Cell-Free and In Vivo Characterization of the Inhibitory Activity of Lavado Cocoa Flavanols on the Amyloid Protein Ataxin-3: Toward New Approaches against Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder characterized by ataxia and other neurological manifestations, with a poor prognosis and a lack of effective therapies. The amyloid aggregation of the ataxin-3 protein is a hallmark of SCA3 and one of the main biochemical events prompting its onset, making it a prominent target for the development of preventive and therapeutic interventions. Here, we tested the efficacy of an aqueous Lavado cocoa extract and its polyphenolic components against ataxin-3 aggregation and neurotoxicity. The combination of biochemical assays and atomic force microscopy morphological analysis provided clear evidence of cocoa flavanols' ability to hinder ATX3 amyloid aggregation through direct physical interaction, as assessed by NMR spectroscopy. The chemical identity of the flavanols was investigated by ultraperformance liquid chromatography-high-resolution mass spectrometry. The use of the preclinical model Caenorhabditis elegans allowed us to demonstrate cocoa flavanols' ability to ameliorate ataxic phenotypes in vivo. To the best of our knowledge, Lavado cocoa is the first natural source whose extract is able to directly interfere with ATX3 aggregation, leading to the formation of off-pathway species.

HspB8 interacts with BAG3 in a "native-like" conformation forming a complex that displays chaperone-like activity.

The HspB8-BAG3 complex plays an important role in the protein quality control acting alone or within multi-components complexes. To clarify the mechanism underlying its activity, in this work we used biochemical and biophysical approaches to study the tendency of both proteins to auto-assemble and to form the complex. Solubility and Thioflavin T assays, Fourier transform infrared spectroscopy and atomic force microscopy analyses clearly showed the tendency of HspB8 to self-assemble at high concentration and to form oligomers in a "native-like" conformation; otherwise, BAG3 aggregates poorly. Noteworthy, also HspB8 and BAG3 associate in a "native-like" conformation, forming a stable complex. Furthermore, the high difference between dissociation constant values of HspB8-HspB8 interaction with respect to the binding to BAG3 obtained by surface plasmon resonance confirms that HspB8 is an obligated partner of BAG3 in vivo. Lastly, both proteins alone or in the complex are able to bind and affect the aggregation of the Josephin domain, the structured domain that triggers the ataxin-3 fibrillation. In particular, the complex displayed higher activity than HspB8 alone. All this considered, we can assert that the two proteins form a stable assembly with chaperone-like activity that could contribute to the physiological role of the complex in vivo.

Cheese-whey permeate improves the fitness of Escherichia coli cells during recombinant protein production.

BACKGROUND: Escherichia coli cells are the most frequently used hosts in recombinant protein production processes and mainly require molecules such as IPTG or pure lactose as inducers of heterologous expression. A possible way to reduce the production costs is to replace traditional inducers with waste materials such as cheese whey permeate (CWP). CWP is a secondary by-product generated from the production of the valuable whey proteins, which are obtained from ultrafiltration of cheese whey, a main by-product of the dairy industry, which is rich in lactose. RESULTS: The effects of CWP collected from an Italian plant were compared with those of traditional inducers on the production of two model proteins (i.e., green fluorescent protein and the toxic Q55 variant of ataxin-3), in E. coli BL21 (DE3) cells. It was found that the high lactose content of CWP (165 g/L) and the antioxidant properties of its micronutrients (vitamins, cofactors and osmolytes) sustain production yields similar to those obtained with traditional inducers, accompanied by the improvement of cell fitness. CONCLUSIONS: CWP has proven to be an effective and low-cost alternative inducer to produce recombinant proteins. Its use thus combines the advantage of exploiting a waste product with that of reducing the production costs of recombinant proteins.

Artichoke (Cynara cardunculus var. scolymus L.) by-products as a source of inulin: how to valorise an agricultural supply chain extracting an added-value compound.

This study is aimed at valorizing artichoke (Cynara cardunculus var. scolymus L.) by-products as source of inulin, a fiber showing relevant prebiotic properties, through the realization of a waste value chain. Starting from artichoke by-products, the inulin fraction was assessed both in terms of total amount and degree of polymerization as a function of the harvest season and storage conditions. These parameters have been found significant at influencing inulin yield of extraction. For the first time, artichoke wastes were proposed to be exploited taking into account the optimal conditions to preserve their high-added chemical value. Our data suggest that Italian farms could obtain from their wastes a total amount of 16 t/year of inulin with an average polymerization degree higher than 40 and would allow the development of a circular economy process within the artichoke supply chain, by exploiting its wastes representing 70% of the total artichoke biomass.

Impact of Tuning the Surface Charge Distribution on Colloidal Iron Oxide Nanoparticle Toxicity Investigated in Caenorhabditis elegans.

Assessing the toxic effect in living organisms remains a major issue for the development of safe nanomedicines and exposure of researchers involved in the synthesis, handling and manipulation of nanoparticles. In this study, we demonstrate that Caenorhabditis elegans could represent an in vivo model alternative to superior mammalians for the collection of several physiological functionality parameters associated to both short-term and long-term effects of colloidally stable nanoparticles even in absence of microbial feeding, usually reported to be necessary to ensure appropriate intake. Contextually, we investigated the impact of surface charge on toxicity of superparamagnetic iron oxide coated with a wrapping polymeric envelop that confers them optimal colloidal stability. By finely tuning the functional group composition of this shallow polymer-obtaining totally anionic, partially pegylated, partially anionic and partially cationic, respectively-we showed that the ideal surface charge organization to optimize safety of colloidal nanoparticles is the one containing both cationic and anionic groups. Our results are in accordance with previous evidence that zwitterionic nanoparticles allow long circulation, favorable distribution in the tumor area and optimal tumor penetration and thus support the hypothesis that zwitterionic iron oxide nanoparticles could be an excellent solution for diagnostic imaging and therapeutic applications in nanooncology.

Superoxide dismutase 1 (SOD1) and cadmium: A three models approach to the comprehension of its neurotoxic effects.

Cadmium (Cd) is a widespread toxic environmental contaminant, released by anthropogenic activities. It interferes with essential metal ions homeostasis and affects protein structures and functions by substituting zinc, copper and iron. In this study, the effect of cadmium on SOD1, a CuZn metalloenzyme catalyzing superoxide conversion into hydrogen peroxide, has been investigated in three different biological models. We first evaluated the effects of cadmium combined with copper and/or zinc on the recombinant GST-SOD1, expressed in E. coli BL21. The enzyme activity and expression were investigated in the presence of fixed copper and/or zinc doses with different cadmium concentrations, in the cellular medium. Cadmium caused a dose-dependent reduction in SOD1 activity, while the expression remains constant. Similar results were obtained in the cellular model represented by the human SH-SY5Y neuronal cell line. After cadmium treatment for 24 and 48 h, SOD1 enzymatic activity decreased in a dose- and time-dependent way, while the protein expression remained constant. Finally, a 16 h cadmium treatment caused a 25 % reduction of CuZn-SOD activity without affecting the protein expression in the Caenorhabditis elegans model. Taken together our results show an inhibitory effect of cadmium on SOD1 enzymatic activity, without affecting the protein expression, in all the biological models used, suggesting that cadmium can displace zinc from the enzyme catalytic site.

Pathological ATX3 Expression Induces Cell Perturbations in E. coli as Revealed by Biochemical and Biophysical Investigations.

Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, which belongs to the class of polyglutamine neurodegenerative disorders. It is widely accepted that the formation of toxic oligomeric species is primarily involved in the onset of the disease. For this reason, to understand the mechanisms underlying toxicity, we expressed both a physiological (ATX3-Q24) and a pathological ATX3 variant (ATX3-Q55) in a simplified cellular model, Escherichia coli. It has been observed that ATX3-Q55 expression induces a higher reduction of the cell growth compared to ATX3-Q24, due to the bacteriostatic effect of the toxic oligomeric species. Furthermore, the Fourier transform infrared microspectroscopy investigation, supported by multivariate analysis, made it possible to monitor protein aggregation and the induced cell perturbations in intact cells. In particular, it has been found that the toxic oligomeric species associated with the expression of ATX3-Q55 are responsible for the main spectral changes, ascribable mainly to the cell envelope modifications. A structural alteration of the membrane detected through electron microscopy analysis in the strain expressing the pathological form supports the spectroscopic results.

Effectiveness of Vigna unguiculata seed extracts in preventing colorectal cancer.

Colorectal cancer (CRC) is one of the most common types of cancer, especially in Western countries, and its incidence rate is increasing every year. In this study, for the first time Vigna unguiculata L. Walp. (cowpea) water boiled seed extracts were found to reduce the viability of different colorectal cancer (CRC) cell lines, such as E705, DiFi and SW480 and the proliferation of Caco-2 line too, without affecting CCD841 healthy cell line. Furthermore, the extracts showed the ability to reduce the level of Epidermal Growth Factor Receptor (EGFR) phosphorylation in E705, DiFi and SW480 cell lines and to lower the EC50 of a CRC common drug, cetuximab, on E705 and DiFi lines from 161.7 ng mL-1 to 0.06 ng mL-1 and from 49.5 ng mL-1 to 0.2 ng mL-1 respectively. The extract was characterized in its protein and metabolite profiles by tandem mass spectrometry and 1H-NMR analyses. A Bowman-Birk protease inhibitor was identified within the protein fraction and was supposed to be the main active component. These findings confirm the importance of a legume-based diet to prevent the outbreak of many CRC and to reduce the amount of drug administered during a therapeutic cycle.

Extraction and Characterization of Inulin-Type Fructans from Artichoke Wastes and Their Effect on the Growth of Intestinal Bacteria Associated with Health.

Globe artichoke is an intriguing source of indigestible sugar polymers such as inulin-type fructans. In this study, the effect of ultrasound in combination with ethanol precipitation to enhance the extraction of long chain fructans from artichoke wastes has been evaluated. The inulin-type fructans content both from bracts and stems was measured using an enzymatic fructanase-based assay, while its average degree of polymerization (DP) was determined by HPLC-RID analysis. Results show that this method provides artichoke extracts with an inulin-type fructans content of 70% with an average DP between 32 and 42 both in bracts and in stems. The prebiotic effect of long chain inulins from artichoke extract wastes was demonstrated by its ability to support the growth of five Lactobacillus and four Bifidobacterium species, previously characterized as probiotics. Besides, we considered the possibility to industrialize the process developing a simpler method for the production of inulin-type fructans from the artichoke wastes so that the artichoke inulin preparation could be suitable for its use in synbiotic formulations in combination with different probiotics for further studies including in vivo trials.

Methacycline displays a strong efficacy in reducing toxicity in a SCA3 Caenorhabditis elegans model.

BACKGROUND: We have previously demonstrated the neuroprotective activity of tetracycline on a Spinocerebellar Ataxia 3 nematode model. Here, we present the screening of a small library of tetracycline congeners in order to identify the most effective compound in preventing ataxin-3 aggregation. METHODS: We performed the assays on the Josephin Domain as it is directly involved in the onset of fibrillation. We used thioflavin T and solubility assays to spot out the most effective tetracycline congeners; Fourier transform infrared and NMR spectroscopies to characterize their mode of action. We employed an ataxic Caenorhabditis elegans model to evaluate the pharmacological efficacy of tetracycline congeners. RESULTS: Methacycline was identified as the most effective compound. Like tetracycline, methacycline neither significantly affected the aggregation kinetics nor did it change the secondary structures of the final aggregates but increased the solubility of the aggregated species. Saturation transfer NMR experiments demonstrated methacycline capability to only bind the oligomeric species of Josephin Domain. Competition assays also showed that methacycline binds to the Josephin Domain more tightly than tetracycline. The treatment with methacycline induced a significant improvement in motility and locomotion of the transgenic C. elegans without changing its lifespan. The efficacy was distinctly stronger than that of tetracycline. Noteworthy, unlike tetracycline, methacycline was able to retard aging-related decline in motility of even the healthy worms used. CONCLUSIONS: The apparent absence of toxic effects displayed by methacycline, along with its stronger efficacy in contrasting expanded ataxin-3 toxicity, makes it a possible candidate for a chronic treatment of the disease.

Valorizing coffee pulp by-products as anti-inflammatory ingredient of food supplements acting on IL-8 release.

Coffee is the second traded food commodity in the world. Beyond roasted seeds, the most part of the original fruit -and in particular pulp- is discarded as waste, with severe environmental and economic consequences in many developing countries. Our research focused on developing an eco-friendly extraction protocol of phytocomplexes from coffee pulp and evaluating their bioactivity and beneficial effects to human health as food supplements. Antioxidant activity assays (Folin-Ciocalteu and DPPH assays) were adopted to select the most effective extraction technique and results show antioxidant activity of coffee pulp extracts. After analysis of cytotoxicity on human epithelial gastric cells, measurements of IL-8 release of treated or pre-treated cells were performed. Results showed that the use of soft technical equipment and sustainable solvents (i.e. maceration process, aqueous extraction) can extract phytocomplexes with antioxidant properties. Moreover, IL-8 measurements showed impairment of this chemokine release at concentrations that may be reached in vivo in the gastrointestinal tract, following consumption of reasonable amount of extract. Pre-treatments analysis demonstrated the ability of coffee pulp extracts to prevent IL-8 release by gastric epithelial cells. Chemical evaluation performed by liquid chromatography mass spectrometry showed that quinic acid derivatives are abundant in coffee pulp extract together with procyanidins derivatives: those compounds might be responsible for the high biological activity. This evidence supports future applications of coffee pulp extracts as food supplement with high added value, starting from a waste that can be valorized through simple yet efficient extraction methods.

Protein Environment: A Crucial Triggering Factor in Josephin Domain Aggregation: The Role of 2,2,2-Trifluoroethanol.

The protein ataxin-3 contains a polyglutamine stretch that triggers amyloid aggregation when it is expanded beyond a critical threshold. This results in the onset of the spinocerebellar ataxia type 3. The protein consists of the globular N-terminal Josephin domain and a disordered C-terminal tail where the polyglutamine stretch is located. Expanded ataxin-3 aggregates via a two-stage mechanism: first, Josephin domain self-association, then polyQ fibrillation. This highlights the intrinsic amyloidogenic potential of Josephin domain. Therefore, much effort has been put into investigating its aggregation mechanism(s). A key issue regards the conformational requirements for triggering amyloid aggregation, as it is believed that, generally, misfolding should precede aggregation. Here, we have assayed the effect of 2,2,2-trifluoroethanol, a co-solvent capable of stabilizing secondary structures, especially α-helices. By combining biophysical methods and molecular dynamics, we demonstrated that both secondary and tertiary JD structures are virtually unchanged in the presence of up to 5% 2,2,2-trifluoroethanol. Despite the preservation of JD structure, 1% of 2,2,2-trifluoroethanol suffices to exacerbate the intrinsic aggregation propensity of this domain, by slightly decreasing its conformational stability. These results indicate that in the case of JD, conformational fluctuations might suffice to promote a transition towards an aggregated state without the need for extensive unfolding, and highlights the important role played by the environment on the aggregation of this globular domain.

Publisher Correction: The polyglutamine protein ataxin-3 enables normal growth under heat shock conditions in the methylotrophic yeast Pichia pastoris.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

A High Sensitivity Biosensor to detect the presence of perfluorinated compounds in environment.

A novel surface plasmon resonance (SPR) optical fiber biosensor, able to bind perfluorooctanoate and perfluorooctanesulfonate compounds, is presented. In the first step, an ad hoc antibody compound has been designed, produced and tested by ELISA, then, in the second step, the gold surface of a plastic optical fiber sensor has been derivatizated and functionalized with this new bio-receptor, able to bind target analytes with high affinity and selectivity. The experimental data have shown that the developed SPR optical fiber biosensor makes it possible to detect these compounds. One advantage of this approach stems from the possibility to monitor the perfluorinated compounds in the environment exploiting the remote sensing capability offered by the optical fibers. The measurements were performed in laboratory, also exploiting matrices mimicking the real environment. The limit of detection of the assay was 0.21ppb, a value that is lower than the maximum residue limit fixed by the European Union regulations.

The polyglutamine protein ataxin-3 enables normal growth under heat shock conditions in the methylotrophic yeast Pichia pastoris.

The protein ataxin-3 carries a polyglutamine stretch close to the C-terminus that triggers a neurodegenerative disease in humans when its length exceeds a critical threshold. A role as a transcriptional regulator but also as a ubiquitin hydrolase has been proposed for this protein. Here, we report that, when expressed in the yeast Pichia pastoris, full-length ataxin-3 enabled almost normal growth at 37 °C, well above the physiological optimum of 30 °C. The N-terminal Josephin domain (JD) was also effective but significantly less, whereas catalytically inactive JD was completely ineffective. Based on MudPIT proteomic analysis, we observed that the strain expressing full-length, functional ataxin-3 displayed persistent upregulation of enzymes involved in mitochondrial energy metabolism during growth at 37 °C compared with the strain transformed with the empty vector. Concurrently, in the transformed strain intracellular ATP levels at 37 °C were even higher than normal ones at 30 °C. Elevated ATP was also paralleled by upregulation of enzymes involved in both protein biosynthesis and biosynthetic pathways, as well as of several stress-induced proteins. A similar pattern was observed when comparing a strain expressing JD with another expressing its catalytically inactive counterpart. We suggest that such effects mostly result from mechanisms of transcriptional regulation.

The Toxic Effects of Pathogenic Ataxin-3 Variants in a Yeast Cellular Model.

Ataxin-3 (AT3) is a deubiquitinating enzyme that triggers an inherited neurodegenerative disorder, spinocerebellar ataxia type 3, when its polyglutamine (polyQ) stretch close to the C-terminus exceeds a critical length. AT3 variants carrying the expanded polyQ are prone to associate with each other into amyloid toxic aggregates, which are responsible for neuronal death with ensuing neurodegeneration. We employed Saccharomyces cerevisiae as a eukaryotic cellular model to better clarify the mechanism by which AT3 triggers the disease. We expressed three variants: one normal (Q26), one expanded (Q85) and one truncated for a region lying from the beginning of its polyQ stretch to the end of the protein (291Δ). We found that the expression of the expanded form caused reduction in viability, accumulation of reactive oxygen species, imbalance of the antioxidant defense system and loss in cell membrane integrity, leading to necrotic death. The truncated variant also exerted a qualitatively similar, albeit milder, effect on cell growth and cytotoxicity, which points to the involvement of also non-polyQ regions in cytotoxicity. Guanidine hydrochloride, a well-known inhibitor of the chaperone Hsp104, almost completely restored wild-type survival rate of both 291Δ- and Q85-expressing strains. This suggests that AT3 aggregation and toxicity is mediated by prion forms of yeast proteins, as this chaperone plays a key role in their propagation.

Natural compounds against neurodegenerative diseases: molecular characterization of the interaction of catechins from green tea with Aß1-42, PrP106-126, and ataxin-3 oligomers.

By combining NMR spectroscopy, transmission electron microscopy, and circular dichroism we have identified the structural determinants involved in the interaction of green tea catechins with Aß1-42, PrP106-126, and ataxin-3 oligomers. The data allow the elucidation of their mechanism of action, showing that the flavan-3-ol unit of catechins is essential for interaction. At the same time, the gallate moiety, when present, seems to increase the affinity for the target proteins. These results provide important information for the rational design of new compounds with anti-amyloidogenic activity and/or molecular tools for the specific targeting of amyloid aggregates in vivo.

Interactions of ataxin-3 with its molecular partners in the protein machinery that sorts protein aggregates to the aggresome.

Ataxin-3 (AT3) is the protein that triggers the inherited neurodegenerative disorder spinocerebellar ataxia type 3 when its polyglutamine (polyQ) stretch close to the C-terminus exceeds a critical length. AT3 consists of the N-terminal globular Josephin domain (JD) and the C-terminal disordered one. It cleaves isopeptide bonds between ubiquitin monomers, an event involved in protein quality control mechanisms. AT3 has been implicated in the pathway that sorts aggregated protein to aggresomes via microtubules, in which dynein and histone deacetylase 6 (HDAC6) also seem to be involved. By taking advantage of small angle X-ray scattering (SAXS) and surface plasmon resonance (SPR), we have investigated the interaction of AT3 with tubulin and HDAC6. Based on SAXS results, the AT3 oligomer, consisting of 6-7 subunits, tightly binds to the tubulin hexameric oligomer in a "parallel" fashion. By SPR analysis we have demonstrated that AT3 binds to tubulin dimer with a 50nM affinity. Binding fits with a Langmuir 1:1 model and involves a single binding interface. Nevertheless, the interaction surface consists of three distinct, discontinuous tubulin-binding regions (TBR), one located in the JD, and the two others in the disordered domain, upstream and downstream of the polyQ stretch. In the absence of any of the three TBRs, the affinity is drastically reduced. By SPR we have also provided the first evidence of direct binding of AT3 to HDAC6, with affinity in the range 0.1-1µM. These results shed light on the interactions among the components of the transport machinery that sorts aggregate protein to the aggresome, and pave the way to in vivo studies aimed at further clarifying their roles.

A conserved loop in polynucleotide phosphorylase (PNPase) essential for both RNA and ADP/phosphate binding.

Polynucleotide phosphorylase (PNPase) reversibly catalyzes RNA phosphorolysis and polymerization of nucleoside diphosphates. Its homotrimeric structure forms a central channel where RNA is accommodated. Each protomer core is formed by two paralogous RNase PH domains: PNPase1, whose function is largely unknown, hosts a conserved FFRR loop interacting with RNA, whereas PNPase2 bears the putative catalytic site, ∼20 Šaway from the FFRR loop. To date, little is known regarding PNPase catalytic mechanism. We analyzed the kinetic properties of two Escherichia coli PNPase mutants in the FFRR loop (R79A and R80A), which exhibited a dramatic increase in Km for ADP/Pi binding, but not for poly(A), suggesting that the two residues may be essential for binding ADP and Pi. However, both mutants were severely impaired in shifting RNA electrophoretic mobility, implying that the two arginines contribute also to RNA binding. Additional interactions between RNA and other PNPase domains (such as KH and S1) may preserve the enzymatic activity in R79A and R80A mutants. Inspection of enzyme structure showed that PNPase has evolved a long-range acting hydrogen bonding network that connects the FFRR loop with the catalytic site via the F380 residue. This hypothesis was supported by mutation analysis. Phylogenetic analysis of PNPase domains and RNase PH suggests that such network is a unique feature of PNPase1 domain, which coevolved with the paralogous PNPase2 domain.