymorphonuclear lymphocytes and committed to the so-called natural immunity. To the contrary, in birds, ovotransferrin remained the only soluble glycoprotein of the transferrin family present both in plasma and egg white. Scope of review Substantial experimental evidences are summarized, illustrating the multiple physiological roles of ovotransferrin in an attempt to overcome the common belief that ovotransferrin is a protein dedicated only to iron transport and to iron withholding antibacterial activity. Major conclusions Similarly to the better known family member protein lactoferrin, ovotransferrin appears to be a multi-functional protein with a major role in avian natural immunity. General significance Biotechnological applications of ovotransferrin and ovotransferrin-related peptides could be considered in the near future, stimulating further research on this remarkable protein. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults, with a median survival of ~12-18 months post-diagnosis. GBM usually recurs within 12 months post-resection, with poor prognosis. Thus, novel therapeutic strategies to target and kill GBM cells are urgently needed. The marked difference of tumour cells with respect to normal brain cells renders glioblastoma a good candidate for selective targeted therapies. Recent experimental strategies focus on over expressed cell surface receptors. Targeted toxins represent a new class of selective molecules composed by a potent protein toxin and a carrier ligand. Targeted toxins approaches against glioblastoma were under investigation in phase I and II clinical trials with several immunotoxins (IT)/ligand toxins such as IL4-Pseudomonas aeruginosa exotoxin A (IL4-PE, NBI-3001), tumour growth factor fused to PE38, a shorter PE variant, (TGF)alpha-TP-38, IL13-PE38, and a transferrin-C diphtheriae toxin mutant (Tf-CRM107). In this work, we studied the effects of the plant ribosome-inactivating saporin and of its chimera transferrin-saporin against two different GBM cell lines. The data obtained here indicate that cell proliferation is affected by the toxin treatments but that different mechanisms are used, directly linked to the presence of an active or inactive p53. A model is proposed for these alternative intracellular pathways.

N-glycosylation is one of the major naturally occurring covalent co-translational modifications of proteins in plants, being involved in proteins structure, folding, stability and biological activity. In the present work the influence of carbohydrate moieties on the structure-function relationships of type 1 ribosome-inactivating proteins (RIPs) was investigated. To this aim, PD-Ls, RIPs isolated from Phytolacca dioica L. leaves, differing for their glycosylation degree, were used as an experimental system. In particular, comparative structural and biological analyses were performed using native and unglycosylated recombinant PD-L1, the most glycosylated P. dioica RIP isoform. The glycans influence on protein synthesis inhibition and adenine polynucleotide glycosidase activity was investigated. The interaction with adenine, the product of the de-adenylation reaction, was also investigated for native and recombinant PD-L1 by fluorescence spectroscopy. Furthermore, the crystal structure of PD-L1 in complex with adenine was determined. Our data confirm that the absence of glycan moieties did not affect the biological activity in terms of protein synthesis inhibition. However, the removal of carbohydrate chains significantly increased the deadenylation capability, likely as a consequence of the increased accessibility of substrates to the active site pocket. Furthermore, preliminary data on cellular uptake showed that all PD-L isoforms were internalized and, for the first time, that the vesicular distribution within cells could be influenced by the protein glycosylation degree. © 2010 The Royal Society of Chemistry.

Many plants produce enzymes with N-glycosidase activity, also known as Ribosome Inactivating Proteins. These proteins remove a specific adenine residue from the ribosomal RNA (28S in eukaryotes) inducing the block of pro- tein synthesis by inhibiting the binding of the Elongation Factor 2. Both eukaryotic and prokaryotic ribosomes (with dif- ferent sensitivity) can irreversibly be damaged by the action of these enzymes, suggesting their use as cytotoxic drugs. In fact several applications of targeted N-glycosidases have been developed (i.e. immunotoxins) for the treatment of human diseases such as leukaemia, but biotechnological development has furthermore suggested new applications of targeted N- glycosidases (i.e. Ig192-saporin) that are now used as powerful tools for cell biology research. The high number of en- zymes available and the possibility to express these proteins as recombinant products, allow to predict new formulations and applications discussed in this paper starting from the example of the model toxins ricin and saporin.

Marek's disease virus (MDV), the causative agent of Marek's disease (MD), is a herpesvirus that infects poultry causing T lymphomas. Although vaccination may prevent lymphomas formation, it is not known whether it controls viral replication and spreading in the environment. Ovotransferrin (Otrf), a member of the transferrin family, is known to exert in vitro antiviral activity in primary cultures of chicken embryo fibroblasts (CEF). In addition Otrf is produced by CEF and by an avian lymphoblastoid cell line (MDCC-MSB1) following infection/reinfection with MDV. The present work was designed to investigate the effects of reinfection and of Otrf and lactoferrin (Lf) on the production by MDCC-MSB1 of nitric oxide (NO), a molecule naturally exerting an antiviral activity. These effects were also tested with two cytokines (IL-8 and IFN-gamma), alone and in association with transferrins. Synergy was found between Otrf and IFN-gamma, thus suggesting a possible role in a complementary or alternative strategy against MDV spreading.

Group A streptococci (GAS) are able to invade cultured epithelial and endothelial cells without evidence of intracellular replication. GAS, like other facultative intracellular bacterial pathogens, evolved such ability to enter and to survive within host cells avoiding the host defences, and bacterial intracellular survival could explain the recurrence of infections. We report here that 1 mg bovine lactoferrin (bLf)/mL significantly hindered the in vitro invasion of cultured epithelial cells by GAS isolated from patients suffering from pharyngitis and completely inhibited the invasiveness of GAS pretreated with subinhibiting concentrations of erythromycin or ampicillin. One milligram of bLf per millilitre was also able to increase the number of epithelial cells undergoing apoptosis following GAS invasion, although the number of intracellular GAS in the presence of bLf decreased by about 10-fold. The ability of bLf to decrease GAS invasion was confirmed by an in vivo trial carried out on 12 children suffering from pharyngitis and already scheduled for tonsillectomy. In tonsil specimens from children treated for 15 days before tonsillectomy with both oral erythromycin (500 mg t.i.d. (three times daily)) and bLf gargles (100 mg t.i.d.), a lower number of intracellular GAS was found in comparison with that retrieved in tonsil specimens from children treated with erythromycin alone (500 mg t.i.d.).Key words: lactoferrin, group A streptococci, invasiveness, anti-invasive activity, apoptosis.

methods must be used to screen for the desired activity. A series of microorganisms have been isolated from sediment of olives maintained in sun-flower oil and tested for lipase production. Specific agar medium containing cattle suet has been used in a preliminary phase. The strains which presented lipolytic activity have later on been cultivated on specific liquid medium and enzymatic activity has been determined. Lipase production yields ranged between12and58ULFIP/mL, comparable to other reported results. The enzymes produced by the strains which developed enhanced lipolytic activity are to be used later in transesterification reactions, including biochemical obtainment of biodiesel. Acknowledgements: The researches performed were developed in the framework of the Romanian POSDRU/107/1.5/S/76888 programme.