Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A

Abstract

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.

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.