Biochemical and Biophysical Research Communications
Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A
Highlights
► Mechanism of small heat shock protein inhibition on fibril formation was studied. ► Peptide SSTSAA with modified ends was used for amyloid fibril formation. ► FRET signal was followed during the fibril formation. ► Mj HSP16.5 inhibits fibril formation when introduced in the lag phase. ► Mj HSP16.5 slows down fibril formation when introduced after the lag phase.
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], [2], [3], [4], [5], [6], [7], [8], [9], [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], [12], [13]. Mj HSP16.5 exists only as a 24mer, with a characteristic hollow, spherical shape evident under EM [12]. The 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-β spine structure, with the formation of a steric zipper between two mating β-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 β-pleated sheet, the steric zipper formed by two mating β-sheets, and the protofibrils [14]. Fragments of the proteins Aβ(1–40) and Aβ(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 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-β 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.
Section snippets
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
The dynamic process of fibril formation monitored by FRET, EM and CD
The fibril formation process of the model peptide dansyl-SSTSAA-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
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.
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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These authors contributed equally to this work.