ABSTRACTS
of CURRENT and RECENT PAPERS
Biochemistry 2006 45(26), 8135-42
Zhu M, Qin ZJ, Hu D, Munishkina
LA, Fink AL
Alpha-synuclein,
a presynaptic protein associated with Parkinson's disease, is found as both
soluble cytosolic and membrane-bound forms. Although the function of
alpha-synuclein is unknown, several observations suggest that its association
with membranes is important. In the present study we investigated the effect of
alpha-synuclein on lipid oxidation in membranes containing phospholipids with
unsaturated fatty acids. The kinetics of lipid oxidation were monitored by the
change in fluorescence intensity of the dye C11-BODIPY. We find that monomeric
alpha-synuclein efficiently prevented lipid oxidation, whereas fibrillar
alpha-synuclein had no such effect. Our data suggest that the prevention of
unsaturated lipid oxidation by alpha-synuclein requires that it bind to the
lipid membrane. The antioxidant function of alpha-synuclein is attributed to
its facile oxidation via the formation of methionine sulfoxide, as shown by
mass spectrometry. These findings suggest that the inhibition of lipid
oxidation by alpha-synuclein may be a physiological function of the protein.
Fibrillation of
human insulin A and B chains.
Biochemistry 2006
Aug 1; 45(30), 9342-53
Hong, DP, Ahmad, A., Fink AL
Human insulin, which consists of disulfide cross-linked A and B polypeptide chains, readily forms amyloid fibrils under slightly destabilizing conditions. We examined whether the isolated A and B chain peptides of human insulin would form fibrils at neutral and acidic pH. Although insulin exhibits a pH-dependent lag phase in fibrillation, the A chain formed fibrils without a lag at both pHs. In contrast, the B chain exhibited complex concentration-dependent fibrillation behavior at acidic pH. At higher concentrations, e.g., >0.2 mg/mL, the B chains preferentially and rapidly formed stable protofilaments rather than mature fibrils upon incubation at 37 degrees C. Surprisingly, these protofilaments did not convert into mature fibrils. At lower B chain concentrations, however, mature fibrils were formed. The explanation for the concentration dependence of B chain fibrillation is as follows. The B chains exist as soluble oligomers at acidic pH, have a beta-sheet rich conformation as determined by CD, and bind ANS strongly, and these oligomers rapidly form dead-end protofilaments. However, under conditions in which the B chain monomer is present, such as low B chain concentration (<0.2 mg/mL) or in the presence of low concentrations of GuHCl, which dissociates the soluble oligomers, mature fibrils were formed. Thus, both A and B chain peptides can form amyloid fibrils, and both are likely to be involved in the interactions leading to the fibrillation of intact insulin.
Independent
Heterologous Fibrillation of Insulin and its B-chain Peptide
Biochemistry
2005,44, 16701-16709
Dong-Pyo Hong, and Anthony L Fink*
Insulin is very prone to form amyloid fibrils under slightly destabilizing conditions, and the B-chain region plays a critical role in the fibrillation. We show here that the isolated B-chain peptide of bovine insulin also forms fibrils at both acidic and neutral pH. When a mixture of insulin and the B-chain peptide was incubated at either acidic or neutral pH, the formation of fibrils was clearly separated into two phases, the faster corresponding to fibrils from the B-chain and the slower to fibrillation of insulin. To further investigate the interaction (or lack thereof) between the two polypeptides, we examined the effects of cross-seeding. The results indicate that seeds of B-chain fibrils accelerate the fibrillation of insulin at pH 1.6, and inhibit the fibrillation at pH 7.5, but seeds of insulin fibrils have little effect on the fibrillation of the B-chain. Our results demonstrate that in the co-aggregation of closely related peptides each peptide species may undergo concurrent homogeneous or heterologous polymerization, and that fibrils of one species may or may not seed fibrillation of the other. The results demonstrate the significant “species” barrier in amyloid fibril formation between fibrillation induced by different fibrils. A model for the fibrillation of the heterogeneous system of insulin and B-chain insulin is proposed.
Concerted Action of Metals and Macromolecular Crowding on
theFibrillation of α-Synuclein
Larissa A. Munishkina,
Vladimir N. Uversky and Anthony L. Fink*
The Oxidation State Of DJ-1 Regulates Its Chaperone Activity
Toward α-Synuclein
Wenbo
Zhou, BS; Min Zhu, PhD; Mark A Wilson, PhD; Gregory A Petsko, PhD; Anthony L
Fink, PhD
Abstract: DJ-1 has been reported to have chaperone activity by preventing
the aggregation of some proteins, and by structural analogy to Hsp31. The L166P
mutation has been linked to a familial early onset form of Parkinson's disease.
Since the aggregation of alpha-synuclein is believed to be a critical step in
the etiology of PD, we have investigated the interaction of wild-type DJ-1 and
its oxidized forms with alpha-synuclein. Native (unoxidized) DJ-1 did not
inhibit alpha-synuclein fibrillation, and no evidence for stable interactions between alpha-synuclein and native DJ-1 was
observed. However, DJ-1 is very susceptible to oxidation by the addition of two
oxygens to form the sulfinic acid of Cys 106 (2O DJ-1) (no 1O oxidized state is
detectable). 2O DJ-1 was readily prepared by the addition of H2O2 at
concentrations up to a 20-fold molar excess. The oxidation of Cys 106 to the
sulfinic acid had minimal effect on the structural properties of DJ-1. However,
2O DJ-1 was very effective in preventing the fibrillation of alpha-synuclein, and
only this form of DJ-1 appears to have significant anti-aggregation properties
against alpha-synuclein. Further oxidation of DJ-1 leads to loss of some
secondary structure, and to loss of the ability to inhibit alpha-synuclein
fibrillation. Our observations confirm the suggestion that DJ-1 may act as an
oxidative-stress-induced chaperone to prevent alpha-synuclein fibrillation.
Since oxidative stress has been associated with PD, this observation may
explain why mutations of DJ-1 could be a contributing factor in PD, and also
indicates that excess oxidative stress could also lead to enhanced
alpha-synuclein aggregation and hence PD.
Journal Biological Chemistry 2005 Dec. 30, 280:52,
42669-42675
Atta Ahmad, Vladimir N. Uversky, Dongpyo Hong and Anthony L. Fink*
Insulin has a
largely α-helical structure, and
exists as a mixture of hexameric, dimeric and monomeric states in solution,
depending on the conditions: the protein is monomeric in 20% acetic acid.
Insulin forms amyloidlike fibrils under a variety of conditions, especially at
low pH. In this study we investigated the fibrillation of monomeric human
insulin by monitoring changes in CD, ATR-FTIR, ANS fluorescence, ThT
fluorescence, dynamic light scattering and H/D exchange during the initial
stages of the fibrillation process in order to provide insight into early
events involving the monomer. The results demonstrate the existence of
structural changes occurring before the onset of fibril formation, which are
detectable by multiple probes. The data indicate at least two major populations
of oligomeric intermediates between the native monomer and fibrils. Both have
significantly non-native conformations, and indicate that fibrillation occurs
from a betarich structure significantly distinct from the native fold.
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J Mol Biol.
2005 Oct 21;353(2):357-72 |
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Kaylor J, Bodner N, Edridge S, Yamin G, Hong DP, Fink AL.
The aggregation of alpha-synuclein is believed to be a
critical step in the etiology of Parkinson's disease. A variety of biophysical
techniques were used to investigate the aggregation and fibrillation of
alpha-synuclein in which one of the four intrinsic Tyr residues was replaced by
Trp, and two others by Phe, in order to permit fluorescence resonance energy
transfer (FRET) between residues 39 (Tyr) and 125 (Trp). The mutant
Y125W/Y133F/Y136F alpha-synuclein (one Tyr, one Trp) showed fibrillation
kinetics similar to that of the wild-type, as did the Y125F/Y133F/Y136F (one
Tyr, no Trp) and Y39F/Y125W/Y133F/Y136F (no Tyr, one Trp) mutants. Time-dependent
changes in FRET, Fourier transform infrared, Trp fluorescence, dynamic
light-scattering and other probes, indicate the existence of a transient
oligomer, whose population reaches a maximum at the end of the lag time. This
oligomer, in which the alpha-synuclein is in a partially folded conformation,
is subsequently converted into fibrils, and has physical properties that are
distinct from those of the monomer and fibrils. In addition, another population
of soluble oligomers was observed to coexist with fibrils at completion of the
reaction. The average distance between Tyr39 and Trp125 decreases from 24.9A in
the monomer to 21.9A in the early oligomer and 18.8A in the late oligomer.
Trp125 remains solvent-exposed in both the oligomers and fibrils, indicating
that the C-terminal domain is not part of the fibril core. No FRET was observed
in the fibrils, due to quenching of Tyr39 fluorescence in the fibril core.
Thus, aggregation of alpha-synuclein involves multiple oligomeric intermediates
and competing pathways.
Agrin Binds a-Synuclein
and Modulates a-Synuclein Fibrillation.
Glycobiology.
2005 15:12, 1320-1331
Liu IH, Uversky VN, Munishkina LA,
Fink AL, Halfter W, Cole GJ.
Recent studies have
begun to investigate the role of agrin in brain, and suggest that agrin's
function likely extends beyond that of a synaptogenic protein. Particularly it
has been shown that agrin is associated with the pathological lesions of
Alzheimer's disease (AD) and may contribute to the formation of ss-amyloid
plaques in AD. We have extended the analysis of agrin's function in
neurodegenerative diseases to investigate its role in Parkinson's disease (PD).
alpha-Synuclein is a critical molecular determinant in familial and sporadic
PD, with the formation of alpha-synuclein fibrils being enhanced by sulfated
macromolecules. In the studies reported here we show that agrin binds to
alpha-synuclein in a heparan sulfate-dependent manner , induces conformational
changes in this protein characterized by ss-sheet structure, and enhances
insolubility of alpha-synuclein. We also show that agrin accelerates the formation
of protofibrils by alpha-synuclein, and decreases the half-time of fibril
formation. The association of agrin with PD lesions was also explored in PD
human brain, and these studies shown that agrin co-localizes with
alpha-synuclein in neuronal Lewy bodies in the substantia nigra of PD brain.
These studies indicate that agrin is capable of accelerating the formation of
insoluble protein fibrils in a second common neurodegenerative disease. These
findings may indicate shared molecular mechanisms leading to the
pathophysiology in these two neurodegenerative disorders.
Forcing
non-amyloidogenic beta-synuclein to fibrillate.
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Biochemistry.
2005 Jun 28;44(25):9096-107. |
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Yamin G, Munishkina LA,
Karymov MA, Lyubchenko YL,
Uversky VN, Fink AL.
The fibrillation and
aggregation of alpha-synuclein is a key process in the formation of
intracellular inclusions, Lewy bodies, in substantia nigral neurons and,
potentially, in the pathology of Parkinson's disease and several other
neurodegenerative disorders. Alpha-synuclein and its homologue beta-synuclein
are both natively unfolded proteins that colocalize in presynaptic terminals of
neurons in many regions of the brain, including those of dopamine-producing
cells of the substantia nigra. Unlike its homologue, beta-synuclein does not
form fibrils and has been shown to inhibit the fibrillation of alpha-synuclein.
In this study, we demonstrate that fast and efficient aggregation and
fibrillation of beta-synuclein can be induced in the presence of a variety of
factors. Certain metals (Zn(2+), Pb(2+), and Cu(2+)) induce a partially folded
conformation of beta-synuclein that triggers rapid fibrillation. In the
presence of these metals, mixtures of alpha- and beta-synucleins exhibited
rapid fibrillation. The metal-induced fibrillation of beta-synuclein was
further accelerated by the addition of glycosaminoglycans or high
concentrations of macromolecular crowding agents. Beta-synuclein also rapidly
formed soluble oligomers and fibrils in the presence of pesticides, whereas the
addition of low concentrations of organic solvents induced formation of
amorphous aggregates. These new findings demonstrate the potential effect of
environmental pollutants in generating an amyloidogenic, and potentially
neurotoxic, conformation, in an otherwise benign protein.
Effects of nitration on the structure and
aggregation of alpha-synuclein.
Brain Res Mol Brain Res. 2005 Mar
24;134(1):84-102.
Uversky
VN, Yamin G, Munishkina LA, Karymov MA, Millett IS, Doniach S, Lyubchenko YL,
Fink AL.
Substantial evidence suggests that the
aggregation of the presynaptic protein alpha-synuclein is a key step in the
etiology of Parkinson's disease (PD). Although the molecular mechanisms underlying
alpha-synuclein aggregation remain unknown, oxidative stress has been
implicated in the pathogenesis of PD. Here, we report the effects of tyrosine
nitration on the propensity of human recombinant alpha-synuclein to fibrillate
in vitro. The properties of nitrated alpha-synuclein were investigated using a
variety of biophysical and biochemical techniques, which revealed that
nitration led to formation of a partially folded conformation with increased
secondary structure relative to the intrinsically disordered structure of the
monomer, and to oligomerization at neutral pH. The degree of self-association
was concentration-dependent, but at 1 mg/mL, nitrated alpha-synuclein was
predominantly an octamer. At low pH, small-angle X-ray scattering data indicated
that the nitrated protein was monomeric. alpha-Synuclein fibrillation at
neutral pH was completely inhibited by nitrotyrosination and is attributed to
the formation of stable soluble oligomers. The presence of heparin or metals
did not overcome the inhibition; however, the inhibitory effect was eliminated
at low pH. The addition of nitrated alpha-synuclein inhibited fibrillation of
non-modified alpha-synuclein at neutral pH. Potential implications of these
findings to the etiology of Parkinson's disease are discussed.
Current Opinion in Structural
Biology Volume 15, Issue 1,
February 2005, Pages 35-41
Anthony L Fink
It is now clear that a significant fraction of eukaryotic genomes encode proteins with substantial regions of disordered structure. In spite of the lack of structure, these proteins nevertheless are functional; many are involved in critical steps of the cell cycle and regulatory processes. In general, intrinsically disordered proteins interact with a target ligand (often DNA) and undergo a structural transition to a folded form when bound. Several features of intrinsically disordered proteins make them well suited to interacting with multiple targets and to cell regulation. New algorithms have been developed to identify disordered regions of proteins and have demonstrated their presence in cancer-associated proteins and proteins regulated by phosphorylation.
Methionine oxidation, α-synuclein and
Parkinson's disease
Biochimica et Biophysica Acta (BBA) - Proteins &
Proteomics
Volume 1703, Issue 2 , 17 January 2005, Pages
157-169
Methionine Oxidation and Methionine Sulfoxide Reductases
Charles B. Glaser, Ghiam Yamin, Vladimir N. Uversky and
Anthony L. Fink
The aggregation of normally soluble α-synuclein in the dopaminergic neurons of the substantia nigra is a crucial step in the pathogenesis of Parkinson's disease. Oxidative stress is believed to be a contributing factor in this disorder. Because it lacks Trp and Cys residues, mild oxidation of α-synuclein in vitro with hydrogen peroxide selectively converts all four methionine residues to the corresponding sulfoxides. Both oxidized and non-oxidized α-synucleins have similar unfolded conformations; however, the fibrillation of α-synuclein at physiological pH is completely inhibited by methionine oxidation. The inhibition results from stabilization of soluble oligomers of Met-oxidized α-synuclein. Furthermore, the Met-oxidized protein also inhibits fibrillation of unmodified α-synuclein. The degree of inhibition of fibrillation by Met-oxidized α-synuclein is proportional to the number of oxidized methionines. However, the presence of metals can completely overcome the inhibition of fibrillation of the Met-oxidized α-synuclein. Since oligomers of aggregated α-synuclein may be cytotoxic, these findings indicate that both oxidative stress and environmental metal pollution could play an important role in the aggregation of α-synuclein, and hence possibly Parkinson's disease. In addition, if the level of Met-oxidized α-synuclein was under the control of methionine sulfoxide reductase (Msr), then this could also be factor in the disease.
Role of Protein-Water Interactions and Electrostatics in
a-Synuclein
Fibril Formation
Biochemistry,
Rifampicin
Inhibits alpha-Synuclein Fibrillation and Disaggregates Fibrils.
Chem Biol. 2004 Nov;11(11):1513-21.
Li J, Zhu M, Rajamani S, Uversky VN, Fink AL.
The aggregation of
alpha-synuclein in dopaminergic neurons of the substantia nigra is a critical
step in the pathogenesis of Parkinson's disease. We show that the antibiotic
rifampicin inhibited alpha-synuclein fibrillation and disaggregated existing
fibrils in a concentration-dependent manner. Size-exclusion chromatography data
indicated that rifampicin stabilized alpha-synuclein as both a monomer and
soluble oligomers comprised of partially folded alpha-synuclein. Experiments
using aged samples of rifampicin indicated that the most active species in
inhibiting fibrillation and disaggregating fibrils is an oxidation product of
rifampicin, which was confirmed in experiments under anaerobic conditions.
These results indicate that rifampicin-mediated inhibition of alpha-synuclein
fibrillation and disaggregation of fibrils involves preferential stabilization
of monomeric and soluble oligomeric forms, and that rifampicin potentially may
have therapeutic application for Parkinson's disease.
The
effect of macromolecular crowding on protein aggregation and amyloid fibril
formation
Munishkina, L.A.,
Cooper, E.M., Uversky, V.N., and Fink, A.L.
Macromolecular crowding is expected to have several significant effects on protein aggregation; the major effects will be those due to excluded volume and increased viscosity. In this report we summarize data demonstrating that macromolecular crowding may lead to a dramatic acceleration in the rate of protein aggregation and formation of amyloid fibrils, using the protein α-synuclein. The aggregation of α-synuclein has been implicated as a critical factor in development of Parkinson’s disease. Various types of polymers, from neutral polyethylene glycols and polysaccharides (Ficolls, dextrans) to inert proteins, are shown to accelerate α-synuclein fibrillation. The stimulation of fibrillation increases with increasing length of polymer, as well as increasing polymer concentration. At lower polymer concentrations (typically up to ~100 mg/ml) the major effect is ascribed to excluded volume, whereas at higher polymer concentrations evidence of opposing viscosity effects become apparent. Pesticides and metals, which are linked to increased risk of Parkinson’s disease by epidemiological studies, are shown to accelerate α-synuclein fibrillation under conditions of molecular crowding.
Conformational prerequisites for formation of amyloid fibrils from histones.
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J Mol Biol. 2004 Sep 24;342(4):1305-24. |
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Munishkina LA, Fink AL, Uversky
VN.
We demonstrate that bovine core histones are
natively unfolded proteins in solutions with low ionic strength due to their
high net positive charge at pH 7.5. Using a variety of biophysical techniques
we characterized their conformation as a function of pH and ionic strength, as
well as correlating the conformation with aggregation and amyloid fibril
formation. Tertiary structure was absent under all conditions except at pH 7.5
and high ionic strength. The addition of trifluoroethanol or high ionic
strength induced significant alpha-helical secondary structure at pH 7.5. At
low pH and high salt concentration, small-angle X-ray scattering and SEC HPLC
indicate the histones are present as a hexadecamer of globular subunits. The
secondary structure at low pH was independent of the ionic strength or presence
of TFE, as judged by FTIR. The data indicate that histones are able to adopt
five different relatively stable conformations; this conformational variability
probably reflects, in part, their intrinsically disordered structure. Under
most of the conditions studied the histones formed amyloid fibrils with typical
morphology as seen by electron microscopy. In contrast to most
aggregation/amyloidogenic systems, the kinetics of fibrillation showed an
inverse dependence on histone concentration; we attribute this to partitioning
to a faster pathway leading to non-fibrillar self-associated aggregates at
higher protein concentrations. The rate of fibril formation was maximal at low pH,
and decreased to zero by pH 10. The kinetics of fibrillation were very
dependent on the ionic strength, increasing with increasing salt concentration,
and showing marked dependence on the nature of the ions; interestingly Gdn.HCl
increased the rate of fibrillation, although much less than NaCl. Different
ions also differentially affected the rate of nucleation and the rate of fibril
elongation.
Conformational Constraints for Amyloid Fibrillation: The Importance of
Being Unfolded.
Biochim
Biophys Acta. (2004) May 6;1698(2):131-53
Uversky, V. N. &
Fink, A. L.,
Recent reports give strong support to the idea that amyloid fibril formation and the subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. In this review, recent findings are surveyed to illustrate that protein fibrillogenesis requires a partially folded conformation. This amyloidogenic conformation is relatively unfolded, and shares many structural properties with the pre-molten globule state, a partially folded intermediate frequently observed in the early stages of protein folding and under some equilibrium conditions. The inherent flexibility of such an intermediate is essential in allowing the conformational rearrangements necessary to form the core cross-beta structure of the amyloid fibril.
The flavonoid baicalein inhibits
fibrillation of alpha-synuclein and disaggregates existing fibrils.
Zhu
M, Rajamani S, Kaylor J, Han S, Zhou F, Fink AL.
The aggregation of alpha-synuclein has been implicated as
a critical step in the development of Parkinson's disease. Parkinson's disease
is a progressive neurodegenerative disorder caused by the loss of dopaminergic neurons
from the substantia nigra; currently, no cure exists. Baicalein is a flavonoid
with antioxidant properties; upon oxidation, it forms several products
including quinones. We show here that low micromolar concentrations of
baicalein, and especially its oxidized forms, inhibit the formation of
alpha-synuclein fibrils. In addition, existing fibrils of alpha-synuclein are
disaggregated by baicalein. The product of the inhibition reaction is
predominantly a soluble oligomer of alpha-synuclein, in which the protein
molecules have been covalently modified by baicalein quinone to form a Schiff
base with a lysine side chain in alpha-synuclein. The binding of baicalein was
abolished by conversion of the Tyr residues into Phe, demonstrating that Tyr is
involved in the interaction of alpha-synuclein with baicalein. In
disaggregation baicalein causes fragmentation throughout the length of the
fibril. These observations suggest that baicalein and similar compounds may
have potential as therapeutic leads in combating Parkinson's disease and that
diets rich in flavonoids may be effective in preventing the disorder.
Annular oligomeric amyloid intermediates observed by In-situ AFM
J
Biol Chem. 2004, Jun 4; 279(2): 24452-9
Zhu,M.; Han,S.;
Zhou,F.; Carter,S.A.; Fink,A.L.
Amyloidoses and related protein deposition diseases involve the
transformation of normally soluble proteins into insoluble deposits, usually
fibrillar in nature. Although it was originally assumed that the fibrils were
the toxic species, this assumption has recently been called into question.
Accumulating evidence in several systems suggests that oligomeric intermediates
on the aggregation pathway may be toxic. In the present study we used in-situ
atomic force microscopy to monitor aggregation in aqueous solution in real
time. The sample used was an amyloidogenic immunoglobulin light chain, involved
in AL or light chain amyloidosis. The nature of the observed oligomeric
intermediates was dependent on the conditions of incubation, especially pH and
ionic strength. Several different aggregation intermediates with a variety of
morphologies, including annular or torus-shaped species, were observed. The
data indicate that protein aggregation can be very complex, involving a variety
of different oligomeric intermediates whose population will be determined by
the kinetic and thermodynamic competition between them
Role of Individual Methionines in the Fibrillation of
Methionine-Oxidized alpha-Synuclein
Biochemistry 2004, 43,
4621-4633
Hokenson,M.J.;
Uversky,V.N.; Goers,J.; Yamin,G.; Munishkina,L.A.; Fink,A.L.
The aggregation of normally soluble alpha-synuclein in the dopaminergic
neurons of the substantia nigra is a crucial step in the pathogenesis of
Parkinson's disease. Oxidative stress is believed to be a contributing factor
in this disorder. We have previously established that oxidation of all four
methionine residues in alpha-synuclein (to the sulfoxide, MetO) inhibits
fibrillation of this protein in vitro and that the MetO protein also inhibits
fibrillation of unmodified alpha-synuclein. Here we show that the degree of
inhibition of fibrillation by MetO alpha-synuclein is proportional to the
number of oxidized methionines. This was accomplished be selectively converting
Met residues into Leu, prior to Met oxidation. The results showed that with one
oxidized Met the kinetics of fibrillation were comparable to those for the
control (nonoxidized), and with increasing numbers of methionine sulfoxides the
kinetics of fibrillation became progressively slower. Electron microscope
images showed that the fibril morphology was similar for all species examined,
although fewer fibrils were observed with the oxidized forms. The presence of
zinc was shown to overcome the Met oxidation-induced inhibition. Interestingly,
substitution of Met by Leu led to increased propensity for aggregation (soluble
oligomers) but slower formation of fibrils
Stimulation
of insulin fibrillation by urea-induced intermediates.
J
Biol Chem. 2004 Apr 9;279(15):14999-5013. Epub 2004 Jan 20
Ahmad A, Millett IS, Doniach S, Uversky VN, Fink
AL.
Fibrillar deposits of insulin
cause serious problems in implantable insulin pumps, commercial production of
insulin, and for some diabetics. We performed a systematic investigation of the
effect of urea-induced structural perturbations on the mechanism of
fibrillation of insulin. The addition of as little as 0.5 m urea to zinc-bound
hexameric insulin led to dissociation into dimers. Moderate concentrations of
urea led to accumulation of a partially unfolded dimer state, which dissociates
into an expanded, partially folded monomeric state. Very high concentrations of
urea resulted in an unfolded monomer with some residual structure. The addition
of even very low concentrations of urea resulted in increased fibrillation. Accelerated
fibrillation correlated with population of the partially folded intermediates,
which existed at up to 8 m urea, accounting for the formation of substantial
amounts of fibrils under such conditions. Under monomeric conditions the
addition of low concentrations of urea slowed down the rate of fibrillation,
e.g. 5-fold at 0.75 m urea. The decreased fibrillation of the monomer was due
to an induced non-native conformation with significantly increased
alpha-helical content compared with the native conformation. The data indicate
a close-knit relationship between insulin conformation and propensity to
fibrillate. The correlation between fibrillation and the partially unfolded
monomer indicates that the latter is a critical amyloidogenic intermediate in
insulin fibrillation.
The
role of protein-water interactions and electrostatics in a-synuclein fibril formation.
Biochemistry (2003) 43,
3289-3300
Munishkina, L. A.,
Henriques, J., Uversky. V. N. and Fink, A. L.
Deposition of misfolded alpha-synuclein
is a critical factor in several neurodegenerative disorders. Filamentous
alpha-synuclein is the major component of Lewy bodies and Lewy neurites, the
intracellular inclusions in the dopaminergic neurons of the substantia nigra,
which are considered the pathological hallmark of Parkinson's disease. We show
here that anions induce partial folding of alpha-synuclein at neutral pH,
forming a critical amyloidogenic intermediate, which leads to significant
acceleration of the rate of fibrillation. The magnitude of the accelerating
effect generally followed the position of the anions in the Hofmeister series,
indicating a major role of protein-water-anion interactions in the process at
salt concentrations above 10 mM. Below this concentration, electrostatic
effects dominated in the mechanism of anion-induced fibrillation. The
acceleration of fibrillation by anions was also dependent on the cation.
Moderate concentrations of anions affected both the rates of nucleation and the
elongation of alpha-synuclein fibrillation, primarily via their effect on the
interaction of the protein with water.
FASEB
J
(2004).Jun; 18(9): 962-4.
Li J, Zhu M,
Manning-Bog, A., Di Monte, D., & Fink, A. L.
Protein deposition diseases
involve the aggregation of normally soluble proteins, leading to both fibrillar
and amorphous deposits. The aggregation of alpha-synuclein is associated with
Parkinson's disease, and the aggregation of the Abeta peptide is associated
with Alzheimer's disease. Here we show that L-dopa, dopamine, and other
catecholamines dissolve fibrils of alpha-synuclein and Abeta peptide generated
in vitro. The catecholamines also inhibited the fibrillation of these proteins.
In addition, intraneuronal alpha-synuclein deposits formed in a mouse model
were dissolved by incubation of tissue slices with L-dopa. These catecholamines
are susceptible to oxidative breakdown, and we show that oxidation products are
more effective than the parent compounds in inhibition. The ability to dissolve
fibrils provides a new approach for studying mechanisms and consequences (e.g.,
the relationship between fibril formation and neurodegeneration) of protein
aggregation. It is also likely to help in the development of strategies for the
prevention and treatment of protein deposition diseases. uclein * Abeta peptide
* catecholamines * oxidation
Spectral properties of Thioflavine T and its complexes with amyloid
fibrils.
Journal of Applied Spectroscopy (Moscow) (2003) 70, 765-773.
Voropay E.S., Samtsov M.P., Kaplevsky K.N., Maskevich A.A.,
Stepuro V.I., Povarova O.I., Kuznetsova I.M., Tutoverov K.K., Fink A.L.,
Uversky V.N.
Comparative analysis of the
absorption and fluorescence spectra and fluorescence excitation spectra of
thioflavin T (ThT) in various solvents and in the composition of amyloid
fibrils has shown that ThT, when excited in the region of the long-wavelength
absorption band, fluoresces in the spectral region with a maximum at 478-484
nm. The appearance in aqueous and
alcohol solutions of a fluorescence band with a maximum near 440 nm has been
attributed to the presence in the composition of the ThT preparations of an
impurity with an absorption band in the 340-350-nm range. The literature data
showing that in glycerol ThT has a wide fluorescence spectrum with two maxima
are due to the artifact connected with the use of a high concentration of the
dye. It has been suggested that the cause of the low quantum yield of ThT aqueous
and alcohol solutions is the breakage of the system of conjugated bonds due to
the reorientation of the benzothiozole and benzaminic rings of ThT in the
excited state with respect to one another. The main factor determining the high
quantum yield of fluorescence of ThT incorporated in fibrils is the steric
restriction of the rotation of the rings about one another under these
conditions. The suggestions made have been verified by the quantum-chemical
calculation of ThT molecule geometry in the ground and excited states.
Partially folded intermediates in insulin
fibrillation.
Biochemistry. 2003 Oct 7;42(39):11404-16
Ahmad A,
Millett IS, Doniach S, Uversky VN, Fink AL.
Native zinc-bound insulin exists as a hexamer at neutral pH. Under destabilizing conditions, the hexamer dissociates, and is very prone to forming fibrils. Insulin fibrils exhibit the typical properties of amyloid fibrils, and pose a problem in the purification, storage, and delivery of therapeutic insulin solutions. We have carried out a systematic investigation of the effect of guanidine hydrochloride (Gdn.HCl)-induced structural perturbations on the mechanism of fibrillation of insulin. At pH 7.4, the addition of as little as 0.25 M Gdn.HCl leads to dissociation of insulin hexamers into dimers. Moderate concentrations of Gdn.HCl lead to formation of a novel partially unfolded dimer state, which dissociates into a partially unfolded monomer state. High concentrations of Gdn.HCl resulted in unfolded monomers with some residual structure. The addition of even very low concentrations of Gdn.HCl resulted in substantially accelerated fibrillation, although the yield of fibrils decreased at high concentrations. Accelerated fibrillation correlated with the population of the expanded (partially folded) monomer, which existed up to >6 M Gdn.HCl, accounting for the formation of substantial amounts of fibrils under such conditions. In the presence of 20% acetic acid, where insulin exists as the monomer, fibrillation was also accelerated by Gdn.HCl. The enhanced fibrillation of the monomer was due to the increased ionic strength at low denaturant concentrations, and due to the presence of the partially unfolded, expanded conformation at Gdn.HCl concentrations above 1 M. The data suggest that under physiological conditions, the fibrillation of insulin involves both changes in the association state (with rate-limiting hexamer dissociation) and conformational changes, leading to formation of the amyloidogenic expanded monomer intermediate.
A model for amyloid fibril formation in
immunoglobulin light chains based on comparison of amyloidogenic and benign
proteins and specific antibody binding.
Amyloid. 2003 Jun;10(2):97-109
Khurana R, Souillac PO, Coats AC, Minert L, Ionescu-Zanetti C, Carter SA,
Solomon A, Fink AL.
In an attempt to understand the mechanism of amyloid fibril formation in light chain amyloidosis, the properties of amyloidogenic (SMA) and benign (LEN) immunoglobulin light chain variable domains (VL) were compared. The conformations of LEN and SMA were measured using secondary and tertiary structural probes over the pH range from 2 and 8. At all pH values, LEN was more stable than SMA. The CD spectra of LEN at pH 2 were comparable to those of SMA at pH 7.5, indicating that the low pH conformation of LEN closely resembles that of SMA at physiological pH. At low pH, a relatively unfolded intermediate conformation is populated for SMA and rapidly leads to amyloid fibrils. The lack of such an intermediate with LEN, is attributed to sequence differences and accounts for the lack of LEN fibrils in the absence of agitation. A kappa IV-specific monoclonal antibody that recognizes the N-terminal of SMA caused unraveling of the fibrils to the protofilaments and was observed to bind to one end of SMA protofilaments by atomic force microscopy. The antibody result indicates that each protofilament is asymmetric with different ends. A model for the formation of fibrils by SMA is proposed.
The association of alpha-synuclein with
membranes affects bilayer structure, stability, and fibril formation.
J Biol Chem. 2003 Oct
10;278(41):40186-97. Epub 2003 Jul 28.
Zhu M, Li
J, Fink AL.
The aggregation of alpha-synuclein is believed to be a critical factor in the etiology of Parkinson's disease. alpha-Synuclein is an abundant neuronal protein of unknown function, which is enriched in the presynaptic terminals of neurons. Although alpha-synuclein is found predominantly in the cytosolic fractions, membrane-bound alpha-synuclein has been suggested to play an important role in fibril formation. The effects of alpha-synuclein on lipid bilayers of different compositions were determined using fluorescent environment-specific probes located at various depths. alpha-Synuclein-membrane interactions were found to affect both protein and membrane properties. Our results indicate that in addition to electrostatic interactions, hydrophobic interactions are important in the association of the protein with the bilayer, and lead to disruption of the membrane. The latter was observed by atomic force microscopy and fluorescent dye leakage from vesicles. The kinetics of alpha-synuclein fibril formation were significantly affected by the protein association and subsequent membrane disruption, and reflected the conformation of alpha-synuclein. The ability of alpha-synuclein to disrupt membranes correlated with the binding affinity of alpha-synuclein for the particular membrane composition, and to the induced helical conformation of alpha-synuclein. Protofibrillar or fibrillar alpha-synuclein caused a much more rapid destruction of the membrane than soluble monomeric alpha-synuclein, indicating that protofibrils (oligomers) or fibrils are likely to be significantly neurotoxic.
A general model for amyloid fibril
assembly based on morphological studies using atomic force microscopy.
Biophys J. 2003 Aug;85(2):1135-44
Khurana
R, Ionescu-Zanetti C, Pope M, Li J, Nielson L, Ramirez-Alvarado M, Regan L,
Fink AL, Carter SA.
Based on atomic force microscopy analysis of the morphology of fibrillar species formed during fibrillation of alpha-synuclein, insulin, and the B1 domain of protein G, a previously described model for the assembly of amyloid fibrils of immunoglobulin light-chain variable domains is proposed as a general model for the assembly of protein fibrils. For all of the proteins studied, we observed two or three fibrillar species that vary in diameter. The smallest, protofilaments, have a uniform height, whereas the larger species, protofibrils and fibrils, have morphologies that are indicative of multiple protofilaments intertwining. In all cases, protofilaments intertwine to form protofibrils, and protofibrils intertwine to form fibrils. We propose that the hierarchical assembly model describes a general mechanism of assembly for all amyloid fibrils.
Nuclear localization of alpha-synuclein
and its interaction with histones.
Biochemistry. 2003 Jul 22;42(28):8465-71
Goers J,
Manning-Bog AB, McCormack AL, Millett IS, Doniach S, Di Monte DA, Uversky VN,
Fink AL.
The aggregation of alpha-synuclein is believed to play an important role in the pathogenesis of Parkinson's disease as well as other neurodegenerative disorders ("synucleinopathies"). However, the function of alpha-synuclein under physiologic and pathological conditions is unknown, and the mechanism of alpha-synuclein aggregation is not well understood. Here we show that alpha-synuclein forms a tight 2:1 complex with histones and that the fibrillation rate of alpha-synuclein is dramatically accelerated in the presence of histones in vitro. We also describe the presence of alpha-synuclein and its co-localization with histones in the nuclei of nigral neurons from mice exposed to a toxic insult (i.e., injections of the herbicide paraquat). These observations indicate that translocation into the nucleus and binding with histones represent potential mechanisms underlying alpha-synuclein pathophysiology.
Tau filaments from human brain and from
in vitro assembly of recombinant protein show cross-beta structure.
Proc Natl Acad Sci U S A. 2003 Jul
22;100(15):9034-8. Epub 2003 Jul 09
Berriman
J, Serpell LC, Oberg KA, Fink AL, Goedert M, Crowther RA.
Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge CB2 2QH, United Kingdom.
Abnormal filaments consisting of hyperphosphorylated microtubule-associated protein tau form in the brains of patients with Alzheimer's disease, Down's syndrome, and various dementing tauopathies. In Alzheimer's disease and Down's syndrome, the filaments have two characteristic morphologies referred to as paired helical and straight filaments, whereas in tauopathies, there is a wider range of morphologies. There has been controversy in the literature concerning the internal molecular fine structure of these filaments, with arguments for and against the cross-beta structure demonstrated in many other amyloid fibers. The difficulty is to produce from brain pure preparations of filaments for analysis. One approach to avoid the need for a pure preparation is to use selected area electron diffraction from small groups of filaments of defined morphology. Alternatively, it is possible to assemble filaments in vitro from expressed tau protein to produce a homogeneous specimen suitable for analysis by electron diffraction, x-ray diffraction, and Fourier transform infrared spectroscopy. Using both these approaches, we show here that native filaments from brain and filaments assembled in vitro from expressed tau protein have a clear cross-beta structure.
Structural transformations of oligomeric
intermediates in the fibrillation of the immunoglobulin light chain LEN.
Biochemistry. 2003 Jul 8;42(26):8094-104
Souillac
PO, Uversky VN, Fink AL.
LEN is a kappaIV immunoglobulin light chain variable domain from a patient suffering from multiple myeloma but with no evidence of amyloid fibrils. However, fibrils are formed when LEN solutions are agitated under mildly destabilizing conditions. Surprisingly, an inverse concentration dependence was observed on the kinetics of fibril formation because of the formation of off-pathway soluble oligomers at high protein concentration. Despite the fact that most of the protein is present in the off-pathway intermediates at relatively early times of aggregation, eventually all the protein forms fibrils. Thus, a structural rearrangement from the non fibril-prone off-pathway oligomers to a more fibril-prone species must occur. A variety of techniques were used to monitor changes in the size, secondary structure, solvent accessibility, and intrinsic stability of the oligomers, as a function of incubation time. The structural rearrangement was accompanied by a significant increase of disordered secondary structure, an increase in solvent accessibility, and a decrease in intrinsic stability of the soluble oligomeric species. We conclude that fibrils arise from the oligomers containing a less stable conformation of LEN, either directly or via dissociation. This is the first fibrillating system in which soluble off-pathway oligomeric intermediates have been shown to be the major transient species and in which fibrillation occurs from a relatively unfolded conformation present in these intermediates.
Certain metals trigger fibrillation of
methionine-oxidized alpha-synuclein.
J Biol Chem. 2003 Jul 25;278(30):27630-5.
Epub 2003 May 16
Yamin G,
Glaser CB, Uversky VN, Fink AL.
The aggregation and fibrillation of alpha-synuclein has been implicated as a key step in the etiology of Parkinson's disease and several other neurodegenerative disorders. In addition, oxidative stress and certain environmental factors, including metals, are believed to play an important role in Parkinson's disease. Previously, we have shown that methionine-oxidized human alpha-synuclein does not fibrillate and also inhibits fibrillation of unmodified alpha-synuclein (Uversky, V. N., Yamin, G., Souillac, P. O., Goers, J., Glaser, C. B., and Fink, A. L. (2002) FEBS Lett. 517, 239-244). Using dynamic light scattering, we show that the inhibition results from stabilization of the monomeric form of Met-oxidized alpha-synuclein. We have now examined the effect of several metals on the structural properties of methionine-oxidized human alpha-synuclein and its propensity to fibrillate. The presence of metals induced partial folding of both oxidized and non-oxidized alpha-synucleins, which are intrinsically unstructured under conditions of neutral pH. Although the fibrillation of alpha-synuclein was completely inhibited by methionine oxidation, the presence of certain metals (Ti3+, Zn2+, Al3+, and Pb2+) overcame this inhibition. These findings indicate that a combination of oxidative stress and environmental metal pollution could play an important role in triggering the fibrillation of alpha-synuclein and thus possibly Parkinson's disease.
Nitration inhibits fibrillation of human
alpha-synuclein in vitro by formation of soluble oligomers.
FEBS Lett. 2003 May 8;542(1-3):147-52
Yamin G,
Uversky VN, Fink AL.
The aggregation of alpha-synuclein in dopaminergic neurons is a critical factor in the etiology of Parkinson's disease (PD). Oxidative and nitrative stress is also implicated in PD. We examined the effect of nitration on the propensity of alpha-synuclein to fibrillate in vitro. Fibril formation of alpha-synuclein was completely inhibited by nitration, due to the formation of stable soluble oligomers (apparently octamers). More importantly the presence of sub-stoichiometric concentrations of nitrated alpha-synuclein led to inhibition of fibrillation of non-modified alpha-synuclein. These observations suggest that nitration of soluble alpha-synuclein may be a protective factor in PD, rather than a causative one.
Prediction of the association state of
insulin using spectral parameters.
J Pharm Sci. 2003 Apr;92(4):847-58
Uversky
VN, Garriques LN, Millett IS, Frokjaer S, Brange J, Doniach S, Fink AL.
Institute for Biological
Instrumentation, Russian Academy of Sciences, 142292 Pushchino, Moscow Region,
Russia. uversky@hydrogen.ucsc.edu
Human insulin exists in different association states, from monomer to hexamer, depending on the conditions. In the presence of zinc the "normal" state is a hexamer. The structural properties of 20 variants of human insulin were studied by near-UV circular dichroism, fluorescence spectroscopy, and small-angle X-ray scattering (SAXS). The mutants showed different degrees of association (monomer, dimers, tetramers, and hexamers) at neutral pH. A correlation was shown between the accessibility of tyrosines to acrylamide quenching and the degree of association of the insulin mutants. The near-UV CD spectra of the insulins were affected by protein association and by mutation-induced structural perturbations. However, the shape and intensity of difference CD spectra, obtained by subtraction of the spectra measured in 20% acetic acid (where all insulin species were monomeric) from the corresponding spectra measured at neutral pH, correlate well with the degree of insulin association. In fact, the near-UV CD difference spectra for monomeric, dimeric, tetrameric, and hexameric insulin are very distinctive, both in terms of intensity and shape. The results show that the spectral properties of the insulins reflect their state of association, and can be used to predict their oligomeric state. Copyright 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:847-858, 2003
Polycation-induced oligomerization and
accelerated fibrillation of human alpha-synuclein in vitro.
Protein Sci. 2003 Apr;12(4):702-7
Goers J,
Uversky VN, Fink AL.
The aggregation and fibrillation of alpha-synuclein has been implicated as a causative factor in Parkinson's disease and several other neurodegenerative disorders known as synucleinopathies. The effect of different factors on the process of fibril formation has been intensively studied in vitro. We show here that alpha-synuclein interacts with different unstructured polycations (spermine, polylysine, polyarginine, and polyethyleneimine) to form specific complexes. In addition, the polycations catalyze alpha-synuclein oligomerization. The formation of alpha-synuclein-polycation complexes was not accompanied by significant structural changes in alpha-synuclein. However, alpha-synuclein fibrillation was dramatically accelerated in the presence of polycations. The magnitude of the accelerating effect depended on the nature of the polymer, its length, and concentration. The results illustrate the potential critical role of electrostatic interactions in protein aggregation, and the potential role of naturally occurring polycations in modulating alpha-synuclein aggregation.
Biophysical
properties of a-synuclein and its role in
Parkinson’s disease.
In
Recent Res. Dev. Proteins, 1
(2002), 153-186.
Uversky, V. N. and Fink, A. L.
Parkinson’s disease (PD) is the second most common neurodegenerative disease, and results from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of α-synuclein in the form of intracellular proteinaceous aggregates (Lewy bodies and Lewy neuritis) has been implicated as a causative factor in this disease, as well as in several other neurodegenerative disorders, including dementia with Lewy bodies Lewy body variant of Alzheimer’s disease, multiple system atrophy and Hallervorden-Spatz disease. Thus, the aggregated forms of α-synuclein play a crucial role in the pathogenesis of the synucleinopathies. However, the molecular mechanisms underlying α-synuclein aggregation into specific filamentous inclusions remained unknown until recently. An intriguing aspect of this problem was that α-synuclein belongs to the class of natively unfolded proteins characterized by little or no ordered structure under physiological conditions. This raised the question of how an essentially disordered protein could be transformed into highly organized fibrils. The first part of this review is devoted to the search for an answer to this question and deals with the description of structural properties of α-synuclein and conformational behavior of this protein under variety of experimental conditions, as well as with the analysis of the effects of these same conditions on fibrillation kinetics of human recombinant α-synuclein.
Two different missense mutations in the α-synuclein gene, corresponding to A30P and A53TL substitutions in the protein, have been identified in a small number of kindreds from human protein at seven positions, including position 53, where the murine protein contains a threonine residue, however neither Parkinsonian symptoms nor Lewy bodies have been observed in aged mice. Besides α-synuclein, the family of human synucleins includes β- and γ-synucleins, with all three proteins showing large sequence homology. Structural consequences of amino substitutions on α-synuclein structure, conformational behavior and aggregation are considered in the second part of review.
The etiology of PD
is unknown, but recent work has shown that, except in rare cases, there appears
to be no direct genetic basis. However, a positive correlation between the
prevalence of PD and industrialization has been recognized. Particularly,
several epidemiological studies have implicated such environmental factors as
pesticides, herbicides and heavy metals in the PD origin and this disorder is
now considered likely to be an “environmental” disease. More specifically,
occupational exposure to specific metals, especially manganese, copper, lead,
iron, mercury, zinc, and aluminum, was shown to be a risk factor for PD.
Moreover, elevated levels of several of these metals have also been reported in
the substantia nigra of PD subjects. Furthermore, farming, rural living,
well-water drinking and exposure to agricultural chemicals are all conditions
that have been associated with an increased risk for PD. The third part of the
review summarizes data showing that the environmental factors can directly
affect the aggregation of α-synuclein,
providing a critical link between toxicant exposures and the pathogenesis of α-synuclein-containing inclusions in
idiopathic PD.
Structural and functional properties of
Yersinia pestis Caf1 capsular antigen and their possible role in fulminant
development of primary pneumonic plague.
J Proteome Res. 2002 Jul-Aug;1(4):307-15
Abramov
VM, Vasiliev AM, Khlebnikov VS, Vasilenko RN, Kulikova NL, Kosarev IV,
Ishchenko AT, Gillespie JR, Millett IS, Fink AL, Uversky VN.
Institute of Immunological
Engineering, 142380 Lyubuchany, Moscow Region, Russia.
Yersinia pestis capsular antigen Caf1 is shown to be a beta-structural protein that in polymeric form possesses very high conformational stability. Different approaches show that a dimer is the minimal cooperative block of Caf1 adhesin. Caf1 dimer interacts effectively with IL-1 receptors of human macrophage and epithelial cells. The specificity of such interaction is confirmed by the inhibition of IL-1alpha binding by Caf1. The Caf1 role in pneumonic plague pathogenesis is discussed.
Effect of zinc and temperature on the
conformation of the gamma subunit of retinal phosphodiesterase: a natively
unfolded protein.
J Proteome Res. 2002 Mar-Apr;1(2):149-59
Uversky
VN, Permyakov SE, Zagranichny VE, Rodionov IL, Fink AL, Cherskaya AM, Wasserman
LA, Permyakov EA.
Institute for Biological Instrumentation, Russian Academy of Sciences, 142290 Pushchino, Russia. uversky@hydrogen.ucsc.edu
The cyclic GMP phosphodiesterase gamma-subunit (PDEgamma) was shown to belong to the family of natively unfolded proteins. Increasing temperature transforms the protein into a more ordered (but still relatively disordered) conformation. The C-terminal part of PDEgamma has a high-affinity zinc-binding site (Kd approximately 1 microM), with His75 and His79 being directly involved into the coordination of Zn2+. Zinc-loaded protein remains effectively unfolded. Possible implications of these findings to the functioning of PDEgamma are discussed.
Lipid binding inhibits alpha-synuclein
fibril formation.
J Biol Chem. 2003 May 9;278(19):16873-7.
Epub 2003 Mar 05
Zhu M,
Fink AL.
Parkinson's disease is the second most common neurodegenerative disorder, and the cause is unknown; however, substantial evidence implicates the aggregation of alpha-synuclein as a critical factor in the etiology of the disease. alpha-Synuclein is a relatively abundant brain protein of unknown function, and the purified protein is intrinsically unfolded. The amino acid sequence has seven repeats with an apolipoprotein lipid-binding motif, which are predicted to form amphiphilic helices. We have investigated the interaction of alpha-synuclein with lipid vesicles of different sizes and properties by monitoring the effects on the conformation of the protein and the kinetics of fibrillation. The nature of the interaction of alpha-synuclein with vesicles was highly dependent on the phospholipid composition, the ratio of alpha-synuclein to phospholipid, and the size of the vesicles. The strongest interactions were between alpha-synuclein and vesicles composed of 1,2-dipalmitoyl-sn-glycero-3-phosphate/1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho-RAC-(1-glycerol)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine and involved formation of helical structure in alpha-synuclein. A strong correlation was observed between the induction of alpha-helix in alpha-synuclein and the inhibition of fibril formation. Thus, helical, membrane-bound alpha-synuclein is unlikely to contribute to aggregation and fibrillation. Given that a significant fraction of alpha-synuclein is membrane-bound in dopaminergic neurons, this observation has significant physiological significance.
Conformational behavior and aggregation
of alpha-synuclein in organic solvents: modeling the effects of membranes.
Biochemistry. 2003 Mar 11;42(9):2720-30
Munishkina
LA, Phelan C, Uversky VN, Fink AL.
Intracellular proteinaceous inclusions (Lewy bodies and Lewy neurites) of alpha-synuclein are pathological hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies (DLB), and multiple systemic atrophy. The molecular mechanisms underlying the aggregation of alpha-synuclein into such filamentous inclusions remain unknown, although many factors have been implicated, including interactions with lipid membranes. To model the effects of membrane fields on alpha-synuclein, we analyzed the structural and fibrillation properties of this protein in mixtures of water with simple and fluorinated alcohols. All solvents that were studied induced folding of alpha-synuclein, with the common first stage being formation of a partially folded intermediate with an enhanced propensity to fibrillate. Protein fibrillation was completely inhibited due to formation of beta-structure-enriched oligomers with high concentrations of methanol, ethanol, and propanol and moderate concentrations of trifluoroethanol (TFE), or because of the appearance of a highly alpha-helical conformation at high TFE and hexafluoro-2-propanol concentrations. At least to some extent, these conformational effects mimic those observed in the presence of phospholipid vesicles, and can explain some of the observed effects of membranes on alpha-synuclein fibrillation.
Disorder in the nuclear pore complex: the
FG repeat regions of nucleoporins are natively unfolded.
Proc Natl Acad Sci U S A. 2003 Mar
4;100(5):2450-5. Epub 2003 Feb 25
Denning
DP, Patel SS, Uversky V, Fink AL, Rexach M.
Department of Biological Sciences,
Stanford University, Stanford, CA 94305, USA.
Nuclear transport proceeds through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope of eukaryotic cells. The Saccharomyces cerevisiae NPC is comprised of 30 nucleoporins (Nups), 13 of which contain phenylalanine-glycine repeats (FG Nups) that bind karyopherins and facilitate the transport of karyopherin-cargo complexes. Here, we characterize the structural properties of S. cerevisiae FG Nups by using biophysical methods and predictive amino acid sequence analyses. We find that FG Nups, particularly the large FG repeat regions, exhibit structural characteristics typical of "natively unfolded" proteins (highly flexible proteins that lack ordered secondary structure). Furthermore, we use protease sensitivity assays to demonstrate that most FG Nups are disordered in situ within the NPCs of purified yeast nuclei. The conclusion that FG Nups constitute a family of natively unfolded proteins supports the hypothesis that the FG repeat regions of Nups form a meshwork of random coils at the NPC through which nuclear transport proceeds
Conformational behavior of human alpha-synuclein
is modulated by familial Parkinson's disease point mutations A30P and A53T.
Neurotoxicology. 2002 Oct;23(4-5):553-67
Li J,
Uversky VN, Fink AL.
Structural properties and response to changes in the environment of wild-type (WT), A30P and A53T alpha-synucleins, as well as their propensity to aggregate orform fibrils, were compared by a variety of biophysical methods, including far-UV CD, FTIR, SAXS, static light scattering and Thioflavin T (TFT) fluorescence. All three proteins were natively unfolded under physiological conditions but adopted identical partially-folded conformations under conditions of acidic pH or high temperature. The initial kinetic event in the fibrillation of all three alpha-synucleins was shown to be the formation of a partially-folded intermediate with properties close to those described for these proteins at acidic pH or at high temperatures. Both mutants showed a greater propensity to form non-fibrillar aggregates than wild-type protein. All three proteins formed fibrils faster in the presence of heparin, although substantially higher concentrations were required for the A30P mutant. In contrast to the wild-type and A53T proteins, in which fibrillation was further accelerated by the presence of the pesticide diethyldithiocarbamate (DDC), the A30P mutant was inhibited by DDC. The mutant proteins had significantly lower affinity for DDC than the WT. A model of the effect of mutations on the aggregation behavior of alpha-synuclein is proposed, which explains the different effects of exogenous agents on the three proteins, based on different kinetic partitioning along pathways leading to fibrils and to non-fibrillar aggregates.
Synergistic effects of pesticides and
metals on the fibrillation of a-synuclein: Implications for Parkinson’s disease
Neurotoxicology (2002) 23(4-5), 527-536
Aggregation of a-synuclein has been implicated in the formation of proteinaceous inclusions in the brain (Lewy bodies, Lewy neurites) that are characteristic of neurodegenerative diseases such as Parkinson’s disease and dementia with Lewy bodies. The etiology of Parkinson’s disease is unknown, but recent work has shown that, except in rare cases, there appears to be no direct genetic basis. However, several studies have implicated environmental factors, especially pesticides and metals. Here we show that certain pesticides and metals induce a conformational change in a-synuclein and directly accelerate the rate of formation of a-synuclein fibrils in vitro. In addition, the simultaneous presence of metal and pesticide led to synergistic effects on the rate of fibrillation. We propose a model in which environmental factors, in conjunction with genetic susceptibility, may form the underlying molecular basis for idiopathic Parkinson’s disease.
Conformational prerequisites for
alpha-lactalbumin fibrillation.
Biochemistry. 2002 Oct 15;41(41):12546-51
Goers J,
Permyakov SE, Permyakov EA, Uversky VN, Fink AL.
Bovine alpha-lactalbumin, a small acidic Ca(2+)-binding milk protein, formed amyloid fibrils at low pH, where it adopted the classical molten globule-like conformation. Fibrillation was accompanied by a dramatic increase in the beta-structure content and a characteristic increase in the thioflavin T fluorescence intensity. S-(Carboxymethyl)-alpha-lactalbumin, a disordered form of the protein with three out of four disulfide bridges reduced, was even more susceptible to fibrillation. Other partially folded conformations induced in the intact protein at neutral pH, either by the removal of Ca(2+) or by the binding of Zn(2+) to the Ca(2+)-protein complex, did not fibrillate, although Zn(2+)-loaded alpha-lactalbumin precipitated out of solution as amorphous aggregates. Our data suggest that the transformation of a protein into an essentially unfolded (thus, highly flexible) conformation is required for successful fibril formation, whereas more rigid (but still flexible) species may form amorphous aggregates.
Surface-catalyzed amyloid fibril formation.
J Biol Chem. 2002 Dec
27;277(52):50914-22. Epub 2002 Sep 27
Zhu M,
Souillac PO, Ionescu-Zanetti C, Carter SA, Fink AL.
Department of Chemistry and
Biochemistry and the Department of Physics, University of California, Santa
Cruz, California 95064, USA.
Light chain (or AL) amyloidosis is characterized by the pathological deposition of insoluble fibrils of immunoglobulin light chain fragments in various tissues, walls of blood vessels, and basement membranes. In the present investigation, the in vitro assembly of a recombinant amyloidogenic light chain variable domain, SMA, on various surfaces was monitored using atomic force microscopy. SMA formed fibrils on native mica at pH 5.0, conditions under which predominantly amorphous aggregates form in solution. Fibril formation was accelerated significantly on surfaces compared with solution; for example, fibrils grew on surfaces at significantly faster rates and at much lower concentrations than in solution. No fibrils were observed on hydrophobic or positively charged surfaces or at pH >7.0. Two novel types of fibril growth were observed on the surface: bidirectional linear assembly of oligomeric units, and linear growth from preformed amorphous cores. In addition to catalyzing the rate of fibrillation, the mechanism of fibril formation on the surfaces was significantly different from in solution, but it may be more physiologically relevant because in vivo the deposits are associated with surfaces.
Amino acid determinants of
alpha-synuclein aggregation: putting together pieces of the puzzle.
FEBS Lett. 2002 Jul 3;522(1-3):9-13
Uversky
VN, Fink AL.
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA. uversky@hydrogen.ucsc.edu
Parkinson's disease is the second most common neurodegenerative disease, and results from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of alpha-synuclein in the form of intracellular proteinaceous aggregates (Lewy bodies and Lewy neurites) have been implicated as a causative factor in this disease, as well as in several other neurodegenerative disorders, including dementia with Lewy bodies, Lewy body variant of Alzheimer's disease, multiple system atrophy and Hallervorden-Spatz disease. Thus, the aggregated forms of alpha-synuclein play a crucial role in the pathogenesis of the synucleinopathies. However, the molecular mechanisms underlying alpha-synuclein aggregation into specific filamentous inclusions remained unknown until recently. Data on the aggregation and fibrillation properties of human alpha-, beta- and gamma-synucleins, mouse alpha-synuclein and familial Parkinson's disease mutants of human alpha-synuclein (A30P and A53T) are an