Site-specific Conformational Studies of Prion Protein (PrP) Amyloid Fibrils Revealed Two Cooperative Folding Domains within Amyloid Structure

Sun, Ying; Breydo, Leonid; Makarava, Natallia; Yang, Qingyuan; Bocharova, Olga V.; Baskakov, Ilia V.

doi:10.1074/jbc.m608623200

Cited by 52 publications

(59 citation statements)

References 48 publications

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“…As suggested by the molecular dynamics simulations, the residues 184-223 have a high tendency to undergo a transition from a-helical to a bÀ or random-coil state and, therefore, might be involved in the initial stages of the conversion from PrP C to PrP Sc (Dima and Thirumalai 2004). Consistent with the simulation results, our recent kinetics studies showed that the C-terminal part encompassing residues 196-230 was involved in the early stages of polymerization (Sun et al 2007). Furthermore, in amyloid fibrils produced in vitro, residue 218 is located within the PK-resistant cross b-sheet core.…”

Section: Discussionsupporting

confidence: 78%

The dominant‐negative effect of the Q218K variant of the prion protein does not require protein X

et al. 2007

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Previous studies identified several single-point mutants of the prion protein that displayed dominantnegative effects on prion replication. The dominant-negative effect was assumed to be mediated by protein X, an as-yet-unknown cellular cofactor that is believed to be essential for prion replication. To gain insight into the mechanism that underlies the dominant-negative phenomena, we evaluated the effect of the Q218K variant of full-length recombinant prion protein (Q218K rPrP), one of the dominant-negative mutants, on cell-free polymerization of wild-type rPrP into amyloid fibrils. We found that both Q218K and wild-type (WT) rPrPs were incorporated into fibrils when incubated as a mixture; however, the yield of polymerization was substantially decreased in the presence of Q218K rPrP. Furthermore, in contrast to fibrils produced from WT rPrP, the fibrils generated in the mixture of WT and Q218K rPrPs did not acquire the proteinase K-resistant core of 16 kDa that was shown previously to encompass residues 97-230 and was similar to that of PrPSc . Our studies demonstrate that the Q218K variant exhibits the dominant-negative effect in cell-free conversion in the absence of protein X, and that this effect is, presumably, mediated by physical interaction between Q218K and WT rPrP during the polymerization process.Keywords: prion protein; dominant-negative effect; prion diseases; amyloid fibrils; cell-free conversion Prion diseases are a group of fatal neurodegenerative maladies that can arise spontaneously or be inherited, and that can also be infectious (Prusiner 1998). All three forms of the prion diseases, sporadic, familial, and infectious, are induced by misfolding and aggregation of the prion protein (PrP). Single-point mutations at several positions within the PrP open reading frame were linked to familial prion diseases. Remarkably, the polymorphisms at other codons of PrP gene were found to protect against scrapie infection or development of sporadic forms of prion diseases. For example, sheep with Q/R polymorphisms at the codon 171 (equivalent to the codon 167 in mouse PrP) were resistant to scrapie, while sheep carrying the Q/Q alleles developed prion disease (Ikeda et al. 1995;Hunter et al. 1997). Human genetic studies revealed that 12% of the Japanese population carries the polymorphism E/K at the codon 219 (equivalent to the codon 218 of mouse PrP) (Shibuya et al. 1998b Abbreviations: PrP, prion protein; PrP C , normal cellular isoform of the prion protein; PrP Sc , abnormal pathological isoform of the prion protein; rPrP, full-length recombinant prion protein; WT, wild type; FTIR, Fourier transform infrared spectroscopy; ThT, Thioflavin T; GdnHCl, guanidine hydrochloride; GdnSCN, guanidine thiocyanate.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

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Section: Discussionsupporting

confidence: 78%

The dominant‐negative effect of the Q218K variant of the prion protein does not require protein X

et al. 2007

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“…In contrast to frequently discussed theoretical models (31,32), available experimental data clearly indicate that formation of rPrP amyloid involves major refolding of the native C-terminal ␣-helices to ␤-sheet structure (22,23,26,27). However, to date, recombinant PrP amyloid has only been observed to form under distinctly nonphysiological conditions, in the presence of chemical denaturants (19 -23, 25-28).…”

Section: Discussionmentioning

confidence: 93%

“…Biophysical and structural aspects of this conversion reaction remain poorly understood, largely because of fundamental difficulties of studies with brain-derived material. In recent years, however, significant advances have been made in elucidating the mechanism of conformational transitions of the recombinant prion protein (18 -21, 24, 27, 28), including structural characterization of PrP Sc -mimicking rPrP amyloid fibrils (22,23,26,27) as well as attempts to generate infectious material from purely synthetic components (25).…”

Section: Discussionmentioning

confidence: 99%

Prion Protein Amyloid Formation under Native-like Conditions Involves Refolding of the C-terminal α-Helical Domain

Cobb

Apetri

Surewicz

2008

Journal of Biological Chemistry

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Transmissible spongiform encephalopathies are associated with conformational conversion of the cellular prion protein, PrP C , into a proteinase K-resistant, amyloid-like aggregate, PrP Sc . Although the structure of PrP Sc remains enigmatic, recent studies have afforded increasingly detailed characterization of recombinant PrP amyloid. However, all previous studies were performed using amyloid fibrils formed in the presence of denaturing agents that significantly alter the folding state(s) of the precursor monomer. Here we report that PrP amyloid can also be generated under physiologically relevant conditions, where the monomeric protein is natively folded. Remarkably, site-directed spin labeling studies reveal that these fibrils possess a ␤-core structure nearly indistinguishable from that of amyloid grown under denaturing conditions, where the C-terminal ␣-helical domain of the PrP monomer undergoes major refolding to a parallel and in-register ␤-structure upon conversion. The structural similarity of fibrils formed under drastically different conditions strongly suggests that the common ␤-sheet architecture within the ϳ160 -220 core region represents a distinct global minimum in the PrP conversion free energy landscape. We also show that the N-terminal region of fibrillar PrP displays conformational plasticity, undergoing a reversible structural transition with an apparent pK a of ϳ5.3. The C-terminal region, on the other hand, retains its ␤-structure over the pH range 1-11, whereas more alkaline buffer conditions denature the fibrils into constituent PrP monomers. This profile of pH-dependent stability is reminiscent of the behavior of brainderived PrP Sc , suggesting a substantial degree of structural similarity within the ␤-core region of these PrP aggregates.

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“…The physical properties of human PrP fibrils such as conformational stability appear to determine to a large extent their biological effects (48). To evaluate the conformational stability, the fibrils produced from human prion protein in the absence and presence of crowding agents were incubated in the presence of increasing concentrations of guanidine thiocyanate (from 0 to 3.5 M) for 1 h, and the amount of intact amyloid structure was analyzed using the ThT binding assay.…”

Section: Effects Of Macromolecular Crowding On Conformational Stabilimentioning

confidence: 99%

Crowded Cell-like Environment Accelerates the Nucleation Step of Amyloidogenic Protein Misfolding

Zhou

Fan

Zhu

et al. 2009

Journal of Biological Chemistry

105

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To understand the role of a crowded physiological environment in the pathogenesis of neurodegenerative diseases, we report the following. 1) The formation of fibrous aggregates of the human Tau fragment Tau-(244 -441), when hyperphosphorylated by glycogen synthase kinase-3␤, is dramatically facilitated by the addition of crowding agents. 2) Fibril formation of nonphosphorylated Tau-(244 -441) is only promoted moderately by macromolecular crowding. 3) Macromolecular crowding dramatically accelerates amyloid formation by human prion protein. A sigmoidal equation has been used to fit these kinetic data, including published data of human ␣-synuclein, yielding lag times and apparent rate constants for the growth of fibrils for these amyloidogenic proteins. These biochemical data indicate that crowded cell-like environments significantly accelerate the nucleation step of fibril formation of human Tau fragment/human prion protein/human ␣-synuclein (a significant decrease in the lag time). These results can in principle be predicted based on some known data concerning protein concentration effects on fibril formation both in vitro and in vivo. Furthermore, macromolecular crowding causes human prion protein to form short fibrils and nonfibrillar particles with lower conformational stability and higher protease resistance activity, compared with those formed in dilute solutions. Our data demonstrate that a crowded physiological environment could play an important role in the pathogenesis of neurodegenerative diseases by accelerating amyloidogenic protein misfolding and inducing human prion fibril fragmentation, which is considered to be an essential step in prion replication.

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Site-specific Conformational Studies of Prion Protein (PrP) Amyloid Fibrils Revealed Two Cooperative Folding Domains within Amyloid Structure

Cited by 52 publications

References 48 publications

The dominant‐negative effect of the Q218K variant of the prion protein does not require protein X

The dominant‐negative effect of the Q218K variant of the prion protein does not require protein X

Prion Protein Amyloid Formation under Native-like Conditions Involves Refolding of the C-terminal α-Helical Domain

Crowded Cell-like Environment Accelerates the Nucleation Step of Amyloidogenic Protein Misfolding

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