Oligomeric State and Holding Activity of Hsp60.

Similar to its bacterial homolog GroEL, Hsp60 in oligomeric conformation is known to work as a folding machine, with the assistance of co-chaperonin Hsp10 and ATP. However, recent results have evidenced that Hsp60 can stabilize aggregation-prone molecules in the absence of Hsp10 and ATP by a different, "holding-like" mechanism. Here, we investigated the relationship between the oligomeric conformation of Hsp60 and its ability to inhibit fibrillization of the Ab40 peptide. The monomeric or tetradecameric form of the protein was isolated, and its effect on beta-amyloid aggregation was separately tested. The structural stability of the two forms of Hsp60 was also investigated using differential scanning calorimetry (DSC), light scattering, and circular dichroism. The results showed that the protein in monomeric form is less stable, but more effective against amyloid fibrillization. This greater functionality is attributed to the disordered nature of the domains involved in subunit contacts.

Stable nanovesicles formed by intrinsically planar bilayers.

HYPOTHESIS: Quatsome nanovesicles, formed through the self-assembly of cholesterol (CHOL) and cetyltrimethylammonium bromide (CTAB) in water, have shown long-term stability in terms of size and morphology, while at the same time exhibiting high CHOL-CTAB intermolecular binding energies. We hypothesize that CHOL/CTAB quatsomes are indeed thermodynamically stable nanovesicles, and investigate the mechanism underlying their formation. EXPERIMENTS: A systematic study was performed to determine whether CHOL/CTAB quatsomes satisfy the experimental requisites of thermodynamically stable vesicles. Coarse-grain molecular dynamics simulations were used to investigate the molecular organization in the vesicle membrane, and the characteristics of the simulated vesicle were corroborated with experimental data obtained by cryo-electron microscopy, small- and wide-angle X-ray scattering, and multi-angle static light scattering. FINDINGS: CHOL/CTAB quatsomes fulfill the requisites of thermodynamically stable nanovesicles, but they do not exhibit the classical membrane curvature induced by a composition asymmetry between the bilayer leaflets, like catanionic nanovesicles. Instead, CHOL/CTAB quatsomes are formed through the association of intrinsically planar bilayers in a faceted vesicle with defects, indicating that distortions in the organization and orientation of molecules can play a major role in the formation of thermodynamically stable nanovesicles.

From Small Peptides to Large Proteins against Alzheimer'sDisease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly. The two cardinal neuropathological hallmarks of AD are the senile plaques, which are extracellular deposits mainly constituted by beta-amyloids, and neurofibrillary tangles formed by abnormally phosphorylated Tau (p-Tau) located in the cytoplasm of neurons. Although the research has made relevant progress in the management of the disease, the treatment is still lacking. Only symptomatic medications exist for the disease, and, in the meantime, laboratories worldwide are investigating disease-modifying treatments for AD. In the present review, results centered on the use of peptides of different sizes involved in AD are presented.

Recombinant mussel protein Pvfp5ß enhances cell adhesion of poly(vinyl alcohol)/k-carrageenan hydrogel scaffolds.

Polymeric hydrogels are increasingly considered as scaffolds for tissue engineering due to their extraordinary resemblance with the extracellular matrix (ECM) of many tissues. As cell adhesion is a key factor in regulating important cell functions, hydrogel scaffolds are often functionalized or loaded with a variety of bioactive molecules that can promote adhesion. Interesting biomimetic approaches exploit the properties of mussel-inspired recombinant adhesive proteins. In this work, we prepared hydrogel scaffolds with a 50%w mixture of k-carrageenan (kC) and polyvinyl alcohol (PVA), by a two-step physical gelation process, and we coated them with Perna viridis foot protein-5ß (Pvfp5ß). The mechanical and morphological properties of hydrogels were investigated both after conditioning with typical cell culture media and also after coating with the Pvfp5ß. The protein resulted strongly adsorbed onto the surface of the hydrogel and also able to penetrate in its interiors to a certain depth, mainly interacting with the kC component of the scaffold as resulted from the confocal analysis. Mouse embryonic fibroblasts NIH-3T3 were seeded on top of the hydrogels and cultured up to two weeks. The role of Pvfp5ß in promoting cell adhesion, spreading and colonization of the scaffold was demonstrated.

Recovery from Food Waste-Biscuit Doughs Enriched with Pomegranate Peel Powder as a Model of Fortified Aliment.

The aim of the present work is the characterization of biscuit doughs enriched with pomegranate peel powder (PPP) at 3 (PPP3) and 5 (PPP5) wt% in the prospect of developing a fortified aliment as a support of the therapy of chronic inflammatory diseases of the intestinal tract. The total phenolic content of the powder was preliminarily evaluated. Then, the main compounds present in the PPP were identified by HPLC-ESI-TOF-MS analysis, being mainly hydrolysable tannins. The PPP was then treated at 180 °C for 20 min to mimic the baking treatment, and its water-soluble fraction (PPPwsf) was then added in the Caco-2 cell culture as a model of the intestinal epithelial barrier to verify its dose-dependent toxicity, ability in counteracting the oxidative stress, and anti-inflammatory action. Rheological experiments were performed to predict the macroscopic behavior of the PPP-added doughs during lamination and biscuit baking. SEM investigations gave their contribution to the microscopic comprehension of the dough structure. Finally, a consumer panel composed by thirty volunteers was enrolled to express its opinion on the sensory agreeableness of the biscuits prepared with two different concentrations of PPP compared with the reference dough. The discussion is focused on the biological effects of the main components found in the PPP.

Synaptosomes: new vesicles for neuronal mitochondrial transplantation.

BACKGROUND: Mitochondrial dysfunction is a critical factor in the onset and progression of neurodegenerative diseases. Recently, mitochondrial transplantation has been advised as an innovative and attractive strategy to transfer and replace damaged mitochondria. Here we propose, for the first time, to use rat brain extracted synaptosomes, a subcellular fraction of isolated synaptic terminal that contains mitochondria, as mitochondrial delivery systems. RESULTS: Synaptosome preparation was validated by the presence of Synaptophysin and PSD95. Synaptosomes were characterized in terms of dimension, zeta potential, polydispersity index and number of particles/ml. Nile Red or CTX-FITCH labeled synaptosomes were internalized in LAN5 recipient cells by a mechanism involving specific protein-protein interaction, as demonstrated by loss of fusion ability after trypsin treatment and using different cell lines. The loading and release ability of the synaptosomes was proved by the presence of curcumin both into synaptosomes and LAN5 cells. The vitality of mitochondria transferred by Synaptosomes was demonstrated by the presence of Opa1, Fis1 and TOM40 mitochondrial proteins and JC-1 measurements. Further, synaptosomes deliver vital mitochondria into the cytoplasm of neuronal cells as demonstrated by microscopic images, increase of TOM 40, cytochrome c, Hexokinase II mitochondrial proteins, and presence of rat mitochondrial DNA. Finally, by using synaptosomes as a vehicle, healthy mitochondria restored mitochondrial function in cells containing rotenone or CCCp damaged mitochondria. CONCLUSIONS: Taken together these results suggest that synaptosomes can be a natural vehicle for the delivery of molecules and organelles to neuronal cells. Further, the replacement of affected mitochondria with healthy ones could be a potential therapy for treating neuronal mitochondrial dysfunction-related diseases.

The degree of compactness of the incipient High Methoxyl Pectin networks. A rheological insight at the sol-gel transition.

Fractal analysis can be properly applied to complex structures, like physical and chemical networks formed by particles or polymers, when they exhibit self-similarity over an extended range of length scales and, hence, can be profitably used not only for their morphological characterization but also for individuating possible relationships between morphology and mechanisms of aggregation and crosslinking, as well as between morphology and physical properties. Several experimental methods are available to determine the fractal dimension of gel networks, including various scattering techniques and microscopies, permeability measurements and rheology. The present study regards the self-assembly kinetics of High Methoxyl Pectin (HMP) solutions with different pectin and sucrose concentrations investigated by rheological measurements to highlight the effects of pectin and sucrose concentrations on the gel point and to evaluate the degree of compactness of the incipient gel networks through an interpretation of the viscoelastic response at the sol-gel transition.

Immunomorphological Pattern of Molecular Chaperones in Normal and Pathological Thyroid Tissues and Circulating Exosomes: Potential Use in Clinics.

The thyroid is a major component of the endocrine system and its pathology can cause serious diseases, e.g., papillary carcinoma (PC). However, the carcinogenic mechanisms are poorly understood and clinical useful biomarkers are scarce. Therefore, we determined if there are quantitative patterns of molecular chaperones in the tumor tissue and circulating exosomes that may be useful in diagnosis and provide clues on their participation in carcinogenesis. Hsp27, Hsp60, Hsp70, and Hsp90 were quantified by immunohistochemistry in PC, benign goiter (BG), and normal peritumoral tissue (PT). The same chaperones were assessed in plasma exosomes from PC and BG patients before and after ablative surgery, using Western blotting. Hsp27, Hsp60, and Hsp90 were increased in PC in comparison with PT and BG but no differences were found for Hsp70. Similarly, exosomal levels of Hsp27, Hsp60, and Hsp90 were higher in PC than in BG, and those in PC were higher before ablative surgery than after it. Hsp27, Hsp60, and Hsp90 show distinctive quantitative patterns in thyroid tissue and circulating exosomes in PC as compared with BG, suggesting some implication in the carcinogenesis of these chaperones and indicating their potential as biomarkers for clinical applications.

Recombinant mussel protein Pvfp-5ß: A potential tissue bioadhesive.

During their lifecycle, many marine organisms rely on natural adhesives to attach to wet surfaces for movement and self-defense in aqueous tidal environments. Adhesive proteins from mussels are biocompatible and elicit only minimal immune responses in humans. Therefore these proteins have received increased attention for their potential applications in medicine, biomaterials, and biotechnology. The Asian green mussel Perna viridis secretes several byssal plaque proteins, molecules that help anchoring the mussel to surfaces. Among these proteins, protein-5ß (Pvfp-5ß) initiates interactions with the substrate, displacing interfacial water molecules before binding to the surface. Here, we established the first recombinant expression in Escherichia coli of Pvfp-5ß. We characterized recombinant Pvfp-5ß, finding that despite displaying a CD spectrum consistent with features of a random coil, the protein is correctly folded as indicated by MS and NMR analyses. Pvfp-5ß folds as a ß-sheet-rich protein as expected for an epidermal growth factor-like module. We examined the effects of Pvfp-5ß on cell viability and adhesion capacity in NIH-3T3 and HeLa cell lines, revealing that Pvfp-5ß has no cytotoxic effects at the protein concentrations used and provides good cell-adhesion strength on both glass and plastic plates. Our findings suggest that the adhesive properties of recombinant Pvfp-5ß make it an efficient surface-coating material, potentially suitable for biomedical applications including regeneration of damaged tissues.

A Multipronged Method for Unveiling Subtle Structural-Functional Defects of Mutant Chaperone Molecules Causing Human Chaperonopathies.

Chaperonopathies are diseases in which abnormal chaperones play an etiopathogenic role. A chaperone is mutated or otherwise abnormal (e.g., modified by an aberrant posttranslational modification) in structure/function. To understand the pathogenic mechanisms of chaperonopathies, it is necessary to elucidate the impact of the pathogenic mutation or posttranslational modification on the chaperone molecule's properties and functions. This impact is usually subtle because if it were more than subtle the overall effect on the cell and organism would be catastrophic, lethal. This is because most chaperones are essential for life and, if damaged in structure/function too strongly, there would be death of the cell/organism, and no phenotype, i.e., there would be no patients with chaperonopathies. Consequently, diagnostic procedures and analysis of defects of the abnormal chaperones require a multipronged method for assessing the chaperone molecule from various angles. Here, we present such a method that includes assessing the intrinsic properties and the chaperoning functions of chaperone molecules.

Biochemical and biophysical characterization of water-soluble pectin from Opuntia ficus-indica and its potential cytotoxic activity.

This work aims to fill the gap in the present knowledge about the structure of pectin from Opuntia ficus-indica. The water-soluble pectin (WSP) fraction, extracted with the Microwave Assisted Extraction (MAE), was further deproteinated (dWSP) and analyzed through several biophysical and biochemical techniques. HPSEC, light scattering and FTIR data showed that dWSP is low methylated high molecular weight pectin. The biochemical structure of dWSP, after methanolysis, silylation, carboxyl reduction showed that dWSP belongs to rhamnogalacturonan I class. Then, dWSP was heat-modified (HM) to obtain small-molecular weight deproteinated fraction (HM-dWSP). Both species, dWSP and HM-dWSP, were tested in LAN5 and NIH 3T3 model cells to study their biological effect. Results indicated that both dWSP and HM-dWSP exerted cytotoxic activity affecting selectively LAN5 cancer cells, without any effect on NIH 3T3 normal cells.

Data concerning the rheological behavior of high methoxyl pectin during gelation process.

The present data concern the structuring kinetics of aqueous high methoxyl pectin (HMP) solutions at acid pH (3.1), constant pectin concentration (0.2% w/w) and sucrose concentrations ranging from 56 to 65% w/w. Consecutive frequency sweep was applied to samples immediately after their preparation. The generalized Maxwell (gM) model was used to describe the change of the mechanical spectra for each different sucrose concentration and to determine the viscoelastic parameters controlling the gelation of the HMP solutions. The viscosities in the sol region are explored in the range 0 to 55% 0 to 40% (w/w) sucrose concentration.

The role of sucrose concentration in self-assembly kinetics of high methoxyl pectin.

Several natural and synthetic polysaccharides are able to form, under appropriate conditions, supramolecular structures, typically physical hydrogels, and, together with their biocompatibility, this explains their wide use in food, pharmaceutical and biomedical sectors. In the case of high methoxyl pectins (HMP) the gel formation is promoted by the presence of cosolutes (sugars or polyols) and low pH. The present investigation mainly regards the structuring kinetics of aqueous HMP solutions at acid pH (3.1) with the same pectin concentration (0.2% w/w) and different sucrose concentrations (from 56 to 65% w/w). Preliminary viscosity tests were performed to individuate the threshold of the sol region. A sequence of consecutive frequency sweeps was applied to each sample immediately after its preparation. The time evolution of the linear viscoelastic behavior is described by the sigmoidal profiles of both moduli at each applied frequency and more thoroughly defined through the change of the mechanical spectrum, i.e. the variation of the parameters of the generalized Maxwell model or the Friedrich-Braun model which are both suitable to provide a satisfactory data fitting. In particular, the equilibrium modulus Ge offers a significant description of the gelation kinetics and its sucrose dependence.

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.

Physico-chemical and mechanical characterization of in-situ forming xyloglucan gels incorporating a growth factor to promote cartilage reconstruction.

The development of growth factors is very promising in the field of tissue regeneration but specifically designed formulations have to be developed in order to enable such new biological entities (NBEs). In particular, the range of therapeutic concentrations is usually very low compared to other active proteins and the confinement in the target site can be of crucial importance. In-situ forming scaffolds are very promising solutions for minimally invasive intervention in cartilage reconstruction and targeting of NBEs. In this work injectable, in-situ forming gels of a temperature responsive partially degalactosylated xyloglucan (Deg-XG) incorporating the growth factor FGF-18 are formulated and characterized. In particular, injectability and shear viscosity at room temperature, time-to-gel at body temperature, morphology and mechanical properties of gels are investigated. The highly hydrophobic growth factor is favorably incorporated and retained by the gel. Gels undergo a slow erosion process when immersed in PBS at 37°C that opens up their porous structure. The prolonged hydrothermal treatment leads to structural rearrangements towards tougher networks with increased dynamic shear modulus. Preliminary biological evaluations confirm absence of cytotoxicity and the ability of these scaffolds to host cells and promote their proliferation.

Chaperonin of Group I: Oligomeric Spectrum and Biochemical and Biological Implications.

Chaperonins play various physiological roles and can also be pathogenic. Elucidation of their structure, e.g., oligomeric status and post-translational modifications (PTM), is necessary to understand their functions and mechanisms of action in health and disease. Group I chaperonins form tetradecamers with two stacked heptameric rings. The tetradecamer is considered the typical functional complex for folding of client polypeptides. However, other forms such as the monomer and oligomers with smaller number of subunits than the classical tetradecamer, also occur in cells. The properties and functions of the monomer and oligomers, and their roles in chaperonin-associated diseases are still incompletely understood. Chaperonin I in eukaryotes occurs in various locations, not just the mitochondrion, which is its canonical place of residence and function. Eukaryotic Chaperonin I, namely Hsp60 (designated HSP60 or HSPD1 in humans) has, indeed, been found in the cytosol; the plasma-cell membrane; on the outer surface of cells; in the intercellular space; in biological liquids such as lymph, blood, and cerebrospinal fluid; and in secretions, for instance saliva and urine. Hsp60 has also been found in cell-derived vesicles such as exosomes. The functions of Hsp60 in all these non-canonical locales are still poorly characterized and one of the questions not yet answered is in what form, i.e., monomer or oligomer, is the chaperonin present in these non-canonical locations. In view of the steady increase in interest on chaperonopathies over the last several years, we have studied human HSP60 to determine its role in various diseases, its locations in cells and tissues and migrations in the body, and its post-translational modifications that might have an impact on its location and function. We also carried out experiments to characterize the oligomeric status of extramitochondrial of HSP60 in solution. Here, we provide an overview of our results, focusing on the oligomeric equilibrium and stability of the various forms of HSP60 in comparison with GroEL. We also discuss post-translational modifications associated with anti-cancer drugs to indicate the potential of Hsp60 in Medicine, as a biomarker and etiopathogenic factor.

Quantitative analysis of the impact of a human pathogenic mutation on the CCT5 chaperonin subunit using a proxy archaeal ortholog.

The human chaperonin complex is a ~ 1 MDa nanomachine composed of two octameric rings formed from eight similar but non-identical subunits called CCT. Here, we are elucidating the mechanism of a heritable CCT5 subunit mutation that causes profound neuropathy in humans. In previous work, we introduced an equivalent mutation in an archaeal chaperonin that assembles into two octameric rings like in humans but in which all subunits are identical. We reported that the hexadecamer formed by the mutant subunit is unstable with impaired chaperoning functions. This study quantifies the loss of structural stability in the hexadecamer due to the pathogenic mutation, using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The disassembly of the wild type complex, which is tightly coupled with subunit denaturation, was decoupled by the mutation without affecting the stability of individual subunits. Our results verify the effectiveness of the homo-hexadecameric archaeal chaperonin as a proxy to assess the impact of subtle defects in heterologous systems with mutations in a single subunit.

Multi-Functional Nanogels for Tumor Targeting and Redox-Sensitive Drug and siRNA Delivery.

(1) Background: A new family of nanosystems able to discern between normal and tumor cells and to release a therapeutic agent in controlled way were synthetized by e-beam irradiation. This technique permits to obtain biocompatible, sterile, carboxyl-functionalized polyvinylpyrrolidone (PVP-co-acrylic acid) nanogels (NGs); (2) Methods: Here, we performed a targeting strategy based on the recognition of over-expressed proteins on tumor cells, like the folate receptor. The selective targeting was demonstrated by co-culture studies and flow cytometry analysis, using folate conjugated NGs. Moreover, nanoparticles were conjugated to a chemotherapeutic drug or to a pro-apoptotic siRNA through a glutathione sensitive spacer, in order to obtain a controlled release mechanism, specific for cancer cells. The drug efficiency was tested on tumor and healthy cells by flow cytometric analysis, confocal and epifluorescence microscopy and cytotoxicity assay; the siRNA effect was investigated by RNAi experiment; (3) Results: The data obtained showed that the use of NGs permits a faster cargo release in cancer cells, in response to high cytosolic glutathione level, also improving their efficacy; (4) Conclusion: The possibility of releasing biological molecules in a controlled way and to recognize a specific tumor target allows overcoming the typical limits of the classic cancer therapy.

Temporal control of xyloglucan self-assembly into layered structures by radiation-induced degradation.

Partially degalactosylated xyloglucan from tamarind seeds (Deg-XG) is a very appealing biopolymer for the production of in situ gelling systems at physiological temperature. In this work, we observe that the morphology of hydrogels evolves towards high degrees of structural organization with time, yielding to dense stacks of thin membranes within 24h of incubation at 37°C. We also explore the possibility offered by gamma irradiation of controlling the time scale of this phenomenon, the final morphology and mechanical properties of the system. Structural and molecular modifications of Deg-XG with dose are investigated by FTIR, dynamic light scattering (DLS) and rotational viscosimetry. The impact on gelation ability and gel strength is studied by rheological analysis. The morphology evolution is investigated by SEM analysis, and absence of cytotoxicity verified by MTS assay and optical microscopy of neuroblastoma cells.

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 Alzheimer's disease (AD). Indeed, in vitro experiments demonstrate that several chaperones are able to significantly slow down or suppress aggregation of Aß 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 Aß fibrillogenesis. RESULTS: We found that Hsp60 powerfully inhibits Aß amyloid aggregation, by closing molecular pathways leading to peptide fibrillogenesis. CONCLUSIONS: We observe that Hsp60 inhibits Aß 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 Aß peptide allowed us to emphasize the important aspects to be taken into consideration when considering the recent promising therapeutic strategies for neurodegeneration.