Elongation of Mouse Prion Protein Amyloid-Like Fibrils: Effect of Temperature and Denaturant Concentration
Smirnovas V (2014) Elongation of Mouse Prion Protein Amyloid-Like Fibrils: Effect of Temperature and Denaturant
Concentration. PLoS ONE 9(4): e94469. doi:10.1371/journal.pone.0094469
Elongation of Mouse Prion Protein Amyloid-Like Fibrils: Effect of Temperature and Denaturant Concentration
Katazyna Milto 0
Ksenija Michailova 0
Vytautas Smirnovas 0
Ilia V. Baskakov, University of Maryland School of Medicine, United States of America
0 Department of Biothermodynamics and Drug Design, Vilnius University Institute of Biotechnology , Vilnius , Lithuania
Prion protein is known to have the ability to adopt a pathogenic conformation, which seems to be the basis for protein-only infectivity. The infectivity is based on self-replication of this pathogenic prion structure. One of possible mechanisms for such replication is the elongation of amyloid-like fibrils. We measured elongation kinetics and thermodynamics of mouse prion amyloid-like fibrils at different guanidine hydrochloride (GuHCl) concentrations. Our data show that both increases in temperature and GuHCl concentration help unfold monomeric protein and thus accelerate elongation. Once the monomers are unfolded, further increases in temperature raise the rate of elongation, whereas the addition of GuHCl decreases it. We demonstrated a possible way to determine different activation energies of amyloid-like fibril elongation by using folded and unfolded protein molecules. This approach separates thermodynamic data for fibril-assisted monomer unfolding and for refolding and formation of amyloid-like structure.
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Prion protein (PrP) plays a big role in a number of lethal
neurological diseases, known as transmissible spongiform
encephalopaties. These disorders are associated with aggregation of
normal cellular prion protein (PrPC) into pathogenic
beta-sheetrich prion isoforms (PrPSc). Although the majority of suspected
cases of human prion diseases are sporadic [1], prions are mostly
known because of their infectivity. The infectious nature of prion
diseases is based on the ability of PrPSc to self-replicate by
converting PrPC into same pathogenic isoform. One of possible
mechanisms of pathogenic prion structure replication is elongation
of amyloid-like fibrils.
Since the discovery of prions, one of the main tasks in the field
was to produce infectious PrP conformation in vitro, as this would
finally prove the hypothesis of protein-only infection [2]. A
number of attempts generated amyloid-like structures [3,4]. Such
prion protein fibrils share some properties of PrPSc (such as
betasheet-rich secondary structure and ability to self-replicate by
addition of native PrP) but have much shorter proteinase K (PK)
resistant cores [5], and show slight infectivity only in mice which
overexpress PrPC 16 fold [6]. Later studies showed that PK
resistance can be extended by annealing at high temperature [7],
or by using protein misfolding cyclic amplification (PMCA) [8].
When examined by hydrogen/deuterium (H/D) exchange,
annealed fibrils showed only slight differences in deuterium
incorporation, when compared to untreated amyloid-like fibrils
[9]. Such fibrils induced disease in hamsters only after the second
passage, thus they are not directly infective [10]. PMCA-generated
recombinant PrP amyloid-like fibrils have an extended region,
protected from H/D exchange [8], and can induce disease in
hamsters, though not as effectively as PrPSc [11]. Studies of H/D
exchange on brain-derived infective PrP show similar highly
packed structure as in case of recombinant PrP amyloid-like fibrils,
but in a much longer region (entire 90230 region versus 160220
region) [12]. It suggests the possibility that brain-derived PrPSc
may be similar to amyloid-like fibrils, just with a much longer
betasheet core region. Recently Ma and co-workers reported a
protocol for de novo generation of infectious prions from
recombinant PrP [13]. It has similar infectivity to brain-derived
prions, but due to low efficiency of the protocol, the structure of
these prions is still unknown.
The structure of recombinant PrP amyloid-like fibrils was
extensively studied by a number of techniques all giving similar
conclusions parallel in-register beta-sheet in the C-terminal
region (starting from residues 160170, up to residues 220225)
and a disordered N-terminus [9,14,15]. The difference in structure
between the native prion protein and the amyloid form requires a
conversion of the native alpha-helices into beta-sheets. Usually,
amyloid-like fibrils are formed in the presence of moderate
denaturant concentration [3,4], under conditions where the native
protein is at least partially unfolded. However in vivo, the
environment does not help in unfolding of native helices. We
tried to test if prion protein amyloid-like fibrils self-replicate using
stable native PrP, and how environmental factors (such as
temperature and denaturant concentration) affect fibril elongation
kinetics.
Results and Discussion
Amyloid (...truncated)