Cytotoxicity of Mutant Huntingtin Fragment in Yeast Can Be Modulated by the Expression Level of Wild Type Huntingtin Fragment

Abstract

Conflicting reports exist in the literature regarding the role of wild-type huntingtin in determining the toxicity of the aggregated, mutant huntingtin in Huntington's disease (HD). Some studies report the amelioration of toxicity of the mutant protein in the presence of the wild-type protein, while others indicate sequestration of the wild-type protein by mutant huntingtin. Over the years, yeast has been established as a valid model organism to study molecular changes associated with HD, especially at the protein level. We have used an inducible system to express human huntingtin fragments harboring normal (25Q) and pathogenic (103Q) polyglutamine lengths under the control of a galactose promoter in a yeast model of HD. We show that the relative expression level of each allele (wild-type/mutant) decides the cellular phenotype. When the expression level of wild-type huntingtin is high, an increase in the solubility of the mutant protein is observed. Fluorescence-recovery-after-photobleaching (FRAP) studies show that solubility reaches ∼94% in these cells. This leads to reduction in oxidative stress and cytotoxicity, and increases cell viability. In-cell FRET studies show that interaction between these proteins does not require the presence of a mediator. When the expression of wild-type huntingtin is low, it is sequestered into aggregates by the mutant protein. Even under these conditions, cytotoxicity is attenuated. Our findings indicate that the presence of wild-type huntingtin has a beneficial role even when its relative expression level is lower than that of the mutant protein.

Does N‐terminal huntingtin function as a ‘holdase’ for inhibiting cellular protein aggregation?

“…Similar sequestration of α‐synuclein by 103Q was also seen in the case of Drosophila . This is important as the concentrations of the peptide employed in this work and earlier are much higher than the cellular concentration of mature huntingtin. Also, the N17‐25Q forms a very short stretch of the complete protein, raising doubts of the role of the excised fragment as a folding aid, if any, in a cellular milieu.…”

“…The ability of 25Q to stabilize aggregation‐prone proteins is a concentration‐dependent phenomenon. While 25Q was able to reduce the aggregation of 103Q when the expression level of the former was high, a relatively higher level of expression of 103Q led to sequestration of 25Q . Even under these conditions, the toxicity due to 103Q was attenuated.…”

“…The amplified sequence was used to replace HSF1‐HA gene sequence in pAD‐ HSF1‐HA (received from Prof. Hiroshi Takagi) digested with the same enzyme pair. Construction of pRS315‐ FLAG‐N17‐25Q‐mRFP and pRS315‐ mRFP (empty vector) has been described earlier. Other plasmids were received as gifts.…”

“…Saccharomyces cerevisiae W303 ( MAT α leu2‐3 112 trp1‐1 can1‐100 ura3‐1 ade2‐1 his3‐11,15 ) cells (transformed with pYES2‐ FLAG‐N17‐25Q‐EGFP, pYES2‐ FLAG‐N17‐25Q , and N17 mutants) were grown and the proteins were expressed as described previously using 2% galactose as an inducer (30 °C/10 h) . Protein purification was carried out using anti‐FLAG M2 affinity gel (Sigma) and monitored by SDS/PAGE, immunoblotting, and dot blot assay.…”

“…On the other hand, m‐htt was able to sequester wt‐htt into SDS‐insoluble aggregates , supporting the loss‐of‐function hypothesis proposed for HD pathogenesis. Sequestration was recently shown to be a dose‐dependent effect of wt‐htt . Expression of wt‐htt from a high copy number plasmid was able to solubilize m‐htt and improve cell function and survival while expression from a low copy number plasmid led to its sequestration by m‐htt aggregates in a yeast model.…”