Disruption of the toxic conformation of the expanded polyglutamine stretch leads to suppression of aggregate formation and cytotoxicity

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Abstract

The polyglutamine (polyQ) diseases are a class of inherited neurodegenerative diseases including Huntington’s disease, caused by the expansion of a polyQ stretch within each disease protein. This expansion is thought to cause a conformational change in the protein leading to aggregation of the protein, resulting in cytotoxicity. To analyze whether disrupting the toxic conformation of the polyQ protein can alter its aggregation propensity and cytotoxicity, we examined the effect of interruption of the expanded polyQ stretch by proline insertion, since prolines cause great alterations in protein conformation. Here, we show that insertion of prolines into the expanded polyQ stretch indeed disrupts its ordered secondary structure, leading to suppression of polyQ protein aggregation both in vitro and in cell culture, and reduction of cytotoxicity in correlation with the number of proline interruptions. Furthermore, we found that a short polyQ stretch with a proline interruption is able to inhibit aggregation of the expanded polyQ protein in trans. These results show that a gain in toxic conformation of the expanded polyQ protein is essential for aggregation and cytotoxicity, providing insight into establishing therapies against the polyQ diseases.

Section snippets

Materials and methods

Introduction of proline coding CCGs into the CAG repeat. Proline coding CCGs were introduced into the CAG repeat by a strategy using polymerase chain reaction (PCR) mutagenesis and digestion with a type IIS restriction enzyme, as shown in Supplementary Fig. 1[16]. The pure polyQ stretches and polyQ stretches with proline interruptions (QPQ) used in this study are listed in Table 1.

Construction of plasmid vectors. Plasmid vectors for expression of polyQ stretches fused with a thioredoxin

Interruption of the expanded polyQ stretch by proline insertion disrupts its conformation

The 1C2 antibody, which specifically recognizes the unique conformation of expanded polyQ stretches, was used to determine the effect of proline insertion on the conformation of the expanded polyQ stretch (Fig. 1A). The immunoreactivities of 1C2 against thio-QPQ decreased in correlation with the number of proline interruptions compared with thio-Q57, while an anti-thioredoxin antibody showed similar immunoreactivity to all the proteins, which is consistent with a previous study showing that an

Acknowledgements

We thank Dr. Kazuhisa Nakayama for helpful discussions and Mr. Timothy Tucker for his technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (C)—Advanced Brain Science Project (to Y.N.), a Grant-in-Aid for Scientific Research on Priority Areas (A)—Life of Proteins (to Y.N.), and the 21st Century COE program (to T.T.), from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a Grant-in-Aid for the Research

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    Supplementary data associated with this article can be found, in the online version, at doi: 10.1016/j.bbrc.2004.03.161.

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    These authors contributed equally to this work.

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