Interruption of CryAB-Amyloid Oligomer Formation by HSP22

Atsushi Sanbe,Mayu Murabe,Akito Tanoue,Yoko Fujiwara,Junji Yamauchi,Yuki Miyamoto

doi:10.1074/jbc.m605481200

Atsushi Sanbe, Mayu Murabe + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m605481200

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Abstract

An R120G missense mutation in alpha-B-crystallin (CryAB), a small heat-shock protein (HSP), causes a desmin-related cardiomyopathy (DRM) that is characterized by the formation of aggregates containing CryAB and desmin. The mutant CryAB protein leads to the formation of inclusion bodies, which contain amyloid oligomer intermediates (amyloid oligomer) in the cardiomyocytes. To further address the underlying mechanism(s) of amyloid oligomer formation in DRM linked to the CryAB R120G, a recombinant CryAB R120G protein was generated. The purified CryAB R120G protein can form a toxic amyloid oligomer, whereas little immunoreactivity was observed in the wild-type CryAB protein. A native PAGE showed that the oligomerized form was present in the CryAB R120G protein, whereas only a high molecular mass was detected in the wildtype CryAB. The oligomerized CryAB R120G of around 240-480 kDa showed strong positive immunoreactivity against an anti-oligomer antibody. The CryAB R120G amyloid oligomer was unstable and easily lost its conformation by beta-mercaptoethanol and SDS. Recombinant HSP25 or HSP22 proteins can directly interrupt oligomer formation by the CryAB R120G protein, whereas the amyloid oligomer is still present in the mixture of the wild-type CryAB and CryAB R120G proteins. This interruption by HSP25 and HSP22 was confirmed in a cardiomyocyte-based study using an adenoviral transfection system. Blockade of amyloid oligomer formation by HSP25 and HSP22 recovered the ubiquitin proteosomal activity and cellular viability. Blockade of oligomer formation by small HSP may be a new therapeutic strategy for treating DRM as well as other types of amyloid-based degenerative diseases.

Highlights

Unfolded proteins in the cells can contribute to or be causal for at least some of these neurodegenerative and systemic diseases [2]
Using a cardiac-specific inducible transgenic system [16], we found that continuous expression of the mutant protein is needed to sustain the high concentrations of the amyloid oligomer that correlate with depressed cardiac function as well as
We showed that the R120G missense mutation in the CryAB protein that is a cause of desmin-related cardiomyopathy (DRM) led to perinuclear aggresome formation and that these aggresomes contained the amyloid oligomer [15]

Summary

The abbreviations used are

A␤, amyloid ␤; amyloid oligomer, positive immunoreactive material against the anti-oligomer antibody; R120G, missense mutation from arginine to glycine at the 120th residue of ␣-␤-crystallin; polyglutamine, expanded polyglutamine tract of n residues; native PAGE, PAGE under a native condition; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; m.o.i., multiplicity of infection; DTT, dithiothreitol; PVDF, polyvinylidene difluoride; HA, hemagglutinin; UPS, ubiquitin-proteosome system; GFP, green fluorescent protein; HSP, heatshock protein; DRM, desmin-related cardiomyopathy; WT, wild type; TBS, Tris-buffered saline. CryAB R120G Oligomer and HSP22 premature lethality [17] These results indicate that the CryABDRM disease is reversible and that the amyloid oligomer is critical for the DRM disease [17]. Potential commonalities due to the presence of the amyloid oligomer may exist in cardiovascular diseases [15] This suggests that the reduction and/or prevention of toxic amyloid oligomer formation may represent a new therapeutic approach for the DRM disease as well as other amyloid-related diseases. To address this therapeutic approach for the DRM disease, it will be important to analyze the molecular mechanisms of amyloid oligomer formation by the CryAB R120G protein. Blockade of oligomer formation by small HSPs, such as HSP22 and HSP25, may be a new therapeutic strategy for treating the DRM disease as well as other types of amyloid-based degenerative diseases

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION