Prion protein interaction with soil humic substances: environmental implications

Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders caused by prions. Animal TSE include scrapie in sheep and goats, and chronic wasting disease (CWD) in cervids. Effective management of scrapie in many parts of the world, and of CWD in North American deer population is complicated by the persistence of prions in the environment. After shedding from diseased animals, prions persist in soil, withstanding biotic and abiotic degradation. As soil is a complex, multi-component system of both mineral and organic components, it is important to understand which soil compounds may interact with prions and thus contribute to disease transmission. Several studies have investigated the role of different soil minerals in prion adsorption and infectivity; we focused our attention on the interaction of soil organic components, the humic substances (HS), with recombinant prion protein (recPrP) material. We evaluated the kinetics of recPrP adsorption, providing a structural and biochemical characterization of chemical adducts using different experimental approaches. Here we show that HS act as potent anti-prion agents in prion infected neuronal cells and in the amyloid seeding assays: HS adsorb both recPrP and prions, thus sequestering them from the prion replication process. We interpreted our findings as highly relevant from an environmental point of view, as the adsorption of prions in HS may affect their availability and consequently hinder the environmental transmission of prion diseases in ruminants

The mechanisms of humic substances self-assembly with biological molecules: The case study of the prion protein

Humic substances (HS) are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP) into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of “salting-out” whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of “prion ecology”. These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment

15N e 13C nella valutazione dell\u2019uso dell\u2019azoto e della conservazione del carbonio da compost

The natural abundance of stable isotopes of nitrogen (15N) and carbon(13C) of four composts (ACV; ACMdig; ACF; ACM) applied at 10 and 20 Mg OM ha-1, was used to estimate the utilization of N in ryegrass and the variation of the soil carbon isotopic signature (\uf04413C\u2030) at the end of a cultivation cycle (112 days). ACMdig and ACF showed the greatest N use efficiency (15NRF: 9-16%), in agreement with their higher mineral-N. At the end of the test the \uf04413C \u2030 ranged between -25.39 and -25.55 (control vs. NPK); between -26.76 and -26.55 in ACV and ACMdig, while ACF and ACM were intermediate (-25.94; -26.06). The \uf04413C \u2030 in the various treatments was described by a linear relationship with CO2 emissions, highlighting a reduction in the carbon's storage capacity in the less stable products, being this influenced by the application level. The greater the application of not completely stable compost, the smaller is the C conserved in the soil

What we talk about when we talk about protein hydrolyzate-based biostimulants

Protein hydrolyzates (PHs) are a well-known group of plant biostimulants that are obtained by thermal, chemical, enzymatic or mixed hydrolysis from animal or plant sources. PHs are mainly applied to plants with the aim to improve their nutrition level, stimulate metabolism, and increase resistance to abiotic stress. Chemically, the PHs are a mixture of free amino acids, oligo- and polypeptides. Considering the differences in the source materials and hydrolytic processes used for their production it is not surprising that PHs are a very complex and inhomogeneous category of biostimulants. This is a critical point that should be considered by both manufacturers and potential users of such products. With the aim to identify a putative correlation among PHs physic-chemical properties, the different source materials and the hydrolytic process used for their production, 22 PHs available on the market were analyzed for: (i) total amino acids concentration, (ii) free amino acids concentration, (iii) degree of racemization, (iv) degree of hydrolysis and (v) apparent average molecular size. The obtained results confirmed the inhomogeneity PHs. However, the chosen parameters were able to provide information about the above-mentioned correlation. Total amino acids in particular were useful for the identification of the original source material. The degree of racemization was useful for the identification of the type of hydrolysis used during production (chemical or enzymatic). The degree of hydrolysis and free amino acid concentrations were useful for the identification of the intensity of the hydrolytic process. The average molecular size was useful for the identification of polydispersity of the peptide size. In conclusion, the PHs can be adequately characterized only through a multi-analytical approach

Changes in soil phenol oxidase activities due to long-term application of compost and mineral N in a walnut orchard

Phenol oxidases (POs) are a group of soil extracellular oxidoreductase enzymes, which are involved in oxidative processes related to nutrient cycling. This class of enzymes has multiple functions at both the organism and ecosystem level and can trigger either positive or negative feedback loops between soil organisms and soil organic matter. The purpose of this study was to evaluate: (i) whether PO activities have a trend different from those of microbial biomass and activity; and (ii) whether PO activities are enhanced or reduced by fertilizer application. Soil samples were taken from plots in a 14-year-old experimental walnut orchard, subjected, since planting, to three fertilizer treatments: organic (compost) or mineral (urea) fertilization at the same rate of nitrogen application (100 kg N ha 12 1), or left unfertilized. Soil samples were divided according to sampling depth (0\u201320 cm and 20\u201340 cm). Results showed that the application of compost significantly increased C and N pools. qCO2 and DHD/Cext data indicated that the biota at 20\u201340 cm soil depth was more stressed or mainly composed of microorganisms with low substrate utilization efficiency. Phenol oxidase, tyrosinase, and catechol oxidase activities were significantly promoted in the surface layer by compost addition. In contrast, laccase activity showed a large increase in the deeper soil layer when supplied with mineral N, whereas compost addition led to increased activity in the surface layer. These findings suggest that soil phenol oxidases play a part in the determination of soil functionality, but they need to be investigated in greater depth in order to understand the mechanisms regulating their activities

Macroscopic characterization of humic substances.

<p>Phase contrast optical images of HS morphology when deposited on a mica surface (20 µg/mL of FA and HA, respectively in A and D) were compared to MoPrP-HS complexes morphology deposited under the same conditions (B shows complexes between 60 µg/mL of MoPrP and FA, E depicts those with HA). Red arrows in B point at “brush-like” structures possibly ascribable to MoPrP clusters. Yellow squares highlight the FA and HA structures analyzed by AFM in panels C and D, representing three-dimensional AFM reconstructions of FA-MoPrP fractal fibers and HA-MoPrP assemblies, respectively.</p

MoPrP adsorption into HS affects protein solubility.

<p>SDS-PAGE gel showing the precipitation of full-length MoPrP induced by increasing amounts of HA and FA. After adding 20 and 10 µg/mL of HS (HA and FA, respectively) the total protein is mainly present in the insoluble fractions (A). Decreased MoPrP solubility in the presence of 1 to 5 µg/mL of FA after 6 hours of incubation, and 1 to 10 µg/mL of HA after 6 and 240 hours of incubation (B). Normalized far-UV CD spectra of MoPrP after 6- and 240-hour incubation with 5 µg/mL of HS (C).</p

Titration of MoPrP(23–230) with a fulvic acid and Zn²⁺ monitored by NMR.

<p>Combined effect upon the addition to MoPrP(23–230) of Zn²⁺ and FABw. Enlarged region of high field shifted methyl resonances of the 1D-NMR spectra of unlabeled MoPrP(23–230) upon addition of Zn²⁺ and FABw. In black: MoPrP(23–230) alone; in red, MoPrP(23–230) with ZnCl₂ (always at 90 μM); in green, MoPrP(23–230) with ZnCl₂ and 60 μg/mL FABw; in violet, MoPrP(23–230) with ZnCl₂ and 120 μg/mL FABw; in yellow, MoPrP(23–230) with ZnCl₂ and 180 μg/mL FABw. In the <i>inset</i>, differences in resonance intensities of MoPrP(23–230) due to the precipitation in the presence of the FABw alone (filled square) or in the presence of both FABw and Zn²⁺ (filled circle).</p

FTIR spectra of the MoPrP(23–230) precipitates in complex with different HS.

<p>The tested HS were at 120 μg/mL, all the results are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188308#pone.0188308.t002" target="_blank">Table 2</a>.</p