Role of molecular chaperones in protein folding diseases

在我们的淀粉样蛋白的形成和溶解的途径的研究中，我们使用酵母作为我们的模型系统，重点是天然存在的朊病毒和哺乳动物亨廷顿蛋白片段与扩展的多聚谷氨酰胺重复区。 有趣的是，朊病毒蛋白具有内在无序的结构域，这是负责这些蛋白质在能量耗尽条件下相分离。Sup 35朊病毒蛋白的相分离已被表征，但Rnq 1和Ure 2的相分离未被表征。我们已经扩展了我们的研究，比较的Rnq 1和Ure 2朊病毒蛋白质的Sup 35在不同条件下的相分离。使用GFP标记的朊病毒蛋白，使用荧光成像研究发现，所有三种朊病毒蛋白促进三种典型朊病毒蛋白Rnq 1，Sup 35和Ure 2的相变。 具体来说，我们想研究三种不同的朊病毒蛋白是否都对变化的条件做出类似的反应，即使除了朊病毒结构域之外，它们的其他结构域并不保守。 我们发现GFP标记Sup 35，Ure 2和Rnq 1在饥饿条件下形成可逆的缩合物，即使在中性的内部pH值下，这表明聚集体的形成可能是对应激的代谢反应。我们还表明，除了2，4-二硝基苯酚，线粒体解偶联剂，使这种响应pH敏感。使用图像分析工具，我们量化了这些结果，表明不到20%的GFP标记的朊病毒形成这些聚集体。我们还表明，应力颗粒标记Pub 1，部分共定位与Sup 35的缩合物，但不与Ure 2或Rnq 1的缩合物。 此外，我们现在发现，通过不对称分离的URE 3的固化受三种分子伴侣Hsp 42，Sis 1和Hsp 70的调节。 Hsp 42通过隔离Ure 2病灶增加聚集体形成，而Sis 1/Ssa 1共同作用以解离团块。 反过来，降低细胞中的Sis 1/Ssa 1水平导致通过不对称分离的治愈，但是在HSP 42缺失菌株中Ure 2聚集点的聚集速率和程度降低。 有趣的是，Sis 1/Ssa 1在解离团块中的伴侣作用独立于它们在切断朊病毒种子中的作用。 种子的切断依赖于Hsp 104，其通过Sis 1和SSa 1的结合而被激活。一旦被激活，Hsp 104就可以切断朊病毒种子。有趣的是，这些分子伴侣在URE 3朊病毒的治疗中的功能与它们在将非淀粉样蛋白错误折叠的蛋白质分类到降解区室中的功能非常不同。 在一个相关的项目中，我们正在研究亨廷顿蛋白（Htt）外显子1片段在酵母中的聚集，这已被用作研究亨廷顿病的系统。 在酵母中，如同在哺乳动物细胞中一样，Htt片段的聚集依赖于多聚谷氨酰胺重复区的长度，但与哺乳动物细胞不同，据报道Htt片段聚集依赖于酵母朊病毒的存在。这反过来又使得聚集依赖于Hsp 104，Hsp 104通过切断朊病毒种子使它们能够繁殖。然而，目前尚不清楚Hsp 104是否具有独立于朊病毒传播的作用。为了研究这个问题，我们在酵母中表达HttQ 103，同时用胍灭活Hsp 104。与朊病毒和活性Hsp 104存在下形成的许多不同大小的病灶相反，对于非活性Hsp 104，存在少得多的较小病灶，并且随着酵母的进一步生长，小聚集体合并形成大聚集体。洗去胍以重新激活Hsp 104导致大量HttQ 103焦点的快速积累，表明Hsp 104切断大聚集体和Htt焦点，从而导致Htt焦点的扩增。 与Hsp 104的这种作用一致，在没有Hsp 104的酵母中，细胞甚至在一周后也不积累Htt聚集体，但在没有朊病毒但有Hsp 104的细胞中，细胞在一周的时间内缓慢积累HttQ 103。 相反，在朊病毒和Hsp 104同时存在的情况下，细胞在几个小时内都积累了大量的聚集体。这些结果表明朊病毒极大地加速了Htt聚集体的形成，其随后充当Hsp 104活性的底物，导致更多聚集体的形成。

In our study of pathways involved in amyloid formation and dissolution, we have used yeast as our model system with a focus on naturally occurring prions and mammalian huntingtin fragments with expanded polyglutamine repeat region. Interestingly, prion proteins have an intrinsically disordered domains, which are responsible for these proteins to phase separate under energy depletion conditions. The phase separation has been characterized for Sup35 prion protein, but not for the Rnq1and Ure2. We have extended our studies to compare the phase separation of the Rnq1 and Ure2 prion proteins to that of Sup35 under different conditions. Using GFP-labeled prion proteins, fluorescence imaging was used study found that all three prion proteins promote phase transition of the three canonical prion proteins, Rnq1, Sup35, and Ure2. Specifically, we wanted to examine whether the three different prion proteins all respond similarly to change conditions even though aside from the prion domain, their other domains are not conserved. We find that that GFP-labeled Sup35, Ure2, and Rnq1 are form reversible condensates under starvation conditions, even at a neutral internal pH, suggesting that aggregate formation may be a metabolic response to stress. We also show that addition of 2,4-dinitrophenol, a mitochondrial uncoupler, renders this response pH-sensitive. Using image analysis tools, we quantify these results, showing that less than 20 percent of the GFP-labeled prion forms these aggregates. We also show that stress granule marker Pub1, partially colocalizes with condensates of Sup35, but not with condensates of either Ure2 or Rnq1. In addition, we now find that the curing of URE3 by asymmetric segregation is regulated by three chaperones, Hsp42, Sis1, and Hsp70. Hsp42 increases aggregate formation by sequestering the Ure2 foci, whereas Sis1/ Ssa1 work together to dissociate the clumps. In turn, reducing Sis1/Ssa1 levels in the cell leads to curing by asymmetric segregation, but the rate and extent of clumping of Ure2 foci is reduced in an HSP42 deletion strain. Interestingly, the chaperone role of Sis1/Ssa1 in dissociating clumps is independent of their role in severing the prion seeds. The severing of the seeds is dependent on Hsp104, which is activated by the binding of Sis1 and SSa1. Once activated, Hsp104 can then sever the prion seeds. Interestingly, the function of these chaperones in the curing of URE3 prion is very different from their function found for the sorting of non-amyloidgenic misfolded proteins to degradative compartments. In a related project, we are examining the aggregation of huntingtin (Htt) exon 1 fragments in yeast, which has been used as a system for studying Huntingtons disease. In yeast as in mammalian cells, aggregation of Htt fragments is dependent on the length of the polyglutamine repeat region, but unlike mammalian cells, Htt fragment aggregation has been reported to be dependent on the presence of a yeast prion. This, in turn, makes aggregation dependent on Hsp104, which by severing the prion seeds enables them to propagate. However, it is not clear whether Hsp104 has a role that is independent of prion propagation. To investigate this question, we expressed HttQ103 in yeast while simultaneously inactivating Hsp104 with guanidine. In contrast to the numerous foci of varying sizes that form in the presence of prion and active Hsp104, with inactive Hsp104, there were much fewer smaller foci and with further growth of the yeast, the small aggregates coalesced to form a large aggregate. Washing out the guanidine to reactivate the Hsp104 led to the rapid accumulation of numerous HttQ103 foci, suggesting that Hsp104 severs both the large aggregates and the Htt foci, thereby leading to amplification of the Htt foci. Consistent with this role of Hsp104, in yeast with no Hsp104, cells did not accumulate Htt aggregates even after a week, but in cells with no prion, but with Hsp104, cells slowly accumulated HttQ103 over a period of a week. In contrast, in the presence of both prion and Hsp104, the cells all accumulated numerous aggregates within several hours. These results show that prion greatly accelerates the formation of Htt aggregates, which then act as substrates for Hsp104 activity, leading to the formation of still more aggregates.