Several different protein and peptide hormones, includ-ing insulin and glucagon [13,14], have long been known to readily form fibrils, and it has recently been proposed that functional am
Trang 1Amyloid structure – one but not the same:
the many levels of fibrillar polymorphism
Jesper S Pedersen1, Christian B Andersen2,3and Daniel E Otzen4
1 Department of Biochemistry, Molecular Biology, and Cell Biology, Rice Institute for Biomedical Research,
Northwestern University, Evanston, IL, USA
2 Protein Structure and Biophysics, Novo Nordisk A⁄ S, Ma˚løv, Denmark
3 Institute of Biophysics, National Research Council, CNR, Palermo, Italy
4 Department of Molecular Biology, Center for Insoluble Protein Structures, Interdisciplinary Nanoscience Centre,
University of Aarhus, Denmark
Amyloid in disease and functional
fibrillar structures
Amyloid and amyloid-like structures are normally
associated with several debilitating diseases, including
Alzheimer’s, Parkinson’s, Huntington’s and transmissible
spongiform encephalopathies [1] In these cases, the formation of amyloid and other aberrant aggregates represents an alternative type of folding that makes
Keywords
aggregation; amyloid; fibrillar polymorphism;
glucagon; mechanism; protein folding
Correspondence
J S Pedersen, Department of
Biochemistry, Molecular Biology, and Cell
Biology, Rice Institute for Biomedical
Research, Northwestern University,
2205 Tech Drive, Hogan 2-100, Evanston,
IL 60208, USA
Tel: +1 847 881 6617
E-mail: jsp@phage.dk
D Otzen, Department of Molecular Biology,
Center for Insoluble Protein Structures,
Interdisciplinary Nanoscience Centre,
University of Aarhus, Gustav Wieds Vej 10,
DK-8000 Aarhus C, Denmark
Fax: +45 8612 3178
Tel: +45 8942 5046
E-mail: dao@inano.au.dk
(Received 16 April 2010, revised 2
September 2010, accepted 17 September
2010)
doi:10.1111/j.1742-4658.2010.07888.x
Many proteins and peptides can form amyloid-like structures both in vivo and in vitro Although strikingly similar fibrillar structures can be observed across a variety of amino acid sequences, the fibrils formed often exhibit a stunning wealth of polymorphisms at the level of electron or atomic force microscopy This appears to violate the Anfinsen principle seen for globu-lar proteins, where each protein sequence codes for just one well-defined fold To a large extent, polymorphism reflects variable packing of a single protofilament structure in the mature fibrils However, we and others have recently demonstrated that polymorphism can also reflect real structural differences in the molecular packing of the polypeptide chains leading to several possible protofilament structures and diverse mature fibrillar struc-tures Glucagon has been a particularly useful model system for studying the fibrillogenesis mechanisms that lead to the formation of structural poly-morphism, thanks to its single tryptophan residue and the availability of large quantities at pharmaceutical-grade quality Combinations of struc-tural investigations and seed extension experiments have revealed the repro-ducible formation of at least five different self-propagating fibril types from subtle variations in growth conditions These reflect the underlying com-plexity of the peptide conformational landscape and provide a link to natively disordered proteins, where structure is dictated by context in the form of different binding partners Here we review some of the latest advances in the study of glucagon fibrillar polymorphism and their implica-tions for mechanisms of fibril formation in general
Abbreviations
AFM, atomic force microscopy; EM, electron microscopy; ITC, isothermic titration calorimetry; SAXS, small angle X-ray scattering;
ThT, thioflavin T.
Trang 2the protein become toxic and⁄ or lose its natural
func-tion [2–4] In systematic amyloidoses, it appears to be
the massive accumulation of amyloid per se that is
pathological, leading to organ failure and eventual
death [5–7] Smaller amounts of amyloids accumulated
in sensitive locations, such as the cornea, can also lead
to severe functional impairment [8] In most
neurode-negerative diseases, however, prefibrillar aggregates are
the toxic species, as detailed by Stefani in the review in
this series [9]
It has been suggested that the ability to form
amy-loid-like structures, given appropriate conditions, is a
general property of the polypeptide chain [10,11] This
ability not only has pathological consequences, but can
be turned into good use as functional amyloid, serving
as structural anchoring material for a range of
pur-poses ranging from biofilm formation to a matrix to
anchor melanin as well as for the reduction of
interfa-cial tension, coating of spores and many other as yet
unknown functions (see [12] and references herein)
Several different protein and peptide hormones,
includ-ing insulin and glucagon [13,14], have long been
known to readily form fibrils, and it has recently been
proposed that functional amyloid structures could
serve as in vivo intracellular storage of these hormones
in pituitary secretory granules, stabilized by
interac-tions with glucosaminoglycans [15,16] Upon release
from the cells, these amyloid structures would
gradu-ally dissociate into active monomers in the
blood-stream [17], which would imply that the amyloid
structures formed by these hormones in vivo should
have evolved to be relatively unstable
Fibrillar polymorphisms reflect
structural ambiguity in amyloid fibrils
The key to the properties of amyloid assemblies lies
in the regularity and repetitiveness of their underlying
molecular architecture of intermolecular b-strands
stacked perpendicular to the fibril axis [18], often
organized in several b-sheets parallel to the axis
Although atomic resolution structures of the
molecu-lar packing of peptides in amyloid-like structures are
beginning to emerge [19], much of our understanding
of the structure of amyloid-like fibrils comes from
electron microscopy (EM) techniques and atomic
force microscopy (AFM) [20] Images produced by
these techniques often show stunningly beautiful,
per-fectly ordered fibrils, some with regular twisting,
oth-ers seemingly smooth and still othoth-ers as parallel
bundles of two or more protofilaments (Fig 1) Fibril
preparations often contain several different
morpho-logies, and for some of these the turn lengths and
morphology are directly correlated to the number of protofilaments they contain [21] Strikingly similar fibrillar morphologies can arise from proteins and peptides with very diverse amino acid sequences [22– 24] In other cases, particular morphologies can be generated by manipulating the specific conditions for fibril formation [25–27]
Initially it was assumed that the protein fold found
in amyloid structures was a single energy minimized structure following Anfinsen’s single-fold principle for native proteins [28], with the different morphologies representing different lateral associations of a single, lowest-energy protofilament structure [22,29] The only exception from this rule was thought to be the prions, which were shown to form several variants [30,31], so-called self-propagating strains, with different
Fig 1 Comparison of representative EM morphologies of the five different glucagon fibril types, type A [27,44,56,63], B unagitated [44,53,56], Bagitated[23,27,71,73], D [27,73] and S [43,61,73], where combinations of spectroscopic, thermostability and seed extension kinetic data indicate distinct protofilament structures Scale bar = 50 nm The emission intensity of ThT staining of type A is
> 40-fold higher than that observed for types B and D Tmapp values represent thermal melting midpoints during 90 C Æ h)1temperature ramping of 25 mgÆL)1fibrils in 25 m M glycine⁄ HCl (pH 2.67) The
CD spectrum shows the reproducible unique fingerprint features of fibrils after sonication that can be used to distinguish between the types [27,43,56].
Trang 3molecular packing of the prion domain into the
amy-loid structure [32] However, the discovery of
self-propagating variants of insulin [33], b2-microglobulin
[34], Ab [35] and glucagon [27] fibrils demonstrated
that strain behavior was not limited to proteins with a
prion-like sequence Recently it has been demonstrated
that even small peptides, including the amylin
frag-ment SNNFGAILSS, have the ability to form amyloid
with both parallel and antiparallel b-sheet structures
[36,37] depending on the structure of the seeds The
peptide GNNQQNY(7-13) forms two different types
of quasifibrillar crystal [38] and three different types of
amyloid according to solid-state NMR [39] that differ
from one another in subtle but distinct ways, such as
variations in the mobility of the aromatic Tyr ring
There is a growing number of similar observations
from other peptide systems [40,41] Remarkably, the
prevailing structure of insulin fibrils formed during
agitation appears to vary randomly between two
optically distinct polymorphisms [42], indicating that
indeterminism in early nucleation events dictates the
final fibril structure We have previously
demon-strated that glucagon is able to form at least five
different fibrillar structures that can be propagated
by seeding in a strain-specific manner [23,27,43,44]
Each type can be identified by a unique combination
of variable characteristics, including thioflavin T
(ThT) staining, CD spectrum fingerprints,
thermosta-bility and morphology in EM (see Fig 1) Type A,
Bagitated, Bunagitated all form in the same 50 mm
gly-cine⁄ HCl buffer at pH 2.5, whereas types D and S
only accumulate when 200 mm Cl) and 1 mm
SO24 are added, respectively Type A forms at high
glucagon concentrations (> 1 gÆL)1), whereas the
other types form at low concentrations (< 0.5 gÆL)1)
We have recently suggested that the prevalence of
different glucagon fibril structures is the result of a
multitude of aggregation pathways, where even small
shifts in environmental conditions can impact the
outcome of the struggle for monomers between
sev-eral types of fibril due to modulation of their
nucle-ation and exponential growth rates [23] Interestingly,
another class of proteins for which environmental
conditions are critical in defining their structure is
the group of intrinsically disordered proteins that are
designed to show great conformational flexibility,
allowing them to interact with multiple binding
partners that can often induce different types of
structure [45,46] In these cases, however, the
poly-morphism has been systematically optimized to
facili-tate heterogeneous contacts with different proteins
rather than the homogeneous assemblies illustrated
by the amyloid folds
Breaking or branching fibril structures allow exponential growth
When glucagon powder is dissolved in acidic buffer, the fibril formation follows a highly reproducible sig-moidal curve [47] Sigsig-moidal curves are observed in a number of biological systems, such as during exponen-tial growth of bacteria in a flask or exponenexponen-tial ampli-fication of DNA in PCR Despite the obvious similarities, a common misconception (as reviewed by Roberts [48]) is that the lag time before the detection
of fibrils can be taken as a direct indicator of the pro-pensity to slowly form a stable nucleus For glucagon, this is illustrated by simple seeding experiments, which clearly demonstrate that the exponential growth phase extends through the apparent lag all the way to the beginning of the experiment [23] (Fig 2C), which means that the observed lag time depends inversely on the growth rate of fibrils, as well as the nucleation rate A compilation of kinetic data from several pro-teins shows a clear inverse correlation between the apparent lag time and growth rates [49,50], suggesting that fibril nucleation may generally take place through-out the apparent lag [51], but exponential amplification from early nucleation events will lessen the effect of later nucleation events The linear nature of fibrils implies that they grow linearly by the addition of pro-tein molecules to their ends [52] Exponential growth can be attributed to the presence of secondary path-ways, which continuously increase the number of fibril ends in proportion to the fibril mass present [51] Recently, we have demonstrated using total internal reflection fluorescence microscopy that a specific type
of glucagon fibril, which we refer to as type Bunagitated [23], grows by branching under unagitated conditions [53] (Fig 2A) Branching in these fibrils is also observed directly in high-resolution EM images [44], which demonstrate that the fibrils consist of two or more protofilaments that twist regularly We speculate that the twists may be necessary for fibrils to send out branches or that cavities created on the surface of these structures catalyze the formation of new fibril nuclei Fibrillogenesis of glucagon as well as a number
of other proteins, including insulin [54], Ab1–40 [35] and prions [55], can be greatly accelerated by agitation
In the case of glucagon, clues to the mechanism of this acceleration can be observed in the morphology of the resulting fibrils, with agitation generally producing type Bagitated fibrils, which are short, nontwisted and nonbranched parallel bundles of two or more filaments [27] We speculate that the type Bagitatedfibrils are brit-tle fibrils that readily break during agitation, thereby doubling the number of fibril ends that can accept
Trang 4monomers (Fig 2B) and providing them with a
selec-tive growth advantage over type Bunagitatedfibrils under
agitated conditions [23] The two types of fibril have
very similar CD spectra with an unusual positive peak
around 203 nm (Fig 1) and two distinct b-sheet
peaks in FTIR spectra and a shoulder at 1664 cm)1,
indicating the presence of b-turns, suggesting that the
molecular packing could be very similar [27,56]
Agita-tion-dependent molecular-level polymorphisms have
also been reported for Ab1–40 and insulin fibrils
[35,57] Moreover, different types of prion fibril form
under shaking and rotating conditions, indicating that
the mode of agitation can also influence the prevailing
pathway of fibrillogenesis [58] Quiescent (unagitated)
and agitated forms of Ab1–40 fibrils exhibit twisted
and striated ribbon morphologies similar to the type
Bunagitated and Bagitated glucagon fibrils, respectively
With rounds of seeded growth it has been possible to
generate homogeneous samples of quiescent and
agi-tated Ab1–40 fibrils that allowed a solid-state NMR
study of underlying structural differences [59] The
structures reveal that the secondary structure of Ab1–40
in the two forms is very similar, but that the quiescent form has a triangular cross-section with three protofil-aments with a narrow cavity in the middle, whereas the agitated striated ribbon form consists of two proto-filaments with a tight interaction between the flat sur-faces between them It has recently been demonstrated that specific types of the Ab1–40fibril can also grow by branching [60] Investigations on the similarities and differences between the type Bunagitated and Bagitated glucagon fibril forms are currently being conducted
Transient off-pathway formation of monofilament type A fibrils allows growth of type B fibrils at high glucagon concentrations
Based on time-lapse EM and AFM studies alone, it has been proposed that formation of the complex multifilament structures of mature fibrils may proceed
by lateral assembly of preformed protofilaments or pro-tofibrils [29,61,62] Even though EM and AFM provide high-quality information about the fibril structures
Fig 2 Secondary pathways in glucagon fibrillogenesis result in exponential growth of mature fibrils (A) Under unagitated conditions, TIRF microscopy directly demonstrates that type B unagitated fibrils increase the number of fibril ends by branching, leading to exponential growth Adapted from [53] (B) Under agitated conditions, type Bagitatedfibrils have selective growth advantage because they continuously break, thereby exposing new fibril ends that adsorb monomeric glucagon [27] (C) Seed extension kinetics of 1 gÆL)1 glucagon in 50 m M
glycine ⁄ HCl pH 2.5 with agitation confirm that type B agitated fibrils grow exponentially, and indicate that a small fraction ( 1 in 10 5
) of glucagon molecules initiate spontaneous nucleation as soon as glucagon is dissolved under the given conditions [23].
Trang 5formed, many images need to be analyzed to quantify
the amounts of various structures formed Moreover,
the images do not provide information about
differ-ences in the molecular packing of fibrils and it is
virtu-ally impossible using these techniques alone to quantify
the fraction of the protein that has become converted
to fibrils at the given time point The hierarchical
build-up model seems to conflict with recent data that
suggest that different types of glucagon fibril grow
exponentially via their own distinct pathways into
structurally distinct entities [23,27,43,44,53,56] Several
studies have revealed that the formation of
monoment type A fibrils (Fig 1), which appear as single
fila-ments in EM and AFM, occurs at glucagon
concentrations above 1 gÆL)1[27,44,63] When agitated
at 1 or 2 gÆL)1, transient formation of type A fibrils
can be observed as a peak in ThT emission, which
dis-appears as type Bagitated fibrils form [27] However,
under unagitated conditions at higher concentrations,
the type A fibrils form a thick gel [47] that appears to
be stable for longer periods, which makes it possible to
study the properties of these otherwise metastable
fibrils [44] A recent study summarized the evidence for
structural differences between type A and Bunagitated
[56]: unlike type B fibrils, type A fibrils have an unusu-ally strong b-sheet CD spectrum (Fig 1) and an FTIR spectrum with only one b-sheet peak Linear dichroism
of aligned fibrils indicates that type Bunagitatedfibrils are less ordered than type A [56] X-ray diffraction patterns reveal that both types exhibit the classical 4.76 A˚ meridional reflection typical for amyloid-like structures [18], but whereas type Bunagitatedonly contains the clas-sical 9.8 A˚ equatorial reflection, type A fibrils exhibit a number of periodic reflections similar to those of a cyl-inder with well-defined edges This suggests that the simple structure of type A fibrils can be aligned more orderly than the branched type Bunagitated fibrils [56] Limited proteolysis results in the release of a different spectrum of peptides, further substantiating the struc-tural differences between the two types of fibril [56] The two fibril types also differ in terms of the mecha-nisms leading to their formation, as evident from kinetic cross-seeding experiments: Fractionated seeds of both type A and Bunagitated fibrils can grow exponen-tially at low concentrations, but type Bunagitated fibrils have a faster exponential growth (a more shallow slope
Fig 3 A proposed mechanism for the
conversion of type A fibrils into type B (A)
Seeding experiments demonstrate that
seeds of type A fibrils can grow at both high
and low glucagon concentrations In
contrast, seeds of type Bunagitatedfibrils
grow exponentially only at low glucagon
concentrations, possibly due to inhibition of
either elongation or branching by a-helical
trimers at high concentrations [44] (B) Once
type A fibrils have consumed > 95% of the
monomers, the concentration is low enough
to allow exponential growth of type
Bunagitatedfibrils A rapid subsequent
equilibrium between monomers and the
relatively unstable type A fibrils ensures that
glucagon monomer concentrations are
maintained at low enough levels to support
the growth of type Bunagitatedfibrils Data
for the graph were taken from the recent
SAXS study [63].
Trang 6on a lag time versus log[seed] plot) [44] In contrast,
only type A fibrils seed exponential growth at high
con-centrations, possibly because either the branching or
elongation of type Bunagitated seeds is inhibited by the
a-helical trimers that form in equilibrium with
mono-mers at these concentrations [64] (Fig 3A) Consistent
with this, the apparent lag time for the formation of
type Bunagitated fibrils actually increases with increasing
glucagon concentrations above 0.3 gÆL)1[44] All of the
abovementioned differences make it difficult to propose
that type Bagitated fibrils could be assembled by simple
lateral associations of several type A protofibrils – the
properties of the structures are simply too different We
therefore hypothesize that conversion from type A to
mature type Bunagitated fibrils over time could occur by
gradual shedding of monomers from unstable type A
fibrils that subsequently adsorb to the more stable
exponentially growing type Bagitated fibril structure
Data from thermal melting suggest that type A fibrils
are relatively unstable compared with type Bagitated
fibrils, with apparent thermal melting midpoints (Tmapp)
of < 32 and 55C, respectively [27] Moreover, linear
extrapolation of urea dissociation kinetics indicates
that type A fibrils have a much faster dissociation rate
of 0.69 h)1compared with the 0.03 h)1 observed for
type Bagitated fibrils [27] This corresponds to a half-life
of only 1 h if type A fibrils were diluted infinitely in
buffer Proteolysis with pepsin, which continuously
degrades flexible monomers more readily than fibrils,
shows nearly the same value [27] Recent developments
in small angle X-ray scattering (SAXS) allows
noninva-sive quantitative analysis of the relative amounts of
fibrils consisting of single (type A) and multiple
proto-filaments (type Bunagitated) (green and orange curves in
Fig 3B, respectively) [63], which is very difficult if not
impossible to achieve using combinations of ThT and
Trp fluorescence alone Data from the SAXS study
indicate that type A fibrils grow until they have
consumed nearly all of the glucagon (blue curve
in Fig 3B) before the exponential growth of type
Bunagitated fibrils reaches detectable levels Interestingly,
the SAXS data show that the lag time for the
forma-tion of type Bunagitated increases from 18 h (5 gÆL)1) to
35 h (10 gÆL)1) [63], indicating that the growth of type
Bunagitated depends on the remaining nonfibrillated
glucagon concentration rather than on the amount of
type A fibrils formed before them [63] This is
inconsis-tent with type A being a structural prerequisite for the
formation of type Bunagitated fibrils, but consistent with
the quantitative cross-seeding data [44] that suggests
type Bunagitated fibrils are unable to grow before
mono-mer concentrations are sufficiently low (i.e due to the
formation of type A) Because type A fibrils have a
half-life of only 1 h, their shedding of monomers apparently keeps concentrations at a sufficient level to facilitate rapid growth of type Bunagitated fibrils Thus, it appears that the transition from type A to B fibrils probably occurs via shedding and adsorption of monomers
It has been reported that multiple distinct assembly pathways may be responsible for the formation of pro-tofibrillar and mature fibrillar structures of Ab [65],
b2-microglobulin [66] and Sup35NM [67] Clearly, future studies should include kinetics experiments and structural data before concluding that fibrils form via
a hierarchical build-up mechanism [68] Nevertheless, it
is unlikely that there will be a single unifying mecha-nism for the build-up of fibrils from its constituents The diversity of possible interactions due to different protein sequences is simply too great [29] There are cases where preformed oligomers can be demonstrated
to be incorporated directly into the fibrils [69], and kinetic data from SAXS also support that insulin fibrils could be built from preformed oligomeric build-ing blocks [70], although the mechanism that leads to
a lag time before the accumulation of the building block has not yet been described in detail As a further example, our SAXS studies of the fibrillation of a-syn-uclein under agitated conditions identify three species, namely a monomer⁄ dimer state, an oligomer with
a central channel and an extended fibril (L Giehm,
D Svergun, D E Otzen and B Vestergaard, submit-ted) Structurally and mechanistically this oligomer appears to be a direct precursor to the fibril
Charge neutrality in type B fibrils
At the acidic pH used for the fibrillogenesis of gluca-gon, histidine residue 1 (His1), the three aspartic acid residues (Asp9, Asp15 and Asp21) and the C-terminus exist mostly in the protonated state This means that glucagon has a net charge of +5 (N-terminus, His1, Lys12, Arg17 and Arg18) If left unshielded, this would lead to high static repulsion, which is irreconcilable with the close packing of glucagon molecules that occurs in fibrils There are two possible mechanisms that would allow glucagon fibrils to exist at low pH: either the pKavalues are shifted so that glucagon mole-cules in fibrils lose some of these charges or counter ions from the solution shield the positive charges The protonation state of type Bagitated glucagon fibrils has been investigated by isothermic titration calorimetry (ITC) during extension of seeds [71] Using a series of buffers with different protonation enthalpies, it was possible to measure how many protons were exchanged with the buffer upon incorporation of a glucagon mole-cule into a seed The data obtained are consistent with
Trang 7the release of five protons upon monomer addition to a
fibril end, indicating that glucagon is charge neutral in
the type Bagitated fibrillated state [71] (Fig 4)
Consis-tently, the stability of type Bagitated depends on pH,
with fibrils dissociating instantly at pH 1.1 and Tapp
m
increasing from 40C at pH 2.1 to 61 C at pH 3.2
[27] Glucagon can also form fibrils in glycine⁄ NaOH
buffer at pH 9.5 [72], where monomeric glucagon is
expected to have a net charge of)1 [73], and
accord-ing to data from ITC experiments, the molecules also
become charge neutral when incorporated into these
fibrils [71] Because of the charge neutrality of fibrils at
both high and low pH, it is conceivable that the type of
molecular packing of glucagon in fibrils formed at these
very different pH values could be identical The
obser-vation that seeds of fibrils formed at pH 9.5 can grow
exponentially at pH 2.5, with kinetics that are virtually
superimposable to kinetics of type Bagitatedseeding
sug-gests that type Bagitated fibrils can also form at pH 9.5
(J S Pedersen, unpublished data)
Shielding of charges by ions allows
fibrils with alternative molecular
structures
Because of the +5 charge on monomeric glucagon
mol-ecules at acidic pH, shielding of charges by anions can
increase the rate of fibril formation by relieving the
charge repulsion between monomers [43] However,
salts appear to favor the growth of fibrils with
alterna-tive properties: in the presence of 150–250 mm Cl), type
D fibrils appear to have a selective growth advantage
over type Bagitatedfibrils, even under agitated conditions
[27], possibly because the salts also stabilize type Bagitated
fibrils, making them less prone to break [27] The
diva-lent anion SO2 is 125-fold more stabilizing than Cl)for
type S fibrils, and every 10-fold increase in salt
concen-tration increases Tapp
m with 22C for, implying that salts are critical to stability of Type S fibrils [43] This explains why adding as little as 1 mm SO24 gives a selec-tive growth advantage to type S fibrils We have previ-ously speculated that the negative charges on SO24 could allow packing of positively charged glucagon mol-ecules into fibrils, as well as lateral associations between several positively charged fibrils or protofilaments thereby increasing stability [43]
A comparison of the X-ray diffraction patterns reveals slight differences in the interstrand distance of type B, S and D fibrils with meridionals at 4.7, 4.8 and 4.9 A˚, respectively [73] Each fibril type also has their own signature of equatorial reflections Moreover, Trp25 appears significantly more exposed to acrylamide quenching in type Bagitatedfibrils compared with type D and S [73] Convincing evidence for structural differ-ences between the three types of fibril also comes from cross-seeding experiments under identical conditions (e.g 1 gÆL)1monomeric glucagon in 50 mm glycine pH 2.5), which lead to propagation of the structure of the seed [27,43] We have used ITC to extend this charac-terization with a thermodynamic comparison of type
Bagitated, S and D fibrils [73] By measuring the enthalpy change, DH, for seeded fibril elongation at a series of temperatures, it is possible to estimate the change in heat capacity for fibril formation (DCp) for the three fibril types [74] Remarkably, the DCp values for the fibril extension of the three types are significantly dif-ferent, with positive values for type D and negative val-ues for type S (Fig 5) For type Bagitated, the enthalpy
Fig 4 Data from ITC experiments during seed extension indicate
that each monomer releases five protons at pH 2.5 and takes up
one proton at pH 9.5 upon addition to a type Bagitatedfibril end [71].
Fig 5 Thermodynamic analysis of fibrillar polymorphism Enthalpy change during extension of various types of glucagon fibril as a function of temperature The slope of these curves corresponds to
DCp values, which are strikingly different for the three types of fibril Adapted from [73].
Trang 8is dominated by buffer deprotonation, making the
intrinsic DCpessentially zero It is difficult to identify a
simple structural basis for this remarkable variation in
DCp values Clearly the predicted change in solvent
accessible area, which correlates strongly with DCp for
globular proteins [75], is not a useful predictor of
fibril-lar DCp It is possible that strong backbone interactions
lead to the unfavorable burial of polar side residues,
water and⁄ or charged groups, which can all have major
influence on the change in DCp
Future perspectives – toxicity of
alternative protein folds
So far we have demonstrated that glucagon is able to
form at least five types of amyloid fibril that appear to
differ at the level of their molecular packing of
gluca-gon Based on some of the structures observed in EM,
it is very likely that several other types of fibril with
unique properties remain to be discovered Judging by
seed extension kinetics and the overall sigmoidal shape
of the growth curves, it appears logical to assume that
they all grow exponentially by monomer addition from
rare thermodynamic nuclei that start to form when
glucagon is dissolved (Fig 6)
In the current research on protein aggregation and
amyloid formation, interest in prefibrillar intermediate
structures and other oligomers is growing, given that
the toxicity of these species appears to surpass that of
mature fibrils [76] It is possible that the toxicity of aggregates is simply correlated directly with the surface
to mass ratio, implying that smaller structures, which have a high surface to mass ratio, are more toxic than large aggregates, which have a small surface to mass ratio However, it is becoming evident that specific folds in oligomers can be significantly more toxic than others [77] It has been reported that mishandling of glucagon solutions of > 2 gÆL)1leads to the formation
of toxic aggregates [78] Moreover, a recent report comparing the toxicity of amyloid structures of several protein hormones indicated that fibril preparations formed during a 14 day incubation at 37C with slight agitation of 2 gÆL)1 glucagon in the presence of 0.4 mm low relative molecular mass heparin and 5%
d-mannitol at pH 5.5 are particularly toxic, resulting
in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction exceeding that caused by Ab1–42
and Ab1–40 aggregates [16] Unfortunately, the study did not reveal what specific type(s) of glucagon aggre-gate(s) cause toxicity Several studies have been aimed
at characterizing prefibrillar intermediate species of glucagon and oligomeric structures have been reported
in AFM studies [61,62] However, according to data from field flow fractionation [79], NMR [80], SAXS [63] and dynamic light scattering [44], the benign a-helical trimer, which is in rapid equilibrium with monomers [44,64,81] and crystallizes readily [14,82], is the only oligomeric structure that forms at detectable levels at pH 2.5 It is possible that a shift to pH 5.5, where the molecules have an average charge of +1, could allow glucagon to form more stable toxic oligo-meric species Another possibility is that the aggre-gated species responsible for toxicity is a type of fibril, which raises the question of what type of fibril is responsible for toxicity With its fibrillation mecha-nisms and fibrillar polymorphisms being so well under-stood, glucagon appears to be an excellent model system for future studies to further our under-standing of the relationship between protein aggregate structures and toxicity
Acknowledgements
We gratefully acknowledge Dr Hans Aage Hjuler and coworkers at Novo Nordisk A⁄ S for extensive fund-ing over the years as well as generously providfund-ing unlimited amounts of the highest possible quality of glucagon samples We are also grateful to Drs Chris-tian Rischel, Peter Westh and James Flink for fruitful collaborations and stimulating discussions JSP is sup-ported by the Carlsberg Research Foundation DEO acknowledges support from the Danish Research
Fig 6 Summary of the five different types of glucagon fibril
inves-tigated in detail and the proposed nucleation-dependent pathways
that lead to their formation On each pathway monomers are in
equilibrium with individual thermodynamic nuclei, which are the
most unstable transition state between monomers and fibrils.
According to the monomer concentration dependence of seeded
fibril elongation, all fibril types grow by monomer addition
[27,43,44] The equilibrium between a-helical trimers and
monomers inhibits exponential growth and ⁄ or nucleation of type
Bunagitated It is possible that the growth of other types of mature
fibril could similarly be inhibited by a-helical trimers.
Trang 9Foundation (inSPIN) CBA is supported by a
postdoc-toral fellowship financed by The Benzon Foundation
and Novo Nordisk
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