Flanking sequences profoundly alter polyglutamine toxicity in yeast

Abstract

Protein misfolding is the molecular basis for several human diseases. How the primary amino acid sequence triggers misfolding and determines the benign or toxic character of the misfolded protein remains largely obscure. Among proteins that misfold, polyglutamine (polyQ) expansion proteins provide an interesting case: Each causes a distinct neurodegenerative disease that selectively affects different neurons. However, all are broadly expressed and most become toxic when the glutamine expansion exceeds approximately 39 glutamine residues. The disease-causing polyQ expansion proteins differ profoundly in the amino acids flanking the polyQ region. We therefore hypothesized that these flanking sequences influence the specific toxic character of each polyQ expansion protein. Using a yeast model, we find that sequences flanking the polyQ region of human huntingtin exon I can convert a benign protein to a toxic species and vice versa. Further, we observe that flanking sequences can direct polyQ misfolding to at least two morphologically distinct types of polyQ aggregates. Very tight aggregates always are benign, whereas amorphous aggregates can be toxic. We thereby establish a previously undescribed systematic characterization of the influence of flanking amino acid sequences on polyQ toxicity.

Fig. 1.

A nontoxic huntingtin exon I construct and construct with graded polyQ length-dependent toxicity. (A) Yeast cells containing huntingtin constructs I (Upper) or II (Lower) with the indicated number polyQ repeats were spotted on plates that either induce (+Induction) or repress (−Induction) huntingtin exon I expression. Four serial 5-fold dilutions of cells are shown. Toxicity is reflected by the reduced growth of cells under inducing conditions. (B) Dot blots prepared with protein extracts from yeast cells expressing the indicated huntingtin constructs 8 h after induction. Four serial dilutions of the protein lysates are shown. Huntingtin constructs were detected with an anti-GFP antibody. (C) Yeast cells containing construct II with an increasing number of polyQ repeats were spotted on plates for either weak expression for constructs under control of the MET promoter (Center) or strong induction for constructs under the control of the GAL1 promoter (Right). (Left) A schematic representation of the huntingtin exon I proteins. The red insert in the polyQ region indicates the 16-aa-long amino-terminal sequence of human huntingtin.

Fig. 2.

PolyQ toxicity depends on amino acids flanking the polyQ region. Yeast cells containing huntingtin constructs II–VII with either 25 or 103 polyQ repeats (except construct III with 104 repeats) were spotted on plates that either induce (+Induction) or repress (−Induction) huntingtin exon I expression (see Materials and Methods for details). Four serial 5-fold dilutions of cells are shown.

Fig. 3.

Flanking amino acids modulate polyQ aggregate morphology. Cells expressing the indicated huntingtin exon I CFP or GFP fusions proteins (constructs I–VII) were analyzed by fluorescence microscopy. (Scale bar: 5 μm.) Below the micrographs, filter retardation assays of protein lysates of cells expressing the indicated huntingtin exon I are shown. Three 5-fold dilutions of the protein extracts are shown.