effect of oculopharyngeal muscular dystrophy-associated extension of seven alanines on the...

Effect of oculopharyngeal muscular dystrophy-associatedextension of seven alanines on the fibrillation propertiesof the N-terminal domain of PABPN1Grit Lodderstedt1, Simone Hess2, Gerd Hause3, Till Scheuermann1,*, Thomas Scheibel2

and Elisabeth Schwarz1

1 Institut fur Biotechnologie, Martin-Luther-Universitat Halle-Wittenberg, Halle, Germany

2 Technische Universitat Munchen, Garching, Germany

3 Biozentrum der Martin-Luther-Universitat Halle-Wittenberg, Halle, Germany

Protein folding to a conformation distinct from the

native fold gives rise to a wide range of diseases. The

most well-known examples are the spongiform encep-

halopathies, Alzheimer’s, Parkinson’s and Hunting-

ton’s diseases [1–4]. These various disorders have all

been traced to individual proteins that undergo alter-

native folding to a conformation, the characteristic

feature of which is a b-cross structure formed by

b-strands lying perpendicular to the fibril axis [5].

However, the molecular processes that either directly

or indirectly cause these highly fatal illnesses are still

under debate.

Huntington’s disease is one of the most prominent

examples of neurodegenerative diseases that are caused

by trinucleotide expansions of CAG repeats and thus

an expansion of a run of glutamine residues [3]. In the

most extreme cases, expansions of up to 180 glutamines

have been described. Besides Huntington’s disease and

Keywords

AFM; alanine expansions; amyloid-like;

kinetics of fibril formation; OPMD

Correspondence

E. Schwartz, Institut fur Biotechnologie,

Martin-Luther-Universitat Halle-Wittenberg,

Kurt-Mothes-Str. 3, 06120 Halle, Germany

Fax: +49 345 55 27 013

Tel. +49 345 55 24 856

E-mail: Elisabeth.Schwarz@biochemtech.

uni-halle.de

*Present address

Roche Diagnostics GmbH, Penzberg,

Germany

(Received 12 September 2006, revised

2 November 2006, accepted 8 November

2006)

doi:10.1111/j.1742-4658.2006.05595.x

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant

disease that usually manifests itself within the fifth decade. The most prom-

inent symptoms are progressive ptosis, dysphagia, and proximal limb mus-

cle weakness. The disorder is caused by trinucleotide (GCG) expansions in

the N-terminal part of the poly(A)-binding protein 1 (PABPN1) that result

in the extension of a 10-alanine segment by up to seven more alanines. In

patients, biopsy material displays intranuclear inclusions consisting primar-

ily of PABPN1. Poly l-alanine-dependent fibril formation was studied using

the recombinant N-terminal domain of PABPN1. In the case of the protein

fragment with the expanded poly l-alanine sequence [N-(+7)Ala], fibril for-

mation could be induced by low amounts of fragmented fibrils serving as

seeds. Besides homologous seeds, seeds derived from fibrils of the wild-type

fragment (N-WT) also accelerated fibril formation of N-(+7)Ala in a con-

centration-dependent manner. Seed-induced fibrillation of N-WT was con-

siderably slower than that of N-(+7)Ala. Using atomic force microscopy,

differences in fibril morphologies between N-WT and N-(+7)Ala were

detected. Furthermore, fibrils of N-WT showed a lower resistance against

solubilization with the chaotropic agent guanidinium thiocyanate than those

from N-(+7)Ala. Our data clearly reveal biophysical differences between

fibrils of the two variants that are likely caused by divergent fibril struc-

tures.

Abbreviations

AFM, atomic force microscopy; ANS, 8-anilinonaphthalene-1-sulfonate; EM, electron microscopy; OPMD, oculopharyngeal muscular

dystrophy; PABPN1, poly(A)-binding protein nuclear; ThT, thioflavine T.

346 FEBS Journal 274 (2007) 346–355 ª 2006 The Authors Journal compilation ª 2006 FEBS

several other poly l-glutamine-linked diseases, exten-

sions of poly l-alanine stretches have also been reported

as a cause of congenital disorders. However, in contrast

to the massive extensions seen in poly l-glutamine-

based disorders, more than 30 consecutive alanines are

rarely observed upon mutation of GCG repeats in

poly l-alanine-caused illnesses [6]. The sensitivity of

protein folds towards poly l-alanine extensions is evi-

dent from genetic analyses, which have revealed that

two additional alanine residues in poly(A)-binding pro-

tein nuclear (PABPN1) are sufficient to elicit dominant

effects [7]. These findings have been confirmed using an

animal model for oculopharyngeal muscular dystrophy

(OPMD), in which hemizygous transgenic mice with

three additional alanine residues in PABPN1 displayed

myopathic changes [8].

PABPN1 [nuclear poly(A)-binding protein, previ-

ously, PABP2] is involved in mRNA processing in the

cell nucleus [9,10]. Together with cleavage and poly-

adenylation specificity factor, PABPN1 induces proces-

sivity of poly(A)polymerase and controls the length of

poly adenine tails [11–13]. PABPN1 is a 306 amino

acid protein with oppositely charged N- and C-ter-

minal domains. An RNP-type RNA-binding domain,

which lies in the middle of the protein, is preceded by

an a-helical segment [13,14]. The poly l-alanine exten-

sions affect the N-terminal fragment of the protein,

which comprises 125 amino acids. In the wild-type

protein, the sequence (Ala)10Gly(Ala)2 follows the

start methionine. In OPMD patients, this natural

poly l-alanine sequence is extended by up to seven

additional alanine residues yielding a total of 17 ala-

nines in the most extreme case [7]. Biochemical

analyses of PABPN1 with extended poly l-alanine

sequences showed that the protein’s activity in poly

adenylation is not affected (B Schulz and E Wahle,

personal communication). Histochemical analysis of

biopsy material from OPMD patients revealed fibrillar

aggregates in muscle fiber nuclei with PABPN1 as a

major constituent [15,16].

The occurrence of aggregates has been confirmed in

both yeast- and cell-culture models of OPMD [17–22]. It

is not clear, however, whether the observed aggregates

in the model systems represent amyloid-like deposits.

Irrespective of the nature of the aggregates (amorphous

or regular b-cross structures), reduction of aggregate

formation by chemical and ⁄or molecular chaperones

has been shown to reduce cytotoxicity both in cell cul-

ture [17,18,22,23] and in animal models [19,24]. How-

ever, the mere fact that no correlation between the

frequency of the inclusions and the severity of the dis-

ease can be observed [25], shows that the molecular pro-

cess(es) that elicits OPMD is to date unknown.

We showed previously that recombinant full-length

PABPN1 tends to form amorphous aggregates in vitro

[26]. The formation of amorphous aggregates was inde-

pendent of the presence or length of the poly l-alanine

sequence. In contrast, no amorphous aggregates were

observed with the N-terminal domain of PABPN1. The

N-terminal fragment of wild-type and the variant carry-

ing the most extreme extension observed in man (seven

additional alanine residues) formed fibrillar structures

with a lag phase that was considerably shorter in the

case of the variant with the poly l-alanine extension

[26]. In this work, we further compare these two N-ter-

minal fragments. Differences on the level of fibril for-

mation kinetics and seeding capacity are observed.

Furthermore, the two variants also differ in their fibril

morphologies and stabilities of the fibrils against solu-

bilization.

Results

Seeding of fibril growth of PABPN1 N-terminal

fragment variants

Previous analyses of poly l-alanine-dependent fibril for-

mation of PABPN1 have revealed that the full-length

protein readily forms amorphous aggregates [26].

Although fibril formation also occurred with full-length

PABPN1 upon storage (data not shown), the simulta-

neous presence of both amorphous aggregates and

fibrils hampered the analysis of fibril formation kinetics.

For this reason, fibril formation was analyzed with the

N-terminal domain of PABPN1 consisting of amino

acids 1–125 of the wild-type protein (N-WT). Because

the aim of this study was to investigate the effect on the

fibrillation properties of the most extreme disease-asso-

ciated extension of seven additional alanines, fibrillation

kinetics and fibril properties of N-WT were compared

with those of the corresponding fragment carrying seven

additional alanines (N-(+7)Ala). Both proteins were

recombinantly produced in Escherichia coli cells and

purified as published previously [26].

Fibrillation kinetics were first followed via fluores-

cence measurements with thioflavine T (ThT), a dye

routinely used to monitor fibril formation [27]. How-

ever, to obtain fluorescence signals of sufficient ampli-

tude, protein concentrations >10 lm had to be used.

We have previously reported unusual tinctorial features

of fibrils of the N-terminal domain of PABPN1 [26]. In

addition, poor staining of fibrils formed by poly alanine

peptides with ThT and resilience against staining with

Congo Red were observed by Shinchuk et al. [28].

Thus, we measured fibril-induced changes of 8-anilino-

naphthalene-1-sulfonate (ANS) fluorescence, a method

G. Lodderstedt et al. Poly L-alanine length dependent fibril properties

FEBS Journal 274 (2007) 346–355 ª 2006 The Authors Journal compilation ª 2006 FEBS 347

that has been described for monitoring fibril formation,

e.g. of the NM domain of the yeast prion protein

Sup35p [29] and also for the detection of a-synucleinfibrils [30]. In fact, ANS signals showed a linear corre-

lation with the concentration of fibrils of N-(+7)Ala in

a range from 1 to 14 lm, whereas no ANS binding was

observed with the monomeric protein (data not shown).

Because ANS fluorescence measurements allowed a

more sensitive quantification of fibrils than with ThT

(data not shown), this spectroscopic detection method

was employed throughout this study.

When the kinetics of N-(+7)Ala fibril formation

were monitored in the absence of seeds, fibril forma-

tion started after a lag phase of � 10 days (Fig. 1A).

In contrast, addition of seeds resulted in an immediate

increase in ANS fluorescence as expected. As a control,

the depletion of the soluble monomeric species was fol-

lowed by RP-HPLC, a method by which the decrease

of monomeric species could be shown to be reciprocal

to the increase in ANS fluorescence (Fig. 1B). Because

we never observed amorphous aggregates with the

N-terminal fragment, we conclude that the increase in

ANS fluorescence correlates with the increase in fibril-

lar species at the expense of monomeric protein.

According to the hypothesis that seeded fibril forma-

tion in the case of the yeast prion protein Sup35p

exhibits a binding equilibrium between soluble interme-

diates and seeding molecules [31,32], an increase in

seed concentration should accelerate fibril formation.

To test this assumption, fibril formation was investi-

gated in the presence of increasing concentrations of

fragmented N-(+7)Ala fibrils acting as seeds. Clearly,

an increase in seed concentration resulted in faster

fibrillation rates (Fig. 2A). Quantification of fibrillation

rates revealed an approximately linear correlation

between seed concentration and fibril growth (Fig. 2B).

A similar dependence of fibrillation rates on seed con-

centration has been demonstrated previously with the

NM domain of Sup35p [31,33].

Induction of fibrillation by seeds was also tested using

N-WT. In the absence of seeds, an increase in ANS

fluorescence was recorded after � 30 days (Fig. 3A).

Seeding with fragmented N-WT fibrils resulted in an

immediate increase in ANS fluorescence. However, the

increase in ANS fluorescence was considerably slower

than in the case of seeded reactions with N-(+7)Ala. To

ensure that the increase in ANS fluorescence reflects

fibril growth, quantification of monomeric species of the

seeded reaction by RP-HPLC was performed. This ana-

lysis reveals that monomeric species decreased very

slowly over an incubation time of � 25 days, indicating

that induction of fibril growth of N-WT by seeds is

significantly slower than in the case of N-(+7)Ala

(Fig. 3B). Subsequent analyses using AFM confirmed

that N-WT seeds had hardly been elongated to longer

fibrils (Fig. 5C,D). In contrast, seeded samples of

N-(+7)Ala, which revealed small seeds to begin with

(data not shown), showed elongated fibrils after 30 days

of incubation (Fig. 5G,H). Possibly, in the case of

N-WT, conformational change to the fibrillar state is so

slow that most of the seeds lose their ability to act as

polymerization points for soluble N-WT.

Because slow fibril formation of N-WT induced by

N-WT seeds may also be due to less active N-WT

seeds, cross-seeding experiments were performed. As

seen upon the addition of homologous seeds, incuba-

tion of N-(+7)Ala with N-WT seeds resulted in an

incubation time (d)

0 1 2 3 4 5 6 7 8 9 100

10

20

30

40

50

60

70

80

90

100

0

2

4

6

8

10

12

14

16

18

20

22

) (A

NS

fluo

resc

ence

(A

U)

% s

olub

le p

rote

in (

)

incubation time (d)0 5 10 15 20 25 30 35 40

AN

S fl

uore

scen

ce (

AU

)

0

2

4

6

8

10

12

14

16A

B

Fig. 1. Fibril formation kinetics of N-(+7)Ala. The kinetics of fibril

formation were followed using ANS fluorescence in arbitrary units

(AU). (A) Fibril growth of N-(+7)Ala was monitored in the absence

(triangles) and presence (circles) of 0.1% seeds (w ⁄ v). For the ana-

lysis, N-(+7)Ala was incubated at 37 �C at a protein concentration

of 1 mM. (B) Quantification of the decrease in monomeric species

by RP-HPLC analysis (squares) as indicated in Experimental proce-

dures. For comparison, the concomitant increase in ANS fluores-

cence (circles) is shown.

Poly L-alanine length dependent fibril properties G. Lodderstedt et al.


increase in ANS fluorescence. The increase in the ANS

signal was reciprocal to the decrease in monomeric

species (data not shown) and depended on the num-

bers of seeds added (Fig. 4). Comparison of the fibril-

lation rates with homologous and heterologous seeds

(Table 1) indicated that fibril growth rates of cross-

seeded samples are slower by a factor of � 2 in com-

parison with experiments using homologous seeds. The

large standard deviations of the growth rates indicate

that absolute fibrillation rates have to be interpreted

cautiously. We assume that these deviations (in each

experimental set-up, the three different seed concentra-

tions originated from an identical seed preparation)

are due to the following technical difficulties: our pre-

vious experiments have indicated that the seeds quickly

lose their activity upon storage (data not shown). For

this reason, seeds had to be freshly prepared for each

test. Seed preparations showed noticeable batch incon-

sistencies that may be due to chemical modification(s)

caused by the long incubation times which were neces-

sary to obtain fibrils.

Fibrils of N-WT and N-(+7)Ala differ in

morphology and stability

CD analysis of soluble monomeric N-WT and

N-(+7)Ala showed that N-(+7)Ala contains more

a-helical secondary structures than N-WT [26]. Thus,

incubation time (d)

0 5 10 15 20

AN

S fl

uore

scen

ce (

AU

)

0

2

4

6

8

10

12

14

16

18A

concentration of seeds (w/v)

0.1% 0.2% 0.4%

rate

s of

fibr

il fo

rmat

ion

(d-1

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6B

Fig. 2. Fibril formation kinetics of N-(+7)Ala at different seed con-

centrations. (A) N-(+7)Ala (1 mM) was incubated at 37 �C with

0.1% (circles), 0.2% (squares) and 0.4% (triangles) seeds (w ⁄ v).

The increase in ANS fluorescence was fitted to a first-order reac-

tion to determine the rates of fibril formation. (B) Rates of fibril for-

mation (per day) as a function of the seed concentration. Error bars

represent variations from two to four separate experiments invol-

ving separate seed preparations.

incubation time (d)0 10 20 30 40 50 60 70 80 90 100

AN

S fl

uore

scen

ce (

AU

)

0

2

4

6

8

10

12

14A


0

1020

30

4050

6070

80

90100

0246810121416182022

AN

S fl

uore

scen

ce (

AU

) (

)

% s

olub

le p

rote

in (

)

B

Fig. 3. Fibril formation kinetics of N-WT. (A) N-WT was incubated

at a protein concentration of 1 mM in the absence (triangles) and

presence (circles) of 0.1% seeds (w ⁄ v). (B) Loss of monomeric

species (squares) in the seeded sample was monitored by

RP-HPLC analysis. For comparison, the concomitant increase in

ANS fluorescence (circles) is shown.



in the as yet unfibrillized form, N-WT and N-(+7)Ala

already display different structural features. Possibly,

these differences at the structural level are reflected in

the different capacities of the two variants to fibrillize

upon seeding. In order to analyze whether structural

differences between N-WT and N-(+7)Ala could also

be detected in the fibrillar forms, fibrils of both vari-

ants were visualized using electron microscopy (EM)

and AFM. Both microscopy techniques revealed fibril

diameters of � 6 nm. Although no differences in fibril

morphology could be detected using EM (Fig. 5A,E),

AFM analysis showed a more pronounced fine struc-

ture in N-WT fibrils than in N-(+7)Ala fibrils

(Figs 5B,F and 6). N-WT fibrils resembled a string of

beads not observed for N-(+7)Ala fibrils. The distan-

ces between the beads ranged from 27 to 43 nm

(Fig. 6). These defined substructures could not be

observed with N-(+7)Ala fibrils. We assume that the

different surface morphologies reflect divergent struc-

tural arrangements of the b-strands inside the fibril.

Whether the bead-like structure is caused by twist

repeats or similar substructures, as reported for the

SH3 domain of phosphatidyl inositol-3¢-kinase and

lysozyme, remains to be clarified [34]. The fact that

beaded fibril morphologies of N-(+7)Ala, which was

seeded with fragmented N-WT fibrils, were not fre-

quently observed (Fig. 5H) may indicate that the seed

structure does not ‘mold’ newly associating molecules

into the existing conformation as has been postulated

for Sup35p [35,36].

Because the different structures detected using AFM

may correlate with different stabilities against solubili-

zation, fibrils of N-WT and N-(+7)Ala were incubated

at increasing concentrations of guanidinium thiocya-

nate, the only denaturing agent identified by us that

can lead to a partial solubilization of the fibrils. First,

solubilization was performed at room temperature

for 6 h. Release of soluble species was quantified by

RP-HPLC and is indicated as the percentage of the

material that had been previously fibrillar (Fig. 7A).

With both variants, 40–50% of the fibrillar protein

was converted into soluble species. Although conver-

sion was not complete, a higher amount of N-WT than

of N-(+7)Ala fibrils was solubilized by guanidinium

thiocyanate concentrations between 3 and 5 m. When

solubilization was performed under more stringent

conditions (16 h at 50 �C), maximal conversion to

monomeric species was obtained with N-WT fibrils

at 1 m guanidinium thiocyanate, whereas those of

N-(+7)Ala required 4 m guanidinium thiocyanate

(data not shown).

In order to detect a possible equilibrium between

fibrils and soluble monomeric species under solubiliz-

ing conditions, the remaining undissolved fibrils were

reincubated with 6 m guanidinium thiocyanate for

24 h. However, no more protein could be dissolved

upon this second incubation ruling out equilibrium

conditions (data not shown). It is currently unclear

whether the remaining guanidinium thiocyanate-resist-

ant material reflects fibrillar core structures that cannot

at all be solubilized or whether heterogeneous fibrils

display differences in resistance against solubilization.

When the kinetics of solubilization were monitored

in the presence of 6 m guanidinium thiocyanate, the

maximal yield of soluble N-WT was obtained after

1 h, whereas for maximal solubilization of N-(+7)Ala

an incubation period of >20 h was required (Fig. 7B).

Clearly, these results indicate differences in stability

between N-WT and N-(+7)Ala fibrils. Distinct solubi-

lization properties in cell culture, depending on the

length of the poly l-alanine sequence, have recently

been reported for PABPN1 fusion proteins [22]. In

general, the results of the solubilization experiments

Table 1. Rates of N-(+7)Ala fibril formation upon addition of seeds.

Fibrillation in the presence of different concentrations of N-(+7)Ala

seeds (seeding) and N-WT seeds (cross-seeding). Rate constants

were calculated by assuming a first order reaction. Standard devia-

tions are based on three independent experiments.

Concentration

of seeds

(w ⁄ v)

Growth rates

with homologous seeds

(d)1)

with heterologous

seeds

(d)1)

0.1% 0.4 (± 0.15) 0.2 (± 0.02)

0.2% 0.7 (± 0.25) 0.3 (± 0.02)

0.4% 1.1 (± 0.29) 0.6 (± 0.11)


AN

S fl

uore

scen

ce (

AU

)

0

5

10

15

20

Fig. 4. Cross-seeding of N-(+7)Ala. N-(+7)Ala (1 mM) was incubated

at 37 �C with 0.1% (circles) or 0.2% (squares) seeds (w ⁄ v). Seeds

were derived from fibrils of N-WT.



underscore the unusually high resistance of the fibrils

against denaturation, which may represent a common

feature of fibrils containing poly l-alanine sequences.

Other well-known examples for extreme mechano-

chemical robustness are spider silks which contain

numerous interspersed poly l-alanine repeats [37,38].

Discussion

Previous experiments have shown that fibril formation

of N-(+7)Ala started after a shorter lag phase than

that of N-WT [26]. This finding can be interpreted as

a higher propensity of the N-(+7)Ala variant to adopt

the b-cross state. Fibrils of N-WT and N-(+7)Ala also

differ with respect to the fibril growth rates. In the

case of N-WT, the conversion to the fibrillar confor-

mation is presumably very slow under the applied

conditions. This assumption, and the fact that seeds

lose their activity upon incubation, may be responsible

for the only moderate acceleration of fibril formation

of N-WT by seeds. Consequently, an increase in the

protein concentration and ⁄or temperature which is

known to reduce the lag phases of fibril formation [26]

may render N-WT fibril growth rates more susceptible

to seeds.

Differences between N-WT and N-(+7)Ala were

also observed at the level of fibril morphology. A likely

interpretation is that the number of alanines deter-

mines the arrangement of the b-strands leading to vari-

ations in the surface structures as well as fibril

stabilities. This assumption would be in good agree-

ment with recent findings by Kirschner and co-workers

who observed poly l-alanine length-dependent differ-

ences in the diffraction patterns in fibrillized peptides

A E

B F

C G

D H

Fig. 5. Visualization of fibrils using EM (A, E) and AFM (B–D, F–H). (A–D), N-WT fibrils; (E,F), N-(+7)Ala fibrils. Fibrils were derived from sam-

ples in which 1 mM soluble protein had been either incubated in the absence of seeds (A, B, E, F) or in the presence of 0.1% seeds (w ⁄ v)

(C, D, G, H) at 37 �C; N-WT (A–D) was incubated for 60 days, N-(+7)Ala (E–H) for 30 days. N-WT incubated with N-WT seeds (C), N-(+7)Ala

seeds (D), N-(+7)Ala with N-(+7)Ala seeds (G) and N-WT seeds (H). Magnification of EM: 50 000, insets: zoom with a 50 000 magnification.

The scale bars represent 250 nm; insets in (A) and (E): scale bars ¼ 50 nm.



[28]. Furthermore, given that even the incubation tem-

perature can influence the conformation and stability

of the Sup-NM domain [35,36], different fibril struc-

tures caused by additional alanines are likely. The

manner in which the number of alanines determines

the atomic structure of the fibril and the b-strandarrangements remains to be determined.

Yet, knowledge of the structure and biophysical

analysis of the fibrils alone will not suffice for

understanding the disease causing mechanism, and

in vitro analyses have to be complemented by cell

biological investigations. From a recent analysis of

PABPN1 toxicity in Drosophila, the authors conclu-

ded that OPMD symptoms such as nuclear inclu-

sions do not result from adverse effects of the

poly l-alanine sequence [39]. Rather, the RNA-bind-

ing function of the protein was suggested to evoke

muscle defects and nuclear inclusions. This conclu-

sion was based on investigations employing mutants

in which the RNA-binding domain of PABPN1 had

been either deleted or inactivated by point muta-

tions. The absence of an intact RNA-binding domain

should, however, significantly reduce local concentra-

tions of PABPN1 close to poly(A) tails. Because

fibril formation, like other aggregation processes, is

known to be a concentration-dependent reaction, the

absence of nuclear inclusions in the case of these

mutants may simply be due to the fact that crit-

ical threshold concentrations of PABPN1 for fibril

formation will not be reached at poly(A) tails. The

argument that high local concentrations of PABPN1

facilitate deposit formation is supported by an earlier

study showing that the ability of PABPN1 to form

oligomers is crucial for the formation of intranuclear

inclusions [20].

Fig. 6. Analysis of AFM images to determine substructure widths.

Longitudinal section of N-WT and N-(+7)Ala fibrils obtained using

Nanoscope Section Analysis.

guanidinium thiocyanate (M)0 1 2 3 4 5 6

% p

rote

in d

isso

lved

0

10

20

30

40

50

60A

incubation time (h)0 10 20 30 40 50

solu

biliz

ed fr

actio

n

0

20

40

60

80

100B

Fig. 7. Stability of fibrils against solubilization with guanidinium thio-

cyanate. (A) Solubilization at the indicated guanidinium thiocyanate

concentrations was performed at room temperature for 6 h. (B) Kin-

etics of conversion to monomeric species. Solubilized material after

the various incubation times is shown as percentage of the max-

imal amount of solubilized material. The increase in soluble protein

was monitored by RP-HPLC analysis. Error bars result from two to

three independent experiments. N-WT, triangles; N-(+7)Ala, circles.



However, an alternative scenario for disease devel-

opment can be envisaged: continuous proteolytic cyto-

solic turn-over may be required to keep nuclear levels

of PABPN1 low. This degradation may be reduced

by the presence of an extended poly l-alanine tract.

Evidence for reduced proteasomal degradation by

glycine–alanine repeats due to impaired substrate

unfolding has been reported recently [40]. Further-

more, imbalance in a-synuclein levels due to muta-

tions, overexpression or inefficient proteasomal

removal of a-synuclein has been proposed to induce

fibril formation and thus a-synucleinopathy [41–43].

The observations that the nuclear inclusions found in

OPMD patients colocalize with ubiquitin and the 20S

proteasomal subunit [16,44] and the findings that pro-

teasome inhibitors increase poly l-alanine-induced

cytotoxicity in cell culture [17] would be in accordance

with this disease-causing cascade.

Experimental procedures

Recombinant protein production and purification

Recombinant constructs have been described previously

[26]. The N-terminal fragments of wild-type (N-WT)

and the variant containing seven additional alanine residues

[N-(+7)Ala] were expressed as fusions with N-terminal

His-tags using the T7 vector system (pET15b) from Nov-

agen (Madison, WI). We showed previously that the His-

tag has no influence on fibril formation [26]. As host cells,

the Escherichia coli strain BL21(DE3)Gold with the vector

pUBS520 was employed to circumvent codon usage prob-

lems. Culture conditions were described previously [26] with

the exception that bacteria were grown in fermentors of 8 L

culture volumes with 5% yeast extract (Roth, Karlsruhe,

Germany). Feeding with yeast extract was started at

D600 ¼ 10. Cells were induced with 1 mm isopropyl thio-

b-d-galactoside at D600 ¼ 20 and harvested 3 h after induc-

tion. Biomass was stored at )80 �C. Per gram biomass,

10 mL disruption buffer (50 mm Tris pH 8, 5 mm EDTA,

1 mm phenylmethylsulfonyl fluoride) was added. All subse-

quent steps were carried out as described previously [26],

with the exception that elution from the Q Sepharose was

achieved by 300 mm NaCl. Relevant fractions were pooled,

diluted 1 : 1 with 8 m guanidinium hydrochloride (NiGU

Chemie, Waldkraiburg, Germany) and loaded on a

Ni-NTA column (His Bind Resin, Novagen, Darmstadt,

Germany) equilibrated with 4 m guanidinium hydrochlo-

ride, 50 mm Tris, pH 8.0. The column was washed with

buffer containing 20 mm imidazol, 4 m guanidinium hydro-

chloride, 50 mm Tris, pH 8.0. Protein was eluted by a linear

imidazol gradient from 20 to 250 mm within 12 column vol-

umes. Relevant fractions were selected, pooled and further

purified by gel filtration as published [26]. Purified protein

was dialyzed against water, lyophillized and stored at

)80 �C.

Fibril formation and fibril analysis by

ANS fluorescence

Lyophilized protein was dissolved in 5 mm KH2PO4

pH 7.5, 150 mm NaCl, 1% (w ⁄ v) NaN3 to a final concen-

tration of 1 mm and then incubated at 37 �C for fibril for-

mation to occur. To determine ANS fluorescence, samples

were briefly mixed and then diluted to a final concentration

of 5 lm with 50 lm ANS in 5 mm KH2PO4, pH 7.5,

150 mm NaCl. Fluorescence spectra were recorded at an

emission wavelength of 480 nm upon excitation at 370 nm

in a Jobin Yvon Spex Fluoromax 2 at 20 �C. Experiments

were performed in 1 cm cuvettes with excitation and emis-

sion slits widths of 5 nm.

Seed preparation

Fibrils were formed by incubation of N-(+7)Ala at a con-

centration of 1 mm for 30 days and N-WT at a concentra-

tion of 2 mm for 100 days. When fibril formation was

completed as monitored by maximal ANS fluorescence

signals, fibrils without further storage were harvested by

centrifugation for 1 h at 260 000 g (OptimaTM TLX

ultracentrifuge), washed with 5 mm KH2PO4, pH 7.5,

150 mm NaCl, and subjected to pulsed ultrasonification

(3 · 20 s) · 5 using UP200S with an amplitude of 50%.

Seeds were added immediately after preparation to protein

solutions.

Quantification of soluble (monomeric) protein

during fibril formation by RP-HPLC

Samples were diluted 1 : 25 with water to a final protein

concentration of 0.5 mgÆmL)1 and centrifuged for 1 h at

260 000 g. The supernatant was loaded on a 1.6 mL

EC125 ⁄ 4 Nucleosil 100-5 C18 column (Machery-Nagel) pre-

equilibrated with 0.05% trifluoroacetic acid in water.

Protein was eluted by an acetonitrile gradient from 0 to

100% with a flow rate of 0.7 mLÆmin)1 over 90 min. Pro-

tein concentrations were determined by integration of peak

areas after calibration with soluble (monomeric) reference

protein.

EM and AFM

For EM analysis, carbonized copper grids (Plano, Wetzlar,

Germany) were pretreated for 1 min with bacitracin

(0.1 mgÆmL)1). After air drying, protein that had been dilu-

ted with water to final concentrations of 0.5 mgÆmL)1 was

applied for 3 min. Subsequently, grids were again air dried.

Protein (fibrils) was negatively stained with 1% (w ⁄ v)



uranyl-acetate and visualized in a Zeiss EM 900 electron

microscope operating at 80 kV. For AFM, samples were

diluted in water to final concentrations of 1.5 mgÆmL)1 and

placed on freshly cleaved mica attached to AFM sample

disks (Ted Pella). After 3 min of adsorption at 25 �C, diskswere rinsed three times with Millipore filtered distilled

water. The samples were then allowed to air dry. Tapping

mode imaging was performed on a multimode scanning

probe microscope (Veeco, Santa Barbara, CA) by using

n+-silicon probes (type NCH-50, Nanosensors, Neuchatel,

Switzerland). Fibril heights and subunit widths were deter-

mined using the nanoscope analysis software.

Chemical stability of fibrils

The chemical stability of fibrils was tested after the fibrilla-

tion process was completed (no further rise of ANS sig-

nals). The sample containing fibrils was split into seven

aliquots that were centrifuged for 1 h at 70 000 r.p.m.

(OptimaTM TLX ultracentrifuge, Beckman, Fullerton,

CA) and washed with 5 mm KH2PO4, pH 7.5, 150 mm

NaCl. Fibrils were resuspended in the same buffer contain-

ing different concentrations of guanidinium thiocyanate.

Samples were incubated under shaking (600 r.p.m.) for

different intervals at room temperature or at 50 �C. Sup-

ernatants obtained after centrifugation were analyzed by

RP-HPLC.

Acknowledgements

We thank Elmar Wahle, Christian Lange, Huma

Yonus, Daniel Huster and David Ferrari for critical

comments on the manuscript. This work was funded

by the Deutsche Forschungsgemeinschaft through

Sonderforschungsbereich 610, subproject A9 (ES) and

Sonderforschungsbereich 596, subproject B14 (TS).

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effect of oculopharyngeal muscular dystrophy-associated extension of seven alanines on the...

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