Effect of oculopharyngeal muscular dystrophy-associated extension of seven alanines on the fibrillation properties of the N-terminal domain of PABPN1 Grit Lodderstedt 1 , Simone Hess 2 , Gerd Hause 3 , Till Scheuermann 1, *, Thomas Scheibel 2 and Elisabeth Schwarz 1 1 Institut fu ¨ r Biotechnologie, Martin-Luther-Universita ¨ t Halle-Wittenberg, Halle, Germany 2 Technische Universita ¨ tMu ¨ nchen, Garching, Germany 3 Biozentrum der Martin-Luther-Universita ¨ t 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 fu ¨ r Biotechnologie, Martin-Luther-Universita ¨ t 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) 10 Gly(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-synuclein fibrils [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) 012345678910 0 10 20 30 40 50 60 70 80 90 100 0 2 4 6 8 10 12 14 16 18 20 22 ) (ANS fluorescence (AU) % soluble protein ( ) incubation time (d) 0 5 10 15 20 25 30 35 40 ANS fluorescence (AU) 0 2 4 6 8 10 12 14 16 A 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 m M. (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. 348 FEBS Journal 274 (2007) 346–355 ª 2006 The Authors Journal compilation ª 2006 FEBS 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 ANS fluorescence (AU) 0 2 4 6 8 10 12 14 16 18 A concentration of seeds (w/v) 0.1% 0.2% 0.4% rates of fibril formation (d -1 ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 B Fig. 2. Fibril formation kinetics of N-(+7)Ala at different seed con- centrations. (A) N-(+7)Ala (1 m M) 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) 0102030405060708090100 ANS fluorescence (AU) 0 2 4 6 8 10 12 14 A incubation time (d) 0 5 10 15 20 25 30 35 40 45 50 0 10 20 30 40 50 60 70 80 90 100 0 2 4 6 8 10 12 14 16 18 20 22 ANS fluorescence (AU) ( ) % soluble protein ( ) B Fig. 3. Fibril formation kinetics of N-WT. (A) N-WT was incubated at a protein concentration of 1 m M 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. 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 349 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) incubation time (d) 02468101214161820 ANS fluorescence (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. Poly L-alanine length dependent fibril properties G. Lodderstedt et al. 350 FEBS Journal 274 (2007) 346–355 ª 2006 The Authors Journal compilation ª 2006 FEBS 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 AE BF CG DH 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 m M 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. 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 351 [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-strand arrangements 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) 0123456 % protein dissolved 0 10 20 30 40 50 60 A incubation time (h) 0 1020304050 solubilized fraction 0 20 40 60 80 100 B 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. Poly L-alanine length dependent fibril properties G. Lodderstedt et al. 352 FEBS Journal 274 (2007) 346–355 ª 2006 The Authors Journal compilation ª 2006 FEBS 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 D 600 ¼ 10. Cells were induced with 1 mm isopropyl thio- b-d-galactoside at D 600 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, 1mm 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 KH 2 PO 4 pH 7.5, 150 mm NaCl, 1% (w ⁄ v) NaN 3 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 KH 2 PO 4 , 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 KH 2 PO 4 , 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 C 18 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) 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 353 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, disks were 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 KH 2 PO 4 , pH 7.5, 150 mm NaCl. Fibrils were resuspended in the same buffer contain- ing different concentrations of guanidinium thiocyanate. 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