Measurement of Chaperone-Mediated Effects on Polyglutamine Protein Aggregation by the Filter Trap Assay.

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).

Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers.

Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol.

HSPB7 is the most potent polyQ aggregation suppressor within the HSPB family of molecular chaperones.

A small number of heat-shock proteins have previously been shown to act protectively on aggregation of several proteins containing an extended polyglutamine (polyQ) stretch, which are linked to a variety of neurodegenerative diseases. A specific subfamily of heat-shock proteins is formed by the HSPB family of molecular chaperones, which comprises 10 members (HSPB1-10, also called small HSP). Several of them are known to act as anti-aggregation proteins in vitro.