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Mammalian prion protein (PrP) forms conformationally different amyloid intracellular aggregates in bacteria.

Macedo B, Sant'Anna R, Navarro S, Cordeiro Y, Ventura S - Microb. Cell Fact. (2015)

Bottom Line: Among them, mammalian prions outstand due to their ability to transmit the pathogenic conformation, becoming thus infectious.The structural conversion of the cellular prion protein (PrP(C)), into its misfolded pathogenic form (PrP(Sc)) is the central event of prion-driven pathologies.Moreover, the inclusions formed by these two PrP proteins display conformational diversity, since they differ in fibril morphology, binding affinity to amyloid dyes, stability, resistance to proteinase K digestion and neurotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Faculdade de Farmácia, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Bloco B, Subsolo, Sala 17, Rio de Janeiro, RJ, 21941-902, Brazil. brunoo_macedo@hotmail.com.

ABSTRACT

Background: An increasing number of proteins are being shown to assemble into amyloid structures that lead to pathological states. Among them, mammalian prions outstand due to their ability to transmit the pathogenic conformation, becoming thus infectious. The structural conversion of the cellular prion protein (PrP(C)), into its misfolded pathogenic form (PrP(Sc)) is the central event of prion-driven pathologies. The study of the structural properties of intracellular amyloid aggregates in general and of prion-like ones in particular is a challenging task. In this context, the evidence that the inclusion bodies formed by amyloid proteins in bacteria display amyloid-like structural and functional properties make them a privileged system to model intracellular amyloid aggregation.

Results: Here we provide the first demonstration that recombinant murine PrP and its C-terminal domain (90-231) attain amyloid conformations inside bacteria. Moreover, the inclusions formed by these two PrP proteins display conformational diversity, since they differ in fibril morphology, binding affinity to amyloid dyes, stability, resistance to proteinase K digestion and neurotoxicity.

Conclusions: Overall, our results suggest that modelling PrP amyloid formation in microbial cell factories might open an avenue for a better understanding of the structural features modulating the pathogenic impact of this intriguing protein.

No MeSH data available.


Related in: MedlinePlus

Aggregation kinetics of PrPWT. The aggregation reactions of 0.5 mg/mL of purified and refolded rPrPWT were carried out under constant agitation at 600 RPM and 37 °C. In vitro preformed fibrils (2 %) or PrPs IBs (final OD350 = 0.1) were used for seeding and cross-seeding assays. PrP fibrilization (black line) as a function of time, exhibits a typical nucleation-elongation profile. The lag phase is reduced in the presence of pre-aggregated homologous protein, either PrPWT fibrils (blue line) or PrPWT IBs (red line). Cross-seeding with PrP90–231 IBs (green line) did not affect the fibrilization extent and kinetics
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Fig10: Aggregation kinetics of PrPWT. The aggregation reactions of 0.5 mg/mL of purified and refolded rPrPWT were carried out under constant agitation at 600 RPM and 37 °C. In vitro preformed fibrils (2 %) or PrPs IBs (final OD350 = 0.1) were used for seeding and cross-seeding assays. PrP fibrilization (black line) as a function of time, exhibits a typical nucleation-elongation profile. The lag phase is reduced in the presence of pre-aggregated homologous protein, either PrPWT fibrils (blue line) or PrPWT IBs (red line). Cross-seeding with PrP90–231 IBs (green line) did not affect the fibrilization extent and kinetics

Mentions: Morphology of PrPWT aggregates at the end of the aggregation kinetics. Aggregates were collected at the end (~19 h) of non-seeded (a), seeded (c–f) and cross-seeded (b) reactions (Fig. 10) and they were monitored by negative staining and transmission electron microscopy. Scale bars 2 μm (a); 0.5 μm (b); 1.0 μm (c); 0.2 μm (d); 2 μm (e); 1 μm (f)


Mammalian prion protein (PrP) forms conformationally different amyloid intracellular aggregates in bacteria.

Macedo B, Sant'Anna R, Navarro S, Cordeiro Y, Ventura S - Microb. Cell Fact. (2015)

Aggregation kinetics of PrPWT. The aggregation reactions of 0.5 mg/mL of purified and refolded rPrPWT were carried out under constant agitation at 600 RPM and 37 °C. In vitro preformed fibrils (2 %) or PrPs IBs (final OD350 = 0.1) were used for seeding and cross-seeding assays. PrP fibrilization (black line) as a function of time, exhibits a typical nucleation-elongation profile. The lag phase is reduced in the presence of pre-aggregated homologous protein, either PrPWT fibrils (blue line) or PrPWT IBs (red line). Cross-seeding with PrP90–231 IBs (green line) did not affect the fibrilization extent and kinetics
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4634817&req=5

Fig10: Aggregation kinetics of PrPWT. The aggregation reactions of 0.5 mg/mL of purified and refolded rPrPWT were carried out under constant agitation at 600 RPM and 37 °C. In vitro preformed fibrils (2 %) or PrPs IBs (final OD350 = 0.1) were used for seeding and cross-seeding assays. PrP fibrilization (black line) as a function of time, exhibits a typical nucleation-elongation profile. The lag phase is reduced in the presence of pre-aggregated homologous protein, either PrPWT fibrils (blue line) or PrPWT IBs (red line). Cross-seeding with PrP90–231 IBs (green line) did not affect the fibrilization extent and kinetics
Mentions: Morphology of PrPWT aggregates at the end of the aggregation kinetics. Aggregates were collected at the end (~19 h) of non-seeded (a), seeded (c–f) and cross-seeded (b) reactions (Fig. 10) and they were monitored by negative staining and transmission electron microscopy. Scale bars 2 μm (a); 0.5 μm (b); 1.0 μm (c); 0.2 μm (d); 2 μm (e); 1 μm (f)

Bottom Line: Among them, mammalian prions outstand due to their ability to transmit the pathogenic conformation, becoming thus infectious.The structural conversion of the cellular prion protein (PrP(C)), into its misfolded pathogenic form (PrP(Sc)) is the central event of prion-driven pathologies.Moreover, the inclusions formed by these two PrP proteins display conformational diversity, since they differ in fibril morphology, binding affinity to amyloid dyes, stability, resistance to proteinase K digestion and neurotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Faculdade de Farmácia, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Bloco B, Subsolo, Sala 17, Rio de Janeiro, RJ, 21941-902, Brazil. brunoo_macedo@hotmail.com.

ABSTRACT

Background: An increasing number of proteins are being shown to assemble into amyloid structures that lead to pathological states. Among them, mammalian prions outstand due to their ability to transmit the pathogenic conformation, becoming thus infectious. The structural conversion of the cellular prion protein (PrP(C)), into its misfolded pathogenic form (PrP(Sc)) is the central event of prion-driven pathologies. The study of the structural properties of intracellular amyloid aggregates in general and of prion-like ones in particular is a challenging task. In this context, the evidence that the inclusion bodies formed by amyloid proteins in bacteria display amyloid-like structural and functional properties make them a privileged system to model intracellular amyloid aggregation.

Results: Here we provide the first demonstration that recombinant murine PrP and its C-terminal domain (90-231) attain amyloid conformations inside bacteria. Moreover, the inclusions formed by these two PrP proteins display conformational diversity, since they differ in fibril morphology, binding affinity to amyloid dyes, stability, resistance to proteinase K digestion and neurotoxicity.

Conclusions: Overall, our results suggest that modelling PrP amyloid formation in microbial cell factories might open an avenue for a better understanding of the structural features modulating the pathogenic impact of this intriguing protein.

No MeSH data available.


Related in: MedlinePlus