Publications by authors named "Alessandro Arcovito"

59 Publications

Structural determinants driving the binding process between PDZ domain of wild type human PALS1 protein and SLiM sequences of SARS-CoV E proteins.

Comput Struct Biotechnol J 2021 18;19:1838-1847. Epub 2021 Mar 18.

Dipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Roma, Italy.

Short Linear Motifs (SLiMs) are functional protein microdomains that typically mediate interactions between a short linear region in one protein and a globular domain in another. Surface Plasmon Resonance assays have been performed to determine the binding affinity between PDZ domain of wild type human PALS1 protein and tetradecapeptides representing the SLiMs sequences of SARS-CoV-1 and SARS-CoV-2 E proteins (E-SLiMs). SARS-CoV-2 E-SLiM binds to the human target protein with a higher affinity compared to SARS-CoV-1, showing a difference significantly greater than previously reported using the F318W mutant of PALS1 protein and shorter target peptides. Moreover, molecular dynamics simulations have provided clear evidence of the structural determinants driving this binding process. Specifically, the Arginine 69 residue in the SARS-CoV-2 E-SLiM is the key residue able to both enhance the specific polar interaction with negatively charged pockets of the PALS1 PDZ domain and reduce significantly the mobility of the viral peptide. These experimental and computational data are reinforced by the comparison of the interaction between the PALS1 PDZ domain with the natural ligand CRB1, as well as the corresponding E-SLiMs of other coronavirus members such as MERS and OCF43. Our results provide a model at the molecular level of the strategies used to mimic the endogenous SLiM peptide in the binding of the tight junctions of the host cell, explaining one of the possible reasons of the severity of the infection and pulmonary inflammation by SARS-CoV-2.
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http://dx.doi.org/10.1016/j.csbj.2021.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7970798PMC
March 2021

Engineered Human Nanoferritin Bearing the Drug Genz-644282 for Cancer Therapy.

Pharmaceutics 2020 Oct 20;12(10). Epub 2020 Oct 20.

CNR-National Research Council of Italy, Institute of Molecular Biology and Pathology, 00185 Rome, Italy.

Gastrointestinal tumors, including pancreatic and colorectal cancers, represent one of the greatest public health issues worldwide, leading to a million global deaths. Recent research demonstrated that the human heavy chain ferritin (HFt) can encapsulate different types of drugs in its cavity and can bind to its receptor, CD71, in several solid and hematological tumors, thus highlighting the potential use of ferritin for tumor-targeting therapies. Here, we describe the development and characterization of a novel nanomedicine based on the HFt that is named The-0504. In particular, this novel system is a nano-assembly comprising an engineered version of HFt that entraps about 80 molecules of a potent, wide-spectrum, non-camptothecin topoisomerase I inhibitor (Genz-644282). The-0504 can be produced by a standardized pre-industrial process as a pure and homogeneously formulated product with favourable lyophilization properties. The preliminary anticancer activity was evaluated in cultured cancer cells and in a mouse model of pancreatic cancer. Overall results reported here make The-0504 a candidate for further preclinical development against CD-71 expressing deadly tumors.
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http://dx.doi.org/10.3390/pharmaceutics12100992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589674PMC
October 2020

Improved binding of SARS-CoV-2 Envelope protein to tight junction-associated PALS1 could play a key role in COVID-19 pathogenesis.

Microbes Infect 2020 Nov - Dec;22(10):592-597. Epub 2020 Sep 4.

Dipartimento di Scienze di Laboratorio e Infettivologiche, Fondazione Policlinico Universitario "A. Gemelli", IRCCS, Largo A. Gemelli 8, 00168 Roma, Italy; Dipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie - Sezione di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Roma, Italy.

The Envelope (E) protein of SARS-CoV-2 is the most enigmatic protein among the four structural ones. Most of its current knowledge is based on the direct comparison to the SARS E protein, initially mistakenly undervalued and subsequently proved to be a key factor in the ER-Golgi localization and in tight junction disruption. We compared the genomic sequences of E protein of SARS-CoV-2, SARS-CoV and the closely related genomes of bats and pangolins obtained from the GISAID and GenBank databases. When compared to the known SARS E protein, we observed a significant difference in amino acid sequence in the C-terminal end of SARS-CoV-2 E protein. Subsequently, in silico modelling analyses of E proteins conformation and docking provide evidences of a strengthened binding of SARS-CoV-2 E protein with the tight junction-associated PALS1 protein. Based on our computational evidences and on data related to SARS-CoV, we believe that SARS-CoV-2 E protein interferes more stably with PALS1 leading to an enhanced epithelial barrier disruption, amplifying the inflammatory processes, and promoting tissue remodelling. These findings raise a warning on the underestimated role of the E protein in the pathogenic mechanism and open the route to detailed experimental investigations.
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http://dx.doi.org/10.1016/j.micinf.2020.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473260PMC
January 2021

Ligand pathways in neuroglobin revealed by low-temperature photodissociation and docking experiments.

IUCrJ 2019 Sep 10;6(Pt 5):832-842. Epub 2019 Jul 10.

Department of Biochemical Sciences 'A. Rossi Fanelli', University of Rome Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy.

A combined biophysical approach was applied to map gas-docking sites within murine neuroglobin (Ngb), revealing snapshots of events that might govern activity and dynamics in this unique hexacoordinate globin, which is most likely to be involved in gas-sensing in the central nervous system and for which a precise mechanism of action remains to be elucidated. The application of UV-visible microspectroscopy , solution X-ray absorption near-edge spectroscopy and X-ray diffraction experiments at 15-40 K provided the structural characterization of an Ngb photolytic intermediate by cryo-trapping and allowed direct observation of the relocation of carbon monoxide within the distal heme pocket after photodissociation. Moreover, X-ray diffraction at 100 K under a high pressure of dioxygen, a physiological ligand of Ngb, unravelled the existence of a storage site for O in Ngb which coincides with Xe-III, a previously described docking site for xenon or krypton. Notably, no other secondary sites were observed under our experimental conditions.
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http://dx.doi.org/10.1107/S2052252519008157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760443PMC
September 2019

Cryo-EM structure of the human ferritin-transferrin receptor 1 complex.

Nat Commun 2019 03 8;10(1):1121. Epub 2019 Mar 8.

Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, P.le A. Moro 5, 00185, Rome, Italy.

Human transferrin receptor 1 (CD71) guarantees iron supply by endocytosis upon binding of iron-loaded transferrin and ferritin. Arenaviruses and the malaria parasite exploit CD71 for cell invasion and epitopes on CD71 for interaction with transferrin and pathogenic hosts were identified. Here, we provide the molecular basis of the CD71 ectodomain-human ferritin interaction by determining the 3.9 Å resolution single-particle cryo-electron microscopy structure of their complex and by validating our structural findings in a cellular context. The contact surfaces between the heavy-chain ferritin and CD71 largely overlap with arenaviruses and Plasmodium vivax binding regions in the apical part of the receptor ectodomain. Our data account for transferrin-independent binding of ferritin to CD71 and suggest that select pathogens may have adapted to enter cells by mimicking the ferritin access gate.
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http://dx.doi.org/10.1038/s41467-019-09098-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6408514PMC
March 2019

Microswitches for the Activation of the Nociceptin Receptor Induced by Cebranopadol: Hints from Microsecond Molecular Dynamics.

J Chem Inf Model 2019 02 30;59(2):818-831. Epub 2019 Jan 30.

Istituto di Biochimica e Biochimica Clinica , Università Cattolica del Sacro Cuore , Rome , 00168 Italy.

Cebranopadol (CBP) is a novel analgesic acting as agonist at the nociceptin (NOP) and μ-opioid (MOP) receptors, exhibiting high potency and efficacy as an antinociceptive and antihypersensitive drug. The binding conformation and the dynamical interactions of CBP with the NOP receptor have been investigated by molecular docking, molecular dynamics (MD) in the microsecond time scale, and hybrid quantum mechanics/molecular mechanics (QM/MM). CBP binds to the NOP receptor as a bidentate ligand of the aspartate D130 by means of both its fluoroindole and dimethyl nitrogens. Starting from the known crystal structure of the inactive state of the receptor, in complex with the antagonist compound-24 (NOP-C24) the comparative analysis of 1 μs MD trajectories of the NOP-C24 complex itself and the NOP_free and NOP-CBP complexes provides new insights on the already known microswitches related to receptor activation, in the frame of the extended ternary complex model. The agonist acts by destabilizing the inactive conformation of the NOP receptor, by inducing a conformational change of M134, which allows W276 to flip around its χ dihedral, going in close proximity to the receptor hydrophobic core (T138, P227, F272), which is known to be fundamental for the activation of the opioid receptors. A complete rational picture is also provided for the role of N133 and W276 undergoing critical conformational changes related to an anticooperativity effect, i.e. the well-known role of sodium as negative modulator of agonist binding. Finally, the movement of residue Y319 belonging to the NPxxY motif is also induced by the binding of the agonist in the inactive state, opening a gate for a water channel just as upon receptor activation.
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http://dx.doi.org/10.1021/acs.jcim.8b00759DOI Listing
February 2019

A protein chimera self-assembling unit for drug delivery.

Biotechnol Prog 2019 03 27;35(2):e2769. Epub 2018 Dec 27.

Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy.

In the modern view of selective drug delivery of bioactive molecules, the attention is moving onto the setup of the perfect carrier more than in the optimization of the active compound. In this respect, virus-like particles constitute bioinspired nanodevices with the intrinsic ability to transport a large class of molecules, ranging from smart drugs to small interfering RNAs. In this work, we demonstrate the efficacy of a novel construct obtained by fusing a self-assembling protein from the human Rotavirus A, VP6, with the Small Ubiquitin Modifier domain, which maintains the ability to form nanoparticles and nanotubes and is able to be used as a drug carrier, even without specific targeting epitopes. The high expression and purification yield, combined with low toxicity of the empty particles, clearly indicate a good candidate for future studies of selective drug delivery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2769, 2019.
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http://dx.doi.org/10.1002/btpr.2769DOI Listing
March 2019

Apixaban Interacts with Haemoglobin: Effects on Its Plasma Levels.

Thromb Haemost 2018 Oct 20;118(10):1701-1712. Epub 2018 Sep 20.

Institute of Internal Medicine & Geriatrics, Haemostasis and Thrombosis Center, Fondazione Policlinico Universitario "A. Gemelli" IRCCS, Area of Hematology, Catholic University of the Sacred Heart School of Medicine, Rome, Italy.

The direct oral anticoagulant apixaban (APX), a strong factor Xa inhibitor, binds also to plasma proteins, especially albumin, and minimally to α-acid glycoprotein. Although APX can cross the red cell membrane, due to its chemical structure, and could bind to haemoglobin (Hb), no investigation was performed on this possible phenomenon that could affect the APX plasma concentration and thus its pharmacokinetics and pharmacodynamics. We addressed this issue by (1) measuring the levels of APX and haematological/biochemical parameters in 90 patients on APX therapy; (2) assessing the effect of APX on oxygen saturation curves of Hb; (3) testing the direct APX binding to Hb by fluorescence spectroscopy and a zinc-induced precipitation of Hb coupled to a reversed-phase high-performance liquid chromatography (HPLC)-based method; and (4) simulating in silico by molecular docking the APX interaction with human Hb. In a multivariable analysis, Hb was the only independent variable significantly and inversely associated in 90 patients with APX peak plasma level, at variance with patients treated with rivaroxaban ( = 86) and dabigatran ( = 34) therapy. APX causes a progressive left-shift of the oxygen dissociation curve of purified Hb solution, with a K ≅300 µM. Fluorescence- and HPLC-based assays concordantly showed that APX binds to Hb with a K ≅350 µM. Finally, docking simulations showed that APX can fit into in the central cavity of Hb. These findings support the hypothesis that APX does bind to Hb, which, due to its millimolar concentration in blood, can act as 'buffer' for the drug and consequently affect its free plasma level.
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http://dx.doi.org/10.1055/s-0038-1669920DOI Listing
October 2018

Insights into the GTP-dependent allosteric control of c-di-GMP hydrolysis from the crystal structure of PA0575 protein from Pseudomonas aeruginosa.

FEBS J 2018 10 7;285(20):3815-3834. Epub 2018 Sep 7.

Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Italy.

Bis-(3'-5')-cyclic diguanylic acid (c-di-GMP) belongs to the class of cyclic dinucleotides, key carriers of cellular information in prokaryotic and eukaryotic signal transduction pathways. In bacteria, the intracellular levels of c-di-GMP and their complex physiological outputs are dynamically regulated by environmental and internal stimuli, which control the antagonistic activities of diguanylate cyclases (DGCs) and c-di-GMP specific phosphodiesterases (PDEs). Allostery is one of the major modulators of the c-di-GMP-dependent response. Both the c-di-GMP molecule and the proteins interacting with this second messenger are characterized by an extraordinary structural plasticity, which has to be taken into account when defining and possibly predicting c-di-GMP-related processes. Here, we report a structure-function relationship study on the catalytic portion of the PA0575 protein from Pseudomonas aeruginosa, bearing both putative DGC and PDE domains. The kinetic and structural studies indicate that the GGDEF-EAL portion is a GTP-dependent PDE. Moreover, the crystal structure confirms the high degree of conformational flexibility of this module. We combined structural analysis and protein engineering studies to propose the possible molecular mechanism guiding the nucleotide-dependent allosteric control of catalysis; we propose that the role exerted by GTP via the GGDEF domain is to allow the two EAL domains to form a dimer, the species competent to enter PDE catalysis.
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http://dx.doi.org/10.1111/febs.14634DOI Listing
October 2018

Human Serum Albumin Is an Essential Component of the Host Defense Mechanism Against Clostridium difficile Intoxication.

J Infect Dis 2018 09;218(9):1424-1435

2nd Infectious Diseases Division, National Institute for Infectious Diseases "L. Spallanzani", Roma, Italy.

Background: The pathogenic effects of Clostridium difficile are primarily attributable to the production of the large protein toxins (C difficile toxins [Tcd]) A (TcdA) and B (TcdB). These toxins monoglucosylate Rho GTPases in the cytosol of host cells, causing destruction of the actin cytoskeleton with cytotoxic effects. Low human serum albumin (HSA) levels indicate a higher risk of acquiring and developing a severe C difficile infection (CDI) and are associated with recurrent and fatal disease.

Methods: We used a combined approach based on docking simulation and biochemical analyses that were performed in vitro on purified proteins and in human epithelial colorectal adenocarcinoma cells (Caco-2), and in vivo on stem cell-derived human intestinal organoids and zebrafish embryos.

Results: Our results show that HSA specifically binds via its domain II to TcdA and TcdB and thereby induces their autoproteolytic cleavage at physiological concentrations. This process impairs toxin internalization into the host cells and reduces the toxin-dependent glucosylation of Rho proteins.

Conclusions: Our data provide evidence for a specific HSA-dependent self-defense mechanism against C difficile toxins and provide an explanation for the clinical correlation between CDI severity and hypoalbuminemia.
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http://dx.doi.org/10.1093/infdis/jiy338DOI Listing
September 2018

BBS9 gene in nonsyndromic craniosynostosis: Role of the primary cilium in the aberrant ossification of the suture osteogenic niche.

Bone 2018 07 17;112:58-70. Epub 2018 Apr 17.

Istituto di Anatomia Umana e Biologia Cellulare, Università Cattolica del Sacro Cuore, 00168 Rome, Italy; Fondazione Policlinico Universitario "Agostino Gemelli", 00168 Rome, Italy. Electronic address:

Nonsyndromic craniosynostosis (NCS) is the premature ossification of skull sutures, without associated clinical features. Mutations in several genes account for a small number of NCS patients; thus, the molecular etiopathogenesis of NCS remains largely unclear. Our study aimed at characterizing the molecular signaling implicated in the aberrant ossification of sutures in NCS patients. Comparative gene expression profiling of NCS patient sutures identified a fused suture-specific signature, including 17 genes involved in primary cilium signaling and assembly. Cells from fused sutures displayed a reduced potential to form primary cilia compared to cells from control patent sutures of the same patient. We identified specific upregulated splice variants of the Bardet Biedl syndrome-associated gene 9 (BBS9), which encodes a structural component of the ciliary BBSome complex. BBS9 expression increased during in vitro osteogenic differentiation of suture-derived mesenchymal cells of NCS patients. Also, Bbs9 expression increased during in vivo ossification of rat sutures. BBS9 functional knockdown affected the expression of primary cilia on patient suture cells and their osteogenic potential. Computational modeling of the upregulated protein isoforms (observed in patients) predicted that their binding affinity within the BBSome may be affected, providing a possible explanation for the aberrant suture ossification in NCS.
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http://dx.doi.org/10.1016/j.bone.2018.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5970090PMC
July 2018

Controlled release of 18-β-glycyrrhetic acid by nanodelivery systems increases cytotoxicity on oral carcinoma cell line.

Nanotechnology 2018 Jul 17;29(28):285101. Epub 2018 Apr 17.

Department of Engineering, University of Rome Niccolò Cusano, Via don Carlo Gnocchi 3, Roma, Italy.

The topical treatment for oral mucosal diseases is often based on products optimized for dermatologic applications; consequently, a lower therapeutic effect may be present. 18-β-glycyrrhetic acid (GA) is extracted from Glycirrhiza glabra. The first aim of this study was to test the cytotoxicity of GA on PE/CA-PJ15 cells. The second aim was to propose and test two different delivery systems, i.e. nanoparticles and fibers, to guarantee a controlled release of GA in vitro. We used chitosan and poly(lactic-co-glycolic) acid based nanoparticles and polylactic acid fibers. We tested both delivery systems in vitro on PE/CA-PJ15 cells and on normal human gingival fibroblasts (HGFs). The morphology of GA-loaded nanoparticles (GA-NPs) and fibers (GA-FBs) was investigated by electron microscopy and dynamic light scattering; GA release kinetics was studied spectrophotometrically. MTT test was used to assess GA cytotoxicity on both cancer and normal cells. Cells were exposed to different concentrations of GA (20-500 μmol l) administered as free GA (GA-f), and to GA-NPs or GA-FBs. ROS production was evaluated using dichlorodihydrofluorescein as a fluorescent probe. Regarding the cytotoxic effect of GA on PE/CA-PJ15 cells, the lowest TC value was 200 μmol l when GA was added as GA-NPs. No cytotoxic effects were observed when GA was administered to HGFs. N-acetyl Cysteine reduced mortality induced by GA-f in PE/CA-PJ15 cells. The specific effect of GA on PE/CA-PJ15 cells is mainly due to the different sensitivity of cancer cells to ROS over-production; GA-NPs and GA-FBs formulations increase, in vitro, this toxic effect on oral cancer cells.
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http://dx.doi.org/10.1088/1361-6528/aabeccDOI Listing
July 2018

The presence of glutamate residues on the PAS sequence of the stimuli-sensitive nano-ferritin improves in vivo biodistribution and mitoxantrone encapsulation homogeneity.

J Control Release 2018 04 20;275:177-185. Epub 2018 Feb 20.

Institute of Molecular Biology and Pathology, CNR - National Research Council of Italy, 00185 Rome, Italy. Electronic address:

A genetically engineered human ferritin heavy chain (HFt)-based construct has been recently shown by our group to efficiently entrap and deliver doxorubicin to cancer cells. This construct, named HFt-MP-PAS, contained a tumor-selective sequence (MP) responsive to proteolytic cleavage by tumor proteases (MMPs), located between each HFt subunit and an outer shielding polypeptide sequence rich in proline (P), serine (S) and alanine (A) residues (PAS). HFt-MP-PAS displayed excellent therapeutic efficacy in xenogenic pancreatic and head and neck cancer models in vivo, leading to a significant increase in overall animal survivals. Here we report a new construct obtained by the genetic insertion of two glutamate residues in the PAS sequence of HFt-MP-PAS. Such new construct, named HFt-MP-PASE, is characterized by improved performances as drug biodistribution in a xenogenic pancreatic cancer model in vivo. Moreover, HFt-MP-PASE efficiently encapsulates the anti-cancer drug mitoxantrone (MIT), and the resulting MIT-loaded nanoparticles proved to be more soluble and monodispersed than the HFt-MP-PAS counterparts. Importantly, in vitro MIT-loaded HFt-MP-PASE kills several cancer cell lines of different origin (colon, breast, sarcoma and pancreas) at least as efficiently as the free drug. Finally, our MIT loaded protein nanocages allowed in vivo an impressive incrementing of the drug accumulation in the tumor with respect to the free drug.
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http://dx.doi.org/10.1016/j.jconrel.2018.02.025DOI Listing
April 2018

"In silico" study of the binding of two novel antagonists to the nociceptin receptor.

J Comput Aided Mol Des 2018 02 16;32(2):385-400. Epub 2018 Jan 16.

Institute of Biochemistry and Clinical Biochemistry, Catholic University of Sacred Heart, Rome, Italy.

Antagonists of the nociceptin receptor (NOP) are raising interest for their possible clinical use as antidepressant drugs. Recently, the structure of NOP in complex with some piperidine-based antagonists has been revealed by X-ray crystallography. In this study, a multi-flexible docking (MF-docking) procedure, i.e. docking to multiple receptor conformations extracted by preliminary molecular dynamics trajectories, together with hybrid quantum mechanics/molecular mechanics (QM/MM) simulations have been carried out to provide the binding mode of two novel NOP antagonists, one of them selective (BTRX-246040, formerly named LY-2940094) and one non selective (AT-076), i.e. able to inactivate NOP as well as the classical µ- k- and δ-opioid receptors (MOP KOP and DOP). According to our results, the pivotal role of residue D130 (upper indexes are Ballesteros-Weinstein notations) is analogous to that enlighten by the already known X-ray structures of opioid receptors: binding of the molecules are predicted to require a slight readjustment of the hydrophobic pocket (residues Y131, M134, I219, Q280 and V283) in the orthosteric site of NOP, accommodating either the pyridine-pyrazole (BTRX-246040) or the isoquinoline (AT-076) moiety of the ligand, in turn allowing the protonated piperidine nitrogen to maximize interaction (salt-bridge) with residue D130 of the NOP, and the aromatic head to be sandwiched in optimal π-stacking between Y131 and M134. The QM/MM optimization after the MF-docking procedure has provided the more likely conformations for the binding to the NOP receptor of BTRX-246040 and AT-076, based on different pharmacophores and exhibiting different selectivity profiles. While the high selectivity for NOP of BTRX-246040 can be explained by interactions with NOP specific residues, the lack of selectivity of AT-076 could be associated to its ability to penetrate into the deep hydrophobic pocket of NOP, while retaining a conformation very similar to the one assumed by the antagonist JDTic into the K-opioid receptor. The proposed binding geometries fit better the binding pocket environment providing clues for experimental studies aimed to design selective or multifunctional opioid drugs.
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http://dx.doi.org/10.1007/s10822-017-0095-5DOI Listing
February 2018

Correlation Between Metabolic Syndrome, Periodontitis and Reactive Oxygen Species Production. A Pilot Study.

Open Dent J 2017 12;11:621-627. Epub 2017 Dec 12.

Institute of Biochemistry and Clinical Biochemistry, Università Cattolica del Sacro Cuore, Largo Francesco Vito - 00168 Rome , Italy.

Background And Objective: Metabolic syndrome (MetS) is associated with an increased risk of periodontitis even if the mechanism is unknown. Since both MetS and periodontitis are characterized by an alteration of inflammation status, the aim of this pilot study was to determine if differences in ROS metabolism of phagocytes isolated from (A) patients with MetS, (B) patients with both MetS and mild periodontitis, (C) healthy subjects and (D) normal weight subjects with mild periodontitis, were present.

Methods: ROS metabolism was studied by a Chemiluminescence (CL) technique: the system was made up of luminol (100 nmol/L) and cells (1 × 10) in the presence or absence of stimulus constituted by opsonized zymosan (0.5 mg). The final volume (1.0 mL) was obtained using modified KRP buffer. ROS production was measured at 25°C for 2 h, using an LB 953 luminometer (Berthold, EG & G Co, Germany). All the experiments were performed in triplicate.

Statistical Analysis: All results are mean ± standard deviation (SD). The group of means was compared by the analysis of variance "(ANOVA)". A value of < 0.05 was considered significant.

Results: Results showed that basal ROS production (both from PMNs and from PBMs) of groups A, B and D was increased with respect to that obtained from group C ( <0.05).

Conclusion: These results are congruent with literature data, although the actual clinical relevance of the phenomenon remains to be evaluated.
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http://dx.doi.org/10.2174/1874210601711010621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738754PMC
December 2017

A Dynamic Picture of the Early Events in Nociceptin Binding to the NOP Receptor by Metadynamics.

Biophys J 2016 Sep;111(6):1203-1213

Institute of Biochemistry and Clinical Biochemistry, Catholic University of Sacred Heart, Rome, Italy.

Nociceptin (NCC, also known as FQ (N/OFQ)) is the 17-amino acid neuropeptide, endogenous ligand for the G-protein-coupled receptor (NOP, also known as ORL-1). In this study, starting from the recently reported x-ray structure at pH 7 of NOP in complex with an antagonist, new insights, to our knowledge, on the binding geometry of NCC to NOP have been provided in silico. After a rigid docking of NCC in an α-helix conformation, molecular dynamics (MD) and metadynamics (METAD), a method for the analysis of free-energy surfaces (FES), were performed on the protein-peptide complex. Free-energy profiles were obtained as a function of the α-helix content of different segments of the 17-mer ligand, and a structural ensemble of conformations of NCC, corresponding to the minimum of the FES, was extracted, thus representing the NCC bound to the inactive form of NOP. The structural features were compared with many known experimental data. The pose of the "message" domain (residues 1-4) of NCC differs from that of the known NOP antagonists, as being slightly slipped deeper inside the protein core. A residual α-helix content in the central part of the peptide (residues 4-9) is maintained, whereas the C-terminal segment (residues 13-17) is unstructured and highly flexible. An important stabilization due to interactions with residues D130 and D110 of the receptor has been found, in agreement with the large decrease in agonist potency reported for the D130A and D110A mutants. The importance of the extracellular domain 2 (ECL2) in the selectivity toward the endogenous ligand has been confirmed. A pivotal role for the conserved residue N133 is suggested and further supported by a study of the N133A in silico mutant. Accordingly, N133 can work as a molecular microswitch driving the change between the inactive and active NOP conformations, in the framework of an extended H-bond and water network rearrangement in the deep binding site.
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http://dx.doi.org/10.1016/j.bpj.2016.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5034302PMC
September 2016

Selective delivery of doxorubicin by novel stimuli-sensitive nano-ferritins overcomes tumor refractoriness.

J Control Release 2016 10 12;239:10-8. Epub 2016 Aug 12.

Institute of Molecular Biology and Pathology, CNR - National Research Council of Italy, 00185 Rome, Italy. Electronic address:

Human ferritin heavy chain (HFt) has been demonstrated to possess considerable potential for targeted delivery of drugs and diagnostic agents to cancer cells. Here, we report the development of a novel HFt-based genetic construct (HFt-MP-PAS) containing a short peptide linker (MP) between each HFt subunit and an outer shielding polypeptide sequence rich in proline (P), serine (S) and alanine (A) residues (PAS). The peptide linker contains a matrix-metalloproteinases (MMPs) cleavage site that permits the protective PAS shield to be removed by tumor-driven proteolytic cleavage within the tumor microenvironment. For the first time HFt-MP-PAS ability to deliver doxorubicin to cancer cells, subcellular localization, and therapeutic efficacy on a xenogeneic mouse model of a highly refractory to conventional chemotherapeutics type of cancer were evaluated. HFt-MP-PAS-DOXO performance was compared with the novel albumin-based drug delivery system INNO-206, currently in phase III clinical trials. The results of this work provide solid evidence indicating that the stimuli-sensitive, long-circulating HFt-MP-PAS nanocarriers described herein have the potential to be exploited in cancer therapy.
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http://dx.doi.org/10.1016/j.jconrel.2016.08.010DOI Listing
October 2016

Structural properties of geminal dicationic ionic liquid/water mixtures: a theoretical and experimental insight.

Phys Chem Chem Phys 2016 Jun 7;18(24):16544-54. Epub 2016 Jun 7.

Dipartimento di Chimica, Università di Roma "La Sapienza", P.le A. Moro 5, 00185 Roma, Italy.

The structural behavior of geminal dicationic ionic liquid 1,n-bis[3-methylimidazolium-1-yl] alkane bromide ([Cn(mim)2]Br2)/water mixtures has been studied using extended X-ray absorption fine structure (EXAFS) spectroscopy in combination with molecular dynamics (MD) simulations. The properties of the mixtures are investigated as a function of both water concentration and alkyl-bridge chain length. The very good agreement between the EXAFS experimental data and the theoretical curves calculated from the MD structural results has proven the validity of the theoretical framework used for all of the investigated systems. In all the solutions the water molecules are preferentially coordinated with the Br(-) ion, even if a complex network of interactions among dications, anions and water molecules takes place. The local molecular arrangement around the bromide ion is found to change with increasing water content, as more and more water molecules are accomodated in the Br(-) first coordination shell. Moreover, with the decrease of the alkyl-bridge chain length, the interactions between dications and anions increase, with Br(-) forming a bridge between the two imidazolium rings of the same dication. On the other hand, in [Cn(mim)2]Br2/water mixtures with long alkyl-bridge chains peculiar internal arrangements of the dications are found, leading to different structural features of geminal dicationic ionic liquids as compared to their monocationic counterparts.
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http://dx.doi.org/10.1039/c6cp01557cDOI Listing
June 2016

Structural studies and SH3 domain binding properties of a human antiviral salivary proline-rich peptide.

Biopolymers 2016 Sep;106(5):714-25

Istituto di Chimica di Riconoscimento Molecolare, C.N.R. Rome, L.Go F. Vito, 1, Rome, 00168, Italy.

Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016.
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http://dx.doi.org/10.1002/bip.22889DOI Listing
September 2016

Improved Doxorubicin Encapsulation and Pharmacokinetics of Ferritin-Fusion Protein Nanocarriers Bearing Proline, Serine, and Alanine Elements.

Biomacromolecules 2016 Feb 30;17(2):514-22. Epub 2015 Dec 30.

Institute of Molecular Biology and Pathology CNR, National Research Council of Italy , 00185 Rome, Italy.

A novel human ferritin-based nanocarrier, composed of 24 modified monomers able to auto-assemble into a modified protein cage, was produced and used as selective carrier of anti-tumor payloads. Each modified monomer derives from the genetic fusion of two distinct modules, namely the heavy chain of human ferritin (HFt) and a stabilizing/protective PAS polypeptide sequence rich in proline (P), serine (S), and alanine (A) residues. Two genetically fused protein constructs containing PAS polymers with 40- and 75-residue lengths, respectively, were compared. They were produced and purified as recombinant proteins in Escherichia coli at high yields. Both preparations were highly soluble and stable in vitro as well as in mouse plasma. Size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy results indicated that PASylated ferritins are fully assembled and highly monodispersed. In addition, yields and stability of encapsulated doxorubicin were significantly better for both HFt-PAS proteins than for wild-type HFt. Importantly, PAS sequences considerably prolonged the half-life of HFt in the mouse bloodstream. Finally, our doxorubicin-loaded nanocages preserved the pharmacological activity of the drug. Taken together, these results indicate that both of the developed HFt-PAS fusion proteins are promising nanocarriers for future applications in cancer therapy.
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http://dx.doi.org/10.1021/acs.biomac.5b01446DOI Listing
February 2016

The non-octarepeat copper binding site of the prion protein is a key regulator of prion conversion.

Sci Rep 2015 Oct 20;5:15253. Epub 2015 Oct 20.

Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy.

The conversion of the prion protein (PrP(C)) into prions plays a key role in transmissible spongiform encephalopathies. Despite the importance for pathogenesis, the mechanism of prion formation has escaped detailed characterization due to the insoluble nature of prions. PrP(C) interacts with copper through octarepeat and non-octarepeat binding sites. Copper coordination to the non-octarepeat region has garnered interest due to the possibility that this interaction may impact prion conversion. We used X-ray absorption spectroscopy to study copper coordination at pH 5.5 and 7.0 in human PrP(C) constructs, either wild-type (WT) or carrying pathological mutations. We show that mutations and pH cause modifications of copper coordination in the non-octarepeat region. In the WT at pH 5.5, copper is anchored to His96 and His111, while at pH 7 it is coordinated by His111. Pathological point mutations alter the copper coordination at acidic conditions where the metal is anchored to His111. By using in vitro approaches, cell-based and computational techniques, we propose a model whereby PrP(C) coordinating copper with one His in the non-octarepeat region converts to prions at acidic condition. Thus, the non-octarepeat region may act as the long-sought-after prion switch, critical for disease onset and propagation.
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http://dx.doi.org/10.1038/srep15253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4651146PMC
October 2015

Dexamethasone-loaded biopolymeric nanoparticles promote gingival fibroblasts differentiation.

Biotechnol Prog 2015 Sep-Oct;31(5):1381-7. Epub 2015 Jul 15.

Inst. of Biochemistry and Clinical Biochemistry, Faculty of Medicine, Università Cattolica Del Sacro Cuore, L.Go F. Vito 1, Rome, 00168, Italy.

Polymer-based nanoparticles (NPs) can be efficiently used for the delivery of bioactive molecules for both in vitro and in vivo applications affording high drug loading and controlled release profiles. Within this framework polylactic-co-glycolic acid (PLGA) NPs with a diameter of 290 ± 41 nm have been fabricated and loaded with dexamethasone (DXM) using a patented procedure. The aim of the project was to setup a controlled delivery system to promote the in vitro differentiation of Human Gingival Fibroblasts (HGFs). First the uptake of fluorescent PLGA NPs by HGFs cells was investigated; then experiments were also addressed to analyze the specific cell response to DXM, in order to evaluate its functional efficiency in comparison with its conventional addition to the culture medium. The results showed that cells treated with DXM-loaded NPs acquired the osteoblast phenotype faster in comparison to those treated with the free drug. The slow and sustained release of DXM from PLGA NPs produced a constant and uniform concentration of drug inside cells with long-term and enhanced biochemical effects.
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http://dx.doi.org/10.1002/btpr.2141DOI Listing
August 2016

Effects of barriers on chemical and biological properties of two dual resin cements.

Eur J Oral Sci 2015 Jun 4;123(3):208-14. Epub 2015 Mar 4.

Facoltà di Medicina e Chirurgia, Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy; Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Rome, Italy.

The aim of this study was to investigate the degree of conversion, monomer release, and cytotoxicity of two dual-cure resin cements (Cement-One and SmartCem2), light-cured across two indirect restorative materials in an attempt to simulate in vitro the clinical conditions. The results obtained show that the degree of conversion was influenced by both barriers, but the effect of the composite material was greater than that of the ceramic one. The amount of monomers released from the polymerized materials in the absence of barriers was significantly lower than that released in the presence of either the ceramic or the composite barrier. However, a higher amount of monomers was released in the presence of the ceramic barrier. All materials, in all the experimental conditions employed, induced slight cytotoxicity (5-10%) on human pulp cells. Our examinations showed that the two resin cements had similar chemical and biological properties. The decreased degree of conversion of the dual-curing self-adhesive composite showed that the light-curing component of these materials has an important role in the polymerization process. In clinical practice, it is therefore important to pay attention to the thickness of the material used for the reconstruction.
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http://dx.doi.org/10.1111/eos.12178DOI Listing
June 2015

The polyamine N-acetyltransferase-like enzyme PmvE plays a role in the virulence of Enterococcus faecalis.

Infect Immun 2015 Jan 10;83(1):364-71. Epub 2014 Nov 10.

Equipe Antibio-Résistance, Unité de Recherche Risques Microbiens (U2RM), Université de Caen, Caen, France

We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyA operon, ef_3001, renamed pmvE (for polyamine metabolism and virulence of E. faecalis). PmvE shares strong homologies with N(1)-spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalis strain carrying the recombinant plasmid pMSP3535-pmvE (V19/p3535-pmvE), which allows the induction of pmvE by addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalis in the Galleria mellonella infection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N-acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis, likely through its involvement in the polyamine metabolism.
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http://dx.doi.org/10.1128/IAI.02585-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288890PMC
January 2015

Intermediate states in the binding process of folic acid to folate receptor α: insights by molecular dynamics and metadynamics.

J Comput Aided Mol Des 2015 Jan 17;29(1):23-35. Epub 2014 Oct 17.

Dipartimento di Medicina Clinica, Sanità Pubblica, Scienze della Vita e dell'Ambiente, Università dell'Aquila, Piazzale S. Tommasi 1, 67100, Coppito, AQ, Italy,

Folate receptor α (FRα) is a cell surface, glycophosphatidylinositol-anchored protein which has focussed attention as a therapeutic target and as a marker for the diagnosis of cancer. It has a high affinity for the dietary supplemented folic acid (FOL), carrying out endocytic transport across the cell membrane and delivering the folate at the acidic pH of the endosome. Starting from the recently reported X-ray structure at pH 7, 100 ns classical molecular dynamics simulations have been carried out on the FRα-FOL complex; moreover, the ligand dissociation process has been studied by metadynamics, a recently reported method for the analysis of free-energy surfaces (FES), providing clues on the intermediate states and their energy terms. Multiple dissociation runs were considered to enhance the configurational sampling; a final clustering of conformations within the averaged FES provides the representative structures of several intermediate states, within an overall barrier for ligand escape of about 75 kJ/mol. Escaping of FOL to solvent occurs while only minor changes affect the FRα conformation of the binding pocket. During dissociation, the FOL molecule translates and rotates around a turning point located in proximity of the receptor surface. FOL at this transition state assumes an "L" shaped conformation, with the pteridin ring oriented to optimize stacking within W102 and W140 residues, and the negatively charged glutamate tail, outside the receptor, interacting with the positively charged R103 and R106 residues, that contrary to the bound state, are solvent exposed. We show that metadynamics method can provide useful insights at the atomistic level on the effects of point-mutations affecting functionality, thus being a very promising tool for any study related to folate-targeted drug delivery or cancer therapies involving folate uptake.
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http://dx.doi.org/10.1007/s10822-014-9801-8DOI Listing
January 2015

Distal-proximal crosstalk in the heme binding pocket of the NO sensor DNR.

Biometals 2014 Aug 10;27(4):763-73. Epub 2014 Jul 10.

Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Istituto Pasteur Fondazione Cenci Bolognetti, Sapienza - Università di Roma, P.le A.Moro 5, 00185, Rome, Italy.

In the opportunistic pathogen Pseudomonas aeruginosa the denitrification process is triggered by nitric oxide (NO) and plays a crucial role for the survival in chronic infection sites as a microaerobic-anaerobic biofilm. This respiratory pathway is transcriptionally induced by DNR, an heme-based gas sensor which positively responds to NO. Molecular details of the NO sensing mechanism employed by DNR are now emerging: we recently reported an in vitro study which dissected, for the first time, the heme-iron environment and identified one of the heme axial ligand (i.e. His187), found to be crucial to respond to NO. Nevertheless, the identification of the second heme axial ligand has been unsuccessful, given that a peculiar phenomenon of ligand switching around the heme-iron presumably occurs in DNR. The unusual heme binding properties of DNR could be due to the remarkable flexibility in solution of DNR itself, which, in turns, is crucial for the sensing activity; protein flexibility and dynamics indeed represent a common strategy employed by heme-based redox sensors, which present features deeply different from those of "canonical" hemeproteins. The capability of DNR to deeply rearrange around the heme-iron as been here demonstrated by means of spectroscopic characterization of the H167A/H187A DNR double mutant, which shows unusual kinetics of binding of NO and CO. Moreover, we show that the alteration (such as histidines mutations) of the distal side of the heme pocket is perceived by the proximal one, possibly via the DNR protein chain.
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http://dx.doi.org/10.1007/s10534-014-9770-3DOI Listing
August 2014

Synergic role of nucleophosmin three-helix bundle and a flanking unstructured tail in the interaction with G-quadruplex DNA.

J Biol Chem 2014 Aug 21;289(31):21230-41. Epub 2014 Jun 21.

Ce.S.I. Centro Scienze dell'Invecchiamento, "Fondazione Università D'Annunzio," 66013 Chieti, Italy, Dipartimento di Scienze Sperimentali e Cliniche, Università di Chieti "G. D'Annunzio," 66013 Chieti, Italy

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a number of functions in ribosome biogenesis and export, cell cycle control, and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia; mutations map to the C-terminal domain of the protein and cause its denaturation and aberrant cytoplasmic translocation. NPM1 C-terminal domain binds G-quadruplex regions at ribosomal DNA and at gene promoters, including the well characterized sequence from the nuclease-hypersensitive element III region of the c-MYC promoter. These activities are lost by the leukemic variant. Here we analyze the NPM1/G-quadruplex interaction, focusing on residues belonging to both the NPM1 terminal three-helix bundle and a lysine-rich unstructured tail, which has been shown to be necessary for high affinity recognition. We performed extended site-directed mutagenesis and measured binding rate constants through surface plasmon resonance analysis. These data, supported by molecular dynamics simulations, suggest that the unstructured tail plays a double role in the reaction mechanism. On the one hand, it facilitates the formation of an encounter complex through long range electrostatic interactions; on the other hand, it directly contacts the G-quadruplex scaffold through multiple and transient electrostatic interactions, significantly enlarging the contact surface.
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http://dx.doi.org/10.1074/jbc.M114.565010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118085PMC
August 2014

Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein.

Int J Nanomedicine 2014 30;9:2727-39. Epub 2014 May 30.

Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy.

In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here - providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures - opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes.
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http://dx.doi.org/10.2147/IJN.S60014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047981PMC
December 2014

Structural and functional insights on folate receptor α (FRα) by homology modeling, ligand docking and molecular dynamics.

J Mol Graph Model 2013 Jul 19;44:197-207. Epub 2013 Jun 19.

Dipartimento di Medicina Clinica, Sanità Pubblica, Scienze della Vita e dell'Ambiente, Università dell'Aquila, Piazzale S. Tommasi 1, 67100, Coppito (AQ), Italy.

Folate receptor α (FRα) is a cell surface, glycophosphatidylinositol (GPI)-anchored protein with a high affinity for its ligand partner, which is highly expressed in malignant cells and has been selected as a therapeutic target and marker for the diagnosis of cancer. No direct structural information is available from either X-ray diffraction or NMR on the post-translational structure of this disulfide-rich protein. Three-dimensional models of the FRα structure have been derived with the recent homology modeling packages, using the crystal structure of the riboflavin-binding protein (RfBP) as a template. Molecular dynamics trajectories have been exploited allowing successfully the formation of a full disulfide bridge network, which was expected based on the similarities between FRα and RfBP. After the selection of the best model, a folic acid molecule was docked "in silico" onto the putative binding site and its binding mode was compared with that of vintafolide, a much larger molecule designed as a chemotherapy agent targeting specifically FRα. In both cases, a 40ns molecular dynamics trajectory was calculated, providing suggestions regarding the key structural determinants driving the affinity and specificity of FRα for folic acid with respect to other folate homologues. Moreover, some other crucial experimental results related to the structure of the receptor are discussed, such as the expected location/accessibility of known immune epitopes, the set of N-linked glycosylation sites and the effect of point mutations on the impairment of folate binding. Our results may provide useful insights for studies related to folate-targeted drug delivery or cancer therapies involving folate uptake.
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http://dx.doi.org/10.1016/j.jmgm.2013.05.012DOI Listing
July 2013