Structural Insights into Alternate Aggregated Prion Protein Forms

Polano, Maurizio; Bek, Alpan; Benetti, Federico; Lazzarino, Marco; Legname, Giuseppe

doi:10.1016/j.jmb.2009.08.056

Cited by 17 publications

(22 citation statements)

References 51 publications

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“…In our experiments we observed a strong increase in fluorescence, which corresponds to the ThT binding to β-sheet rich structures like amyloid aggregates (Figure 9). Our results are consistent with previous fibrilization experiments performed with MoPrP(89-230) expressed in E. coli as inclusion bodies [28,29] and provide a good indication about the ability of soluble MoPrP(89-230) to form ThT-positive fibrils. Furthermore, to gain insight into the morphology and structure of amyloid fibrils formed using solubly expressed MoPrP(89-230) we performed atomic force microscopy and X-ray fibril diffraction (Figure 10).…”

Section: Resultssupporting

confidence: 92%

A novel expression system for production of soluble prion proteins in E. coli

et al. 2012

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Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP) and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Sulfhydryl OXidase (QSOX), a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies.

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Section: Resultssupporting

confidence: 92%

A novel expression system for production of soluble prion proteins in E. coli

et al. 2012

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“…The amount of proteinase K-resistant prion protein was not significantly different between wild-type and T187I samples (not shown). Higher ThT fluorescence intensity could thus originate from different types of amyloid structures formed as previously observed [24], [25].…”

Section: Resultssupporting

confidence: 56%

Effect of Hydrophobic Mutations in the H2-H3 Subdomain of Prion Protein on Stability and Conversion In Vitro and In Vivo

Hafner-Bratkovič

Gaedtke²,

Ondračka

et al. 2011

PLoS ONE

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Prion diseases are fatal neurodegenerative diseases, which can be acquired, sporadic or genetic, the latter being linked to mutations in the gene encoding prion protein. We have recently described the importance of subdomain separation in the conversion of prion protein (PrP). The goal of the present study was to investigate the effect of increasing the hydrophobic interactions within the H2-H3 subdomain on PrP conversion. Three hydrophobic mutations were introduced into PrP. The mutation V209I associated with human prion disease did not alter protein stability or in vitro fibrillization propensity of PrP. The designed mutations V175I and T187I on the other hand increased protein thermal stability. V175I mutant fibrillized faster than wild-type PrP. Conversion delay of T187I was slightly longer, but fluorescence intensity of amyloid specific dye thioflavin T was significantly higher. Surprisingly, cells expressing V209I variant exhibited inefficient proteinase K resistant PrP formation upon infection with 22L strain, which is in contrast to cell lines expressing wild-type, V175I and T187I mPrPs. In agreement with increased ThT fluorescence at the plateau T187I expressing cell lines accumulated an increased amount of the proteinase K-resistant prion protein. We showed that T187I induces formation of thin fibrils, which are absent from other samples. We propose that larger solvent accessibility of I187 in comparison to wild-type and other mutants may interfere with lateral annealing of filaments and may be the underlying reason for increased conversion efficiency.

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“…The peculiar structural plasticity of FA was clearly visible in AFM experiments showing sponge-like structures and globular assemblies at low MoPrP and FA concentrations, respectively, while at high MoPrP concentration FA appeared assembled in fibrillar structures. Although AFM provides only morphological information, these fibrils are reasonably made only of FA, as their height is not compatible with current studies on PrP fibrils dimension [43], and as FA cannot generate ThT-positive MoPrP aggregates. Taken together, these findings indicate that protein encapsulation by HS causes the formation of large insoluble aggregates, as is the case with HA, or ordered structures made of FA.…”

Section: Discussionmentioning

confidence: 73%

“…The polymerization process was monitored for 110 hours by reading the ThT-fluorescence intensity at 444 nm excitation and 485 nm emission. The time of fibrillization (lag phase) was estimated according to [43], data were analyzed using Origin 8.6 software.…”

Section: Methodsmentioning

confidence: 99%

Prion Protein Interaction with Soil Humic Substances: Environmental Implications

et al. 2014

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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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Structural Insights into Alternate Aggregated Prion Protein Forms

Cited by 17 publications

References 51 publications

A novel expression system for production of soluble prion proteins in E. coli

A novel expression system for production of soluble prion proteins in E. coli

Effect of Hydrophobic Mutations in the H2-H3 Subdomain of Prion Protein on Stability and Conversion In Vitro and In Vivo

Prion Protein Interaction with Soil Humic Substances: Environmental Implications

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