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Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A
Biochemical and Biophysical Research Communications 416 (2011) 130–134
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A Dong Xi a,b,1, Xiao Dong a,1, Wei Deng a, Luhua Lai a,b,⇑ a b
BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Center for Theoretical Biology, Peking University, Beijing 100871, China
a r t i c l e
i n f o
Article history: Received 24 October 2011 Available online 11 November 2011 Keywords: Amyloid fibril formation Inhibition Mj HSP16.5 Small heat shock protein FRET Electron microscopy
a b s t r a c t Small heat shock proteins, a class of molecular chaperones, are reported to inhibit amyloid fibril formation in vitro, while the mechanism of inhibition remains unknown. In the present study, we investigated the mechanism by which Mj HSP16.5 inhibits amyloid fibril formation of a small peptide (SSTSAA) from RNase A. A model peptide (dansyl-SSTSAA-W) was designed by introducing a pair of fluorescence resonance energy transfer (FRET) probes into the peptide, allowing for the monitoring of fibril formation by this experimental model. Mj HSP16.5 completely inhibited fibril formation of the model peptide at a molar ratio of 1:120. The dynamic process of fibril formation, revealed by FRET, circular dichroism, and electron microscopy, showed a lag phase of about 2 h followed by a fast growth period. The effect of Mj HSP16.5 on amyloid fibril formation was investigated by adding it into the incubation solution during different growth phases. Adding Mj HSP16.5 to the incubating peptide before or during the lag phase completely inhibited fibril formation. However, introducing Mj HSP16.5 after the lag phase only slowed down the fibril formation process by adhering to the already formed fibrils. These findings provide insight into the inhibitory roles of small heat shock proteins on amyloid fibril formation at the molecular level. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Amyloid deposition is found in the amyloid diseases such as Alzheimer’s disease and Type II diabetes. Small heat shock proteins (sHSPs), a class of molecular chaperons, suppress aggregation of misfolded proteins, acting as a protective mechanism in cells under stress conditions. Diverse members of the sHSP family are reported to inhibit fibril formation in vitro [1–10]. In the present study, we used Mj HSP16.5, a sHSP from Methanococcus jannaschii [11], to study the effect of small heat shock proteins on the growth of amyloid fibrils by an amyloidogenic peptide, SSTSAA, derived from RNase A. Both the crystal structure of Mj HSP16.5 [11] and the microcrystal formed by the peptide SSTSAA [14] have been solved. The chaperone activity of Mj HSP16.5 has been reported for various substrates in vitro [11–13]. Mj HSP16.5 exists only as a 24mer, with a characteristic hollow, spherical shape evident under EM [12]. The Abbreviations: FRET, fluorescence resonance energy transfer; CD, circular dichroism; EM, electron microscopy; sHSPs, small heat shock proteins; TTR, transthyretin; ESI-MS, electrospray ionization mass spectrometry; PBS, phosphate buffer saline. ⇑ Corresponding author at: BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China. Fax: +86 10 62751725. E-mail address: [email protected] (L. Lai). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.11.010
synthesized peptide SSTSAA is an amyloidogenic segment derived from RNase A, and it forms amyloid fibrils itself. X-ray crystallography studies examining the microcrystals formed by this peptide aggregate reveal a cross-b spine structure, with the formation of a steric zipper between two mating b-sheets, which has been suggested to be a common structural feature shared by amyloid fibrils at the molecular level [14]. Amyloid fibril formation of small peptides, from monomer into the mature fibril is a multi-stage process, during which different kinds of intermediates form, such as the oligomers, the b-pleated sheet, the steric zipper formed by two mating b-sheets, and the protofibrils [14]. Fragments of the proteins Ab(1–40) and Ab(1– 42), which are the primary component of senile plaques in Alzheimer’s disease, experience a similar stepwise process on the way to aggregation from monomer to oligomer to protofibril into mature fibrils [15,16]. A slow lag phase is commonly observed in this process, during which no fibrils form and the slow nucleation process is underway. After the lag phase, the fast elongation phase of fibrils begins, and the protofibrils are further twisted and cross-linked to form mature fibrils during the late stage [14]. The main aim of this study was to determine at which stage of fibril formation sHSPs play an inhibitory role. Diverse methods, such as surface plasmon resonance [16], Congo red binding and turbidity [8], sedimentation equilibrium and velocity [4], quartz crystal microbalance, and NMR [17] have been used by other groups to study this problem in vitro. However, much remains to
D. Xi et al. / Biochemical and Biophysical Research Communications 416 (2011) 130–134
be done in order to fully understand the inhibitory mechanism by using different systems and methods. Previously, we developed a fluorescence resonance energy transfer (FRET)-based method to detect the fibril formation process by attaching a dansyl group at the N-terminal and a tryptophan residue at the C-terminal of the studied peptide [18]. This method can be used to follow the dynamic process of amyloid fibril growth simply by using a fluorometer. We have used it to distinguish different cross-b spine arrangements in fibril structures [19]. In the present study, we used the same method and attached a dansyl group to the N-terminal and a tryptophan residue to the C-terminal of the SSTSAA peptide. Then we used FRET, electron microscopy (EM), and circular dichroism (CD) to follow the fibril formation process. We investigated the inhibition effect of Mj HSP16.5 on amyloid fibril formation by adding Mj HSP16.5 to the amyloid fibril incubation solution during different phases of growth, exploiting both FRET detection and EM sample analysis. 2. Material and methods The pET21a plasmid containing the Mj sHSP16.5 gene was a generous gift from Professor Sung-Hou Kim at the University of California, Berkeley. The rink resin and the amino acid with Fmoc protection were from GL Biochem (Shanghai) Ltd. (Shanghai, China). Water was of Millipore quality. All other chemicals were of analytical grade or higher. The expression and purification of Mj HSP16.5 was performed as previously described [20]. The concentration of Mj HSP16.5 was determined by absorbance at 280 nm using a Lambda 45 UV/Vis spectrometer (Perkin–Elmer, Waltham, MA) with an extinction coefficient of 0.565 mg 1 cm2. 2.1. Model peptide synthesis The amyloidogenic peptide SSTSAA from RNase A was labeled with a dansyl group at the N-terminal and a tryptophan residue at the C-terminal. The solid-based synthesis of the dual-labeled peptide was performed with the same procedures as in our previous study [18]. The purity and the validity of the synthesized peptide were determined by electrospray ionization mass spectrometry. 2.2. Amyloid fibril incubation The lyophilized peptide dansyl-SSTSAA-W was dissolved in Millipore water by sonication for 5 s, and then filtered with a filter membrane (0.22 lm, Millipore, Billerica, MA) for a homogeneous sample. The start of the incubation was the time when the peptide dissolved. To ensure reproducibility among different experiments, the concentration of the model peptide was determined by absorbance at 280 nm using a Lambda 45 UV/Vis spectrometer (Perkin– Elmer, Waltham, MA). The dissolved peptide was then mixed with 2 PBS (100 mM PBS, pH 7.3, 300 mM NaCl) in equal volume to make a final incubation buffer of physiological PBS (50 mM PBS, pH 7.3, 150 mM NaCl). The final concentration of the peptide for incubation was about 150 lM with an absorbance of 0.15 at 280 nm. To study the inhibitory mechanism of Mj HSP16.5 on the dynamic process of fibril formation, the peptide solution was mixed with Mj HSP16.5 at a final molar ratio of 1:120 (Mj HSP16.5 to dansyl-SSTSAA-W) before and after different incubation times at 25 °C. 2.3. CD spectra CD spectra were recorded for the model peptide on a Jobin Yvon CD6 at 25 °C, with 1-mm pathlength cylinder quartz cuvettes for
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far UV. The concentration of the peptide was about 150 lM, the same as the incubation condition. The buffer condition was 20 mM PBS buffer (pH 7.3), which differed from the incubation condition only in salt concentration. CD spectra were recorded for the samples after different incubation times to follow the dynamic process of fibril formation. 2.4. Fluorescence spectra Amyloid fibril formation was monitored by the change of the fluorescence emission spectrum of the reaction mixture before and after different incubation times in physiological PBS buffer at room temperature. The emission spectrum from 340 to 570 nm with the excitation wavelength of 295 nm was scanned by a FluoroLog 3 Spectrofluorometer (HORIBA Jobin Yvon Inc., Longjumeau, France). The reaction mixture was diluted to 1/50 for the fluorescence spectrum measurement. The spectra shown in the results were all for the model peptide alone. The Mj HSP16.5 spectrum was subtracted from the spectrum when it was present in the mixture. 2.5. EM study Samples were directly removed from the incubation vessels for the EM study. A 6-ll sample was transferred to a 300-mesh carbon-coated grid and 4 min were allowed for absorption. The grid was washed with three drops of water, then stained with 1% phosphotungstic acid (pH 6.8) for 4 min, washed again with three drops of water, and then blotted dry with filter paper. Samples were viewed on a transmission electron microscope (JEM-100cx; JEOL, Tokyo, Japan). Ten photographs at different locations on the same grid for each sample were taken and the diameters of at least 100 fibrils were measured for each sample to provide the statistics of fibril diameter. 3. Results and discussion 3.1. The dynamic process of fibril formation monitored by FRET, EM and CD The fibril formation process of the model peptide dansylSSTSAA-W was detected by changes in the fluorescence spectrum of the incubated peptide. A strong FRET signal was seen after 6 h of incubation with an increase of the peak at 487 nm corresponding to the emission of the dansyl group, while the peak at 360 nm, corresponding to the emission of the tryptophan, decreased (Fig. 1A). From the crystal structure, we know that the SSTSAA forms a steric zipper structure of Class 1, sharing a basic unit of two parallel, crosslinked b-sheets with their chains facing each other. Its fibril structure has the following features: (1) the distance between the two mating sheets is 10 Å; (2) the distance between the two neighboring strands in the same sheet is 4.8 Å; (3) the length of the extended peptide is approximately 26 Å [14]. As the Förster distance of the dansyl–tryptophan FRET pair is 21 Å, the FRET signal is triggered between the tryptophan group from a strand of one sheet and the dansyl group from another strand of the neighboring sheet, as suggested in our previous study [19]. Here, we used the change in FRET signal to follow the formation of the steric zipper structure by two mating b-sheets in the process of fibril formation. This has been suggested to be a critical step in the slow fibril nucleation process [14]. As can be seen from Fig. 1A, the fluorescence signal did not change much in the first 3 h but began to change markedly after 3 h of incubation. Samples of the model peptide at different incubation times, monitored by the fluorescent emission spectra, were also analyzed
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sponding to the disordered structure) continuously decreased during the whole process of incubation. A new local minimum emerged around 216 nm after 2 h of incubation, and this spectra at 216 nm was typical (corresponding to the b structure) after 6 h of incubation (Fig. 1C). This indicates a continuous change in the secondary structure of the model peptide from a disordered structure to the b-strand structure during the whole process of fibril formation, with the b structure emerging after 2 h of incubation. We used FRET, CD, and EM to study the dynamic process of fibril formation of the model peptide. FRET detection showed a lag phase of about 3 h before the mating of the neighboring sheets in the steric zipper structure, whereas the EM results showed that fibrils were evident in samples after incubation for 3 h, suggesting a lag phase less than 3 h. The inconsistency between these results might be due to the fact that the samples used for the fluorescence measurements taken after 3 h of incubation were a mixture of monomers, oligomers, and newly-formed fibrils, and the amount of fibrils was rather small relative to the monomer. Therefore, we consider the lag phase to have lasted for about 2 h. The lag phase detected was sensitive to the state of the lyophilized peptide and to the shaking conditions in the experiment, so it differed slightly between batches. However, all the results shown in this paper were from the same batch of lyophilized peptide under the same shaking conditions, and the differences between batches did not affect the final qualitative conclusions of the inhibitory mechanism. 3.2. Mj HSP16.5 inhibited amyloid fibril formation at a molar ratio of 1:120 The model peptide was mixed with Mj HSP16.5 at different molar ratios before incubation. The fluorescence signal of the peptide did not change after 1 day of incubation at the molar ratio of 1:120 (Mj HSP16.5 to the model peptide, Fig. 2A). This mixture was also analyzed by EM. No fibrils were seen, and Mj HSP16.5 alone was observed (Fig. 2B). All these findings demonstrate that Mj HSP16.5 inhibits fibril formation by the model peptide. The inhibitory activity of Mj HSP16.5 was concentration-dependent. A molar ratio less than 1:120 was not enough to completely suppress the fibril formation process, but did slow it down (data not shown). In this study, we show that Mj HSP16.5 completely suppresses the amyloid fibril formation of a model peptide, dansyl-SSTSAA-W, at a molar ratio of 1:120 (Mj HSP16.5 to dansyl-SSTSAA-W). The model peptide has the potential to be used as another substrate to measure the in vitro chaperon activity of Mj HSP16.5 in physiological PBS buffer at room temperature, in addition to the conventional substrate, insulin [13]. 3.3. Mj HSP16.5 influenced the dynamic process of fibril formation
Fig. 1. The dynamic process of fibril formation monitored by FRET, EM, and CD. (A) Fluorescence emission spectra of the model peptide sampled at different incubation times. (B) Diameter distribution of amyloid fibrils of the model peptide sampled after 3–6 h of incubation. X-axis: fibril diameters measured from electron micrographs. Y-axis: percentage of the number of fibrils of each diameter to the total number of fibrils in each sample. (C) Far UV CD spectra of the model peptide sampled at different incubation times(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
by EM to check for fibril formation. Amyloid fibrils were found in all samples incubated for 3–6 h, whereas no fibrils were found in the samples incubated for 1 or 2 h. The distribution of fibril diameters tended to be thicker after longer incubation (Fig. 1B). The far UV CD spectra of the samples at different incubation times showed that the negative trough around 200 nm (corre-
We have shown that fibril formation of the model peptide can be completely suppressed when incubating with Mj HSP16.5 from the beginning and at a molar ratio of 1:120 (Mj HSP16.5 to dansylSSTSAA-W). Next, we tested how the same ratio of Mj HSP16.5 might influence the fibril formation process if introduced at varying time points during the incubation process. When Mj HSP16.5 was mixed with the peptide sample before fibrils can be detected by EM (1 and 2 h of incubation), the fluorescence spectra remained the same even after 6 h of incubation, indicating that it can completely suppress fibril formation. However, when Mj HSP16.5 was mixed with the peptide samples incubated for at least 3 h, noticeable FRET signal was observed and there was a clear tendency of the FRET signal to increase along with the waiting time for introducing Mj HSP16.5 (Fig. 3A). The results also show that the addition of Mj HSP16.5 after 3 h of incubation slowed down the fibril
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Fig. 2. Inhibitory activity of Mj HSP16.5 on fibril formation of the model peptide shown by FRET and EM. (A) Fluorescence emission spectra of the model peptide (150 lM) before and after incubation with Mj HSP16.5 (1.25 lM) for 24 h at room temperature. (B) Electron micrograph of the sample in A showing Mj HSP16.5 alone and no fibrils.
formation process, as suggested by the weaker FRET signal compared to the sample without Mj HSP16.5 addition. We show evidence that the earlier Mj HSP16.5 was added, the weaker the final FRET signal was (Fig. 3A). We then monitored fibril formation by EM. After introducing Mj HSP16.5 at different incubation times, the mixtures were further incubated at room temperature for 3 days to allow the fibrils to mature and to be analyzed by EM. Amyloid fibrils were detected in the samples with Mj HSP16.5 addition after 3, 4, and 5 h of incubation, while no fibrils were seen in the samples with Mj HSP16.5 addition after 1 and 2 h of incubation. The statistical distribution of fibril diameters measured by electron micrographs showed a tendency to shift from thinner fibrils to thicker ones the longer the introduction of Mj HSP16.5 was delayed (Fig. 3B). Notably, Mj HSP16.5 was seen to accumulate and adhere to the formed fibrils (Fig. 4). In the present study, we used a model system to investigate the molecular mechanisms underlying the inhibition of the dynamic process of fibril formation. Taken together, both FRET and EM results suggest that the addition of Mj HSP16.5 during the first 2 h completely suppresses fibril formation, which equates to the lag phase of this process as detected by EM. This implies that Mj HSP16.5 completely inhibits fibril formation by interfering with the nucleation process. According to the FRET and EM results, once the fibrils formed after the lag phase, Mj HSP16.5 failed to completely suppress subsequent fibril formation, but it did slow this process down. Samples with Mj HSP16.5 added after the lag phase showed that thinner fibrils formed compared to that formed with
Fig. 3. Influence of Mj HSP16.5 on the dynamic process of fibril formation monitored by FRET and EM. (A) Fluorescence spectra of the model peptide mixed with Mj HSP16.5 after different incubation times. The spectra were all taken at 6 h after the beginning of the incubation. The fluorescence spectra of the model peptide alone, before, and after incubation for 6 h are also shown for comparison. (B) Diameter distribution of amyloid fibrils formed by the model peptide with the addition of Mj HSP16.5 after different incubation times. X-axis: fibril diameters from electron micrographs. Y-axis: percentage of the number of the fibrils of each diameter to the total number of fibrils in each sample(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Fig. 4. Electron micrograph of a sample with Mj HSP16.5 addition after a 5-h incubation of the model peptide alone. Mj HSP16.5 molecules that accumulated and adhered to the fibrils are indicated by black arrows.
the peptide alone. The mature fibrils were, on average, thinner in samples to which Mj HSP16.5 was added earlier in the incubation process. This was similar to the statistical distribution of fibril
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diameters formed by the peptide alone, where thinner fibrils occupied a larger proportion during the earlier stages of fibril formation. A similar phenomenon was previously reported showing that Mj HSP16.5 significantly inhibits the growth and maturation of WTTR fibrils, resulting in much thinner fibrils compared to those under normal conditions [9]. Taken together, these results suggest that Mj HSP16.5 slows down fibril growth by protecting the thin fibrils and preventing them from further cross-linking and forming thicker fibrils. For the first time, we show electron micrographs directly illustrating that sHSPs accumulate and adhere to the fibrils. This is further evidence of the mechanism that Mj HSP16.5 adheres to the fibrils and prevents further cross-linking. Similarly, it has been shown that alpha B-crystallin interacts with and inhibits alpha Syn amyloid fibril formation by binding along the length of mature amyloid fibrils using an antibody against alpha B-crystallin [17]. In conclusion, we studied the inhibition activity of Mj HSP16.5 on the amyloid fibril formation of a model peptide, dansylSSTSAA-W, and found that Mj HSP16.5 interferes with fibril nucleation during the lag phase to prevent fibril formation and adheres to the fibrils that have already formed to prevent further growth and cross-linking between them. Authors contribution Dong Xi, Wei Deng and Luhua Lai conceived the project; Dong Xi designed the experiments; Xiao Dong and Dong Xi carried out the experiments and analyzed the data; Dong Xi, Xiao Dong, Wei Deng and Luhua Lai wrote the paper.
[4]
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[13]
[14]
[15]
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Acknowledgments This work was supported in part by the National Natural Science Foundation of China and the Ministry of Science and Technology of China.
[17]
[18]
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Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A Dong Xi a,b,1, Xiao Dong a,1, Wei Deng a, Luhua Lai a,b,⇑ a b
BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Center for Theoretical Biology, Peking University, Beijing 100871, China
a r t i c l e
i n f o
Article history: Received 24 October 2011 Available online 11 November 2011 Keywords: Amyloid fibril formation Inhibition Mj HSP16.5 Small heat shock protein FRET Electron microscopy
a b s t r a c t Small heat shock proteins, a class of molecular chaperones, are reported to inhibit amyloid fibril formation in vitro, while the mechanism of inhibition remains unknown. In the present study, we investigated the mechanism by which Mj HSP16.5 inhibits amyloid fibril formation of a small peptide (SSTSAA) from RNase A. A model peptide (dansyl-SSTSAA-W) was designed by introducing a pair of fluorescence resonance energy transfer (FRET) probes into the peptide, allowing for the monitoring of fibril formation by this experimental model. Mj HSP16.5 completely inhibited fibril formation of the model peptide at a molar ratio of 1:120. The dynamic process of fibril formation, revealed by FRET, circular dichroism, and electron microscopy, showed a lag phase of about 2 h followed by a fast growth period. The effect of Mj HSP16.5 on amyloid fibril formation was investigated by adding it into the incubation solution during different growth phases. Adding Mj HSP16.5 to the incubating peptide before or during the lag phase completely inhibited fibril formation. However, introducing Mj HSP16.5 after the lag phase only slowed down the fibril formation process by adhering to the already formed fibrils. These findings provide insight into the inhibitory roles of small heat shock proteins on amyloid fibril formation at the molecular level. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Amyloid deposition is found in the amyloid diseases such as Alzheimer’s disease and Type II diabetes. Small heat shock proteins (sHSPs), a class of molecular chaperons, suppress aggregation of misfolded proteins, acting as a protective mechanism in cells under stress conditions. Diverse members of the sHSP family are reported to inhibit fibril formation in vitro [1–10]. In the present study, we used Mj HSP16.5, a sHSP from Methanococcus jannaschii [11], to study the effect of small heat shock proteins on the growth of amyloid fibrils by an amyloidogenic peptide, SSTSAA, derived from RNase A. Both the crystal structure of Mj HSP16.5 [11] and the microcrystal formed by the peptide SSTSAA [14] have been solved. The chaperone activity of Mj HSP16.5 has been reported for various substrates in vitro [11–13]. Mj HSP16.5 exists only as a 24mer, with a characteristic hollow, spherical shape evident under EM [12]. The Abbreviations: FRET, fluorescence resonance energy transfer; CD, circular dichroism; EM, electron microscopy; sHSPs, small heat shock proteins; TTR, transthyretin; ESI-MS, electrospray ionization mass spectrometry; PBS, phosphate buffer saline. ⇑ Corresponding author at: BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China. Fax: +86 10 62751725. E-mail address: [email protected] (L. Lai). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.11.010
synthesized peptide SSTSAA is an amyloidogenic segment derived from RNase A, and it forms amyloid fibrils itself. X-ray crystallography studies examining the microcrystals formed by this peptide aggregate reveal a cross-b spine structure, with the formation of a steric zipper between two mating b-sheets, which has been suggested to be a common structural feature shared by amyloid fibrils at the molecular level [14]. Amyloid fibril formation of small peptides, from monomer into the mature fibril is a multi-stage process, during which different kinds of intermediates form, such as the oligomers, the b-pleated sheet, the steric zipper formed by two mating b-sheets, and the protofibrils [14]. Fragments of the proteins Ab(1–40) and Ab(1– 42), which are the primary component of senile plaques in Alzheimer’s disease, experience a similar stepwise process on the way to aggregation from monomer to oligomer to protofibril into mature fibrils [15,16]. A slow lag phase is commonly observed in this process, during which no fibrils form and the slow nucleation process is underway. After the lag phase, the fast elongation phase of fibrils begins, and the protofibrils are further twisted and cross-linked to form mature fibrils during the late stage [14]. The main aim of this study was to determine at which stage of fibril formation sHSPs play an inhibitory role. Diverse methods, such as surface plasmon resonance [16], Congo red binding and turbidity [8], sedimentation equilibrium and velocity [4], quartz crystal microbalance, and NMR [17] have been used by other groups to study this problem in vitro. However, much remains to
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be done in order to fully understand the inhibitory mechanism by using different systems and methods. Previously, we developed a fluorescence resonance energy transfer (FRET)-based method to detect the fibril formation process by attaching a dansyl group at the N-terminal and a tryptophan residue at the C-terminal of the studied peptide [18]. This method can be used to follow the dynamic process of amyloid fibril growth simply by using a fluorometer. We have used it to distinguish different cross-b spine arrangements in fibril structures [19]. In the present study, we used the same method and attached a dansyl group to the N-terminal and a tryptophan residue to the C-terminal of the SSTSAA peptide. Then we used FRET, electron microscopy (EM), and circular dichroism (CD) to follow the fibril formation process. We investigated the inhibition effect of Mj HSP16.5 on amyloid fibril formation by adding Mj HSP16.5 to the amyloid fibril incubation solution during different phases of growth, exploiting both FRET detection and EM sample analysis. 2. Material and methods The pET21a plasmid containing the Mj sHSP16.5 gene was a generous gift from Professor Sung-Hou Kim at the University of California, Berkeley. The rink resin and the amino acid with Fmoc protection were from GL Biochem (Shanghai) Ltd. (Shanghai, China). Water was of Millipore quality. All other chemicals were of analytical grade or higher. The expression and purification of Mj HSP16.5 was performed as previously described [20]. The concentration of Mj HSP16.5 was determined by absorbance at 280 nm using a Lambda 45 UV/Vis spectrometer (Perkin–Elmer, Waltham, MA) with an extinction coefficient of 0.565 mg 1 cm2. 2.1. Model peptide synthesis The amyloidogenic peptide SSTSAA from RNase A was labeled with a dansyl group at the N-terminal and a tryptophan residue at the C-terminal. The solid-based synthesis of the dual-labeled peptide was performed with the same procedures as in our previous study [18]. The purity and the validity of the synthesized peptide were determined by electrospray ionization mass spectrometry. 2.2. Amyloid fibril incubation The lyophilized peptide dansyl-SSTSAA-W was dissolved in Millipore water by sonication for 5 s, and then filtered with a filter membrane (0.22 lm, Millipore, Billerica, MA) for a homogeneous sample. The start of the incubation was the time when the peptide dissolved. To ensure reproducibility among different experiments, the concentration of the model peptide was determined by absorbance at 280 nm using a Lambda 45 UV/Vis spectrometer (Perkin– Elmer, Waltham, MA). The dissolved peptide was then mixed with 2 PBS (100 mM PBS, pH 7.3, 300 mM NaCl) in equal volume to make a final incubation buffer of physiological PBS (50 mM PBS, pH 7.3, 150 mM NaCl). The final concentration of the peptide for incubation was about 150 lM with an absorbance of 0.15 at 280 nm. To study the inhibitory mechanism of Mj HSP16.5 on the dynamic process of fibril formation, the peptide solution was mixed with Mj HSP16.5 at a final molar ratio of 1:120 (Mj HSP16.5 to dansyl-SSTSAA-W) before and after different incubation times at 25 °C. 2.3. CD spectra CD spectra were recorded for the model peptide on a Jobin Yvon CD6 at 25 °C, with 1-mm pathlength cylinder quartz cuvettes for
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far UV. The concentration of the peptide was about 150 lM, the same as the incubation condition. The buffer condition was 20 mM PBS buffer (pH 7.3), which differed from the incubation condition only in salt concentration. CD spectra were recorded for the samples after different incubation times to follow the dynamic process of fibril formation. 2.4. Fluorescence spectra Amyloid fibril formation was monitored by the change of the fluorescence emission spectrum of the reaction mixture before and after different incubation times in physiological PBS buffer at room temperature. The emission spectrum from 340 to 570 nm with the excitation wavelength of 295 nm was scanned by a FluoroLog 3 Spectrofluorometer (HORIBA Jobin Yvon Inc., Longjumeau, France). The reaction mixture was diluted to 1/50 for the fluorescence spectrum measurement. The spectra shown in the results were all for the model peptide alone. The Mj HSP16.5 spectrum was subtracted from the spectrum when it was present in the mixture. 2.5. EM study Samples were directly removed from the incubation vessels for the EM study. A 6-ll sample was transferred to a 300-mesh carbon-coated grid and 4 min were allowed for absorption. The grid was washed with three drops of water, then stained with 1% phosphotungstic acid (pH 6.8) for 4 min, washed again with three drops of water, and then blotted dry with filter paper. Samples were viewed on a transmission electron microscope (JEM-100cx; JEOL, Tokyo, Japan). Ten photographs at different locations on the same grid for each sample were taken and the diameters of at least 100 fibrils were measured for each sample to provide the statistics of fibril diameter. 3. Results and discussion 3.1. The dynamic process of fibril formation monitored by FRET, EM and CD The fibril formation process of the model peptide dansylSSTSAA-W was detected by changes in the fluorescence spectrum of the incubated peptide. A strong FRET signal was seen after 6 h of incubation with an increase of the peak at 487 nm corresponding to the emission of the dansyl group, while the peak at 360 nm, corresponding to the emission of the tryptophan, decreased (Fig. 1A). From the crystal structure, we know that the SSTSAA forms a steric zipper structure of Class 1, sharing a basic unit of two parallel, crosslinked b-sheets with their chains facing each other. Its fibril structure has the following features: (1) the distance between the two mating sheets is 10 Å; (2) the distance between the two neighboring strands in the same sheet is 4.8 Å; (3) the length of the extended peptide is approximately 26 Å [14]. As the Förster distance of the dansyl–tryptophan FRET pair is 21 Å, the FRET signal is triggered between the tryptophan group from a strand of one sheet and the dansyl group from another strand of the neighboring sheet, as suggested in our previous study [19]. Here, we used the change in FRET signal to follow the formation of the steric zipper structure by two mating b-sheets in the process of fibril formation. This has been suggested to be a critical step in the slow fibril nucleation process [14]. As can be seen from Fig. 1A, the fluorescence signal did not change much in the first 3 h but began to change markedly after 3 h of incubation. Samples of the model peptide at different incubation times, monitored by the fluorescent emission spectra, were also analyzed
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sponding to the disordered structure) continuously decreased during the whole process of incubation. A new local minimum emerged around 216 nm after 2 h of incubation, and this spectra at 216 nm was typical (corresponding to the b structure) after 6 h of incubation (Fig. 1C). This indicates a continuous change in the secondary structure of the model peptide from a disordered structure to the b-strand structure during the whole process of fibril formation, with the b structure emerging after 2 h of incubation. We used FRET, CD, and EM to study the dynamic process of fibril formation of the model peptide. FRET detection showed a lag phase of about 3 h before the mating of the neighboring sheets in the steric zipper structure, whereas the EM results showed that fibrils were evident in samples after incubation for 3 h, suggesting a lag phase less than 3 h. The inconsistency between these results might be due to the fact that the samples used for the fluorescence measurements taken after 3 h of incubation were a mixture of monomers, oligomers, and newly-formed fibrils, and the amount of fibrils was rather small relative to the monomer. Therefore, we consider the lag phase to have lasted for about 2 h. The lag phase detected was sensitive to the state of the lyophilized peptide and to the shaking conditions in the experiment, so it differed slightly between batches. However, all the results shown in this paper were from the same batch of lyophilized peptide under the same shaking conditions, and the differences between batches did not affect the final qualitative conclusions of the inhibitory mechanism. 3.2. Mj HSP16.5 inhibited amyloid fibril formation at a molar ratio of 1:120 The model peptide was mixed with Mj HSP16.5 at different molar ratios before incubation. The fluorescence signal of the peptide did not change after 1 day of incubation at the molar ratio of 1:120 (Mj HSP16.5 to the model peptide, Fig. 2A). This mixture was also analyzed by EM. No fibrils were seen, and Mj HSP16.5 alone was observed (Fig. 2B). All these findings demonstrate that Mj HSP16.5 inhibits fibril formation by the model peptide. The inhibitory activity of Mj HSP16.5 was concentration-dependent. A molar ratio less than 1:120 was not enough to completely suppress the fibril formation process, but did slow it down (data not shown). In this study, we show that Mj HSP16.5 completely suppresses the amyloid fibril formation of a model peptide, dansyl-SSTSAA-W, at a molar ratio of 1:120 (Mj HSP16.5 to dansyl-SSTSAA-W). The model peptide has the potential to be used as another substrate to measure the in vitro chaperon activity of Mj HSP16.5 in physiological PBS buffer at room temperature, in addition to the conventional substrate, insulin [13]. 3.3. Mj HSP16.5 influenced the dynamic process of fibril formation
Fig. 1. The dynamic process of fibril formation monitored by FRET, EM, and CD. (A) Fluorescence emission spectra of the model peptide sampled at different incubation times. (B) Diameter distribution of amyloid fibrils of the model peptide sampled after 3–6 h of incubation. X-axis: fibril diameters measured from electron micrographs. Y-axis: percentage of the number of fibrils of each diameter to the total number of fibrils in each sample. (C) Far UV CD spectra of the model peptide sampled at different incubation times(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
by EM to check for fibril formation. Amyloid fibrils were found in all samples incubated for 3–6 h, whereas no fibrils were found in the samples incubated for 1 or 2 h. The distribution of fibril diameters tended to be thicker after longer incubation (Fig. 1B). The far UV CD spectra of the samples at different incubation times showed that the negative trough around 200 nm (corre-
We have shown that fibril formation of the model peptide can be completely suppressed when incubating with Mj HSP16.5 from the beginning and at a molar ratio of 1:120 (Mj HSP16.5 to dansylSSTSAA-W). Next, we tested how the same ratio of Mj HSP16.5 might influence the fibril formation process if introduced at varying time points during the incubation process. When Mj HSP16.5 was mixed with the peptide sample before fibrils can be detected by EM (1 and 2 h of incubation), the fluorescence spectra remained the same even after 6 h of incubation, indicating that it can completely suppress fibril formation. However, when Mj HSP16.5 was mixed with the peptide samples incubated for at least 3 h, noticeable FRET signal was observed and there was a clear tendency of the FRET signal to increase along with the waiting time for introducing Mj HSP16.5 (Fig. 3A). The results also show that the addition of Mj HSP16.5 after 3 h of incubation slowed down the fibril
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Fig. 2. Inhibitory activity of Mj HSP16.5 on fibril formation of the model peptide shown by FRET and EM. (A) Fluorescence emission spectra of the model peptide (150 lM) before and after incubation with Mj HSP16.5 (1.25 lM) for 24 h at room temperature. (B) Electron micrograph of the sample in A showing Mj HSP16.5 alone and no fibrils.
formation process, as suggested by the weaker FRET signal compared to the sample without Mj HSP16.5 addition. We show evidence that the earlier Mj HSP16.5 was added, the weaker the final FRET signal was (Fig. 3A). We then monitored fibril formation by EM. After introducing Mj HSP16.5 at different incubation times, the mixtures were further incubated at room temperature for 3 days to allow the fibrils to mature and to be analyzed by EM. Amyloid fibrils were detected in the samples with Mj HSP16.5 addition after 3, 4, and 5 h of incubation, while no fibrils were seen in the samples with Mj HSP16.5 addition after 1 and 2 h of incubation. The statistical distribution of fibril diameters measured by electron micrographs showed a tendency to shift from thinner fibrils to thicker ones the longer the introduction of Mj HSP16.5 was delayed (Fig. 3B). Notably, Mj HSP16.5 was seen to accumulate and adhere to the formed fibrils (Fig. 4). In the present study, we used a model system to investigate the molecular mechanisms underlying the inhibition of the dynamic process of fibril formation. Taken together, both FRET and EM results suggest that the addition of Mj HSP16.5 during the first 2 h completely suppresses fibril formation, which equates to the lag phase of this process as detected by EM. This implies that Mj HSP16.5 completely inhibits fibril formation by interfering with the nucleation process. According to the FRET and EM results, once the fibrils formed after the lag phase, Mj HSP16.5 failed to completely suppress subsequent fibril formation, but it did slow this process down. Samples with Mj HSP16.5 added after the lag phase showed that thinner fibrils formed compared to that formed with
Fig. 3. Influence of Mj HSP16.5 on the dynamic process of fibril formation monitored by FRET and EM. (A) Fluorescence spectra of the model peptide mixed with Mj HSP16.5 after different incubation times. The spectra were all taken at 6 h after the beginning of the incubation. The fluorescence spectra of the model peptide alone, before, and after incubation for 6 h are also shown for comparison. (B) Diameter distribution of amyloid fibrils formed by the model peptide with the addition of Mj HSP16.5 after different incubation times. X-axis: fibril diameters from electron micrographs. Y-axis: percentage of the number of the fibrils of each diameter to the total number of fibrils in each sample(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Fig. 4. Electron micrograph of a sample with Mj HSP16.5 addition after a 5-h incubation of the model peptide alone. Mj HSP16.5 molecules that accumulated and adhered to the fibrils are indicated by black arrows.
the peptide alone. The mature fibrils were, on average, thinner in samples to which Mj HSP16.5 was added earlier in the incubation process. This was similar to the statistical distribution of fibril
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diameters formed by the peptide alone, where thinner fibrils occupied a larger proportion during the earlier stages of fibril formation. A similar phenomenon was previously reported showing that Mj HSP16.5 significantly inhibits the growth and maturation of WTTR fibrils, resulting in much thinner fibrils compared to those under normal conditions [9]. Taken together, these results suggest that Mj HSP16.5 slows down fibril growth by protecting the thin fibrils and preventing them from further cross-linking and forming thicker fibrils. For the first time, we show electron micrographs directly illustrating that sHSPs accumulate and adhere to the fibrils. This is further evidence of the mechanism that Mj HSP16.5 adheres to the fibrils and prevents further cross-linking. Similarly, it has been shown that alpha B-crystallin interacts with and inhibits alpha Syn amyloid fibril formation by binding along the length of mature amyloid fibrils using an antibody against alpha B-crystallin [17]. In conclusion, we studied the inhibition activity of Mj HSP16.5 on the amyloid fibril formation of a model peptide, dansylSSTSAA-W, and found that Mj HSP16.5 interferes with fibril nucleation during the lag phase to prevent fibril formation and adheres to the fibrils that have already formed to prevent further growth and cross-linking between them. Authors contribution Dong Xi, Wei Deng and Luhua Lai conceived the project; Dong Xi designed the experiments; Xiao Dong and Dong Xi carried out the experiments and analyzed the data; Dong Xi, Xiao Dong, Wei Deng and Luhua Lai wrote the paper.
[4]
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[8]
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[13]
[14]
[15]
[16]
Acknowledgments This work was supported in part by the National Natural Science Foundation of China and the Ministry of Science and Technology of China.
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