Abstract
The formation of aggregates by polyglutamine-containing (polyQ) proteins in neurons is a key to the pathogenesis of several progressive neurodegenerative diseases such as Huntington’s disease (HD) spinocerebellar ataxias (SCAs), and spinal and bulbar muscular atrophy (SBMA). In order to study whether the members of the heat shock protein (HSP) families, by virtue of their molecular chaperone activity, can inhibit the formation of polyQ aggregates, we developed a cell culture model expressing the GFP tagged fragment of exon1 of the huntingtin gene with an expanded polyQ chain and tetracycline inducible chaperones. Expression of mutated Huntington’s protein leads to the formation of 2% SDS insoluble high molecular weight polyQ aggregates that are retarded on a cellulose acetate membrane in the so-called filter trap assay (FTA). This chapter explains in detail the protocols of the FTA and how it can be a useful tool to study the effect of HSPs or their functional mutants on aggregation of polyglutamine proteins. Moreover, the assay is useful to investigate how externally added polyQ peptides can act as nucleation seeds for internally expressed polyQ proteins.
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Acknowledgments
This work was supported by a grant from Senter Novem (IOP-IGE07004) and a Stimulation Grant from the Nederlandse Hersenstichting (project 15F07(2)-58) awarded to H.H.K. The authors wish to thank Dr. M.A. Rujano and Dr. J. Hageman for their contribution to the introduction of the Filter Trap Assay in our lab. The corrections and suggestions after careful reading of the manuscript by E. Preusser de Mattos are very much appreciated.
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van Waarde-Verhagen, M.A.W.H., Kampinga, H.H. (2018). Measurement of Chaperone-Mediated Effects on Polyglutamine Protein Aggregation by the Filter Trap Assay. In: Calderwood, S., Prince, T. (eds) Chaperones. Methods in Molecular Biology, vol 1709. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7477-1_5
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DOI: https://doi.org/10.1007/978-1-4939-7477-1_5
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