The aggregation propensities of this repeat peptide and its corresponding phosphorylated form were investi-gated using turbidity, thioflavin T fluorescence and electron microscopy.. Notabl
Trang 1self-aggregation ability of a peptide from the fourth tau microtubule-binding repeat
Jin-Tang Du1, Chun-Hui Yu1, Lian-Xiu Zhou1, Wei-Hui Wu1, Peng Lei1, Yong Li1, Yu-Fen Zhao1, Hiroshi Nakanishi2and Yan-Mei Li1
1 Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing, China
2 Research Institute of Instrumentation Frontier, National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan
Post-translational phosphorylation serves as a control
mechanism in a myriad of cellular processes including
metabolic pathway regulation, extracellular signal
transduction, ion channel regulation and cell-cycle
pro-gression [1] In other cases, abnormal phosphorylation
may be harmful; for example, hyperphosphorylation
causes the aggregation of microtubule-associated
pro-tein tau, which is implicated in Alzheimer’s disease
(AD) [2,3]
AD is the main form of dementia in today’s ageing
population [2] It is characterized by the presence of
two aberrant structures, senile plaques and
neurofibril-lary tangles The main components of neurofibrilneurofibril-lary
tangles are paired helical filaments (PHFs) [4], which
are mainly comprised of the protein tau in an
abnor-mally phosphorylated form [3] Tau protein, whose
main function is to stimulate and stabilize microtubule
assembly from tubulin subunits, is abundant in both the central and peripheral nervous systems [5] Tau stabilizes microtubules and regulates the transport of vesicles or organelles along them, it supports the outgrowth of axons and serves as an anchor for enzymes [6] Tau binds to microtubules via the micro-tubule-binding domain, which contains four copies of
a highly conserved 18-amino acid repeat, namely, R1, R2, R3 and R4, each of which is separated from another repeat by less conserved 13- or 14-amino acid inter-repeat domains [7] Although tau protein is water soluble and shows little tendency to aggregate under physiological conditions, it dissociates from microtu-bules and aggregates into PHFs in the brains of AD patients [8–12] Functionally, tau binds to tubulin, whereas PHF-tau does not [10–15] Because this aggre-gation leads to toxicity in neurons due to damage to
Keywords
aggregation; Alzheimer’s disease;
microtubule-binding repeat; phosphorylation;
tau
Correspondence
Y.-M Li, Key Laboratory of Bioorganic
Phosphorus Chemistry & Chemical Biology
(Ministry of Education), Department of
Chemistry, Tsinghua University,
Beijing 100084, China
Fax: +86 10 6278 1695
Tel: +86 10 6279 6197
E-mail: liym@mail.tsinghua.edu.cn
(Received 27 May 2007, revised 24 July
2007, accepted 30 July 2007)
doi:10.1111/j.1742-4658.2007.06018.x
Phosphorylation of tau protein modulates both its physiological role and its aggregation into paired helical fragments, as observed in Alzheimer’s diseased neurons It is of fundamental importance to study paired helical fragment formation and its modulation by phosphorylation This study focused on the fourth microtubule-binding repeat of tau, encompassing an abnormal phosphorylation site, Ser356 The aggregation propensities of this repeat peptide and its corresponding phosphorylated form were investi-gated using turbidity, thioflavin T fluorescence and electron microscopy There is evidence for a conformational change in the fourth microtubule-binding repeat of tau peptide upon phosphorylation, as well as changes in aggregation activity Although both tau peptides have the ability to aggre-gate, this is weaker in the phosphorylated peptide This study reveals that both tau peptides are capable of self-aggregation and that phosphorylation
at Ser356 can modulate this process
Abbreviations
AD, Alzheimer’s disease; PHF, paired helical filament; ThT, thioflavin T.
Trang 2the cell interior, it is important to clarify the
mecha-nism of aggregation of tau protein and develop ways
to prevent this pathological assembly
The microtubule-binding domain, located in the
C-terminal of tau protein, has been reported to assume
the core structure of PHFs and promote tau
aggrega-tion in vitro [16–18] We previously studied the
metal-binding properties and the effects of phosphorylation
on tau protein fragments Several tentative
explana-tions for PHF formation have been proposed [19–21]
It is also observed that the second (R2) and third (R3)
microtubule-binding repeats can aggregate with the
help of heparin [22] However, little such data
concern-ing the contribution of the R4 repeat to the formation
of PHFs and the modulation by phosphorylation have
been reported
Here, we investigate the aggregation propensity of
the R4 repeat using turbidity measurements,
thiofla-vin T (ThT) fluorescence and electron microscopy
Tur-bidity measurements are an excellent, widely adopted
method of quantifying aggregation in solution [23,24]
In addition, it has been suggested that phosphorylation
of some specific tau sites may be a prerequisite for
aggregation [25,26] Ser356, which is located in the R4
repeat, is one likely abnormal phosphorylation sites
[27] It is not clear how abnormal phosphorylation of
tau protein modulates aggregation In vitro, the
stimu-latory effects of phosphorylation on the aggregation of
tau have been reported [28] However, different
phos-phorylation sites may have different effects on filament
formation, and it is advantageous to study the effect of
only one confined phosphorylation site on a tau
pep-tide However, analysis of the effect of phosphorylation
at defined sites is hampered by the low specificity of
protein kinases and the highly dynamic turnover of
phosphorylation in vivo Site-directed mutagenesis,
which converts serine and threonine to aspartic acid
and glutamic acid, has been used to imitate
phosphory-lation [29] In our study, synthetic phosphopeptide was
used and the effect of phosphorylation on the tau
repeat fragment assembly was also studied
Recently, Wang et al showed that AD P-tau
dephosphorylated by protein phosphatase did not
aggre-gate into filaments, whereas several protein kinases
and their combinations can abnormally
hyperphos-phate protein phosphatase dephosphorylation of AD
P-tau and induce its self-aggregation into PHF similar
to those seen in AD It is, thus, important to learn how phosphorylation modulates the self-aggregation of tau This study focused on the aggregation propensity
of the fourth microtubule-binding repeat of tau peptide
in its unphosphorylated (R4) and also phosphorylated (pR4) form [30], to try and explain how phosphoryla-tion modulates the process of aggregaphosphoryla-tion at the molecular level Peptide R4 and phosphopeptide pR4 relating to the human tau protein (Table 1) were syn-thesized according to a solid-phase synthetic strategy Phosphopeptide pR4 was phosphorylated at Ser356 The structural differences between phosphopeptide and nonphosphopeptide were analysed using CD and high-resolution NMR spectroscopy The aggregation behav-ior of peptide R4 and phosphopeptide pR4 and the structural differences between them were then exam-ined using turbidity, ThT fluorescence and electron microscopy The results from turbidity measurements, ThT fluorescence and electron microscopy show that the R4 repeat and its phosphorylated form pR4 are capable of self-aggregation It is proposed that this repeat plays an important role in the aggregation of tau protein and phosphorylation is able to modulate the process of aggregation
Results
Phosphorylation of Ser356-induced conforma-tional change in peptide R4
To investigate the effect of phosphorylation at Ser356
on the native structure of peptide R4, both CD and NMR spectroscopy were performed
In NMR spectroscopy, TOCSY and NOESY spectra
of the two peptides were recorded and compared Changes in the backbone NH and aH chemical shifts upon phosphorylation (dphosphorylated–dnonphosphorylated) were shown for each residue A comparison of the chemical shifts of NH and aH between the non-phosphorylated and non-phosphorylated peptides is sum-marized in Fig 1 The chemical shift deviations of NH and aH reflect changes in the electrostatic state and molecular structure Upon phosphorylation at Ser356, the largest proton chemical shift deviation of NH and
aH was observed for Ser356 (downfield 0.41 p.p.m for
Table 1 Synthetic peptides corresponding to the repeat domain of the human tau441 sequence p, phosphorylation.
Trang 3NH and 0.10 p.p.m for aH) In general, the chemical
shift of NH deviates was more than that of aH upon
phosphorylation Notable chemical shift deviation of
NH and aH occurs mainly at the phosphorylation site
and sites proximal to it, and may reflect both intrinsic
effects through the covalent bond and the formation
of a hydrogen bond between phosphate and the amide
group [31,32], indicating that phosphorylation may
affect the local structure in the vicinity of the
phos-phorylated site
Identification of the hydrogen-bonding partners depends on detailed investigations into the pH depen-dence of their NMR parameters over the pH range 3–8 Obviously, the pH titration curve of the amide proton and the titration curve of 31P of phosphory-lated serine residue have the almost identical pK values, which indicate hydrogen-bonding interactions between phosphate and the amide group (Fig 2) An important result is that the titration parameters of the backbone amide proton of Ser356 remain virtually
Fig 1 Comparison of chemical shift differences of NH (white bar) and aH (black bar) between peptide R4 and phosphopeptide pR4 at pH 5.6 and 278 K Changes in chemical shifts upon phosphory-lation (dphosphorylated–dnonphosphorylated) are shown for each residue, positive values are downfield shifts and negative values are upfield shifts.
Fig 2 One-dimensional 31 P NMR spectra of pR4 with pH titration at 295K: (A) pH 3.0, (B) pH 3.9, (C) pH 4.9, (D) pH 5.6, (E) pH 6.6, (F) pH 7.5 (left) Changes with d1H NMR of amide protons in Ser356 and d31P NMR of the phosphate group during the pH titration of R4 and pR4 (right).
Trang 4unchanged in nonphosphopeptide R4 compared with
phosphopeptide pR4
In phosphopeptide, a phosphorylated serine acid
side chain contains a single titratable group
(phos-phate) with pKa1 and pKa2 The pKa value is
deter-mined from a fit of the phosphorus chemical shifts of
the without ionic, monoionic and diionic forms of the
phosphate group as a function of pH [33,34] The
phosphorus chemical shift change reflecting the
equi-librium between without ionic and monoionic form
(pKa1) is not observed in the pH range 3–8 [31,35]
The pKavalues of phosphate group for the equilibrium
between the monoionic and diionic form (pKa2) are
obtained from the changes of the phosphorus chemical
shift with pH titration (Fig 2)
Under acidic conditions, the phosphopeptide had
one negative charge and an intraresidue hydrogen bond
between the nearby amide proton and the phosphate
group As the pH increased, deprotonation began at
the phosphate group, and the hydrogen bond began to
surpass weakened electrostatic repulsion and led to the
amide proton chemical shift downfield Thus, the
Ser356 amide proton chemical shift downfield in the
phosphopeptide could be explained by the hydrogen
bond between the nearby amide proton and the
phos-phate group and deprotonation in the phosphos-phate
In phosphorylated peptide pR4, a hydrogen bond
between the nearby amide proton and the phosphate
group appears to be the driving force behind the
struc-tural changes that occur upon phosphorylation of
Ser356 In addition, the NMR spectra in water
sug-gested the presence, except for the major conformer, of
one or more minor conformations for the R4 peptide,
as evidenced by the appearance of additional
reso-nances of lower intensity than those in the major
con-former [21] However, only one major conformation
was observed in phosphopeptide pR4 CD spectra for
R4 and pR4 are characterized by a strong negative
apex at 198 nm (Fig 3), which indicates a large
amount of random coil structure [36] No remarkable
structural perturbation is suggested upon the
phos-phorylation of Ser356
Effect of phosphorylation on assembly
of the tau repeat
An aggregating study was performed in NaCl⁄ Pi, a
buffer widely used to mimic physiological conditions
[37,38] Electron microscopy, turbidity and ThT
fluo-rescence measurements confirm that both R4 and pR4
are capable of self-aggregation
The aggregation kinetics process was derived from
the time dependence of turbidity at 405 nm As shown
in Fig 4, both peptides showed little aggregation on day 1 However, the turbidity of both peptides increased sharply on the day 2, indicative of a nucle-ation step involved in the aggregnucle-ation Once the seed is formed, the filaments can form quickly In addition, peptide R4 aggregated more quickly than phospho-peptide pR4 on days 2–4 During day 5, phospho-peptide R4 aggregated at almost the same speed as on day 4, whereas the aggregation speed of phosphopeptide pR4 increased On day 6, the turbidity of both pep-tides had decreased somewhat, indicating that the aggregation had reached equilibrium According to the kinetic turbidity curve, a different intrinsic rate of nucleation of aggregation is suggested Filibration of R4 is considerably easier than that of pR4
In addition, the aggregation kinetics was also derived from the time dependence of the relative ThT
fluores-Fig 3 CD spectra of peptide R4 and phosphopeptide pR4.
Fig 4 Aggregation of peptide R4 and phosphopeptide pR4 as mon-itored by turbidity Peptides were dissolved in NaCl ⁄ P i , pH 7.4 (137.0 m M NaCl, 3.0 m M KCl, 10.0 m M Na2HPO4 and 2.0 m M
KH 2 PO 4 , ionic strength 160.0 m M ) to a final concentration of 1.0 mgÆmL)1 and incubated at room temperature The assembly time course of peptide R4 and phosphopeptide pR4 is plotted ver-sus the incubation time according to the turbidity at 405 nm.
Trang 5cence intensity at 485 nm in NaCl⁄ Pi (Fig 5) As
shown in Fig 5, the rate of filament formation was
much greater for R4 than for pR4, indicating
non-iden-tical filament formation for the R4 and pR4 peptides
Electron microscopy was also used to evaluate the
aggregation of peptide R4 and the effect of
phosphory-lation moduphosphory-lation on the process In contrast to the
typical long filament of peptide R4, negatively stained
images of polymerized phosphopeptide pR4 revealed
some thinner filaments (Fig 6) Electron microscopy
confirmed that phosphorylation in Ser356 was able to
modulate the aggregation form of R4 in vitro
Discussion
Knowing what regions of the protein tau are involved
in its aggregation into aberrant filaments and what
molecular structure is induced by aggregation are
criti-cal steps towards understanding the mechanisms
involved in the pathological aggregation of tau Tau
protein purified from brain extracts or
recombin-ant tau is able to aggregate in vitro at high protein
concentrations [39–41] However, it is difficult to study the mechanism of tau aggregation using the full-length tau molecule because some regions act as inhibitors of polymerization Furthermore, even if full-length tau obtained by recombinant means was used, it does not mimic the phosphorylation state of tau molecules com-prising PHFs [42] Therefore, despite the growing body
of data suggesting that different domains of the pro-tein may have different secondary structures [43], we decided to approach the problem by studying these factors in small tau fragments It has reported that fragments from the tubulin-binding motif of tau can assemble into filaments in vitro
At present, the contribution of the R4 repeat to PHF formation remains to be elucidated, even though the roles of the R2 and R3 repeats in the aggregation of tau have been reported [22] Moreover, there is no firm con-clusion concerning the effect of phosphorylation on aggregation In this study, we have shown that both peptide R4 and phosphopeptide pR4 are capable of self-aggregation without the need to add aggregation inducer in NaCl⁄ Pi, according to the results of electron microscopy, ThT fluorescence and turbidity experi-ments This leads to the suggestion that the R4 repeat might also play an essential role in PHF formation
in vivo The ability of the R4 repeat to self-aggregate implies that R4 repeats in the microtubule-binding domain might recognize each other and facilitate aggre-gation of the tau protein
To better understand the mechanism of PHF for-mation, the effects of phosphorylation on the confor-mation and aggregation of the R4 repeat were studied Introduction of the phosphate ion, which predominantly carries a double negative charge at neutral pH, affects the electrostatic potential and quite often the conformation of the modified protein Even in the absence of rearrangement, the change
in the electric field and steric hindrance from a phosphate group can have biologically significant
Fig 6 Electron microscopy images of
in vitro filaments of peptide R4 (left) and phosphopeptide pR4 (right) The black bar in the figure represents 100 nm.
Fig 5 Time profiles of peptide R4 and phosphopeptide pR4
aggre-gations in NaCl ⁄ P i as monitored by relative ThT fluorescence
inten-sity at 485 nm.
Trang 6consequences, e.g promoting or opposing protein–
protein interactions Phosphorylated side chains
typi-cally carry a )2 charge at physiological pH, although
the pKa of the phosphate group is 6, and the )1
species may be present in certain proteins or at low
pH Phosphorylation is a key cause of modification
in cellular regulation There is increasing evidence
that phosphorylation may influence filament
forma-tion in peptides and proteins [28,29] In this study,
phosphorylation at Ser356 exhibited a modulated
effect on aggregation compared with peptide R4 This
modulated effect of phosphorylation on aggregation
from the tubulin-binding motif might offer some clues
on its role in the progression of AD The modulation
of phosphorylation on aggregation might be essential
for the aggregation of tau protein in vivo The peptide
concentration used in this study is much higher than
in vivo, so the process of assembly is very clear in our
experiments, that is, the time needed for aggregation
is much shorter than in vivo It is likely that
phos-phorylation exerts its toxic effects in AD via different
aggregation behavior of the tau protein
The different aggregation behavior of peptide R4
and phosphopeptide pR4 might be explained by
con-sidering the different conformations of R4 and pR4 It
has been reported that phosphorylation can modulate
the structure of the first and third tau
microtubule-binding repeat, which in turn results in a change in
aggregation behavior [42,44] For R4 and pR4, a local
conformational difference was deduced from the
pro-ton chemical shift deviation of NH and aH However,
there was no remarkable structural perturbation from
the CD spectra Furthermore, our study has confirmed
that a hydrogen bond is formed between the phosphate
and the amide proton of the phosphorylated serine
res-idue in phosphopeptide pR4 [21] The hydrogen bond
is supposed to be the driving force behind the
struc-tural changes that occur upon phosphorylation So
how does phosphorylation alter the aggregation
behav-ior of peptide R4? A possible explanation is that
phos-phorylation might affect the kinetics of conversion of
the native structure to a filament-like structure [45] In
other words, phosphorylation might act through the
hydrogen bond to alter the structural proclivity among
different conformational states, which results in
differ-ent aggregation behavior
In conclusion, it is shown that the R4 repeat is
capa-ble of self-aggregation and phosphorylation at Ser356
can modulate the aggregation in the process of
assem-bly, implying that R4 repeat might also play an
impor-tant role in PHFs formation and phosphorylation at
Ser356 might serve as an aggregation modulation in
the progression of AD Study such as this may be
valuable in future research undertaken to clarify the pathophysiology of AD
Experimental procedures
Peptide synthesis
Peptides were synthesized on Fmoc-Wang resin using the standard Fmoc⁄ tBu chemistry and HBTU ⁄ HOBt protocol [46] For phosphopeptide, phosphoserine was incorporated
as Fmoc-Ser(PO3HBzl)-OH [47] Peptides and all protecting groups were cleaved from the resin with trifluoroacetic acid containing phenol (5%), thioanisole (5%), ethanedithiol (2.5%) and water (5%) for 120 min [48] Crude peptides were purified by reverse-phase HPLC using an ODS-UG-5 column (Develosil) with a linear gradient of 20–50% aceto-nitrile containing 0.06% trifluoroacetic acid as an ionpairing reagent The integrity of the peptide and phosphopeptide was verified by ESI-MS and NMR spectroscopy The synthetic peptides are listed in Table 1
CD
The peptides (1.0 mgÆmL)1) were dissolved in phosphate buffer, pH 7.6 (10.0 mm Na2HPO4) CD spectra were recorded on a Jasco model J-715 spectropolarimeter (Jasco, Tokyo, Japan) at 298 K under a constant flow of nitrogen gas Typically, a quartz cell with a 0.1 cm path length was used for spectra recorded between 190 and 250 nm with a 1-nm scan interval CD intensities reported in the figure are expressed in mdeg
NMR spectroscopy
Peptide samples for NMR measurements were dissolved in
H2O⁄ D2O 9 : 1 (v⁄ v) in 10.0 mm phosphate or sodium d4 -acetic acid buffer The pH value was adjusted by adding HCl or NaOH Sodium d4-2,2-dimethyl-2-silapentonate in a capillary tube was used as the external standard for 1
H NMR chemical shifts Standard NOESY [49] and
TOC-SY [50] experiments were collected on a Varian Inova-600 spectrometer (Palo Alto, CA, USA) or a Jeol ECA-600 spectrometer (Tokyo, Japan) The 31P NMR spectra were acquired on a Bruker ACP200 spectrometer with 85% phosphoric acid as the external reference Two-dimensional NMR data were processed using the nmrpipe⁄ nmrdraw program [51] A sinesquared window function shifted by
p⁄ 4) p ⁄ 2 was applied in both dimensions, with zero filling
in f1–2K points Quadrature detection in f1 was achieved using time proportional phase incrementation [52] H2O res-onance was suppressed either by presaturation of the sol-vent peak during the relaxation delay (and the mixing time
in the NOESY spectra) or by using a pulsed-field gradient technique with a WATER-GATE sequence [53,54] In
Trang 7general, spectra were collected with 2K points in f2 and 512
in f1
Identification of hydrogen bond using pH
titration experiments
The protocol to identify hydrogen-bonding interactions
between phosphate and the amide group was based on
changing the pH from acidic to basic [33,34] When a
cer-tain amide proton was involved in such a hydrogen bond
and downfield chemical shifts were observed during the pH
variation, which indicated the hydrogen bond between the
amide proton and phosphate pH values were measured
with a microcombination pH⁄ sodium electrode (Orion
Research, Inc., Beverly, MA) attached to an Orion 520A
pH meter Calibration of the pH meter was carried out at
room temperature using pH 4.00 ± 0.01, pH 7.01 ± 0.01,
and pH 10.00 ± 0.01 calibration buffers
Monitoring the aggregation of R4 and pR4 using
turbidity
Peptides (1.0 mgÆmL)1) were dissolved in NaCl⁄ Pi, pH 7.4
(137.0 mm NaCl, 3.0 mm KCl, 10.0 mm Na2HPO4, and
2.0 mm KH2PO4, ionic strength 160.0 mm) Identical
methods were used to prepare peptide samples utilized for
electron microscopy and ThT fluorescence experiments To
study aggregation, peptides (1.0 mgÆmL)1) were incubated
at room temperature in a nonbinding surface 96-well plate
The aggregation was monitored each day at the same time
via turbidity measurements at 405 nm on Wellscan MK3
instrument (Labsystems Dragon Co., MA, USA)
Monitoring of aggregation of R4 and pR4 using
ThT fluorescence
Peptides (1.0 mgÆmL)1) were dissolved in NaCl⁄ Pi and
incubated at room temperature During the incubation,
20.0 lL aliquots of the reaction solutions were added to
sodium phosphate buffer (700.0 lL) containing ThT
(10.0 lm) Fluorescence spectra were collected using a
Hit-achi F-4500 fluorescence spectrophotometer (Tokyo,
Japan) An excitation frequency of 440 nm was used, and
data were collected over the range of 450–600 nm Samples
were placed in a four-sided quartz fluorescence cuvette
(Mu¨llheim, Germany), and data were recorded at room
temperature The excitation slit width was set at 5 nm and
the emission slit width was set at 5 nm The background
fluorescence of the sample was subtracted when necessary
Transmission electron microscopy
Filaments were viewed by electron microscopy Negative
staining of the sample was performed on formvar- and
carbon-coated 300-mesh copper grids Samples were loaded
on the grid and left for 2 min for absorption and then stained with 1% tungstophosphoric acid for another 2 min After drying in a desiccator overnight, the samples were viewed on a JEOL-1200EX electron microscope at 100 kV
Acknowledgements
The authors are grateful for financial support from National Natural Science Foundation of China (Nos
20672067, 20532020, 20475032, and NSFCBIC 20320130046), and Innovative Research Team in Uni-versity (IRT0404)
References
1 Matter N, Herrlich P & Konig H (2002) Signal depen-dent regulation of splicing via phosphorylation of Sam68 Nature 420, 691–695
2 Mandelkow E (1999) Alzheimer’s disease: the tangled tale of tau protein Nature 402, 588–589
3 Grundke-Iqbal I, Iqbal K, Tung YC, Quinlan M, Wisniewski HM & Binder LI (1986) Abnormal phos-phorylation of the microtubule-associate protein tau in Alzheimer cytoskeletal pathology Proc Natl Acad Sci USA 83, 4913–4917
4 Kidd M (1963) Paired helical filaments in electron microscopy of Alzheimer’s disease Nature 197, 192–193
5 Gong CX, Liu F, Grundke-Iqbal I & Iqbal K (2005) Post-translational modifications of tau protein in Alz-heimer’s disease J Neural Transm 112, 813–838
6 Mandelkow EM & Mandelkow E (1998) Tau in Alzhei-mer’s disease Trends Cell Biol 8, 425–427
7 Lee G, Cowan N & Kirschner M (1988) The primary structure and heterogeneity of tau protein from mouse brain Science 239, 285–288
8 Buee L, Bussiere T, Buee-Scherrer V, Delacourte A & Hof PR (2000) Tau protein isoforms, phosphorylation and role in neurodegenerative disorders Brain Res Rev
33, 95–130
9 Friedhoff P, von Bergen M, Mandelkow EM & Man-delkow E (2000) Structure of tau protein and assembly into paired helical filaments Biochim Biophys Acta
1502, 122–132
10 Iqbal K, Zaidi T, Wen GY, Grundke-Iqbal I, Merz PA, Shaikh SS, Wisniewski HM, Alafuzoff I & Winblad B (1986) Defective brain microtubule assembly in Alzhei-mer disease Lancet 2, 421–426
11 Iqbal K, Zaidi T, Bancher C & Grundke-Iqbal I (1994) Alzheimer paired helical filaments Restoration of the biological activity by dephosphorylation FEBS Lett
349, 104–108
12 Alonso AD, Grundkeiqbal I & Iqbal K (1994) Role of abnormally phosphorylated tau in the breakdown of
Trang 8microtubules in Alzheimer disease Proc Natl Acad Sci
USA 91, 5562–5566
13 Drechsel DN, Hyman AA, Cobb MH & Kirschner MW
(1992) Modulation of the dynamic instability of tubulin
assembly by the microtubule-associated protein tau
Mol Cell Biol 3, 1141–1154
14 Biernat J, Gustke N, Drewes G, Mandelkow EM &
Mandelkow E (1993) Phosphorylation of Ser262
strongly reduces binding of tau to microtubules:
distinc-tion between PHF-like immunoreactivity and
microtu-bule binding Neuron 11, 153–163
15 Goedert M & Jakes R (1990) Expression of separate
isoforms of human tau protein: correlation with the tau
pattern in brain and effects on tubulin polymerization
EMBO J 9, 4225–4230
16 Friedhoff P, von Bergen M, Mandelkow E & Davies P
(1998) A nucleated assembly mechanism of Alzheimer
paired helical filaments Proc Natl Acad Sci USA 95,
15712–15717
17 Wille H, Drewes G, Biernat J, Mandelkow EM &
Mandelkow E (1992) Alzheimer-like paired helical
filaments and antiparallel dimers formed from
microtubule-associated protein tau in vitro J Cell Biol
118, 573–584
18 Hiraoka S, Yao TM, Minoura K, Tomoo K, Sumida
M, Taniguchi T & Ishida T (2004) Conformational
transition state is responsible for assembly of
micro-tubule-binding domain of tau protein Biochem Biophys
Res Commun 315, 659–663
19 Du JT, Li YM, Wei W, Wu GS, Zhao YF, Kanazawa
K, Nemoto T & Nakanishi H (2005) Low barrier
hydrogen bond between phosphate and the amide group
in phosphopeptide J Am Chem Soc 127, 16350–16351
20 Ma QF, Du Li YMJT, Liu HD, Kanazawa K, Nemoto
T, Nakanishi H & Zhao YF (2006) Copper binding
properties of a tau peptide associated with Alzheimer’s
disease studied by CD, NMR, and MALDI-TOF MS
Peptides 27, 841–849
21 Du JT, Li YM, Ma QF, Qiang W, Zhao YF, Abe H,
Kanazawa K, Qin XR, Aoyaqi R, Ishizuka Y et al
(2005) Synthesis and conformational properties of
phos-phopeptides related to the human tau protein Regul
Peptides 130, 48–56
22 Minoura K, Yao TM, Tomoo K, Sumida M, Sasaki M,
Taniguchi T & Ishida T (2004) Different associational
and conformational behaviors between the second and
third repeat fragments in tau microtubule-binding
domain Eur J Biochem 277, 545–552
23 Gestwicki JE, Crabtree GR & Graef IA (2004)
Harness-ing chaperones to generate small-molecule inhibitors of
amyloid aggregation Science 304, 865–869
24 Soto C, Castano EM, Frangione B & Inestrosa NC
(1995) The a-helical to b-strand transition in the
amino-terminal fragment of the amyloid b-peptide modulates
amyloid formation J Biol Chem 270, 3063–3067
25 Bancher C, Braunner C, Lassmann H, Budka H, Jellin-ger KA, Wiche G, SeitelberJellin-ger F, Grundke-Iqbal I, Iqbal K & Wisniewski HM (1989) Accumulation of abnormally phosphorylated tau precedes the formation
of neurofibrillary tangles in Alzheimer’s disease Brain Res 477, 90–99
26 Gordon-Krajcer W, Yang L & Ksiezak-Reding H (1993) Conformation of paired helical filaments blocks dephosphorylation of epitopes shared with fetal tau except Ser202⁄ Thr205 Brain Rev 268, 1166–1173
27 Litersky JM, Johnson GVM, Jakes R, Goedert M, Lee
M & Seubert P (1996) tau protein is phosphorylated by cyclic AMP-dependent protein kinase and calcium⁄ cal-modulin-dependent protein kinase II within its microtu-bule-binding domains at Ser262 and Ser356 Biochem J
316, 655–660
28 Alonso AC, Zaidi T, Novak M, Grundke-Iqbal I & Iqbal K (2001) Hyperphosphorylation induced self-assembly of tau into tangles of paired helical fila-ments⁄ straight filaments Proc Natl Acad Sci USA 98, 6923–6928
29 Haase C, Stieler JT, Arendt T & Holzer M (2004) Pseudophosphorylation of tau protein alters its ability for self-aggregation J Neurochem 88, 1509–1520
30 Wang JZ, Grundke-Iqbal I & Iqbal K (2007) Kinases and phosphatases and tau sites involved in Alzheimer neurofibrillary degeneration Eur J Neurosci
25, 59–68
31 Coadou G, Evrard-Todeschi N, Gharbi-Benarous J, Benarous R & Girault JP (2002) HIV-1 encoded virus protein U (Vpu) solution structure of the 41–62 hydro-philic region containing the phosphorylated sites Ser52 and Ser56 Int J Biol Macromol 30, 23–40
32 Gschwind RM, Armbruster M & Zubrzycki IZ (2004) NMR detection of intermolecular NHÆÆÆOP hydrogen bonds between guanidinium protons and bisposphonate moieties in an artificial arginine receptor J Am Chem Soc 126, 10228–10229
33 Szyperski T, Antuch W, Schick M, Betz A, Stone SR & Wuthrich K (1994) Transient hydrogen bonds identified
on the surface of the NMR solution structure of Hiru-din Biochemistry 33, 9303–9310
34 Haruyama H, Qian YQ & Wuthrich K (1989) Static and transient hydrogen-bonding interactions in recombi-nant desulfatohirudin studied by1H nuclear magnetic resonance measurements of amide proton exchange rates and pH-dependent chemical shifts Biochemistry
28, 4312–4317
35 Kyte J (1995) Structure in Protein Chemistry Garland, New York, NY
36 Yi HQ, Gruszczynska-Biegala J, Wood D, Zhao ZF & Zolkiewska A (2005) Cooperation of the metalloprotease, disintegrin, and cysteine-rich domains of ADAM12 during of myogenic differentiation J Biol Chem 280, 23475–23488
Trang 937 Scaramozzino F, Peterson DW, Farmer P, Gerig JT,
Graves DJ & Lew J (2006) TMAO promotes
fibrilli-zation and microtubule assembly activity in the
C-terminal repeat region of tau Biochemistry 45,
3684–3694
38 Barghorn S & Mandelkow E (2002) Toward a unified
scheme for the aggregation of tau into Alzheimer paired
helical filaments Biochemistry 41, 14885–14896
39 Crowther RA, Olesen OF, Smith MJ, Jakes R &
Goed-ert M (1994) Assembly of Alzheimer-like filaments from
full-length tau protein FEBS Lett 337, 135–138
40 Montejo de Garcini E & Avila J (1987) In vitro
condi-tions for the self polymerization of the
microtubule-associated protein, tau factor J Biochem (Tokyo) 102,
1415–1421
41 Montejo de Garcini E, Carrascosa JL, Correas I, Nieto
A & Avila J (1988) Tau factor polymers are similar to
paired helical filaments of Alzheimer’s disease FEBS
Lett 236, 150–154
42 Mendieta J, Fuertes MA, Kunjishapatham R,
Santa-Maria I, Moreno FJ, Alonso C, Gaqo F, Munoz V,
Avila J & Hernandez F (2005) Phosphorylation
modu-lates the alpha-helical structure and polymerization of a
peptide from the third tau microtubule-binding repeat
Biochim Biophys Acta 1721, 16–26
43 Gamblin TC, Berry RW & Binder LI (2003) Modeling
tau polymerization in vitro: a review and synthesis
Biochemistry 42, 15009–15017
44 Zhou LX, Du Zeng ZYJT, Zhao YF & Li YM (2006)
The self-assembly ability of the first microtubule-binding
repeat from tau and it modulation by phosphorylation
Biochem Biophys Res Commun 348, 637–642
45 Liang FC, Chen RPY, Lin CC, Huang KT & Chan SI
(2006) Tuning the conformation properties of a peptide
by glycosylation and phosphorylation Biochem Biophys
Res Commun 342, 482–488
46 Fields GB & Noble RL (1990) Solid phase peptide syn-thesis utilizing 9-fluorenylmethoxycarbonyl amino acids Int J Peptide Protein Res 35, 161–214
47 Wakamiya T, Saruta K, Yasuoka J & Kusumoto S (1994) An efficient procedure for solid-phase synthesis
of phosphopeptides by the Fmoc strategy Chem Lett 6, 1099–1102
48 Muhlradt PF, Kie M, Meyer H, Suuth R & Jung G (1997) Isolation, structure elucidation, and synthesis of
a macrophage stimulatory lipopeptide from mycoplasma fermentans acting at picomolar concentration J Exp Med 185, 1951–1958
49 Macura S & Ernst RR (1980) Elucidation of cross-relax-ation in liquids by two-dimensional NMR spectroscopy Mol Phys 41, 95–117
50 Bax AD & Davis DG (1985) Practical aspects of twodi-menssional transverse NOE spectroscopy J Magn Reson
63, 207–213
51 Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J & Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes J Biomol NMR 6, 277–293
52 Marion D & Wuthrich K (1983) Application of phase sensitive two dimensional correlated spectroscopy (COSY) for measurements of 1H)1H spin–spin cou-pling constants in proteins Biochem Biophys Res Commun 113, 967–974
53 Piotto M, Saudek V & Sklenar V (1992) Gradient-tailored excitation for single quantum NMR spectroscopy of aqueous solutions J Biomol NMR 2, 661–665
54 Kay LE (1995) Pulsed field gradient multi-dimensional NMR methods for the study of protein structure and dynamics in solution Prog Biophys Mol Biol 63, 277– 299