Ataxin-1 fusion partners alter polyQ lethality and aggregation.

Abstract

Intranuclear inclusion bodies (IBs) are the histopathologic markers of multiple protein folding diseases. IB formation has been extensively studied using fluorescent fusion products of pathogenic polyglutamine (polyQ) expressing proteins. These studies have been informative in determining the cellular targets of expanded polyQ protein as well as the methods by which cells rid themselves of IBs. The experimental thrust has been to intervene in the process of polyQ aggregation in an attempt to alleviate cytotoxicity. However new data argues against the notion that polyQ aggregation and cytotoxicity are inextricably linked processes. We reasoned that changing the protein context of a disease causing polyQ protein could accelerate its precipitation as an IB, potentially reducing its cytotoxicity. Our experimental strategy simply exploited the fact that conjoined proteins influence each others folding and aggregation properties. We fused a full-length pathogenic ataxin-1 construct to fluorescent tags (GFP and DsRed1-E5) that exist at different oligomeric states. The spectral properties of the DsRed1-E5-ataxin-1 transfectants had the additional advantage of allowing us to correlate fluorochrome maturation with cytotoxicity. Each fusion protein expressed a distinct cytotoxicity and IB morphology. Flow cytometric analyses of transfectants expressing the greatest fluorescent signals revealed that the DsRed1-E5-ataxin-1 fusion was more toxic than GFP fused ataxin-1 (31.8+/-4.5% cell death versus 12.85+/-3%), although co-transfection with the GFP fusion inhibited maturation of the DsRed1-E5 fluorochrome and diminished the toxicity of the DsRed1-E5-ataxin-1 fusion. These data show that polyQ driven aggregation can be influenced by fusion partners to generate species with different toxic properties and provide new opportunities to study IB aggregation, maturation and lethality.