Heterologous Stacking of Prion Protein Peptides Reveals Structural
Details of Fibrils and Facilitates Complete Inhibition of Fibril
Growth |
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Authors: | Ronald S Boshuizen Veronica Schulz Michela Morbin Giulia Mazzoleni Rob H Meloen and Johannes P M Langedijk |
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Institution: | ‡Pepscan Therapeutics B.V., Zuidersluisweg 2, 8243 RC AB Lelystad, The Netherlands, §Fondazione Istituto Di Ricovero e Cura a Carattere Scientifico-Instituto Neurologico “Carlo Besta,” via Celoria 11, 20133 Milan, Italy, and ¶Academic Biomedical Centre, University of Utrecht, Yalelaan 1, Utrecht, The Netherlands |
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Abstract: | Fibrils play an important role in the pathogenesis of amyloidosis; however,
the underlying mechanisms of the growth process and the structural details of
fibrils are poorly understood. Crucial in the fibril formation of prion
proteins is the stacking of PrP monomers. We previously proposed that the
structure of the prion protein fibril may be similar as a parallel left-handed
β-helix. The β-helix is composed of spiraling rungs of parallel
β-strands, and in the PrP model residues 105–143 of each PrP
monomer can contribute two β-helical rungs to the growing fibril. Here we
report data to support this model. We show that two cyclized human PrP
peptides corresponding to residues 105–124 and 125–143, based on
two single rungs of the left-handed β-helical core of the human
PrPSc fibril, show spontaneous cooperative fibril growth in
vitro by heterologous stacking. Because the structural model must have
predictive value, peptides were designed based on the structure rules of the
left-handed β-helical fold that could stack with prion protein peptides
to stimulate or to block fibril growth. The stimulator peptide was designed as
an optimal left-handed β-helical fold that can serve as a template for
fibril growth initiation. The inhibiting peptide was designed to bind to the
exposed rung but frustrate the propagation of the fibril growth. The single
inhibitory peptide hardly shows inhibition, but the combination of the
inhibitory with the stimulatory peptide showed complete inhibition of the
fibril growth of peptide huPrP-(106–126). Moreover, the unique strategy
based on stimulatory and inhibitory peptides seems a powerful new approach to
study amyloidogenic fibril structures in general and could prove useful for
the development of therapeutics.Transmissible spongiform encephalopathies are neurodegenerative disorders
in a wide range of mammalian species, including Creutzfeldt-Jacob disease in
man, scrapie in sheep, and bovine spongiform encephalopathy in cattle. The
deposition of aggregated prion protein fibrils on and in neurons is regarded
to be the source of these neurodegenerative diseases and is frequently
associated with occurrence of Congo red positivity
(1–3).
The fibrils are formed by the conformational change of the prion protein
(PrPc)2
into the scrapie form (PrPSc). The misfolded conformer of the prion
protein (PrPSc) is considered as the causative agent in these
diseases according to the protein-only hypothesis
(4). Studies have shown the
toxicity of fibrils of the full-length recombinant mammalian prion protein as
well as soluble β-rich oligomers to cultured cells and primary neurons
(5).It is still unknown how much of the whole PrPSc molecule is
involved in the fibril growth. It is shown that the N-terminal part of PrP,
specifically residues 112–141, can go through conformational changes
involving β-strand formation, which subsequently triggers fibril growth
(6–8),
and solid state NMR studies showed that residues 112–141 are part of the
highly ordered core of huPrP-(23–144)
(9). It was previously shown
that peptides based on the 89–143 region of the human PrP protein can
form fibrils rich in β-sheet structure which are biologically active in
transgenic mice (10). Within
this region it is the huPrP-(106–126) peptide that is the smallest known
region of PrP that forms fibrils that are toxic and resemble the physiological
properties of PrPSc
(11–16).
The formation of PrPSc is considered to be a two-step event; first,
there is the binding between PrPc and PrPSc and
subsequently the conformational conversion from PrPc into
PrPSc occurs. Mutation studies in a prion-infected neuroblastoma
cell line showed that in mouse PrP the regions 101–110 and 136–158
are crucial for the binding and conversion events, respectively
(17). Because prevention of
fibril growth is the prime therapeutic target, detailed structural knowledge
of the fibril is essential for understanding the mechanism of fibril growth.
However, structural analysis of amyloid fibrils is hampered by insolubility,
isomorphism, and aggregation. X-ray diffraction of several amyloid fibrils
revealed a so-called cross-β diffraction pattern which indicates that the
fibrils contain β-strands perpendicular to the fibril axis and hydrogen
bonds in parallel (18,
19). Thus, for fibril growth
the β-strands have to stack on top of each other. Several structures have
been suggested to explain the structure of the stacked β-strands;
e.g. a parallel in register organization of stacked β hairpins
(24) or the comparable dry
steric zipper structure (25).
Previously, we and other groups suggested that the β-sheet structures in
the PrPSc fibril may be similar to the topologically most simple
class of β-sheets; that is, the parallel left-handed β-helix
()
(6,
20,
21). The left-handed β
helix is formed by triangular progressive coils (rungs) of 18–20
residues. Each rung is formed by three hexapeptide motifs, which results in an
approximate 3-fold symmetry. Backbone-backbone hydrogen bonding and stacking
of the side chains in adjacent rungs contribute to the folding of
β-helical rungs. We suggested that each PrPSc monomer
contributes two left-handed β-helical rungs to the fibril, comprising
residues 105–124 and 125–143
(). This
two-rung structural model was recently confirmed for amyloid fibrils of the
HET-s prion by NMR analysis
(22). In contrast to fibrils
which are composed of homologous stacks of identical peptides, e.g.
the Aβ peptide (23), the
PrPSc fibril is more complex because it is composed of heterologous
stacks of at least two peptides. For homologous stacking of two identical
peptides, the complementarity issue is relatively simple because the identical
side chains are in register (e.g. Ile-Ile, Val-Val stacking, and Asn
ladders). However, in the case of heterologous stacking, the side chains of
the additional heterologous peptide needs to be complementary with the other
peptide to allow fibril growth.Open in a separate windowA, theoretical model of the fibrillogenic core of
PrPSc. In the PrPSc model based on the left-handed
β-helix structure, each PrPSc monomer contributes two stacked
rungs to the fibril (different color for each monomer). The protofibril is
formed by consecutive stacking of the two windings. The stack of two rungs
provides enough elevation to accommodate the remaining part (residues ∼
146–253) of the PrPSc molecule
(20). B, the
left-handed β-helix structure of LpxA-based on x-ray crystallography. In
the left-handed β-helix structure of LpxA (PDB code 1LXA) rungs 6 and 7
are indicated (red) that were used for the heterologous stacking
studies. Linear and cyclized peptides based on rung 6 and rung 7 were modified
to satisfy the ideal left-handed β-helix motif (see “LpxA
Peptides” under “Results”) and tested for their intrinsic
and cooperative fibrillogenicity. C, left-handed β-helical rung
based on rung 6 of LpxA. The rung is formed by three hexapeptide motifs, which
results in an approximate 3-fold symmetry. A left-handed β-helical rung
can be cyclized by a disulfide bridge after the introduction of a cysteine at
position 2 of the first hexapeptide and position 1 of the fourth hexapeptide
(according to the numbering used for the hexapeptide repeats in the
left-handed β-helix).To investigate whether the suggested rungs 105–123 and 125–143
from human PrP could be complementary
(20), we studied the
homologous stacking and the heterologous stacking of linear and cyclized prion
protein peptides comprising the huPrP-(105–143) region
(KTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGS). Qualitative and semiquantitative
analysis were done by electron microscopy and Congo red staining. The
quantification of the fibril formation was assessed by thioflavin S staining,
in which the addition of polyanions (e.g. heparin) enhance the
β-sheet formation of peptides comprising the 82–143 region of PrP
and improve the reproducibility of the fibril growth
(24). This study provides
first evidence of heterologous stacking by two isolated putative β-strand
layers (or rungs) of the human prion protein with fibril formation as a
result. The left-handed β-helix structure provided insight for the
“stack-and-stop” approach. With this approach a mix of a
stimulatory peptide and an inhibitory peptide could completely block fibril
formation. The stimulatory peptide was based on the 125–143 region that
was optimized to serve as a folding template for the consecutive stacking of
the 106–126 peptide. This cooperative fibril growth was completely
inhibited by the inhibitory peptide based on peptides 106–126 with
strategic d-amino acid and/or proline substitutions. The findings
in this study support models in which the sequential strands in a fibril must
somehow spiral up- or downward along the fibril axis, e.g. like the
hypothetical left-handed β-helical structure of PrPSc fibrils
(20). Furthermore, it allows
the development of well defined small protein modules which can be used for
structure studies of the 82–143 domain of PrPSc and the
development of therapeutics. |
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