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How does domain replacement affect fibril formation of the rabbit/human prion proteins.

Yan X, Huang JJ, Zhou Z, Chen J, Liang Y - PLoS ONE (2014)

Bottom Line: It is known that in vivo human prion protein (PrP) have the tendency to form fibril deposits and are associated with infectious fatal prion diseases, while the rabbit PrP does not readily form fibrils and is unlikely to cause prion diseases.As evidenced by circular dichroism, Fourier transform infrared spectroscopy, and proteinase K digestion assays, amyloid fibrils formed by human chimera have secondary structures and proteinase K-resistant features similar to those formed by the human PrP.Our findings can help to explain why amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and why macromolecular crowding has different effects on fibrillization of PrPs from different species.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.

ABSTRACT

Background: It is known that in vivo human prion protein (PrP) have the tendency to form fibril deposits and are associated with infectious fatal prion diseases, while the rabbit PrP does not readily form fibrils and is unlikely to cause prion diseases. Although we have previously demonstrated that amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and macromolecular crowding has different effects on fibril formation of the rabbit/human PrPs, we do not know which domains of PrPs cause such differences. In this study, we have constructed two PrP chimeras, rabbit chimera and human chimera, and investigated how domain replacement affects fibril formation of the rabbit/human PrPs.

Methodology/principal findings: As revealed by thioflavin T binding assays and Sarkosyl-soluble SDS-PAGE, the presence of a strong crowding agent dramatically promotes fibril formation of both chimeras. As evidenced by circular dichroism, Fourier transform infrared spectroscopy, and proteinase K digestion assays, amyloid fibrils formed by human chimera have secondary structures and proteinase K-resistant features similar to those formed by the human PrP. However, amyloid fibrils formed by rabbit chimera have proteinase K-resistant features and secondary structures in crowded physiological environments different from those formed by the rabbit PrP, and secondary structures in dilute solutions similar to the rabbit PrP. The results from transmission electron microscopy show that macromolecular crowding caused human chimera but not rabbit chimera to form short fibrils and non-fibrillar particles.

Conclusions/significance: We demonstrate for the first time that the domains beyond PrP-H2H3 (β-strand 1, α-helix 1, and β-strand 2) have a remarkable effect on fibrillization of the rabbit PrP but almost no effect on the human PrP. Our findings can help to explain why amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and why macromolecular crowding has different effects on fibrillization of PrPs from different species.

No MeSH data available.


Related in: MedlinePlus

Concentration-dependent proteinase K-digestion assays of human (A) and rabbit (B) chimera PrP fibrils.Samples were treated with PK for 1 h at 37°C at PK: PrP molar ratios as follows: 1∶100 (lane 1) and 1∶50 (lane 2). PK concentration: 0.4 µg/ml (lane 1) and 0.8 µg/ml (lane 2). The controls with zero protease in the absence of a crowding agent were loaded in lane 0. Protein molecular weight markers were loaded on lane M: restriction endonuclease Bsp98 I (25.0 kDa), β-lactoglobulin (18.4 kDa), and lysozyme (14.4 kDa). Amyloid fibrils were produced from human chimera and rabbit chimera in the absence of a crowding agent. Protein fragments were separated by SDS-PAGE and detected by silver staining.
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pone-0113238-g006: Concentration-dependent proteinase K-digestion assays of human (A) and rabbit (B) chimera PrP fibrils.Samples were treated with PK for 1 h at 37°C at PK: PrP molar ratios as follows: 1∶100 (lane 1) and 1∶50 (lane 2). PK concentration: 0.4 µg/ml (lane 1) and 0.8 µg/ml (lane 2). The controls with zero protease in the absence of a crowding agent were loaded in lane 0. Protein molecular weight markers were loaded on lane M: restriction endonuclease Bsp98 I (25.0 kDa), β-lactoglobulin (18.4 kDa), and lysozyme (14.4 kDa). Amyloid fibrils were produced from human chimera and rabbit chimera in the absence of a crowding agent. Protein fragments were separated by SDS-PAGE and detected by silver staining.

Mentions: PK resistance activity has been widely used to distinguish PrPC from PrPSc since the pioneering studies of Prusiner and co-workers [38]. As shown in Fig. 6A, amyloid fibrils produced from human chimera generated PK-resistant fragments of 15–16-kDa after PK digestion for 1 h, which are similar to those of the human PrP [25], [27]. Human chimera fibrils also generated three short fragments (12-, 10-, and 8-kDa bands), which are similar to those of the human PrP reported previously [25], [27]. Taken together, our CD, FTIR and PK digestion data demonstrate that amyloid fibrils formed by human chimera have secondary structures and PK-resistant features similar to those formed by the human PrP.


How does domain replacement affect fibril formation of the rabbit/human prion proteins.

Yan X, Huang JJ, Zhou Z, Chen J, Liang Y - PLoS ONE (2014)

Concentration-dependent proteinase K-digestion assays of human (A) and rabbit (B) chimera PrP fibrils.Samples were treated with PK for 1 h at 37°C at PK: PrP molar ratios as follows: 1∶100 (lane 1) and 1∶50 (lane 2). PK concentration: 0.4 µg/ml (lane 1) and 0.8 µg/ml (lane 2). The controls with zero protease in the absence of a crowding agent were loaded in lane 0. Protein molecular weight markers were loaded on lane M: restriction endonuclease Bsp98 I (25.0 kDa), β-lactoglobulin (18.4 kDa), and lysozyme (14.4 kDa). Amyloid fibrils were produced from human chimera and rabbit chimera in the absence of a crowding agent. Protein fragments were separated by SDS-PAGE and detected by silver staining.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4234653&req=5

pone-0113238-g006: Concentration-dependent proteinase K-digestion assays of human (A) and rabbit (B) chimera PrP fibrils.Samples were treated with PK for 1 h at 37°C at PK: PrP molar ratios as follows: 1∶100 (lane 1) and 1∶50 (lane 2). PK concentration: 0.4 µg/ml (lane 1) and 0.8 µg/ml (lane 2). The controls with zero protease in the absence of a crowding agent were loaded in lane 0. Protein molecular weight markers were loaded on lane M: restriction endonuclease Bsp98 I (25.0 kDa), β-lactoglobulin (18.4 kDa), and lysozyme (14.4 kDa). Amyloid fibrils were produced from human chimera and rabbit chimera in the absence of a crowding agent. Protein fragments were separated by SDS-PAGE and detected by silver staining.
Mentions: PK resistance activity has been widely used to distinguish PrPC from PrPSc since the pioneering studies of Prusiner and co-workers [38]. As shown in Fig. 6A, amyloid fibrils produced from human chimera generated PK-resistant fragments of 15–16-kDa after PK digestion for 1 h, which are similar to those of the human PrP [25], [27]. Human chimera fibrils also generated three short fragments (12-, 10-, and 8-kDa bands), which are similar to those of the human PrP reported previously [25], [27]. Taken together, our CD, FTIR and PK digestion data demonstrate that amyloid fibrils formed by human chimera have secondary structures and PK-resistant features similar to those formed by the human PrP.

Bottom Line: It is known that in vivo human prion protein (PrP) have the tendency to form fibril deposits and are associated with infectious fatal prion diseases, while the rabbit PrP does not readily form fibrils and is unlikely to cause prion diseases.As evidenced by circular dichroism, Fourier transform infrared spectroscopy, and proteinase K digestion assays, amyloid fibrils formed by human chimera have secondary structures and proteinase K-resistant features similar to those formed by the human PrP.Our findings can help to explain why amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and why macromolecular crowding has different effects on fibrillization of PrPs from different species.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.

ABSTRACT

Background: It is known that in vivo human prion protein (PrP) have the tendency to form fibril deposits and are associated with infectious fatal prion diseases, while the rabbit PrP does not readily form fibrils and is unlikely to cause prion diseases. Although we have previously demonstrated that amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and macromolecular crowding has different effects on fibril formation of the rabbit/human PrPs, we do not know which domains of PrPs cause such differences. In this study, we have constructed two PrP chimeras, rabbit chimera and human chimera, and investigated how domain replacement affects fibril formation of the rabbit/human PrPs.

Methodology/principal findings: As revealed by thioflavin T binding assays and Sarkosyl-soluble SDS-PAGE, the presence of a strong crowding agent dramatically promotes fibril formation of both chimeras. As evidenced by circular dichroism, Fourier transform infrared spectroscopy, and proteinase K digestion assays, amyloid fibrils formed by human chimera have secondary structures and proteinase K-resistant features similar to those formed by the human PrP. However, amyloid fibrils formed by rabbit chimera have proteinase K-resistant features and secondary structures in crowded physiological environments different from those formed by the rabbit PrP, and secondary structures in dilute solutions similar to the rabbit PrP. The results from transmission electron microscopy show that macromolecular crowding caused human chimera but not rabbit chimera to form short fibrils and non-fibrillar particles.

Conclusions/significance: We demonstrate for the first time that the domains beyond PrP-H2H3 (β-strand 1, α-helix 1, and β-strand 2) have a remarkable effect on fibrillization of the rabbit PrP but almost no effect on the human PrP. Our findings can help to explain why amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and why macromolecular crowding has different effects on fibrillization of PrPs from different species.

No MeSH data available.


Related in: MedlinePlus