Role of molecular chaperones in protein folding diseases

研究最广泛的酵母蛋白是PSI+、URE3和PIN+，它们分别是Sup35、Ure2和Rnq1错误折叠的蛋白质形式。虽然已经建立了正确折叠的Sup35的凝聚体形成，但尚不清楚正确折叠的Ure2和Rnq1是否在与Sup35相同的条件下形成凝聚体，因为除了它们的Prion结构域外，其他结构域不是保守的。与Sup35类似，Ure2和Rnq1在饥饿条件下形成凝聚体，同时消耗ATP和降低pH。加入葡萄糖后，这三种蛋白形成的凝集物很容易逆转，从而将ATP和pH恢复到生理水平。然而，当pH升高而不补充ATP时，只有Ure2和Rnq1冷凝物容易溶解。此外，在生理pH下耗尽ATP会产生Sup35的凝聚体，而Rnq1和Ure2蛋白在这些条件下保持高度流动性，不形成凝聚体。有趣的是，只有Sup35有一个M结构域，据报道这是一个pH传感器结构域，可以在较高的pH下抑制冷凝物的形成。这些结果表明，Pron蛋白对pH的感知以及Pron凝聚体的形成和溶解的条件，特别是关于Sup35，尚不清楚。 酵母蛋白URE3是通过Hsp42、btn2、Cur1和Ydj1的过度表达而治愈的，这些蛋白质也参与了非淀粉样聚集体的清除。有趣的是，这些蛋白质定位于不同的细胞隔间。Ydj1和Hsp42是胞浆蛋白，而Cur1是细胞核蛋白，btn2在细胞核和胞浆之间洗牌。Btn2、Hsp42、Cur1或Ydj1在URE3酵母中的过表达聚集了URE3种子，这导致了细胞分裂过程中Pron种子的不对称分离。Btn2、Cur1或Ydj1的过表达导致内源Hsp42形成聚集体，从而结合URE3种子。当Hsp42缺失时，过表达Cur1或Ydj1仍可治愈URE3，但速度明显减慢，而过表达btn2则不能治愈。Hsp42对URE3种子的聚集仅与全长Hsp42一起发生，截断Hsp42的N-末端Prion结构域或固有无序结构域都消除了Ure2种子的聚集。我们的结果表明，全长Hsp42作为结合普恩种子的支架，从而通过种子的不对称分离有助于URE3普恩的固化。 我们还在研究HSP104在治愈普恩和在酵母中聚集亨廷顿蛋白外显子1片段中的作用。为了在一代又一代中保持稳定数量的PrP种子，必须有Pron种子的持续繁殖，这依赖于Hsp104的切割活性。矛盾的是，Hsp104的过度表达以及Hsp104活性的丧失可以治愈PSI+Prion。已经提出了两种非常不同的固化模式：通过Hsp104过表达来溶解普鲁恩种子和通过不对称分离普鲁恩种子来固化。为了更好地了解Hsp104过表达治疗PSI+的机制，我们研究了Hsp70家族不同成员是否影响治疗速度。在只表达Ssa1的酵母中，Hsp104过表达对PSI+的固化速度明显快于只表达Ssa2的酵母，即使这些同源基因有98%的同源性。同时，无论酵母是表达Ssa1还是表达Ssa2，PSI+种子的数量都没有显著差异。由于Ssa1/Ssa2通过过表达Hsp104而不影响种子数来影响PSI+的固化速率，这些结果表明Hsp104过表达的固化机制与Hsp104的切割活性无关。 在酵母中，与哺乳动物细胞不同，亨廷顿蛋白片段与扩展的聚谷氨酰胺重复区域(HttPolyQ)的聚集已被发现依赖于Pron。这反过来又使HttPolyQ聚集依赖于Hsp104，这是传播Pron种子所必需的。然而，目前还不清楚Hsp104在HttPolyQ聚集中是否有其他作用。在本研究中，我们发现Hsp104对HttQ103片段的聚集有显著的影响，无论有无病毒。在Prion和Hsp104存在的情况下，细胞有大量的聚集体，但当Hsp104被灭活时，细胞有一个大的HttQ103聚集体，由较小的聚集体合并形成。在没有Prion和Hsp104的情况下，HttQ103没有显著的聚集，但在没有Prion的情况下，有活性的Hsp104，细胞缓慢地积累聚集；推测，Hsp104正在放大已知发生的少量自发成核的聚集。一周后，有HSP104的细胞，但没有普恩病毒的细胞，每个细胞有大量的HttQ103聚集体，这些聚集体的生物物理性质与既有普恩蛋白又有HSP104的聚集体没有区别。这些结果表明，无论HttQ103是自发成核还是普恩模板化成核，Hsp104都会切断初始的HttQ103聚集体，从而增加它们的数量并增强它们向子代细胞的传递。与HttQ103不同的是，HttQ103P在聚谷氨酰胺区域下游有一个多脯氨酸区，它需要Pron来积累聚集体；聚集体不会单独在Hsp104存在下聚集。此外，由Pron成核的HttQ103P聚集体不会被Hsp104切断，无论是否存在Hsp104，这些小聚集体都会聚集成一个大的HttQ103P聚集体。我们的结论是，Hsp104能够自发地切割HttQ103的核或PrP模板的核，但不能切割HttQ103P的等效核，这是HttQ103和HttQ103P聚集性质不同的原因。

The most widely studied yeast prions are the PSI+, URE3, and PIN+, which are the misfolded protein forms of Sup35, Ure2, and Rnq1, respectively. Although condensate formation of properly folded Sup35 has been established, it is not clear whether the properly folded Ure2 and Rnq1 form condensates under the same conditions as Sup35 since aside from their prion domain, the other domains are not conserved. Similar to Sup35, Ure2 and Rnq1 form condensates under starvation conditions that simultaneously deplete ATP and lower pH. The condensates formed from the three prion proteins are readily reversible upon addition of upon addition of glucose, which restores both ATP and pH to physiological levels. However, when pH is raised without replenishing ATP, only Ure2 and Rnq1 condensates readily dissolve. Furthermore, depletion of ATP at physiological pH produced condensates of Sup35, whereas the Rnq1 and Ure2 proteins remain highly mobile under these conditions and do not form condensates. Interestingly only Sup35 has a M-domain, which has been reported to be a pH sensor domain that inhibits condensate formation at higher pH. These results suggest that the sensing of pH by prion proteins and the conditions for formation and dissolution of prion condensates, in particular with regards to Sup35, are not understood. The yeast prion, URE3, is cured by overexpression of Hsp42, Btn2, Cur1, and Ydj1, proteins that are also involved in the clearance of non-amyloid aggregates. Interestingly, these proteins are localized to different cellular compartments. Ydj1 and Hsp42 are cytosolic proteins, whereas Cur1 is nuclear, Btn2 shuffles between the nucleus and the cytosol. The overexpression of Btn2, Hsp42, Cur1, or Ydj1 in URE3 yeast aggregates the URE3 seeds, which causes asymmetric segregation of the prion seeds during cell division. Overexpression of Btn2, Cur1 or Ydj1 causes the endogenous Hsp42 to form an aggregate, which binds the URE3 seeds. When Hsp42 is deleted, overexpression of Cur1 or Ydj1 still cures URE3, but at a significantly slower rate, whereas overexpression of Btn2 does not cure. Aggregation of the URE3 seeds by Hsp42 only occurred with full-length Hsp42, truncation of either the N-terminal prion domain or the intrinsically disorder domain of the Hsp42 eliminated aggregation of the Ure2 seeds. Our results suggest that the full-length Hsp42 acts as a scaffold that binds the prion seeds, thereby contributing to the curing of URE3 prion by asymmetric segregation of the seeds. We are also investigating the role of Hsp104 in curing prion and in aggregating huntingtin exon 1 fragments in yeasts. To maintain a steady state number of prion seeds from generation to generation, there must be constant propagation of the prion seeds, which is dependent on the severing activity of Hsp104. Paradoxically, Hsp104 overexpression as well as loss of Hsp104 activity cures the PSI+ prion. Two very different models of curing have been proposed: dissolution of the prion seeds by Hsp104 overexpression and curing by asymmetric segregation of the prion seeds. To better understand the mechanism of curing PSI+ by Hsp104 overexpression, we examined whether the rate of curing is affected by different members of the Hsp70 family. The rate of curing of PSI+ by Hsp104 overexpression is significantly faster in yeast expressing only Ssa1 than in yeast expressing only Ssa2 even though these homologs are 98% identical. At the same time, the number of PSI+ seeds are not significantly different whether the yeast are expressing Ssa1 or Ssa2. Since Ssa1/SSa2 affected the rate of curing by PSI+ by overexpression of Hsp104 without affecting seed number these results show that the mechanism of curing by Hsp104 overexpression is independent of the severing activity of Hsp104. In yeast, unlike in mammalian cells, the aggregation of huntingtin fragments with expanded polyglutamine repeat regions (HttpolyQ) has been found to be dependent on prion. This, in turn, makes HttpolyQ aggregation dependent on Hsp104, which is required to propagate the prion seeds. However, it is not clear whether Hsp104 has any other role in HttpolyQ aggregation. In the present study, we find that Hsp104 has a marked effect on the aggregation of HttQ103 fragments both in the presence and absence of prion. In the presence of both prion and Hsp104 the cells have numerous aggregates, but when Hsp104 is inactivated, the cells have a large HttQ103 aggregate formed from coalescence of the smaller aggregates. In the absence of both prion and Hsp104, there is no significant aggregation of HttQ103, but with active Hsp104 in the absence of prion, the cells slowly accumulate aggregates; presumably, the Hsp104 is amplifying aggregates from the small amount of spontaneous nucleation that is known to occur. After a week, the cells with Hsp104, but without prion, have numerous HttQ103 aggregates per cell and the biophysical properties of these aggregates are indistinguishable from the aggregates that form with both prion and Hsp104. These results show, that regardless of whether there is spontaneous or prion-templated nucleation of HttQ103, Hsp104 severs the initial HttQ103 aggregates, thus increasing their number and enhancing their transmission to the daughter cells. In contrast to HttQ103, HttQ103P, which has a polyproline region downstream of the polyglutamine region, requires prion to accumulate aggregates; aggregates do not accumulate in the presence of Hsp104 alone. Furthermore, the HttQ103P aggregates that are nucleated by prion are not severed by Hsp104, whether Hsp104 is present or not, these small aggregates coalesce into one large HttQ103P aggregate. We conclude that the ability of Hsp104 to sever either the spontaneously or prion-templated nuclei of HttQ103, but not the equivalent nuclei of HttQ103P, accounts for the differences in the aggregation properties of HttQ103 and HttQ103P.