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Prion protein interaction with soil humic substances: environmental implications.

Giachin G, Narkiewicz J, Scaini D, Ngoc AT, Margon A, Sequi P, Leita L, Legname G - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy.

ABSTRACT
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.

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Effectiveness of HS in inhibiting prion replication.ASA showing the kinetics of MoPrP fibrillization in the presence of 1 µg/mL of HS (A) and the corresponding mean value of the lag phases (in hours) for MoPrP treated with HS (**P<0.01) (B). In (C) Western-blot results showing the dose-dependent removal of PrPSc from ScGT1 cells. In the upper panel, total PrP expression detected by immunoblotting after the addition of increasing concentrations of HA and FA (here used as non-PK controls); the lower panel shows PrPSc levels after digestion with PK.
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pone-0100016-g005: Effectiveness of HS in inhibiting prion replication.ASA showing the kinetics of MoPrP fibrillization in the presence of 1 µg/mL of HS (A) and the corresponding mean value of the lag phases (in hours) for MoPrP treated with HS (**P<0.01) (B). In (C) Western-blot results showing the dose-dependent removal of PrPSc from ScGT1 cells. In the upper panel, total PrP expression detected by immunoblotting after the addition of increasing concentrations of HA and FA (here used as non-PK controls); the lower panel shows PrPSc levels after digestion with PK.

Mentions: To investigate the possible role of HS in reducing prion infectivity, first we used the amyloid seeding assay (ASA). In contrast to the original protocol employing denaturants and high ionic strength to accelerate the fibrillization reaction [37], we used MoPrP in native conditions in the presence of 1 µg/mL of HS. This concentration has a minor impact on protein solubility (Figure 1) and does not interfere with Thioflavin-T (ThT) fluorescence, the dye commonly used in ASA to monitor the formation of newly generated β-strand-enriched structures. When the protein was pre-incubated with PrPSc and then exposed to HA and FA, the lag-phases of fibrillization were prolonged up to 88.2±4.7 and 84.5±9.4 hours, respectively, while the control started to polymerize after 59.4±7.4 hours. To exclude that the observed lag-phase shifts were due to a partial MoPrP removal from the reaction induced by HS encapsulation, we preincubated the seed with HS before adding MoPrP. Interestingly, in these conditions we did not detect any ThT-fluorescence sigmoidal increases in the reactions seeded by PrPSc complexed by HS (Figure 5A and 5B). We obtained similar results, showing no ThT-fluorescence increase, in control experiments performed in the presence of HS without PrPSc seed (Figure S4). To investigate whether HS have a significant effect in reducing PrPSc content in more physiological conditions, we added HS to ScGT1 cells medium and evaluated PK-resistant PrPSc levels, a test currently employed to assess whether a compound displays anti-prion activity [38]. As shown in Figure 5C, the treatment with HA and FA induced a clearance of pre-existing PrPSc from ScGT1 cells in a dose-dependent manner (lower panel) without altering the total PrPC expression (upper panel). The half maximal effective concentrations (EC50) were 7.8±0.4 and 12.3±0.7 µg/mL for HA and FA, respectively, as determined by ELISA [42]. None of the compounds tested was cytotoxic after HS exposure (Figure S5). These results provide first evidence that HS may play a role in PrPSc adsorption, thus limiting prion transmission.


Prion protein interaction with soil humic substances: environmental implications.

Giachin G, Narkiewicz J, Scaini D, Ngoc AT, Margon A, Sequi P, Leita L, Legname G - PLoS ONE (2014)

Effectiveness of HS in inhibiting prion replication.ASA showing the kinetics of MoPrP fibrillization in the presence of 1 µg/mL of HS (A) and the corresponding mean value of the lag phases (in hours) for MoPrP treated with HS (**P<0.01) (B). In (C) Western-blot results showing the dose-dependent removal of PrPSc from ScGT1 cells. In the upper panel, total PrP expression detected by immunoblotting after the addition of increasing concentrations of HA and FA (here used as non-PK controls); the lower panel shows PrPSc levels after digestion with PK.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061048&req=5

pone-0100016-g005: Effectiveness of HS in inhibiting prion replication.ASA showing the kinetics of MoPrP fibrillization in the presence of 1 µg/mL of HS (A) and the corresponding mean value of the lag phases (in hours) for MoPrP treated with HS (**P<0.01) (B). In (C) Western-blot results showing the dose-dependent removal of PrPSc from ScGT1 cells. In the upper panel, total PrP expression detected by immunoblotting after the addition of increasing concentrations of HA and FA (here used as non-PK controls); the lower panel shows PrPSc levels after digestion with PK.
Mentions: To investigate the possible role of HS in reducing prion infectivity, first we used the amyloid seeding assay (ASA). In contrast to the original protocol employing denaturants and high ionic strength to accelerate the fibrillization reaction [37], we used MoPrP in native conditions in the presence of 1 µg/mL of HS. This concentration has a minor impact on protein solubility (Figure 1) and does not interfere with Thioflavin-T (ThT) fluorescence, the dye commonly used in ASA to monitor the formation of newly generated β-strand-enriched structures. When the protein was pre-incubated with PrPSc and then exposed to HA and FA, the lag-phases of fibrillization were prolonged up to 88.2±4.7 and 84.5±9.4 hours, respectively, while the control started to polymerize after 59.4±7.4 hours. To exclude that the observed lag-phase shifts were due to a partial MoPrP removal from the reaction induced by HS encapsulation, we preincubated the seed with HS before adding MoPrP. Interestingly, in these conditions we did not detect any ThT-fluorescence sigmoidal increases in the reactions seeded by PrPSc complexed by HS (Figure 5A and 5B). We obtained similar results, showing no ThT-fluorescence increase, in control experiments performed in the presence of HS without PrPSc seed (Figure S4). To investigate whether HS have a significant effect in reducing PrPSc content in more physiological conditions, we added HS to ScGT1 cells medium and evaluated PK-resistant PrPSc levels, a test currently employed to assess whether a compound displays anti-prion activity [38]. As shown in Figure 5C, the treatment with HA and FA induced a clearance of pre-existing PrPSc from ScGT1 cells in a dose-dependent manner (lower panel) without altering the total PrPC expression (upper panel). The half maximal effective concentrations (EC50) were 7.8±0.4 and 12.3±0.7 µg/mL for HA and FA, respectively, as determined by ELISA [42]. None of the compounds tested was cytotoxic after HS exposure (Figure S5). These results provide first evidence that HS may play a role in PrPSc adsorption, thus limiting prion transmission.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus