Publications by authors named "Matteo Ardini"

19 Publications

  • Page 1 of 1

Taking Advantage of the Morpheein Behavior of Peroxiredoxin in Bionanotechnology.

Bioconjug Chem 2021 Jan 7;32(1):43-62. Epub 2021 Jan 7.

Department of Life, Health, and Environmental Sciences, University of L'Aquila, Piazzale Salvatore Tommasi 1, 67100 L'Aquila, Italy.

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.
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http://dx.doi.org/10.1021/acs.bioconjchem.0c00621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8023583PMC
January 2021

Antibody-Drug Conjugates: The New Frontier of Chemotherapy.

Int J Mol Sci 2020 Jul 31;21(15). Epub 2020 Jul 31.

Department of Life, Health and Environmental Sciences, University of L'Aquila, I-67100 L'Aquila, Italy.

In recent years, antibody-drug conjugates (ADCs) have become promising antitumor agents to be used as one of the tools in personalized cancer medicine. ADCs are comprised of a drug with cytotoxic activity cross-linked to a monoclonal antibody, targeting antigens expressed at higher levels on tumor cells than on normal cells. By providing a selective targeting mechanism for cytotoxic drugs, ADCs improve the therapeutic index in clinical practice. In this review, the chemistry of ADC linker conjugation together with strategies adopted to improve antibody tolerability (by reducing antigenicity) are examined, with particular attention to ADCs approved by the regulatory agencies (the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA)) for treating cancer patients. Recent developments in engineering Immunoglobulin (Ig) genes and antibody humanization have greatly reduced some of the problems of the first generation of ADCs, beset by problems, such as random coupling of the payload and immunogenicity of the antibody. ADC development and clinical use is a fast, evolving area, and will likely prove an important modality for the treatment of cancer in the near future.
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http://dx.doi.org/10.3390/ijms21155510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432430PMC
July 2020

A ring-shaped protein clusters gold nanoparticles acting as molecular scaffold for plasmonic surfaces.

Biochim Biophys Acta Gen Subj 2020 08 15;1864(8):129617. Epub 2020 Apr 15.

Department of Life, Health and Environmental Sciences, University of L'Aquila, Piazzale Salvatore Tommasi 1, 67100 Coppito, L'Aquila, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbagen.2020.129617DOI Listing
August 2020

A ring-shaped protein clusters gold nanoparticles acting as molecular scaffold for plasmonic surfaces.

Biochim Biophys Acta Gen Subj 2020 08 15;1864(8):129617. Epub 2020 Apr 15.

Department of Life, Health and Environmental Sciences, University of L'Aquila, Piazzale Salvatore Tommasi 1, 67100 Coppito, L'Aquila, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbagen.2020.129617DOI Listing
August 2020

On-Demand Intracellular Delivery of Single Particles in Single Cells by 3D Hollow Nanoelectrodes.

Nano Lett 2019 02 23;19(2):722-731. Epub 2019 Jan 23.

Istituto Italiano di Tecnologia , Via Morego 30 , 16163 Genova , Italy.

Delivery of molecules into intracellular compartments is one of the fundamental requirements in molecular biology. However, the possibility of delivering a precise number of nano-objects with single-particle resolution is still an open challenge. Here we present an electrophoretic platform based on 3D hollow nanoelectrodes to enable delivery of single nanoparticles into single selected cells and monitoring of the single-particle delivery by surface-enhanced Raman scattering (SERS). The gold-coated hollow nanoelectrode capable of confinement and enhancement of electromagnetic fields upon laser illumination can distinguish the SERS signals of a single nanoparticle flowing through the nanoelectrode. Tight wrapping of cell membranes around the nanoelectrodes allows effective membrane electroporation such that single gold nanorods are delivered on demand into a living cell by electrophoresis. The capability of the 3D hollow nanoelectrodes to porate cells and reveal single emitters from the background in continuous flow is promising for the analysis of both intracellular delivery and sampling.
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http://dx.doi.org/10.1021/acs.nanolett.8b03764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378653PMC
February 2019

Author Correction: Live Intracellular Biorthogonal Imaging by Surface Enhanced Raman Spectroscopy using Alkyne-Silver Nanoparticles Clusters.

Sci Rep 2018 Oct 23;8(1):15910. Epub 2018 Oct 23.

Istituto Italiano di Tecnologia, Via Morego 30, 16163, Genova, Italy.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-33285-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6198011PMC
October 2018

Hybrid plasmonic nanostructures based on controlled integration of MoS flakes on metallic nanoholes.

Nanoscale 2018 Sep;10(36):17105-17111

Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy.

Here, we propose an easy and robust strategy for the versatile preparation of hybrid plasmonic nanopores by means of controlled deposition of single flakes of MoS2 directly on top of metallic holes. The device is realized on silicon nitride membranes and can be further refined by TEM or FIB milling to achieve the passing of molecules or nanometric particles through a pore. Importantly, we show that the plasmonic enhancement provided by the nanohole is strongly accumulated in the 2D nanopore, thus representing an ideal system for single-molecule sensing and sequencing in a flow-through configuration. Here, we also demonstrate that the prepared 2D material can be decorated with metallic nanoparticles that can couple their resonance with the nanopore resonance to further enhance the electromagnetic field confinement at the nanoscale level. This method can be applied to any gold nanopore with a high level of reproducibility and parallelization; hence, it can pave the way to the next generation of solid-state nanopores with plasmonic functionalities. Moreover, the controlled/ordered integration of 2D materials on plasmonic nanostructures opens a pathway towards new investigation of the following: enhanced light emission; strong coupling from plasmonic hybrid structures; hot electron generation; and sensors in general based on 2D materials.
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http://dx.doi.org/10.1039/c8nr05026kDOI Listing
September 2018

Live Intracellular Biorthogonal Imaging by Surface Enhanced Raman Spectroscopy using Alkyne-Silver Nanoparticles Clusters.

Sci Rep 2018 08 23;8(1):12652. Epub 2018 Aug 23.

Istituto Italiano di Tecnologia, Via Morego 30, 16163, Genova, Italy.

Live intracellular imaging is a valuable tool in modern diagnostics and pharmacology. Surface Enhanced Raman Spectroscopy (SERS) stands out as a non-destructive and multiplexed technique, but intracellular SERS imaging still suffers from interfering background from endogenous components. Here we show the assembly of small colloidal SERS probes with Raman signal in the cell-silent window of 1800-2900 cm for biorthogonal intracellular SERS imaging of dopamine that was undistinguishable from the endogenous cell background. By linking colloidal silver nanoparticles with alkyne-dopamine adducts, clusters are formed by 2-6 nanoparticles spaced by tight interparticle gaps that exhibited high electric field enhancement and strong SERS signals of alkyne and dopamines. Due to the cell-silent signals of the alkyne, intracellular in-vitro Raman imaging shows that the dopamines on the internalized clusters remain distinguishable across the cytoplasm with good spatial resolution. Our method can be a general-purpose method for real-time imaging of biomolecules, such as proteins, peptides, DNA and drugs.
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http://dx.doi.org/10.1038/s41598-018-31165-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107644PMC
August 2018

Fragment-Based Discovery of a Regulatory Site in Thioredoxin Glutathione Reductase Acting as "Doorstop" for NADPH Entry.

ACS Chem Biol 2018 08 11;13(8):2190-2202. Epub 2018 Jun 11.

Department of Life, Health and Environmental Sciences , University of L'Aquila , 67100 L'Aquila , Italy.

Members of the FAD/NAD-linked reductase family are recognized as crucial targets in drug development for cancers, inflammatory disorders, and infectious diseases. However, individual FAD/NAD reductases are difficult to inhibit in a selective manner with off-target inhibition reducing usefulness of identified compounds. Thioredoxin glutathione reductase (TGR), a high molecular weight thioredoxin reductase-like enzyme, has emerged as a promising drug target for the treatment of schistosomiasis, a parasitosis afflicting more than 200 million people. Taking advantage of small molecules selected from a high-throughput screen and using X-ray crystallography, functional assays, and docking studies, we identify a critical secondary site of the enzyme. Compounds binding at this site interfere with well-known and conserved conformational changes associated with NADPH reduction, acting as a doorstop for cofactor entry. They selectively inhibit TGR from Schistosoma mansoni and are active against parasites in culture. Since many members of the FAD/NAD-linked reductase family have similar catalytic mechanisms, the unique mechanism of inhibition identified in this study for TGR broadly opens new routes to selectively inhibit homologous enzymes of central importance in numerous diseases.
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http://dx.doi.org/10.1021/acschembio.8b00349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905387PMC
August 2018

A peroxiredoxin-based proteinaceous scaffold for the growth and differentiation of neuronal cells and tumour stem cells in the absence of prodifferentiation agents.

J Tissue Eng Regen Med 2017 09 8;11(9):2462-2470. Epub 2016 Feb 8.

Department of Life Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.

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.
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http://dx.doi.org/10.1002/term.2144DOI Listing
September 2017

Study of manganese binding to the ferroxidase centre of human H-type ferritin.

J Inorg Biochem 2018 05 9;182:103-112. Epub 2018 Feb 9.

INSTM, Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019 Sesto Fiorentino, Florence, Italy. Electronic address:

Ferritins are ubiquitous and conserved proteins endowed with enzymatic ferroxidase activity, that oxidize Fe(II) ions at the dimetal ferroxidase centre to form a mineralized Fe(III) oxide core deposited within the apo-protein shell. Herein, the in vitro formation of a heterodimetal cofactor constituted by Fe and Mn ions has been investigated in human H ferritin (hHFt). Namely, Mn and Fe binding at the hHFt ferroxidase centre and its effects on Fe(II) oxidation have been investigated by UV-Vis ferroxidation kinetics, fluorimetric titrations, multifrequency EPR, and preliminary Mössbauer spectroscopy. Our results show that in hHFt, both Fe(II) and Mn(II) bind the ferroxidase centre forming a Fe-Mn cofactor. Moreover, molecular oxygen seems to favour Mn(II) binding and increases the ferroxidation activity of the Mn-loaded protein. The data suggest that Mn influences the Fe binding and the efficiency of the ferroxidation reaction. The higher efficiency of the Mn-Fe heterometallic centre may have a physiological relevance in specific cell types (i.e. glia cells), where the concentration of Mn is the same order of magnitude as iron.
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http://dx.doi.org/10.1016/j.jinorgbio.2018.02.003DOI Listing
May 2018

Biocompatibility of composites based on chitosan, apatite, and graphene oxide for tissue applications.

J Biomed Mater Res A 2018 06 21;106(6):1585-1594. Epub 2018 Feb 21.

Dipartimento di Medicina Clinica, Sanità Pubblica, Scienze della Vita e dell'Ambiente/Università degli Studi dell'Aquila, L'Aquila, Italy.

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.
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http://dx.doi.org/10.1002/jbm.a.36361DOI Listing
June 2018

Typical 2-Cys peroxiredoxins in human parasites: Several physiological roles for a potential chemotherapy target.

Mol Biochem Parasitol 2016 Mar-Apr;206(1-2):2-12. Epub 2016 Mar 18.

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

Peroxiredoxins (Prxs) are ubiquitary proteins able to play multiple physiological roles, that include thiol-dependent peroxidase, chaperone holdase, sensor of H2O2, regulator of H2O2-dependent signal cascades, and modulator of the immune response. Prxs have been found in a great number of human pathogens, both eukaryotes and prokaryotes. Gene knock-out studies demonstrated that Prxs are essential for the survival and virulence of at least some of the pathogens tested, making these proteins potential drug targets. However, the multiplicity of roles played by Prxs constitutes an unexpected obstacle to drug development. Indeed, selective inhibitors of some of the functions of Prxs are known (namely of the peroxidase and holdase functions) and are here reported. However, it is often unclear which function is the most relevant in each pathogen, hence which one is most desirable to inhibit. Indeed there are evidences that the main physiological role of Prxs may not be the same in different parasites. We here review which functions of Prxs have been demonstrated to be relevant in different human parasites, finding that the peroxidase and chaperone activities figure prominently, whereas other known functions of Prxs have rarely, if ever, been observed in parasites, or have largely escaped detection thus far.
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http://dx.doi.org/10.1016/j.molbiopara.2016.03.005DOI Listing
July 2017

Supramolecular self-assembly of graphene oxide and metal nanoparticles into stacked multilayers by means of a multitasking protein ring.

Nanoscale 2016 Mar;8(12):6739-53

Dept. of Life, Health and Environmental Sciences, University of L'Aquila, Italy.

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.
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http://dx.doi.org/10.1039/c5nr08632aDOI Listing
March 2016

One ring (or two) to hold them all – on the structure and function of protein nanotubes.

FEBS J 2015 Aug 23;282(15):2827-45. Epub 2015 Jun 23.

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

Understanding the structural determinants relevant to the formation of supramolecular assemblies of homo-oligomeric proteins is a traditional and central scope of structural biology. The knowledge thus gained is crucial both to infer their physiological function and to exploit their architecture for bionanomaterials design. Protein nanotubes made by one-dimensional arrays of homo-oligomers can be generated by either a commutative mechanism, yielding an 'open' structure (e.g. actin), or a noncommutative mechanism, whereby the final structure is formed by hierarchical self-assembly of intermediate 'closed' structures. Examples of the latter process are poorly described and the rules by which they assemble have not been unequivocally defined. We have collected and investigated examples of homo-oligomeric circular arrangements that form one-dimensional filaments of stacked rings by the noncommutative mechanism in vivo and in vitro. Based on their quaternary structure, circular arrangements of protein subunits can be subdivided into two groups that we term Rings of Dimers (e.g. peroxiredoxin and stable protein 1) and Dimers of Rings (e.g. thermosome/rosettasome), depending on the sub-structures that can be identified within the assembly (and, in some cases, populated in solution under selected experimental conditions). Structural analysis allowed us to identify the determinants by which ring-like molecular chaperones form filamentous-like assemblies and to formulate a novel hypothesis by which nanotube assembly, molecular chaperone activity and macromolecular crowding may be interconnected.
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http://dx.doi.org/10.1111/febs.13336DOI Listing
August 2015

Metal-induced self-assembly of peroxiredoxin as a tool for sorting ultrasmall gold nanoparticles into one-dimensional clusters.

Nanoscale 2014 Jul;6(14):8052-61

Dept. of Life, Health and Environmental Sciences, University of L'Aquila, Piazzale Salvatore Tommasi 1, 67100 L'Aquila, Italy.

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.
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http://dx.doi.org/10.1039/c4nr01526fDOI Listing
July 2014

Kineococcus radiotolerans Dps forms a heteronuclear Mn-Fe ferroxidase center that may explain the Mn-dependent protection against oxidative stress.

Biochim Biophys Acta 2013 Jun 8;1830(6):3745-55. Epub 2013 Feb 8.

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

Background: The ferroxidase center of DNA-binding protein from starved cells (Dps) is a major player in the iron oxidation/detoxification process that leads to a decreased reactive oxygen species production. The possible Mn(II) participation in this process has been studied in Dps from Kineococcus radiotolerans, a radiation-resistant bacterium with a high cytosolic Mn/Fe ratio and a high capacity to survive ionizing and stress conditions.

Methods: The X-ray structure of recombinant K. radiotolerans Dps loaded with Mn(II) has been solved at 2.0Å resolution. Mn(II) binding to K. radiotolerans Dps and its effect on Fe(II) oxidation have been characterized in spectroscopic measurements.

Results: In K. radiotolerans Dps, the Fe-Fe ferroxidase center can have a Mn-Fe composition. Mn(II) binds only at the high affinity, so-called A site, whereas Fe(II) binds also at the low affinity, so-called B site. The Mn-Fe and Fe-Fe centers behave distinctly upon iron oxidation by O2. A site-bound Mn(II) or Fe(II) plays a catalytic role, while B site-bound Fe(II) behaves like a substrate and can be replaced by another Fe(II) after oxidation. When H2O2 is the Fe(II) oxidant, single electrons are transferred to aromatic residues near the ferroxidase center and give rise to intra-protein radicals thereby limiting OH release in solution. The presence of the Mn-Fe center results in significant differences in the development of such intra-protein radicals.

Conclusions: Mn(II) bound at the Dps ferroxidase center A site undergoes redox cycling provided the B site contains Fe.

General Significance: The results provide a likely molecular mechanism for the protective role of Mn(II) under oxidative stress conditions as it participates in redox cycling in the hetero-binuclear ferroxidase center.
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http://dx.doi.org/10.1016/j.bbagen.2013.02.003DOI Listing
June 2013

Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations.

PLoS One 2012 28;7(2):e32555. Epub 2012 Feb 28.

UT-BIORAD-FARM CR Casaccia ENEA, Rome, Italy.

Protein tyrosine phosphatase ρ (PTPρ) belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032555PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3289658PMC
July 2012

Dps proteins prevent Fenton-mediated oxidative damage by trapping hydroxyl radicals within the protein shell.

Free Radic Biol Med 2010 Jan 3;48(2):292-7. Epub 2009 Nov 3.

CNR Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi-Fanelli, Sapienza University of Rome, 00185 Rome, Italy.

Dps (DNA-binding proteins from starved cells) proteins belong to a widespread bacterial family of proteins expressed under nutritional and oxidative stress conditions. In particular, Dps proteins protect DNA against Fenton-mediated oxidative stress, as they catalyze iron oxidation by hydrogen peroxide at highly conserved ferroxidase centers and thus reduce significantly hydroxyl radical production. This work investigates the possible generation of intraprotein radicals during the ferroxidation reaction by Escherichia coli and Listeria innocua Dps, two representative members of the family. Stopped-flow analyses show that the conserved tryptophan and tyrosine residues located near the metal binding/oxidation center are in a radical form after iron oxidation by hydrogen peroxide. DNA protection assays indicate that the presence of both residues is necessary to limit release of hydroxyl radicals in solution and the consequent oxidative damage to DNA. In general terms, the demonstration that conserved protein residues act as a trap that dissipates free electrons generated during the oxidative process brings out a novel role for the Dps protein cage.
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http://dx.doi.org/10.1016/j.freeradbiomed.2009.10.053DOI Listing
January 2010