Maria Rosalia Mangione

Human Hsp60 with Its Mitochondrial Import Signal Occurs in Solution as Heptamers and Tetradecamers Remarkably Stable over a Wide Range of Concentrations

It has been established that Hsp60 can accumulate in the cytosol in various pathological conditions, including cancer and chronic inflammatory diseases. Part or all of the cytosolic Hsp60 could be naïve, namely, bear the mitochondrial import signal (MIS), but neither the structure nor the in solution oligomeric organization of this cytosolic molecule has still been elucidated. Here we present a detailed study of the structure and self-organization of naïve cytosolic Hsp60 in solution. Results were obtained by different biophysical methods (light and X ray scattering, single molecule spectroscopy and hydrodynamics) that all together allowed us to assay a wide range of concentrations of Hsp60. We found that Naïve Hsp60 in aqueous solution is assembled in very stable heptamers and tetradecamers at all concentrations assayed, without any trace of monomer presence.

The Tempered Polymerization of Human Neuroserpin

Neuroserpin, a member of the serpin protein superfamily, is an inhibitor of proteolytic activity that is involved in pathologies such as ischemia, Alzheimers disease, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). The latter belongs to a class of conformational diseases, known as serpinopathies, which are related to the aberrant polymerization of serpin mutants. Neuroserpin is known to polymerize, even in its wild type form, under thermal stress. Here, we study the mechanism of neuroserpin polymerization over a wide range of temperatures by different techniques. Our experiments show how the onset of polymerization is dependent on the formation of an intermediate monomeric conformer, which then associates with a native monomer to yield a dimeric species. After the formation of small polymers, the aggregation proceeds via monomer addition as well as polymer-polymer association. No further secondary mechanism takes place up to very high temperatures, thus resulting in the formation of neuroserpin linear polymeric chains. Most interesting, the overall aggregation is tuned by the co-occurrence of monomer inactivation (i.e. the formation of latent neuroserpin) and by a mechanism of fragmentation. The polymerization kinetics exhibit a unique modulation of the average mass and size of polymers, which might suggest synchronization among the different processes involved. Thus, fragmentation would control and temper the aggregation process, instead of enhancing it, as typically observed (e.g.) for amyloid fibrillation.

Pectin from Opuntia ficus indica: Optimization of microwave-assisted extraction and preliminary characterization

Optimization of microwave-assisted extraction (MAE) of water-soluble pectin (WSP) from Opuntia ficus indica cladodes was performed using Response Surface Methodology. The effect of extraction time (X1), microwave power (X2), pH (X3) and solid-to-liquid ratio (X4) on the extraction yield was examined. The optimum conditions of MAE were as follows: X1=2.15min; X2=517W; X3=2.26 and X4=2g/30.6mL. The maximum obtained yield of pectin extraction was 12.57%. Total carbohydrate content of WSP is about 95.5% including 34.4% of Galacturonic acid. Pectin-related proteins represent only the 0.66% of WSP mass. HPSEC and light scattering analyses reveal that WSP is mostly constituted of high molecular pectin and FTIR measurements show that the microwave treatment does not alter the chemical structure of WSP, in which Galacturonic acid content and yield are 34.4% and 4.33%, respectively. Overall, application of MAE can give rise to high quality pectin.

On the origin of functionalization in one-pot radiation synthesis of nanogels from aqueous polymer solutions

Radiation-engineered poly(N-vinyl pyrrolidone) nanogels are very interesting biocompatible nanocarriers for i.v. administration of therapeutics and contrast agents for bioimaging. The manufacturing process is fast and effective, it grants excellent control of particle size and simultaneous sterilization of the formed nanogels. Interestingly, primary amino groups and carboxyl groups, useful for (bio)conjugation, are also formed in a dose-dependent fashion. In this paper, by means of both numerical simulations and experiments, the origin of nanogel size control and functionalization is investigated. This understanding offers a new dimension for the design and production of radiation-sculptured multifunctional nanocarriers from aqueous solutions of polymers.

Photo-inhibition of Aβ fibrillation mediated by a newly designed fluorinated oxadiazole

Uncontrolled aggregation of amyloid beta peptide (Aβ) is the main cause of Alzheimers disease. Therapeutic approaches to intervention in amyloid diseases include the use of small molecules able to stabilize the soluble Aβ conformation, or to redirect the amyloidogenic pathway towards non-toxic and non-fibrillar states. Fluorometric measurements revealed that 3-(4′-trifluoromethylphenyl)-5-(4′-methoxyphenyl)-1,2,4-oxadiazole, when irradiated, is able to interact with the monomeric Aβ peptide readdressing the aggregation pathway toward the formation of amorphous aggregates as evidenced by CD, AFM, and SAXS measurements. We hypothesize that this compound, under radiation, forms a reactive intermediate that sticks on the Aβ peptide by interfering with its fibrillation process. Cytotoxicity assays performed on LAN5 neuroblastoma cells suggest that the presence of oxadiazole reduces the toxicity of Aβ. This finding might be the start of innovative therapies against Alzheimers disease.

Hsp60, amateur chaperone in amyloid-beta fibrillogenesis.

BACKGROUND Molecular chaperones are a very special class of proteins that play essential roles in many cellular processes like folding, targeting and transport of proteins. Moreover, recent evidence indicates that chaperones can act as potentially strong suppressor agents in Alzheimers disease (AD). Indeed, in vitro experiments demonstrate that several chaperones are able to significantly slow down or suppress aggregation of Aβ peptide and in vivo studies reveal that treatment with specific chaperones or their overexpression can ameliorate some distinct pathological signs characterizing AD. METHODS Here we investigate using a biophysical approach (fluorescence, circular dichroism (CD), transmission electron (TEM) and atomic force (AFM) microscopy, size exclusion chromatography (SEC)) the effect of the human chaperonin Hsp60 on Aβ fibrillogenesis. RESULTS We found that Hsp60 powerfully inhibits Aβ amyloid aggregation, by closing molecular pathways leading to peptide fibrillogenesis. CONCLUSIONS We observe that Hsp60 inhibits Aβ aggregation through a more complex mechanism than a simple folding chaperone action. The action is specifically directed toward the early oligomeric species behaving as aggregation seeds for on-pathway amyloid fibrillogenesis. GENERAL SIGNIFICANCE Understanding the specificity of the molecular interactions of Hsp60 with amyloid Aβ peptide allowed us to emphasize the important aspects to be taken into consideration when considering the recent promising therapeutic strategies for neurodegeneration.

Entrapment of A?1?40 peptide in unstructured aggregates

Recognizing the complexity of the fibrillogenesis process provides a solid ground for the development of therapeutic strategies aimed at preventing or inhibiting protein-protein aggregation. Under this perspective, it is meaningful to identify the possible aggregation pathways and their relative products. We found that Aβ-peptide dissolved in a pH 7.4 solution at small peptide concentration and low ionic strength forms globular aggregates without typical amyloid β-conformation. ThT binding kinetics was used to monitor aggregate formation. Circular dichroism spectroscopy, AFM imaging, static and dynamic light scattering were used for structural and morphological characterization of the aggregates. They appear stable or at least metastable with respect to fiber growth, therefore appearing as an incidental product in the pathway of fibrillogenesis.

On the molecular structure of human neuroserpin polymers

The polymerization of serpins is at the root of a large class of diseases; the molecular structure of serpin polymers has been recently debated. In this work, we study the polymerization kinetics of human neuroserpin by Fourier Transform Infra Red spectroscopy and by time‐lapse Size Exclusion Chromatography. First, we show that two distinct neuroserpin polymers, formed at 45 and 85°C, display the same isosbestic points in the Amide I′ band, and therefore share common secondary structure features. We also find a concentration independent polymerization rate at 45°C suggesting that the polymerization rate‐limiting step is the formation of an activated monomeric species. The polymer structures are consistent with a model that predicts the bare insertion of portions of the reactive center loop into the A β‐sheet of neighboring serpin molecule, although with different extents at 45 and 85°C. Proteins 2012;

Functional and dysfunctional conformers of human neuroserpin characterized by optical spectroscopies and molecular dynamics

Neuroserpin (NS) is a serine protease inhibitor (SERPIN) involved in different neurological pathologies, including the Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), related to the aberrant polymerization of NS mutants. Here we present an in vitro and in silico characterization of native neuroserpin and its dysfunctional conformation isoforms: the proteolytically cleaved conformer, the inactive latent conformer, and the polymeric species. Based on circular dichroism and fluorescence spectroscopy, we present an experimental validation of the latent model and highlight the main structural features of the different conformers. In particular, emission spectra of aromatic residues yield distinct conformational fingerprints, that provide a novel and simple spectroscopic tool for selecting serpin conformers in vitro. Based on the structural relationship between cleaved and latent serpins, we propose a structural model for latent NS, for which an experimental crystallographic structure is lacking. Molecular Dynamics simulations suggest that NS conformational stability and flexibility arise from a spatial distribution of intramolecular salt-bridges and hydrogen bonds.

Curcumin-like compounds designed to modify amyloid beta peptide aggregation patterns

Curcumin is a natural polyphenol able to bind the amyloid beta peptide, which is related to Alzheimer’s disease, and modify its self-assembly pathway. This paper focuses on a multi-disciplinary study that starts from the design of curcumin-like compounds with the key chemical features required for inhibiting amyloid beta aggregation, and reports the effects of these compounds on the in vitro aggregation of amyloid beta peptides. Chemoinformatic screening was performed through the calculation of molecular descriptors that were able to highlight the drug-like profile, followed by docking studies with an amyloid beta peptide fibril. The computational design underlined two different scaffolds that were easily synthesized in good yields. In vitro experiments, ranging from fluorescence spectroscopy and confocal microscopy up to small angle X-ray scattering, provided evidence that the synthesized compounds are able to modify the aggregation pattern of amyloid beta peptides both in the secondary structures, and in terms of the overall structure dimensions. The cytotoxic potential of the synthesized compounds was finally tested in vitro with a model neuronal cell line (LAN5). The overall view of this study suggests new concepts and potential difficulties in the design of novel drugs against diverse amyloidoses, including Alzheimer’s disease.