The role of the glycerol transporter channel Fps1p in cellular proteostasis during enhanced proteotoxic stress.

In response to osmotic shock, the components of high-osmolarity glycerol (HOG) pathway regulate the level of intracellular glycerol in yeast and ensure cell survival. Glycerol is a compatible solute and a stabiliser of proteins. Its role in maintaining proteostasis is less explored. We show that mild stress in the form of dietary restriction leads to increased glycerol level which increases cell viability. However, dietary restriction coupled with protein aggregation decreases intracellular glycerol level and attenuates cell viability. The transcript level of FPS1, the glycerol transporter channel, remains unchanged. However, its activity is altered under enhanced proteotoxic stress. Our results provide evidence for a probable role of the Fps1p channel in the cellular proteostasis network. KEY POINTS: • Dietary restriction led to increased accumulation of glycerol in Fps1-deleted yeast cells. • This led to lower protein aggregation in these cells. • Increased production of glycerol under dietary restriction was not linked to increased level of Fps1.

Stabilization of elongated polyglutamine tracts by a helical peptide derived from N-terminal huntingtin.

In Huntington's disease, the length of the polyglutamine tract in the mutant protein correlates positively with the formation of aggregates and disease symptoms and severity of the disease. Some disease-modifying factors exist. However, no organized study has been carried out to investigate the effect of polyglutamine length in the mutant protein on the efficacy of a therapeutic strategy. We had shown earlier that the helical peptide arising out of the N-terminal stretch of normal huntingtin is able to inhibit aggregation of a number of proteins, including luciferase, α-synuclein, p53, and Rnq1. In this work, we show that polyglutamine stretches of differing lengths, namely 51Q, 72Q, and 103Q, form a mixture of aggregates at different rates, with the rate increasing in a polyQ length-dependent manner. The helical peptide is able to inhibit the rate of aggregation. The extent of inhibition was different when measuring either total aggregation or only fibrillar aggregates, suggesting that the helical peptide with benign polyQ stretch alters the aggregation landscape of different elongated polyQ lengths differently. Our results suggest that designing a therapeutic approach to inhibit protein aggregation must take note of polyQ length of the protein.

Discrete roles of trehalose and Hsp104 in inhibition of protein aggregation in yeast cells.

Heat shock response (HSR) is an important element of cellular homeostasis. In yeast, HSR comprises of the heat shock proteins (Hsps) and the osmolytes trehalose and glycerol. The respective roles of trehalose and Hsp104 in regulating protein aggregation remain ambiguous. We report that trehalose and Hsp104 are important during the early stages of protein aggregation, i.e. when the process is still reversible. This corroborates the earlier reported role of trehalose being an inhibitor of protein folding. Under in vitro conditions, trehalose is able to restore the GdHCl-induced loss of ATPase activity of recombinant Hsp104 to almost its original level. As the saturation phase of aggregation approaches, neither of the two components is able to exert any effect. Inactivation of Hsp104 at the stage when oligomers have already been formed increases the rate of formation of aggregates by inhibiting disaggregation of oligomers. In the absence of an active disaggregase, the oligomers are converted to mature irreversible aggregates, accelerating their formation. Our results suggest that the disaccharide may have a marginally stronger influence than Hsp104 in inhibiting protein aggregation in yeast cells.

Does N-terminal huntingtin function as a 'holdase' for inhibiting cellular protein aggregation?

Proteolytic cleavage of huntingtin gives rise to N-terminal fragments. While the role of truncated mutant huntingtin is described in Huntington's disease (HD) pathogenesis, the function of N-terminal wild-type protein is less studied. The yeast model of HD is generated by the presence of FLAG tag and absence of polyproline tract as flanking sequences of the elongated polyglutamine stretch. We show that the same sequence derived from wild-type huntingtin exon1 is able to inhibit the aggregation of proteins in vitro and in yeast cells. It is able to stabilize client proteins as varied as luciferase, α-synuclein, and p53 in a soluble but non-native state. This is somewhat similar to the 'holdase' function of small heat shock proteins and 'nonchaperone proteins' which are able to stabilize partially unfolded client proteins in a nonspecific manner, slowing down their aggregation. Mutagenesis studies show this property to be localized at the N17 domain preceding the polyglutamine tract. Distortion of this ordered segment, either by deletion of this segment or mutation of a single residue (L4A), leads to decreased stability and increased aggregation of client proteins. It is interesting to note that the helical conformation of the N17 domain is also essential for aggregation of the N-terminal mutant protein. Our results provide evidence for a novel function for the amphipathic helix derived from exon1 of wild-type huntingtin.

Nicotine slows down oligomerisation of α-synuclein and ameliorates cytotoxicity in a yeast model of Parkinson's disease.

Several retrospective epidemiological reports have indicated an inverse correlation between smoking and development of Parkinson's disease (PD). This has mostly been attributed to the neuroprotective role of nicotine in stimulating nicotinic acetylcholine receptors and dopaminergic neurons which are damaged in PD. One of the characteristic features of PD is the intraneuronal deposition of globular inclusions of the intrinsically disordered protein α-synuclein as Lewy bodies. Using in vitro and the well-validated yeast cell models, we show that nicotine also exerts a beneficial effect on aggregation of α-synuclein. The alkaloid increases the lag time of the nucleation step and reduces the build-up of the more toxic oligomeric species in a concentration-dependent manner. This results in lower oxidative stress in the cell, reduced cytotoxicity and increased cell survival. Structural studies using CD spectroscopy and fluorescence quenching showed that α-synuclein forms a transient complex with nicotine, distorting its native structure and altering its aggregation landscape such that the formation of oligomers is inhibited. As soluble oligomers are believed to modulate the mechanism of PD pathogenesis mainly by formation of pores in neuronal membranes, resulting in leaching of vital components of the cytoplasm with deleterious effects for the cell, our results provide a mechanistic rationale for the observed beneficial role of nicotine on the progression of the disease.

Challenges with osmolytes as inhibitors of protein aggregation: Can nucleic acid aptamers provide an answer?

Protein aggregation follows some common motifs. Whether in the formation of inclusion bodies in heterologous overexpression systems or inclusions in protein conformational diseases, or aggregation during storage or transport of protein formulations, aggregates form cross beta-sheet structures and stain with amyloidophilic dyes like Thioflavin T and Congo Red, irrespective of the concerned protein. Traditionally, osmolytes are used to stabilize proteins against stress conditions. They are employed right from protein expression, through production and purification, to formulation and administration. As osmolytes interact with the solvent, the differential effect of the stress condition on the solvent mostly determines the effect of the osmolyte on protein stability. Nucleic acid aptamers, on the other hand, are highly specific for their targets. When selected against monomeric, natively folded proteins, they bind to them with very high affinity. This binding inhibits the unfolding of the protein and/or monomer-monomer interaction which are the initial common steps of protein aggregation. Thus, by changing the approach to a protein-centric model, aptamers are able to function as universal stabilizers of proteins. The review discusses cases where osmolytes were unable to provide stabilization to proteins against different stress conditions, a gap which the aptamers seem to be able to fill.

Cellular toxicity of yeast prion protein Rnq1 can be modulated by N-terminal wild type huntingtin.

Aggregation of the N-terminal human mutant huntingtin and the consequent toxicity in the yeast model of Huntington's disease (HD) requires the presence of Rnq1 protein (Rnq1p) in its prion conformation [RNQ1(+)]. The understanding of interaction of wild-type huntingtin (wt-Htt) with the amyloidogenic prion has some gaps. In this work, we show that N-terminal fragment of wt-Htt (N-wt-Htt) ameliorated the toxic effect of [RNQ1(+)] depending on expression levels of both proteins. When the expression of N-wt-Htt was high, it increased the expression and delayed the aggregation of [RNQ1(+)]. As the expression of N-wt-Htt was reduced, it formed high molecular weight aggregates along with the prion. Even when sequestered by [RNQ1(+)], the beneficial effect of N-wt-Htt on expression of Rnq1p and on cell survival was evident. Huntingtin protein ameliorated toxicity due to the prion protein [RNQ1(+)] in yeast cells in a dose-dependent manner, resulting in increase in cell survival, hinting at its probable role as a component of the proteostasis network of the cell. Taking into account the earlier reports of the beneficial effect of expression of N-wt-Htt on the aggregation of mutant huntingtin, the function of wild-type huntingtin as an inhibitor of protein aggregation in the cell needs to be explored.