Role of molecular chaperones in protein folding diseases

我的实验室正在使用细胞生物学技术来研究Pron在哺乳动物细胞中的繁殖机制，Pron在酵母细胞中的固化，以及Huntingtin(Htt)在哺乳动物和酵母细胞中的毒性机制。现在人们了解到，当PrPc在质膜上或沿着其在哺乳动物细胞中的运输路线暴露于错误折叠的PrPsc时，正确折叠的Prion蛋白PrPc转换为错误折叠的淀粉样构象PrPsc。然而，我们仍然不了解转换的机制，也不知道细胞中的转换发生在哪里。为了确定这一点，我们使用了不同的Rab突变体，并敲除了特定的蛋白质来阻止沿着特定路径的运输。我们的结果表明，抑制MVB的成熟可以清除PrPsc，这表明MVB是PrPsc转化的主要内部部位。MVB是一个细胞器，它是将货物从早期的内体运送到溶酶体的中间体，在溶酶体中蛋白质被降解。有趣的是，MVB有一个不同寻常的几何结构，腔内囊泡含有针对溶酶体降解的货物。这种不同寻常的几何构型允许两组膜的结合，可能正是这种几何构型在质膜和MVB中都诱导了Pron转换。 我实验室一直在研究的另一个项目是酵母PSI+Prion的固化，它是由错误折叠的Sup35淀粉样蛋白聚集体形成的。与其他酵母蛋白一样，PSI+是通过灭活热休克蛋白104来治愈的，而热休克蛋白104是切断蛋白种子所必需的。令人惊讶的是，PSI+也是通过Hsp104的过度表达而治愈的，尽管这一过程已经被许多不同的实验室研究过，但其机制尚不清楚。Hsp104过表达的一种治疗模式是由于种子在母细胞和子细胞之间的不对称分离。然而，通过使用流式细胞术根据年龄分离酵母，我们排除了这一模型。然后，通过使用GFP标记的Sup35的活细胞成像，我们观察到Hsp104导致Prion种子的大小减小，这表明多余的Hsp104可能正在从Prion纤维的末端移除分子。然而，我们发现Hsp104的这种活性，我们称之为修剪，本身并不足以治愈PSI+。此外，修剪活动留下的淀粉样蛋白核心由多余的Hsp104和其他伴侣蛋白提供给蛋白酶体进行降解。这种通过过度表达来治愈的机制已经展示了一种新的方法，让细胞自己摆脱普恩聚集体。 最后，我们还检查了Htt片段在酵母和哺乳动物细胞中引起的毒性，试图了解它们为什么会导致神经退化。我们首先发现，在酵母中，带有扩展的聚谷氨酰胺重复序列的Htt片段的毒性取决于酵母中与Htt片段一起存在的Prion。我们发现，尽管HttQP103在PSI+和PIN+酵母中都形成了多个聚集体，但HttQP103在多聚Q重复区域的C末端具有明显的毒性，在PSI+酵母中比在PIN+酵母中更具毒性。此外，通过表达Sup35的可溶性C末端片段，HttQP103聚合体的毒性被有效地拯救。这表明，HttQP103对含有PSI+Prion的酵母的毒性主要是由于隔离了必要的蛋白质Sup35。有趣的是，HttQ103的表达在PSI+和PIN+酵母中都是有毒的，并且不能通过表达Sup35的可溶C末端片段来拯救，HttQ103不包含多聚脯氨酸重复区域。这表明了Htt毒性的复杂性，这可能是由于不同细胞类型的不同蛋白质的隔离造成的。在哺乳动物细胞的相关工作中，我们研究了不同长度的多聚Q重复区域的Htt片段的聚集情况，通过使用荧光相关光谱发现，非病理性和病理性Htt片段都在胞浆中形成了可溶的寡聚体。因此，造成毒性的不是可溶低聚物本身，而是低聚物的性质决定了它们是否会导致神经病理，可能是因为不同的低聚物隔离了不同的蛋白质。

My laboratory is using cell biological techniques to examine the mechanism of prion propagation in mammalian cells, prion curing in yeast cells, and the mechanism of huntingtin (Htt) toxicity in mammalian and yeast cells. It is now understood that conversion of the properly folded prion protein, PrPc, to the misfolded amyloid conformation, PrPsc, occurs upon exposure of PrPc to misfolded PrPsc either at the plasma membrane or along its trafficking itinerary in mammalian cells. However, we still do not understand the mechanism of conversion or where in the cell conversion takes place. To determine this, we have used different Rab mutants and knocked out specific proteins to block trafficking along particular pathways. Our results show that inhibiting maturation of the MVB clears PrPsc, which indicates that the MVB is the major internal site of PrPsc conversion. The MVB is an organelle that is an intermediate in the trafficking of cargo from the early endosome to the lysosome where proteins are degraded. Interestingly, the MVB has an unusual geometry with intralumenal vesicles that contain cargo targeted for lysosomal degradation. This unusual geometry allows apposition of two sets of membranes and it may be this geometry that induces prion conversion both at the plasma membrane and in the MVB. Another project in my laboratory has been examining is the curing of the yeast PSI+ prion, which is formed from misfolded Sup35 amyloid aggregates. Similar to other yeast prions, PSI+ is cured by inactivation of Hsp104, which is required for the severing of the prion seeds. Surprisingly, PSI+ is also cured by overexpression of Hsp104 by a mechanism that is not yet understood although this process has been investigated by many different laboratories. One suggested model for curing by Hsp104 overexpression is due to asymmetric segregation of the seeds between mother and daughter cells. However, we ruled out this model by using flow cytometry to separate yeast based on age. Then by using live cell imaging of GFP-labeled Sup35, we observed that Hsp104 causes a reduction in the size of the prion seeds, suggesting that the excess Hsp104 might be removing molecules from the ends of the prion fibers. However, we find that this activity of Hsp104, which we have termed trimming, is not by itself sufficient to cure PSI+. In addition, the amyloid core that is left by the trimming activity is presented by the excess Hsp104 and other chaperones to the proteasome for degradation. This mechanism of curing by overexpression has demonstrated a new way for the cell to rid itself of prion aggregates. Finally, we have also examined the toxicity caused by Htt fragments in both yeast and mammalian cells in an attempt to understand why they cause neurodegeneration. We first found that in yeast, the toxicity of the Htt fragments with expanded polyglutamine repeats depends on the prion that is present in the yeast along with the Htt fragment. We found that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, was significantly more toxic in PSI+ yeast than in PIN+ even though HttQP103 formed multiple aggregates in both PSI+ and PIN+ yeast. Furthermore, the toxicity caused by HttQP103 aggregates was effectively rescued by expressing the soluble C-terminal fragment of Sup35. This shows that the toxicity of HttQP103 in yeast containing the PSI+ prion is primarily due to sequestration of the essential protein, Sup35. Interestingly, expression of HttQ103, which does not contain the polyproline repeat region, was toxic in both PSI+ and PIN+ yeast and poorly rescued by expressing the soluble C-terminal fragment of Sup35. This shows the complex nature of Htt toxicity, which may be caused by the sequestration of different proteins depending on the cell type. In related work in mammalian cells where we examined the aggregation of Htt fragments with different lengths polyQ repeat regions, we found by using fluorescence correlation spectroscopy that both non-pathological and pathological Htt fragments form soluble oligomers in the cytosol. Therefore, it is not the presence of soluble oligomers per se that causes toxicity, but the nature of the oligomers that determines whether they cause neuropathology, perhaps because different oligomers sequester different proteins.