Research Article: Cu/Zn-superoxide dismutase forms fibrillar hydrogels in a pH-dependent manner via a water-rich extended intermediate state

Date Published: October 5, 2018

Publisher: Public Library of Science

Author(s): Noriko Fujiwara, Michiru Wagatsuma, Naoto Oba, Daisaku Yoshihara, Eiichi Tokuda, Haruhiko Sakiyama, Hironobu Eguchi, Motoko Ichihashi, Yoshiaki Furukawa, Tadashi Inoue, Keiichiro Suzuki, Esmaiel Jabbari.


Under certain conditions, amyloid-like fibrils can develop into three-dimensional networks and form hydrogels by a self-assembly process. When Cu/Zn superoxide dismutase (SOD1), an anti-oxidative enzyme, undergoes misfolding, fibrillar aggregates are formed, which are a hallmark of a certain form of familial amyotrophic lateral sclerosis (ALS). However, the issue of whether SOD1 fibrils can be assembled into hydrogels remains to be tested. Here, we show that the SOD1 polypeptides undergo hydrogelation accompanied by the formation of thioflavin T-positive fibrils at pH 3.0 and 4.0, but not at pH 5.0 where precipitates are formed. The results of viscoelastic analyses indicate that the properties of SOD1 hydrogels (2%) were similar to and slightly more fragile than a 0.25% agarose gel. In addition, monitoring by a quartz crystal microbalance with admittance analysis showed that the denaturing of immobilized SOD1 on a sensor under the hydrogelation conditions at pH 3.0 and 4.0 resulted in an increase in the effective acoustic thickness from ~3.3 nm (a folded rigid form) to ~50 and ~100 nm (an extended water-rich state), respectively. In contrast, when SOD1 was denatured under the same conditions at pH 5.0, a compact water-poor state with an effective acoustic thickness of ~10 nm was formed. The addition of physiological concentrations of NaCl to the pH 4.0 sample induced a further extension of the SOD1 with larger amounts of water molecules (with an effective acoustic thickness of ~200 nm) but suppressed hydrogel formation. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates and that a conformational change to a suitable level of extended water-rich intermediate state before and/or during intermolecular assembling is required for fibrillation and hydrogelation in the case of globular proteins.

Partial Text

Fibrillation is considered to be an innate property that is common to all polypeptides, and amyloid-like fibrillar aggregates have been found in a number of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, prion diseases and amyotrophic lateral sclerosis (ALS). Under certain conditions, fibrous proteins such as gelatin [1] and fibrils derived from a specific peptide motif such as elastin-like pentapeptides, VPGXG (where X is any residue but proline) [2] and Ac-C(FKFE)2CG-NH2 [3] can develop into three-dimensional networks and form hydrogels by self-assembly. Amyloidogenic peptides, such as the β-amyloid diphenylalanine [4], islet amyloid polypeptide [5], were also reported to produce viscoelastic hydrogels consisting of fibrillar meshworks. Furthermore, globular proteins, such as insulin [6], β-lactoglobulin [7, 8], bovine serum albumin (BSA) [9] and α-synuclein [10] also are known to form hydrogels. Intriguingly, fused in sarcoma (FUS), one of the proteins that causes ALS also forms fibrillar hydrogel-like assemblies that impair the function of ribonucleoprotein granules [11, 12]. Hydrogelation is, therefore, thought to be another important property that is inherently registered in polypeptides. Furthermore, it has been proposed that the primary cause of cell death in amyloid neurodegenerative diseases is the physical effect of the amyloid gels and not chemical toxicity, since amyloid gels would halt the convection essential to the transport of dissolved molecules in and out of cells [13]. Therefore, an understanding of the mechanism responsible for the formation of protein gels becomes important. However, the mechanism is still unclear as to whether the amyloid-like fibrils proceeds to hydrogelation or proceeds to aggregation.

The findings reported herein indicate, for the first time, that thioflavin T-positive fibrils of wild-type SOD1 undergoes hydrogelation at pH 3.0 (2.2)−4.0, but with precipitates being formed at pH 5.0. The QCM-A analysis showed that the immobilized SOD1 on the sensor under the hydrogelation conditions at pH 3.0−4.0 resulted in an extension accompanied by the binding of large amounts of water molecules. In contrast, at pH 5.0, the denatured SOD1 hardly bound water molecules and remained compact. On the other hand, the addition of NaCl to the pH 4.0 sample caused a further water-rich intermediate, which resulted in the formation of an aggregation like emulsion but not a hydrogel. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates, such as fibrillar hydrogels, precipitates or aggregates like emulsions. An approach using the viscoelastic properties to monitor protein unfolding and water-protein interactions would be promising in terms of developing a further understanding of protein fibrils and the development of therapeutic strategies for the treatment of neurodegenerative diseases.




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