The effects of histidine substitution of aromatic residues on the amyloidogenic properties of the fragment 264-277 of nucleophosmin 1.

Histidine (His) plays a key role in mediating protein interactions and its unique side chain determines pH responsive self-assembling processes and thus in the formation of nanostructures. In this study, To identify novel self-assembling bioinspired sequences, we analyzed a series of peptide sequences obtained through the point mutation of aromatic residues of 264-277 fragment of nucleophosmin 1 (NPM1) with single and double histidines. Through several orthogonal biophysical techniques and under different pH and ionic strength conditions we evaluated the effects of these substitutions in the amyloidogenic features of derived peptides. The results clearly indicate that both the type of aromatic mutated residue and its position can have different effect on amyloid-like behaviors. They corroborate the crucial role exerted by Tyr271 in the self-assembling process of CTD of NPM1 in AML mutated form and add novel insights in the accurate investigation of how side chain orientations can determine successful design of innovative bioinspired materials.

Ruthenium complexes bearing glucosyl ligands are able to inhibit the amyloid aggregation of short histidine-peptides.

Neurodegenerative diseases are often characterized by the formation of aggregates of amyloidogenic peptides and proteins, facilitating the formation of neurofibrillary plaques. In this study, we investigate a series of Ru-complexes sharing three-legged piano-stool structures based on the arene ring and glucosylated carbene ligands. The ability of these complexes to bind amyloid His-peptides was evaluated by ESI-MS, and their effects on the aggregation process were investigated through ThT and Tyr fluorescence emission. The complexes were demonstrated to bind the amyloidogenic peptides even with different mechanisms and kinetics depending on the chemical nature of the ligands around the Ru(II) ion. TEM analysis detected the disaggregation of typical fibers caused by the presence of Ru-compounds. Overall, our results show that the Ru-complexes can modulate the aggregation of His-amyloids and can be conceived as good lead compounds in the field of novel anti-aggregating agents in neurodegeneration.

PLGA microparticle formulations for tunable delivery of a nano-engineered filamentous bacteriophage-based vaccine: in vitro and in silico-supported approach.

Bacteriophages have attracted great attention in the bioengineering field in diverse research areas from tissue engineering to therapeutic and clinical applications. Recombinant filamentous bacteriophage, carrying multiple copies of foreign peptides on protein capsid has been successfully used in the vaccine delivery setting, even if their plasma instability and degradation have limited their use on the pharmaceutical market. Encapsulation techniques in polymeric materials can be applied to preserve bacteriophage activity, extend its half-life, and finely regulate their release in the target environment. The main goal of this study was to provide tunable formulations of the bacteriophage encapsulated in polymeric microparticles (MPs). We used poly (lactic-co-glycolic-acid) as a biocompatible and biodegradable polymer with ammonium bicarbonate as a porogen to encapsulate bacteriophage expressing OVA (257-264) antigenic peptide. We demonstrate that nano-engineered fdOVA bacteriophages encapsulated in MPs preserve their structure and are immunologically active, inducing a strong immune response towards the delivered peptide. Moreover, MP encapsulation prolongs bacteriophage stability over time also at room temperature. Additionally, in this study, we show the ability of in silico-supported approach to predict and tune the release of bacteriophages. These results lay the framework for a versatile bacteriophage-based vaccine delivery system that could successfully generate robust immune responses in a sustained manner, to be used as a platform against cancer and new emerging diseases. Graphical abstract: Synopsis: administration of recombinant bacteriophage-loaded PLGA microparticles for antigen delivery. PLGA microparticles release the bacteriophages, inducing activation of dendritic cells and enhancing antigen presentation and specific T cell response. Bacteriophage-encapsulated microneedles potentially can be administered into human body and generate robust immune responses.

Modulation of hydrogel networks by metal ions.

Self-assembling hydrogels are receiving great attention for both biomedical and technological applications. Self-assembly of protein/peptides as well as organic molecules is commonly induced in response to external triggers such as changes of temperature, concentration, or pH. An interesting strategy to modulate the morphology and mechanical properties of the gels implies the use of metal ions, where coordination bonds regulate the dynamic cross-linking in the construction of hydrogels, and coordination geometries, catalytic, and redox properties of metal ions play crucial roles. This review aims to discuss recent insights into the supramolecular assembly of hydrogels involving metal ions, with a focus on self-assembling peptides, as well as applications of metallogels in biomedical fields including tissue engineering, sensing, wound healing, and drug delivery.

Insights into Network of Hot Spots of Aggregation in Nucleophosmin 1.

In a protein, point mutations associated with diseases can alter the native structure and provide loss or alteration of functional levels, and an internal structural network defines the connectivity among domains, as well as aggregate/soluble states' equilibria. Nucleophosmin (NPM)1 is an abundant nucleolar protein, which becomes mutated in acute myeloid leukemia (AML) patients. NPM1-dependent leukemogenesis, which leads to its aggregation in the cytoplasm (NPMc+), is still obscure, but the investigations have outlined a direct link between AML mutations and amyloid aggregation. Protein aggregation can be due to the cooperation among several hot spots located within the aggregation-prone regions (APR), often predictable with bioinformatic tools. In the present study, we investigated potential APRs in the entire NPM1 not yet investigated. On the basis of bioinformatic predictions and experimental structures, we designed several protein fragments and analyzed them through typical aggrsegation experiments, such as Thioflavin T (ThT), fluorescence and scanning electron microscopy (SEM) experiments, carried out at different times; in addition, their biocompatibility in SHSY5 cells was also evaluated. The presented data clearly demonstrate the existence of hot spots of aggregation located in different regions, mostly in the N-terminal domain (NTD) of the entire NPM1 protein, and provide a more comprehensive view of the molecular details potentially at the basis of NPMc+-dependent AML.

Hydrogelation tunability of bioinspired short peptides.

Supramolecular assemblies of short peptides are experiencing a stimulating flowering. Herein, we report a novel class of bioinspired pentapeptides, not bearing Phe, that form hydrogels with fibrillar structures. The inherent sequence comes from the fragment 269-273 of nucleophosmin 1 protein, that is normally involved in liquid-liquid phase separation processes into the nucleolus. By means of rheology, spectroscopy, and scanning microscopy the crucial roles of the extremities in the modulation of the mechanical properties of hydrogels were elucidated. Three of four peptide showed a typical shear-thinning profile and a self-assembly into hierarchical nanostructures fibers and two of them resulted biocompatible in MCF7 cells. The presence of an amide group at C-terminal extremity caused the fastest aggregation and the major content of structured intermediates during gelling process. The tunable mechanical and structural features of this class of hydrogels render derived supramolecular systems versatile and suitable for future biomedical applications.

Type C mutation of nucleophosmin 1 acute myeloid leukemia: Consequences of intrinsic disorder.

BACKGROUND: Nucleophosmin 1 (NPM1) protein is a multifunctional nucleolar chaperone and its gene is the most frequently mutated in Acute Myeloid Leukemia (AML). AML mutations cause the unfolding of the C-terminal domain (CTD) and the protein delocalizing in the cytosol (NPM1c+). Marked aggregation endowed with an amyloid character was assessed as consequences of mutations. SCOPE: Herein we analyzed the effects of type C mutation on two protein regions: i) a N-terminal extended version of the CTD, named Cterm_mutC and ii) a shorter polypeptide including the sequences of the second and third helices of the CTD, named H2_mutC. MAJOR CONCLUSIONS: Both demonstrated able to self-assembly with different kinetics and conformational intermediates and to provide fibers presenting large flexible regions. GENERAL SIGNIFICANCE: The present study adds a new piece of knowledge to the effects of AML-mutations on structural biology of Nucleophosmin 1, that could be exploited in therapeutic interventions targeting selectively NPMc+.

Targeting Ship2-Sam with peptide ligands: Novel insights from a multidisciplinary approach.

The lipid phosphatase Ship2 binds the EphA2 receptor through a heterotypic Sam-Sam (Sterile alpha motif) interaction. Inhibitors of the Ship2-Sam/EphA2-Sam complex hold a certain potential as novel anticancer agents. The previously reported "KRI3" peptide binds Ship2-Sam working as a weak antagonist of the EphA2-Sam/Ship2-Sam interaction. Herein, the design and functional evaluation of KRI3 analogues, both linear and cyclic, are described. A multidisciplinary study was conducted through computational docking techniques, and conformational analyses by CD and NMR spectroscopies. The ability of new peptides to bind Ship2-Sam was analysed by NMR, MST and SPR assays. Studies on linear KRI3 analogues pointed out that aromatic interactions through tyrosines are important for the association with Ship2-Sam whereas, an increase of the net positive charge of the sequence or peptide cyclization through a disulfide bridge can favour unspecific interactions without a substantial improvement of the binding affinity to Ship2-Sam. Interestingly, preliminary cell-based assays demonstrated KRI3 cellular uptake even without the conjugation to a cell penetrating sequence with a main cytosolic localization. This work highlights important features of the KRI3 peptide that can be further exploited to design analogues able to hamper Sam-Sam interactions driven by electrostatic contacts.

Engineered Bacterial Cellulose Nanostructured Matrix for Incubation and Release of Drug-Loaded Oil in Water Nanoemulsion.

Bacterial cellulose (BC) is a highly pure form of cellulose produced by bacteria, which possesses numerous advantages such as good mechanical properties, high chemical flexibility, and the ability to assemble in nanostructures. Thanks to these features, it achieved a key role in the biomedical field and in drug delivery applications. BC showed its ability to modulate the release of several drugs and biomolecules to the skin, thus improving their clinical outcomes. This work displays the loading of a 3D BC nanonetwork with an innovative drug delivery nanoemulsion system. BC was optimized by static culture of SCOBY (symbiotic colony of bacteria and yeast) and characterized by morphological and ultrastructural analyses, which indicate a cellulose fiber diameter range of 30-50 nm. BC layers were then incubated at different time points with a nanocarrier based on a secondary nanoemulsion (SNE) previously loaded with a well-known antioxidant and anti-inflammatory agent, namely, coenzyme-Q10 (Co-Q10). Incubation of Co-Q10-SNE in the BC nanonetwork and its release were analyzed by fluorescence spectroscopy.

Self-Assembling Peptides: From Design to Biomedical Applications.

Self-assembling peptides could be considered a novel class of agents able to harvest an array of micro/nanostructures that are highly attractive in the biomedical field. By modifying their amino acid composition, it is possible to mime several biological functions; when assembled in micro/nanostructures, they can be used for a variety of purposes such as tissue regeneration and engineering or drug delivery to improve drug release and/or stability and to reduce side effects. Other significant advantages of self-assembled peptides involve their biocompatibility and their ability to efficiently target molecular recognition sites. Due to their intrinsic characteristics, self-assembled peptide micro/nanostructures are capable to load both hydrophobic and hydrophilic drugs, and they are suitable to achieve a triggered drug delivery at disease sites by inserting in their structure's stimuli-responsive moieties. The focus of this review was to summarize the most recent and significant studies on self-assembled peptides with an emphasis on their application in the biomedical field.

Type F mutation of nucleophosmin 1 Acute Myeloid Leukemia: A tale of disorder and aggregation.

Protein aggregation is suggested as a reversible, wide-spread physiological process used by cells to regulate their growth and adapt to different stress conditions. Nucleophosmin 1(NPM1) protein is an abundant multifunctional nucleolar chaperone and its gene is the most frequently mutated in Acute Myeloid Leukemia (AML) patients. So far, the role of NPM1 mutations in leukemogenesis has remained largely elusive considering that they have the double effect of unfolding the C-terminal domain (CTD) and delocalizing the protein in the cytosol (NPM1c+). This mislocalization heavily impacts on cell cycle regulation. Our recent investigations unequivocally demonstrated an amyloid aggregation propensity introduced by AML mutations. Herein, employing complementary biophysical assays, we have characterized a N-terminal extended version of type F AML mutation of CTD and proved that it is able to form assemblies with amyloid character and fibrillar morphology. The present study represents an additional phase of knowledge to deepen the roles exerted by different types of cytoplasmatic NPM1c+ forms to develop in the future potential therapeutics for their selective targeting.

Cell Membrane-Coated Oil in Water Nano-Emulsions as Biomimetic Nanocarriers for Lipophilic Compounds Conveyance.

Recently, we developed ultra-stable oil in water nano-emulsions (O/W NEs), able to carry both internal and external cargos (Somes), such as lipophilic compounds and hydrophilic coatings, respectively, that we call here NEsoSomes. O/W NEs are an excellent bioengineering tool for drug and molecules delivery, due to their ability to dissolve a large number of hydrophobic compounds and protect them from hydrolysis and degradation under biological conditions. At present, no report is available on the combination of cell membrane coatings with such nanocarriers, probably due to their typical instability feature. Since then, we have reported, for the first time, a new cell membrane (CM)-coated nanomaterial composed of membranes extracted from glioblastoma cancer cells (U87-MG) deposited on NEsoSomes, through a liquid-liquid interface method, to produce highly controllable membrane caked nano-capsules, namely CM-NEsoSomes. CM-NEsoSomes were physically characterized by dynamic light scattering (DLS) over time and their correct morphology was analyzed by confocal and transmission electron microscopy (TEM) microscopy. Moreover, CM-NEsoSomes biocompatibility was tested on the healthy model cell line, performing cell cytotoxicity and uptake assay, showing nanocarriers uptake by cells with no induced cytotoxicity.

Easy Surface Functionalization and Bioconjugation of Peptides as Capture Agents of a Microfluidic Biosensing Platform for Multiplex Assay in Serum.

The development of assays for protein biomarkers in complex matrices is a demanding task that still needs implementation of new approaches. Antibodies as capture agents have been largely used in bioassays but their low stability, low-efficiency production, and cross-reactivity in multiplex approaches impairs their larger applications. Instead, synthetic peptides, even with higher stability and easily adapted amino acid sequences, still remain largely unexplored in this field. Here, we provide a proof-of-concept of a microfluidic device for direct detection of biomarker overexpression. The multichannel microfluidic polydimethylsiloxane (PDMS) device was first derivatized with PAA (poly(acrylic acid)) solution. CRP-1, VEGF-114, and ΦG6 peptides were preliminarily tested to respectively bind the biomarkers, C-reactive protein (CRP), vascular endothelial growth factor (VEGF), and tumor necrosis factor-alpha (TNF-α). Each PDMS microchannel was then respectively bioconjugated with a specific peptide (CRP-1, VEGF-114, or ΦG6) to specifically capture CRP, VEGF, and TNF-α. With such microdevices, a fluorescence bioassay has been set up with sensitivity in the nanomolar range, both in buffered solution and in human serum. The proposed multiplex assay worked with a low amount of sample (25 µL) and detected biomarker overexpression (above nM concentration), representing a noninvasive and inexpensive screening platform.

Conformational consequences of NPM1 rare mutations: An aggregation perspective in Acute Myeloid Leukemia.

Often proteins association is a physiological process used by cells to regulate their growth and to adapt to different stress conditions, including mutations. In the case of a subtype of Acute Myeloid Leukemia (AML), mutations of nucleophosmin 1 (NPM1) protein cause its aberrant cytoplasmatic mislocalization (NPMc+). We recently pointed out an amyloidogenic propensity of protein regions including the most common mutations of NPMc+ located in the C-terminal domain (CTD): they were able to form, in vitro, amyloid cytotoxic aggregates with fibrillar morphology. Herein, we analyzed the conformational characteristics of several peptides including rare AML mutations of NPMc+. By means of different spectroscopic, microscopic and cellular assays we evaluated the importance of amino acid composition, among rare AML mutations, to determine amyloidogenic propensity. This study could add a piece of knowledge to the structural consequences of mutations in cytoplasmatic NPM1c+.

Effects of surface nanopatterning on internalization and amyloid aggregation of the fragment 264-277 of Nucleophosmin 1.

The mechanical interpretation of the plethora of factors that governs cellular localization of amyloid aggregates is crucial for planning novel therapeutical interventions in neurodegenerative diseases since these aggregates exert a primary role in the proteostasis machinery. The uptake of Cell Penetrating Peptides (CPPs) conjugated with different amyloid polypeptides occurs via different endocytic processes regulated by cytoskeleton organization and cell morphology. Herein, we deepened the internalization of an amyloid system in cells cultured on nanopatterned surfaces that represent a powerful tool to shape cell and regulate its contractility. We analyzed the behavior of an amyloid model system, employing NPM1264-277 sequence, covalently conjugated to Tat fragment 48-60 as CPP. To investigate its internalization mechanism, we followed the formation of aggregates on two kinds of substrates: a flat and a nanopatterned surface. Herein, investigations during time were carried out by employing both confocal and second harmonic generation (SHG) microscopies. We showed that modifications of cellular environment affect peptide localization, its cytoplasmic translocation and the size of amyloid aggregates.

Proteostasis unbalance of nucleophosmin 1 in Acute Myeloid Leukemia: An aggregomic perspective.

The role exerted by the nucleus in the regulation of proteostasis in both health and disease is recognized of outmost importance, even though not fully understood. Many recent investigations are focused on its ability to modulate and coordinate protein quality control machineries in mammalian cells. Nucleophosmin 1 (NPM1) is one of the most abundant nucleolar proteins and its gene is mutated in ~30% of Acute Myeloid Leukemia (AML) patients. Mutations are localized in the C-terminal domain of the protein and cause cytoplasmatically delocalized and possibly aggregated forms of NPM1 (NPM1c+). Therapeutic interventions targeted on NPM1c+ are in demand and, to this end, deeper knowledge of NPM1c+ behavior in the blasts' cytosol is required. Here by means of complementary biophysical techniques we compared the conformational and aggregative behavior of the entire C-terminal domains of NPM1wt and type A NPM1c+ (bearing the most common mutation). Overall data show that only Cterm_mutA is able to form amyloid-like assemblies with fibrillar morphology and that the oligomers are toxic in human neuroblastoma SHSY cells. This study adds a novel piece of knowledge to the comprehension of the molecular roles exerted by cytoplasmatic NPM1c+ and suggests the exploitation of the amyloidogenic propensity of NPM1c+ as a new strategy for targeting AML with NPM1 mutations.

Recombinant Filamentous Bacteriophages Encapsulated in Biodegradable Polymeric Microparticles for Stimulation of Innate and Adaptive Immune Responses.

Escherichia coli filamentous bacteriophages (M13, f1, or fd) have attracted tremendous attention from vaccinologists as a promising immunogenic carrier and vaccine delivery vehicle with vast possible applications in the development of vaccines. The use of fd bacteriophage as an antigen delivery system is based on a modification of bacteriophage display technology. In particular, it is designed to express multiple copies of exogenous peptides (or polypeptides) covalently linked to viral capsid proteins. This study for the first time proposes the use of microparticles (MPs) made of poly (lactic-co-glycolic acid)(PLGA) to encapsulate fd bacteriophage. Bacteriophage-PLGA MPs were synthesized by a water in oil in water (w1/o/w2) emulsion technique, and their morphological properties were analyzed by confocal and scanning electron microscopy (SEM). Moreover, phage integrity, encapsulation efficiency, and release were investigated. Using recombinant bacteriophages expressing the ovalbumin (OVA) antigenic determinant, we demonstrated the immunogenicity of the encapsulated bacteriophage after being released by MPs. Our results reveal that encapsulated bacteriophages are stable and retain their immunogenic properties. Bacteriophage-encapsulated PLGA microparticles may thus represent an important tool for the development of different bacteriophage-based vaccine platforms.

Tunable Release of Curcumin with an In Silico-Supported Approach from Mixtures of Highly Porous PLGA Microparticles.

In recent years, drug delivery systems have become some of the main topics within the biomedical field. In this scenario, polymeric microparticles (MPs) are often used as carriers to improve drug stability and drug pharmacokinetics in agreement with this kind of treatment. To avoid a mere and time-consuming empirical approach for the optimization of the pharmacokinetics of an MP-based formulation, here, we propose a simple predictive in silico-supported approach. As an example, in this study, we report the ability to predict and tune the release of curcumin (CUR), used as a model drug, from a designed combination of different poly(d,l-lactide-co-glycolide) (PLGA) MPs kinds. In detail, all CUR-PLGA MPs were synthesized by double emulsion technique and their chemical-physical properties were characterized by Mastersizer and scanning electron microscopy (SEM). Moreover, for all the MPs, CUR encapsulation efficiency and kinetic release were investigated through the UV-vis spectroscopy. This approach, based on the combination of in silico and experimental methods, could be a promising platform in several biomedical applications such as vaccinations, cancer-treatment, diabetes therapy and so on.

Exploring the Ability of Cyclic Peptides to Target SAM Domains: A Computational and Experimental Study.

Sterile alpha motif (SAM) domains are protein interaction modules with a helical fold. SAM-SAM interactions often adopt the mid-loop (ML)/end-helix (EH) model, in which the C-terminal helix and adjacent loops of one SAM unit (EH site) bind the central regions of another SAM domain (ML site). Herein, an original strategy to attack SAM-SAM associations is reported. It relies on the design of cyclic peptides that target a region of the SAM domain positioned at the bottom side of the EH interface, which is thought to be important for the formation of a SAM-SAM complex. This strategy has been preliminarily tested by using a model system of heterotypic SAM-SAM interactions involving the erythropoietin-producing hepatoma kinase A2 (EphA2) receptor and implementing a multidisciplinary plan made up of computational docking studies, experimental interaction assays (by NMR spectroscopy and surface plasmon resonance techniques) and conformational analysis (by NMR spectroscopy and circular dichroism). This work further highlights how only a specific balance between flexibility and rigidity may be needed to generate modulators of SAM-SAM interactions.

The acute myeloid leukemia-associated Nucleophosmin 1 gene mutations dictate amyloidogenicity of the C-terminal domain.

Nucleophosmin 1 (NPM1) is a nucleus-cytoplasm shuttling protein ubiquitously expressed and highly conserved. It is involved in many cellular processes and its gene is mutated in ~ 50-60% of Acute Myeloid Leukemia (AML) patients. These mutations cause its cytoplasmic mislocation and accumulation (referred to as NPM1c+) and open the door to rational targeted therapy for AML diseases with mutated NPM1. Currently, there is limited knowledge on the mechanism of action of NPM1c+ and on structural determinants of the leukemogenic potential of AML mutations. Numerous previous studies outlined an unexpected amyloid-like aggregation tendency of several regions located in the C-terminal domain that, in wild-type form, fold as a three-helical-bundle. Here, using a combination of different techniques including Thioflavin T fluorescence, congo red absorbance, CD spectroscopy, Scanning Electron Microscopy (SEM) and wide-angle X-ray scattering on a series of peptides bearing mutations, we evidence that the amyloidogenicity of NPM1 mutants is directly linked to AML. Noticeably, AML point mutations strongly affect the amyloid cytotoxic effects in neuroblastoma cells and the morphologies of deriving fibrils. This study paves the way to deepen our understanding of AML-associated NPM1 mutants, and could help to break new ground for the identification of novel drugs targeting NPM1c+ for treatment of AML.