Role of molecular chaperones in protein folding diseases

National Heart, Lung, And Blood Institute

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Abstract

Our research has been examining different mechanisms of curing of two canonical yeast prions, the PSI+ and URE3 prions, which are cured by perturbing the balance of molecular chaperones. Inactivation of the molecular chaperone, Hsp104, which inhibits the severing of the prion seeds, cures both prions. However, overexpression of Hsp104 cures only the PSI+ prion and the mechanism of curing is still not well understood. It is known that the N-terminal domain of Hsp104 is necessary for the curing of PSI+ by Hsp104 overexpression. Since an Hsp70 binding site has been identified in the N-terminal domain of Hsp104, we mutated this binding site. Mutating the N-terminal Hsp70 binding site of Hsp104 does not affect prion propagation or severing of the prion seeds by Hsp104. However, it does inhibit the curing of PSI+ by Hsp104 overexpression, along with inhibiting the trimming activity of Hsp104. The trimming activity of Hsp104 causes a reduction in the size of the prion seeds by removing monomers from the ends of the amyloid fibers. We previously found that trimming of the prion seeds is necessary for the curing of PSI+ by Hsp104 overexpression. Furthermore, we find that the binding of different Ssa isozymes, which are members of Hsp70, regulates the trimming activity of Hsp104 by binding to the N-terminal domain Hsp70 binding site and, in turn, the rate of PSI+ curing by Hsp104 overexpression. Therefore, the Hsp70 binding site in the N-terminal domain of Hsp104 is important for PSI+ curing by Hsp104 overexpression and may also function in the dissolution of other misfolded substrates in yeast. The N-terminal domain Hsp70 binding site has provided insight into the phenotype of yeast expressing Ssa1-21, a mutant of Ssa1, which was identified in a screen of Ssa1 mutants that cause mitotic instability of PSI+. Even though genetic screens showed a link between Ssa1-21 and the curing of PSI+ by Hsp104 overexpression, the mode of action of Ssa1-21 was not yet understood. We now find that Ssa1-21 binds to the N-terminal Hsp70 binding site on Hsp104. This causes an increase in the trimming activity of Hsp104, which produces a decrease in seed number and faster curing of PSI+ by Hsp104 overexpression. Increasing the expression of Ssa1-21 causes curing of PSI+ by endogenous levels of Hsp104. Furthermore, the latter curing is dependent on the cochaperone, Sti1, just like the curing by Hsp104 overexpression, indicating a common mechanism of curing. These results further support the role of trimming in the curing of PSI+ both by endogenous levels of Hsp104 and by overexpression of Hsp104. To further our understanding of the mechanism of PSI+ curing by Hsp104 overexpression, we examined whether curing by Hsp104 overexpression could be achieved without cell division. If curing occurs independent of cell division, this would not be consistent with a model in which curing is due to asymmetric segregation of the seeds. To determine whether this model is compatible with curing of PSI+ by Hsp104 overexpression, we have examined different conditions to achieve curing independent of cell division. We find that when cell division is arrested by ethanol, overexpression of Hsp104 cures 80% of the cells of the PSI+ prion in about one-third of a generation. In fact, ethanol, itself, causes a heat shock response, which, in turn, increases the expression of Hsp104. Therefore, these results indicate that curing of PSI+ prion by Hsp104 overexpression is independent of cell division, which is not compatible with a model of asymmetric segregation. Instead, these data, along with imaging data, support a model in which Hsp104 overexpression cures PSI+ by dissolution of the prion seeds, which is dependent on the trimming activity of Hsp104. We also have been studying the curing of URE3 by overexpression of Btn2, Hsp42, Cur1 or Ydj1. The latter proteins cause aggregation of the Ure2 seeds, which leads to curing of URE3 by asymmetric segregation of the seeds. When these proteins are overexpressed, there is upregulation of endogenous Hsp42, which forms an oligomeric aggregate in the cytosol that binds the Ure2 seeds. This suggests that Hsp42 is acting as a scaffold to bind the seeds, but it is not clear how these proteins promote aggregation, especially since the Ure2 seeds only colocalize with Btn2 and Hsp42 in the cytosol. Furthermore, the seeds do not bind directly to Hsp42 since the curing by overexpression of Hsp42 is dependent on expression of Btn2. To further understand how these proteins promote aggregation of the Ure2 seeds, we overexpressed Ydj1 or Ydj1 truncations in different URE3 deletion strains. Ydj1, a member of the Hsp40 family with a J-domain that binds Hsp70, is a cytosolic protein . The curing of URE3 by overexpression of Ydj1 is dependent on a functional J-domain, which indicates curing is due to cytosolic depletion of Hsp70. We find that compared to the wild-type strain, both an HSP42 deletion strain and a BTN2/CUR1 double deletion strain reduced the rate of aggregation of the Ure2 seeds and the rate of URE3 curing. Interestingly, there is a further reduction in rate of aggregation of Ure2 seeds and rate of URE3 curing in an HSP42/BTN2/CUR1 triple deletion strain, which shows there are two independent protein quality control pathways, the Hsp42 and the Btn2/Cur1 pathways, that promote the aggregation of the Ure2 sees and the curing of the URE3 prion. Another property of prion proteins is that they contain an intrinsically disordered domain, which drives phase separation of the proteins in condensates upon energy depletion. Sup35 has been used as a model system for this phase separation. Sup35 phase separates into condensates upon energy depletion at low pH, but not at neutral pH. At neutral pH, sup35 is immobilized in the cytosol even though it is not in condensates. We find that Sup45, which is the binding partner of Sup35, also forms condensates upon energy depletion at low pH, but when energy depleted at neutral pH, Sup45 does not form condensates, but is immobilized in the cytosol. In contrast to Sup35, the URE3 prion, which has a similar domain structure as Sup35, does not share the same properties as Sup35. Upon energy depletion, it does not form condensates. Nor is it immobilized at higher pH, condition in which Sup35 does not form condensates. Pab1, another protein that binds Sup35, forms stress granules in energy depleted cells with different properties from the Sup35 condensates. The Sup35 condensates show only partial colocalization with Pab1 stress granules and different conditions cause the phase separation of Pab1 and Sup35. The Sup35 condensates also appear to contain RNA since addition of cycloheximide during energy depletion inhibits the formation of Sup35 condensates. This suggests that the Sup35 condensates contain Sup45 and RNA, but not Pab1.