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Sarah?L. Shammas Christopher?A. Waudby Shuyu Wang Alexander?K. Buell Tuomas?P.J. Knowles Heath Ecroyd Mark?E. Welland John?A. Carver Christopher?M. Dobson Sarah Meehan 《Biophysical journal》2011,101(7):1681-1689
The molecular chaperone αB-crystallin is a small heat-shock protein that is upregulated in response to a multitude of stress stimuli, and is found colocalized with Aβ amyloid fibrils in the extracellular plaques that are characteristic of Alzheimer''s disease. We investigated whether this archetypical small heat-shock protein has the ability to interact with Aβ fibrils in vitro. We find that αB-crystallin binds to wild-type Aβ42 fibrils with micromolar affinity, and also binds to fibrils formed from the E22G Arctic mutation of Aβ42. Immunoelectron microscopy confirms that binding occurs along the entire length and ends of the fibrils. Investigations into the effect of αB-crystallin on the seeded growth of Aβ fibrils, both in solution and on the surface of a quartz crystal microbalance biosensor, reveal that the binding of αB-crystallin to seed fibrils strongly inhibits their elongation. Because the lag phase in sigmoidal fibril assembly kinetics is dominated by elongation and fragmentation rates, the chaperone mechanism identified here represents a highly effective means to inhibit fibril proliferation. Together with previous observations of αB-crystallin interaction with α-synuclein and insulin fibrils, the results suggest that this mechanism is a generic means of providing molecular chaperone protection against amyloid fibril formation. 相似文献
3.
Ronald S. Boshuizen Veronica Schulz Michela Morbin Giulia Mazzoleni Rob H. Meloen Johannes P. M. Langedijk 《The Journal of biological chemistry》2009,284(19):12809-12820
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
(Fig. 1A)
(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
(Fig. 1A). 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 windowFIGURE 1.A, 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. 相似文献
4.
Using implicit solvent molecular dynamics and replica exchange simulations, we study the impact of ibuprofen on the growth of wild-type Aβ fibrils. We show that binding of ibuprofen to Aβ destabilizes the interactions between incoming peptides and the fibril. As a result, ibuprofen interference modifies the free energy landscape of fibril growth and reduces the free energy gain of Aβ peptide binding to the fibril by ≃2.5 RT at 360 K. Furthermore, ibuprofen interactions shift the thermodynamic equilibrium from fibril-like locked states to disordered docked states. Ibuprofen''s anti-aggregation effect is explained by its competition with incoming Aβ peptides for the same binding site located on the fibril edge. Although ibuprofen impedes fibril growth, it does not significantly change the mechanism of fibril elongation or the structure of Aβ peptides bound to the fibril. 相似文献
5.
Sharad P. Adekar Igor Klyubin Sally Macy Michael J. Rowan Alan Solomon Scott K. Dessain Brian O'Nuallain 《The Journal of biological chemistry》2010,285(2):1066-1074
We have previously shown that a subpopulation of naturally occurring human IgGs were cross-reactive against conformational epitopes on pathologic aggregates of Aβ, a peptide that forms amyloid fibrils in the brains of patients with Alzheimer disease, inhibited amyloid fibril growth, and dissociated amyloid in vivo. Here, we describe similar anti-amyloidogenic activity that is a general property of free human Ig γ heavy chains. A γ1 heavy chain, F1, had nanomolar binding to an amyloid fibril-related conformational epitope on synthetic oligomers and fibrils as well as on amyloid-laden tissue sections. F1 did not bind to native Aβ monomers, further indicating the conformational nature of its binding site. The inherent anti-amyloidogenic activity of Ig γ heavy chains was demonstrated by nanomolar amyloid fibril and oligomer binding by polyclonal and monoclonal human heavy chains that were isolated from inert or weakly reactive antibodies. Most importantly, the F1 heavy chain prevented in vitro fibril growth and reduced in vivo soluble Aβ oligomer-induced impairment of rodent hippocampal long term potentiation, a cellular mechanism of learning and memory. These findings demonstrate that free human Ig γ heavy chains comprise a novel class of molecules for developing potential therapeutics for Alzheimer disease and other amyloid disorders. Moreover, establishing the molecular basis for heavy chain-amyloidogenic conformer interactions should advance understanding on the types of interactions that these pathologic assemblies have with biological molecules. 相似文献
6.
Deposition of amyloid fibrils, consisting primarily of Aβ40 and Aβ42 peptides, in the extracellular space in the brain is a major characteristic of Alzheimer''s disease (AD). We recently developed new (to our knowledge) drug candidates for AD that inhibit the fibril formation of Aβ peptides and eliminate their neurotoxicity. We performed all-atom molecular-dynamics simulations on the Aβ42 monomer at its α-helical conformation and a pentamer fibril fragment of Aβ42 peptide with or without LRL and fluorene series compounds to investigate the mechanism of inhibition. The results show that the active drug candidates, LRL22 (EC50 = 0.734 μM) and K162 (EC50 = 0.080 μM), stabilize hydrophobic core I of Aβ42 peptide (residues 17–21) to its α-helical conformation by interacting specifically in this region. The nonactive drug candidates, LRL27 (EC50 > 10 μM) and K182 (EC50 > 5 μM), have little to no similar effect. This explains the different behavior of the drug candidates in experiments. Of more importance, this phenomenon indicates that hydrophobic core I of the Aβ42 peptide plays a major mechanistic role in the formation of amyloid fibrils, and paves the way for the development of new drugs against AD. 相似文献
7.
S. Fabio Falsone Andreas J. Kungl Angelika Rek Roberto Cappai Klaus Zangger 《The Journal of biological chemistry》2009,284(45):31190-31199
α-Synuclein is an intrinsically unstructured protein that binds to membranes, forms fibrils, and is involved in neurodegeneration. We used a reconstituted in vitro system to show that the molecular chaperone Hsp90 influenced α-synuclein vesicle binding and amyloid fibril formation, two processes that are tightly coupled to α-synuclein folding. Binding of Hsp90 to monomeric α-synuclein occurred in the low micromolar range, involving regions of α-synuclein that are critical for vesicle binding and amyloidogenesis. As a consequence, both processes were affected. In the absence of ATP, the accumulation of non-amyloid α-synuclein oligomers prevailed over fibril formation, whereas ATP favored fibril growth. This suggests that Hsp90 modulates the assembly of α-synuclein in an ATP-dependent manner. We propose that Hsp90 affects these folding processes by restricting conformational fluctuations of α-synuclein. 相似文献
8.
Shuichi Karasaki 《The Journal of cell biology》1963,18(1):135-151
The yolk platelets of mature eggs and young embryonic cells of all amphibian species studied (Rana pipiens, Triturus pyrrhogaster, Diemictylus viridescens, Rana nigromaculata, and Bufo vulgaris) have a superficial layer of fine particles or fibrils (ca. 50 A in diameter), a central main body with a crystalline lattice structure, and an enclosing membrane approximately 70 A in thickness. Electron micrographs of the main body reveal hexagonal net (spacing ca. 70 A), square net (spacing ca. 80 A), and parallel band (spacing from 35 to 100 A but most frequent at ca. 70 A) patterns. The crystalline structure is believed to be a simple hexagonal lattice made of closely packed cylindrical rods. Each rod is estimated to be about 80 A in diameter and 160 A in length. 相似文献
9.
THE USE OF SHADOW-CASTING TECHNIQUE FOR MEASUREMENT OF THE WIDTH OF ELONGATED PARTICLES 总被引:3,自引:3,他引:0 下载免费PDF全文
A method is described for the estimation of the true width of fibrillar or rod-like structures from electron micrographs of metal-shadowed preparations. The method is based on variations in the image width as a function of the angle (β) between the long axis of the fibril and the direction of the shadow in the plane of the preparation. The image width when β = 0° practically represents the real width of the elongated particle but is often indistinguishable from the background. The fibril image width is conveniently measured at β values between 15° and 90°. The true width is obtained by plotting the image width versus sin β and extrapolating to β = 0°. Latex spheres are sprayed with the fibrils or rods to indicate the direction of shadow. Tobacco mosaic virus (TMV) was used as a model structure because of its known constant diameter of 150 A (5). The width (in the case of TMV equal to the diameter) found by the present method was 150 A ± 8 A. 相似文献
10.
Natallia Makarava Valeriy G. Ostapchenko Regina Savtchenko Ilia V. Baskakov 《The Journal of biological chemistry》2009,284(21):14386-14395
A key structural component of amyloid fibrils is a highly ordered,
crystalline-like cross-β-sheet core. Conformationally different amyloid
structures can be formed within the same amino acid sequence. It is generally
assumed that individual fibrils consist of conformationally uniform
cross-β-structures. Using mammalian recombinant prion protein (PrP), we
showed that, contrary to common perception, amyloid is capable of
accommodating a significant conformational switching within individual
fibrils. The conformational switch occurred when the amino acid sequence of a
PrP variant used as a precursor substrate in a fibrillation reaction was not
compatible with the strain-specific conformation of the fibrillar template.
Despite the mismatch in amino acid sequences between the substrate and
template, individual fibrils recruited the heterologous PrP variant; however,
the fibril elongation proceeded through a conformational adaptation, resulting
in a change in amyloid strain within individual fibrils. This study
illustrates the high adaptation potential of amyloid structures and suggests
that conformational switching within individual fibrils may account for
adaptation of amyloid strains to a heterologous substrate. This work proposes
a new mechanistic explanation for the phenomenon of strain conversion and
illustrates the direction in evolution of amyloid structures. This study also
provides a direct illustration that catalytic activity of self-replicating
amyloid structures is not ultimately coupled with their templating effect.The ability to form amyloid structures is considered to be one of the most
general properties of a polypeptide backbone
(1). Regardless of the specific
peptides or proteins involved in fibril formation, all types of amyloid
fibrils share a common structural motif that consists of a
cross-β-structure (2).
Cross-β-structures are comprised of highly ordered, nearly anhydrous,
crystalline-like β-sheets stabilized by hydrogen bonding and densely
packed side chains (3,
4). Growing evidence indicates
that multiple amyloid structures referred to as amyloid strains could be
formed within the same amino acid sequence
(5–7).Amyloids are capable of self-replicating
(8). Self-replicating
properties of amyloid fibrils are attributed to the unique arrangement of
cross-β-strands that are assembled perpendicular to the fibrillar axis,
where β-strands at the growing edge provide a template for recruiting and
converting a monomeric precursor. The self-replicating property of the amyloid
cross-β-structure consists of two activities: catalytic (i.e.
the ability to convert a monomeric precursor into an amyloid state) and
templating (i.e. the ability to accurately imprint the
strain-specific conformation onto a newly recruited polypeptide). The
templating activity is believed to be intimately coupled to the catalytic
activity and accounts for the high fidelity of amyloid replication. High
fidelity of replication requires identity or high homology between the amino
acid sequences of a fibrillar template and a precursor substrate. The species
specificity of a template-substrate interaction is believed to account for the
species barrier in prion transmission and species specificity of in
vitro cross-seeded fibrillation reactions. Local perturbations arising
due to mismatches in packing of amino acid side chains within the
crystalline-like cross-β-structures could prevent efficient replication
of amyloid fibrils.It is generally assumed that individual fibrils are structurally uniform,
i.e. maintain the same structure of a cross-β-core throughout
the fibrillar length. In the current study, we showed that, contrary to the
common perception, amyloid fibrils are capable of accommodating significant
conformational switching within individual fibrils. The conformational switch
occurred when the amino acid sequence of the precursor substrate was not
compatible with the conformation of the template. Despite mismatched amino
acid sequences, individual fibrils were able to recruit the heterologous
recombinant prion protein
(PrP)2 variant;
however, fibril elongation proceeded through switching to a new conformational
state. The implications of these studies are multifold. First, our work
illustrates the high adaptation potential of amyloid structures and suggests
that the conformational switch accounts for adaptation of amyloid strains to
the heterologous substrate. Second, the current studies propose a new
molecular explanation for the phenomenon referred to as convergence of
strains. Third, this work illustrates the directionality in evolution of
amyloid structures, showing that the species-specific amyloid structures
(i.e. structures that exist only within a single PrP sequence) can
give rise to promiscuous or indiscriminative structures (structures compatible
with several PrP variants), but not vice versa. Finally, our studies provide
direct illustration that catalytic activity of self-replicating amyloid
structures is not ultimately coupled with their templating effect. 相似文献
11.
The dynamics of amyloid fibrils, including their formation and dissociation, could be of vital importance in life. We studied the kinetics of dissociation of the amyloid fibrils from wild-type hen lysozyme at 25°C in vitro as a function of pressure using Trp fluorescence as a probe. Upon 100-fold dilution of 8 mg ml−1 fibril solution in 80 mM NaCl, pH 2.2, no immediate change occurred in Trp fluorescence, but at pressures of 50–450 MPa the fluorescence intensity decreased rapidly with time (kobs = 0.00193 min−1 at 0.1 MPa, 0.0348 min−1 at 400 MPa). This phenomenon is attributable to the pressure-accelerated dissociation of amyloid fibrils into monomeric hen lysozyme. From the pressure dependence of the rates, which reaches a plateau at ∼450 MPa, we determined the activation volume ΔV0‡ = −32.9 ± 1.7 ml mol(monomer)−1 and the activation compressibility Δκ‡ = −0.0075 ± 0.0006 ml mol(monomer)−1 bar−1 for the dissociation reaction. The negative ΔV0‡ and Δκ‡ values are consistent with the notion that the amyloid fibril from wild-type hen lysozyme is in a high-volume and high-compressibility state, and the transition state for dissociation is coupled with a partial hydration of the fibril. 相似文献
12.
Claire J. Sarell Lucy A. Woods Yongchao Su Galia T. Debelouchina Alison E. Ashcroft Robert G. Griffin Peter G. Stockley Sheena E. Radford 《The Journal of biological chemistry》2013,288(10):7327-7337
Amyloid fibrils can be generated from proteins with diverse sequences and folds. Although amyloid fibrils assembled in vitro commonly involve a single protein precursor, fibrils formed in vivo can contain more than one protein sequence. How fibril structure and stability differ in fibrils composed of single proteins (homopolymeric fibrils) from those generated by co-polymerization of more than one protein sequence (heteropolymeric fibrils) is poorly understood. Here we compare the structure and stability of homo and heteropolymeric fibrils formed from human β2-microglobulin and its truncated variant ΔN6. We use an array of approaches (limited proteolysis, magic angle spinning NMR, Fourier transform infrared spectroscopy, and fluorescence) combined with measurements of thermodynamic stability to characterize the different fibril types. The results reveal fibrils with different structural properties, different side-chain packing, and strikingly different stabilities. These findings demonstrate how co-polymerization of related precursor sequences can expand the repertoire of structural and thermodynamic polymorphism in amyloid fibrils to an extent that is greater than that obtained by polymerization of a single precursor alone. 相似文献
13.
Keiichi Yamaguchi Kenshiro Hasuo Masatomo So Kensuke Ikenaka Hideki Mochizuki Yuji Goto 《The Journal of biological chemistry》2021,297(5)
Amyloid fibrils, crystal-like fibrillar aggregates of proteins associated with various amyloidoses, have the potential to propagate via a prion-like mechanism. Among known methodologies to dissolve preformed amyloid fibrils, acid treatment has been used with the expectation that the acids will degrade amyloid fibrils similar to acid inactivation of protein functions. Contrary to our expectation, treatment with strong acids, such as HCl or H2SO4, of β2-microglobulin (β2m) or insulin actually promoted amyloid fibril formation, proportionally to the concentration of acid used. A similar promotion was observed at pH 2.0 upon the addition of salts, such as NaCl or Na2SO4. Although trichloroacetic acid, another strong acid, promoted amyloid fibril formation of β2m, formic acid, a weak acid, did not, suggesting the dominant role of anions in promoting fibril formation of this protein. Comparison of the effects of acids and salts confirmed the critical role of anions, indicating that strong acids likely induce amyloid fibril formation via an anion-binding mechanism. The results suggest that although the addition of strong acids decreases pH, it is not useful for degrading amyloid fibrils, but rather induces or stabilizes amyloid fibrils via an anion-binding mechanism. 相似文献
14.
Frank Shewmaker Ryan P. McGlinchey Kent R. Thurber Peter McPhie Fred Dyda Robert Tycko Reed B. Wickner 《The Journal of biological chemistry》2009,284(37):25065-25076
The extracellular curli proteins of Enterobacteriaceae form fibrous structures that are involved in biofilm formation and adhesion to host cells. These curli fibrils are considered a functional amyloid because they are not a consequence of misfolding, but they have many of the properties of protein amyloid. We confirm that fibrils formed by CsgA and CsgB, the primary curli proteins of Escherichia coli, possess many of the hallmarks typical of amyloid. Moreover we demonstrate that curli fibrils possess the cross-β structure that distinguishes protein amyloid. However, solid state NMR experiments indicate that curli structure is not based on an in-register parallel β-sheet architecture, which is common to many human disease-associated amyloids and the yeast prion amyloids. Solid state NMR and electron microscopy data are consistent with a β-helix-like structure but are not sufficient to establish such a structure definitively.Interest in amyloid is largely because of its association with many late onset human diseases, including Alzheimer disease (Aβ),2 Parkinson disease (α-synuclein), type II diabetes (amylin), and the transmissible spongiform encephalopathies (PrP). In each case a particular endogenous protein becomes incorporated into large aggregates known as amyloid, which was originally defined by pathologists as a tissue deposit staining like starch (1). However, the term amyloid has come to mean a filamentous protein aggregate with cross-β secondary structure (cross-β means that the β-strands that form β-sheets in the amyloid fibrils run approximately perpendicular to the long axis of the fibril with interstrand hydrogen bonds that run approximately parallel to the long axis) and protease resistance. Morphologically amyloid fibrils may vary in length from tens of nanometers to micrometers and have diameters in the range of 3–10 nm, although lateral association can produce much larger apparent diameters.Proteins from a variety of organisms can form amyloid both in vitro and in vivo, and the propensity to form amyloid may be a common property of many proteins (2). In addition to disease-associated amyloids, there are several confirmed cases of functional amyloid (for a review, see Ref. 3). For example, hydrophobins are amyloid-like proteins that coat the surface of fungal cells, and amyloid fibrils coating fish eggs protect them from dehydration (4, 5). The [Het-s] prion of Podospora anserina is involved in heterokaryon incompatibility, a recognition of non-self reaction believed to be important as a defense against fungal virus infection (6).Curli are extracellular filamentous structures of Enterobacteriaceae (7) that are integral to biofilm formation and are the major protein component of the extracellular matrix of these organisms (8). Curli of Escherichia coli are composed of the secreted proteins CsgA and CsgB. The latter is believed to prime the polymerization of the former and anchor the fibrils to the outer membrane (9). Both CsgA and CsgB fibrils are β-sheet-rich and, like amyloids, stain with the dye Congo red (10, 11).Because amyloid fibrils are non-crystalline and insoluble, solution NMR and x-ray crystallography are not directly applicable in structural studies. Solid state NMR and electron spin resonance have both been useful in obtaining constraints on amyloid structures and, in some cases, determining detailed structural information. The disease-associated amyloids formed by Aβ, amylin, α-synuclein, and tau along with the infectious amyloids of several yeast prions each have in-register parallel β-sheet structure (12–19).Here we confirm that the fibrils formed in vitro by CsgA and CsgB proteins are amyloids and explore their structure using solid state NMR and electron microscopy. Our results indicate that, unlike the pathogenic amyloids of humans and yeast, CsgA and CsgB amyloids are not in-register parallel β-sheet structures. Solid state NMR and electron microscopy data are consistent with a β-helix-like structure but do not establish such a structure definitively. 相似文献
15.
Carol L. Ladner Min Chen David P. Smith Geoffrey W. Platt Sheena E. Radford Ralf Langen 《The Journal of biological chemistry》2010,285(22):17137-17147
β2-microglobulin (β2m) is a 99-residue protein with an immunoglobulin fold that forms β-sheet-rich amyloid fibrils in dialysis-related amyloidosis. Here the environment and accessibility of side chains within amyloid fibrils formed in vitro from β2m with a long straight morphology are probed by site-directed spin labeling and accessibility to modification with N-ethyl maleimide using 19 site-specific cysteine variants. Continuous wave electron paramagnetic resonance spectroscopy of these fibrils reveals a core predominantly organized in a parallel, in-register arrangement, by contrast with other β2m aggregates. A continuous array of parallel, in-register β-strands involving most of the polypeptide sequence is inconsistent with the cryoelectron microscopy structure, which reveals an architecture based on subunit repeats. To reconcile these data, the number of spins in close proximity required to give rise to spin exchange was determined. Systematic studies of a model protein system indicated that juxtaposition of four spin labels is sufficient to generate exchange narrowing. Combined with information about side-chain mobility and accessibility, we propose that the amyloid fibrils of β2m consist of about six β2m monomers organized in stacks with a parallel, in-register array. The results suggest an organization more complex than the accordion-like β-sandwich structure commonly proposed for amyloid fibrils. 相似文献
16.
Jessica W. Wu Leonid Breydo J. Mario Isas Jerome Lee Yurii G. Kuznetsov Ralf Langen Charles Glabe 《The Journal of biological chemistry》2010,285(9):6071-6079
Soluble amyloid oligomers are potent neurotoxins that are involved in a wide range of human degenerative diseases, including Alzheimer disease. In Alzheimer disease, amyloid β (Aβ) oligomers bind to neuronal synapses, inhibit long term potentiation, and induce cell death. Recent evidence indicates that several immunologically distinct structural variants exist as follows: prefibrillar oligomers (PFOs), fibrillar oligomers (FOs), and annular protofibrils. Despite widespread interest, amyloid oligomers are poorly characterized in terms of structural differences and pathological significance. FOs are immunologically related to fibrils because they react with OC, a conformation-dependent, fibril-specific antibody and do not react with antibodies specific for other types of oligomers. However, fibrillar oligomers are much smaller than fibrils. FOs are soluble at 100,000 × g, rich in β-sheet structures, but yet bind weakly to thioflavin T. EPR spectroscopy indicates that FOs display significantly more spin-spin interaction at multiple labeled sites than PFOs and are more structurally similar to fibrils. Atomic force microscopy indicates that FOs are approximately one-half to one-third the height of mature fibrils. We found that Aβ FOs do not seed the formation of thioflavin T-positive fibrils from Aβ monomers but instead seed the formation of FOs from Aβ monomers that are positive for the OC anti-fibril antibody. These results indicate that the lattice of FOs is distinct from the fibril lattice even though the polypeptide chains are organized in an immunologically identical conformation. The FOs resulting from seeded reactions have the same dimensions and morphology as the initial seeds, suggesting that the seeds replicate by growing to a limiting size and then splitting, indicating that their lattice is less stable than fibrils. We suggest that FOs may represent small pieces of single fibril protofilament and that the addition of monomers to the ends of FOs is kinetically more favorable than the assembly of the oligomers into fibrils via sheet stacking interaction. These studies provide novel structural insight into the relationship between fibrils and FOs and suggest that the increased toxicity of FOs may be due to their ability to replicate and the exposure of hydrophobic sheet surfaces that are otherwise obscured by sheet-sheet interactions between protofilaments in a fibril. 相似文献
17.
Alessandra Corazza Enrico Rennella Paul Schanda Maria Chiara Mimmi Thomas Cutuil Sara Raimondi Sofia Giorgetti Federico Fogolari Paolo Viglino Lucio Frydman Maayan Gal Vittorio Bellotti Bernhard Brutscher Gennaro Esposito 《The Journal of biological chemistry》2010,285(8):5827-5835
β2-microglobulin (β2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified β2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G β2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G β2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the β2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type β2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G β2m mutant. 相似文献
18.
Jiann-Jiu Wu Mary Ann Weis Lammy S. Kim Bryan G. Carter David R. Eyre 《The Journal of biological chemistry》2009,284(9):5539-5545
Collagen type V/XI is a minor but essential component of collagen fibrils
in vertebrates. We here report on age- and tissue-related variations in
isoform usage in cartilages. With maturation of articular cartilage, the
α1(V) chain progressively replaced the α2(XI) chain. A mix of the
molecular isoforms, α1(XI)α1(V)α3(XI) and
α1(XI)α2(XI)α3(XI), best explained this finding. A
prominence of α1(V) chains is therefore characteristic and a potential
biomarker of mature mammalian articular cartilage. Analysis of cross-linked
peptides showed that the α1(V) chains were primarily cross-linked to
α1(XI) chains in the tissue and hence an integral component of the V/XI
polymer. From nucleus pulposus of the intervertebral disc (in which the bulk
collagen monomer is type II as in articular cartilage), type V/XI collagen
consisted of a mix of five genetically distinct chains, α1(XI),
α2(XI), α3(XI), α1(V), and α2(V). These presumably
were derived from several different molecular isoforms, including
α1(XI)α2(XI)α3(XI), (α1(XI))2α2(V),
and others. Meniscal fibrocartilage shows yet another V/XI phenotype. The
findings support and extend the concept that the clade B subfamily of COL5 and
COL11 gene products should be considered members of the same collagen
subfamily, from which, in combination with clade A gene products (COL2A1 or
COL5A2), a range of molecular isoforms has evolved into tissue-dependent
usage. We propose an evolving role for collagen V/XI isoforms as an adaptable
polymeric template of fibril macro-architecture.The collagen framework of hyaline cartilages is based on a covalently
cross-linked heteropolymeric network of types II, IX, and XI collagens. During
development, collagen type IX molecules are covalently linked to the surface
of thin, new fibrils of type II collagen polymerized on a template of type XI
collagen
(1–5).
In fetal cartilage, type XI collagen is a heterotrimer of three genetically
distinct chains, α1(XI), α2(XI), and α3(XI) in a 1:1:1 ratio
(6–9).
The α3(XI) chain has the same primary sequence as α1(II), but the
chains differ in their post-translational processing and cross-linking
properties
(7–9).
All three collagen subunits, II, IX, and XI, are heavily cross-linked in the
same fibril through a lysyl oxidase-mediated mechanism
(2,
5,
9). The location of the
cross-links determined by sequence analysis of peptides prepared from
proteolytically degraded fibrils reveals a high degree of chain specificity
(9). Collagen XI molecules are
linked to each other in a head-to-tail fashion by
N-telopeptide2 to
helix cross-links and laterally to type II collagen molecules through
α1(II) C-telopeptides
(9). Isolated from mature
articular cartilage, type XI collagen includes a significant pool of
α1(V) chains (6),
implying the presence of V/XI hybrid molecules. The ratio of type XI collagen
to type II collagen is about 1 to 10 in fetal bovine and human epiphyseal
cartilage when compared with 1 to 30 in adult articular cartilage. Similarly,
the ratio of collagen IX to collagen II falls from about 1 to 10 to 1 to 100
between fetal and adult. In adult articular cartilage, most of the collagen IX
is located in the immediate pericellular matrix
(10–12).The intervertebral disc has a unique collagen architecture that combines
features of ligament and cartilage in its morphology, function, and matrix
biochemistry. The lamellar fabric of the outer annulus fibrosus combines
collagens I and II fibrils in a complex weave with a radial gradient from
mostly type I in the outermost layers and mostly type II in the interior.
Nucleus pulposus, the gel-like center of the young intervertebral disc, has a
similar collagen molecular phenotype to hyaline cartilage in which types II,
IX, and XI collagens are the principal cross-linked fibrillar components
(13–16).
Collagen IX in the disc has a different protein isoform to that of hyaline
cartilages. The α1(IX) chain is expressed as a short form that lacks the
amino-terminal NC4 domain
(16). One of the aims of the
present study was to determine whether a unique pattern of type V/XI hybrid
molecules is present in disc tissue when compared with articular cartilage and
a more typical fibrocartilage, the knee meniscus.The results show an accumulation of collagen α1(V) chains as
articular cartilage matures. A related but distinct complexity in chain usage
in the type V/XI collagen of nucleus pulposus is also revealed. Such tissue
diversity suggests that the different molecular isoforms produce functional
differences in the type V/XI polymeric template on which the bulk fibril
architecture of a tissue is built. 相似文献
19.
Johann Schredelseker Anamika Dayal Thorsten Schwerte Clara Franzini-Armstrong Manfred Grabner 《The Journal of biological chemistry》2009,284(2):1242-1251
The paralyzed zebrafish strain relaxed carries a null mutation for
the skeletal muscle dihydropyridine receptor (DHPR) β1a
subunit. Lack of β1a results in (i) reduced membrane
expression of the pore forming DHPR α1S subunit, (ii)
elimination of α1S charge movement, and (iii) impediment of
arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine
receptor (RyR1), a structural prerequisite for skeletal muscle-type
excitation-contraction (EC) coupling. In this study we used relaxed
larvae and isolated myotubes as expression systems to discriminate specific
functions of β1a from rather general functions of β
isoforms. Zebrafish and mammalian β1a subunits quantitatively
restored α1S triad targeting and charge movement as well as
intracellular Ca2+ release, allowed arrangement of DHPRs in
tetrads, and most strikingly recovered a fully motile phenotype in
relaxed larvae. Interestingly, the cardiac/neuronal
β2a as the phylogenetically closest, and the ancestral
housefly βM as the most distant isoform to β1a
also completely recovered α1S triad expression and charge
movement. However, both revealed drastically impaired intracellular
Ca2+ transients and very limited tetrad formation compared with
β1a. Consequently, larval motility was either only partially
restored (β2a-injected larvae) or not restored at all
(βM). Thus, our results indicate that triad expression and
facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common
features of all tested β subunits, whereas the efficient arrangement of
DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the
β1a isoform. Consequently, we postulate a model that presents
β1a as an allosteric modifier of α1S
conformation enabling skeletal muscle-type EC coupling.Excitation-contraction
(EC)3 coupling in
skeletal muscle is critically dependent on the close interaction of two
distinct Ca2+ channels. Membrane depolarizations of the myotube are
sensed by the voltage-dependent 1,4-dihydropyridine receptor (DHPR) in the
sarcolemma, leading to a rearrangement of charged amino acids (charge
movement) in the transmembrane segments S4 of the pore-forming DHPR
α1S subunit
(1,
2). This conformational change
induces via protein-protein interaction
(3,
4) the opening of the
sarcoplasmic type-1 ryanodine receptor (RyR1) without need of Ca2+
influx through the DHPR (5).
The release of Ca2+ from the sarcoplasmic reticulum via RyR1
consequently induces muscle contraction. The protein-protein interaction
mechanism between DHPR and RyR1 requires correct ultrastructural targeting of
both channels. In Ca2+ release units (triads and peripheral
couplings) of the skeletal muscle, groups of four DHPRs (tetrads) are coupled
to every other RyR1 and hence are geometrically arranged following the
RyR-specific orthogonal arrays
(6).The skeletal muscle DHPR is a heteromultimeric protein complex, composed of
the voltage-sensing and pore-forming α1S subunit and
auxiliary subunits β1a, α2δ-1, and
γ1 (7). While
gene knock-out of the DHPR γ1 subunit
(8,
9) and small interfering RNA
knockdown of the DHPR α2δ-1 subunit
(10-12)
have indicated that neither subunit is essential for coupling of the DHPR with
RyR1, the lack of the α1S or of the intracellular
β1a subunit is incompatible with EC coupling and accordingly
null model mice die perinatally due to asphyxia
(13,
14). β subunits of
voltage-gated Ca2+ channels were repeatedly shown to be responsible
for the facilitation of α1 membrane insertion and to be
potent modulators of α1 current kinetics and voltage
dependence (15,
16). Whether the loss of EC
coupling in β1-null mice was caused by decreased DHPR membrane
expression or by the lack of a putative specific contribution of the β
subunit to the skeletal muscle EC coupling apparatus
(17,
18) was not clearly resolved.
Recently, other β-functions were identified in skeletal muscle using the
β1-null mutant zebrafish relaxed
(19,
20). Like the
β1-knock-out mouse
(14) zebrafish
relaxed is characterized by complete paralysis of skeletal muscle
(21,
22). While
β1-knock-out mouse pups die immediately after birth due to
respiratory paralysis (14),
larvae of relaxed are able to survive for several days because of
oxygen and metabolite diffusion via the skin
(23). Using highly
differentiated myotubes that are easy to isolate from these larvae, the lack
of EC coupling could be described by quantitative immunocytochemistry as a
moderate ∼50% reduction of α1S membrane expression
although α1S charge movement was nearly absent, and, most
strikingly, as the complete lack of the arrangement of DHPRs in tetrads
(19). Thus, in skeletal muscle
the β subunit enables EC coupling by (i) enhancing α1S
membrane targeting, (ii) facilitating α1S charge movement,
and (iii) enabling the ultrastructural arrangement of DHPRs in tetrads.The question arises, which of these functions are specific for the skeletal
muscle β1a and which ones are rather general properties of
Ca2+ channel β subunits. Previous reconstitution studies made
in the β1-null mouse system
(24,
25) using different β
subunit constructs (26) did
not allow differentiation between β-induced enhancement of non-functional
α1S membrane expression and the facilitation of
α1S charge movement, due to the lack of information on
α1S triad expression levels. Furthermore, the β-induced
arrangement of DHPRs in tetrads was not detected as no ultrastructural
information was obtained.In the present study, we established zebrafish mutant relaxed as
an expression system to test different β subunits for their ability to
restore skeletal muscle EC coupling. Using isolated myotubes for in
vitro experiments (19,
27) and complete larvae for
in vivo expression studies
(28-31)
and freeze-fracture electron microscopy, a clear differentiation between the
major functional roles of β subunits was feasible in the zebrafish
system. The cloned zebrafish β1a and a mammalian (rabbit)
β1a were shown to completely restore all parameters of EC
coupling when expressed in relaxed myotubes and larvae. However, the
phylogenetically closest β subunit to β1a, the
cardiac/neuronal isoform β2a from rat, as well as the
ancestral βM isoform from the housefly (Musca
domestica), could recover functional α1S membrane
insertion, but led to very restricted tetrad formation when compared with
β1a, and thus to impaired DHPR-RyR1 coupling. This impairment
caused drastic changes in skeletal muscle function.The present study shows that the enhancement of functional
α1S membrane expression is a common function of all the
tested β subunits, from β1a to even the most distant
βM, whereas the effective formation of tetrads and thus proper
skeletal muscle EC coupling is an exclusive function of the skeletal muscle
β1a subunit. In context with previous studies, our results
suggest a model according to which β1a acts as an allosteric
modifier of α1S conformation. Only in the presence of
β1a, the α1S subunit is properly folded to
allow RyR1 anchoring and thus skeletal muscle-type EC coupling. 相似文献
20.
Satoru Fujiwara Katsuya Araki Tatsuhito Matsuo Hisashi Yagi Takeshi Yamada Kaoru Shibata Hideki Mochizuki 《PloS one》2016,11(4)
α-synuclein (αSyn) is a protein consisting of 140 amino acid residues and is abundant in the presynaptic nerve terminals in the brain. Although its precise function is unknown, the filamentous aggregates (amyloid fibrils) of αSyn have been shown to be involved in the pathogenesis of Parkinson''s disease, which is a progressive neurodegenerative disorder. To understand the pathogenesis mechanism of this disease, the mechanism of the amyloid fibril formation of αSyn must be elucidated. Purified αSyn from bacterial expression is monomeric but intrinsically disordered in solution and forms amyloid fibrils under various conditions. As a first step toward elucidating the mechanism of the fibril formation of αSyn, we investigated dynamical behavior of the purified αSyn in the monomeric state and the fibril state using quasielastic neutron scattering (QENS). We prepared the solution sample of 9.5 mg/ml purified αSyn, and that of 46 mg/ml αSyn in the fibril state, both at pD 7.4 in D2O. The QENS experiments on these samples were performed using the near-backscattering spectrometer, BL02 (DNA), at the Materials and Life Science Facility at the Japan Accelerator Research Complex, Japan. Analysis of the QENS spectra obtained shows that diffusive global motions are observed in the monomeric state but largely suppressed in the fibril state. However, the amplitude of the side chain motion is shown to be larger in the fibril state than in the monomeric state. This implies that significant solvent space exists within the fibrils, which is attributed to the αSyn molecules within the fibrils having a distribution of conformations. The larger amplitude of the side chain motion in the fibril state than in the monomeric state implies that the fibril state is entropically favorable. 相似文献