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朊病毒在哺乳动物细胞中的繁殖机制，朊病毒在酵母细胞中的治愈机制，以及亨廷顿蛋白（Htt）在哺乳动物和酵母细胞中的毒性机制。现在可以理解，正确折叠的朊病毒蛋白PrPc转化为错误折叠的淀粉样蛋白构象PrPsc，发生在PrPc在质膜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。然而，我们仍然不了解转换的机制或在细胞中转换发生的位置。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，以阻断沿着特定途径的运输。我们的结果表明，抑制MVB的成熟清除PrPsc，这表明MVB是PrPsc转化的主要内部位点。MVB是一种细胞器，其是将货物从早期内体运输到蛋白质降解的溶酶体的中间体。有趣的是，MVB具有不寻常的几何形状，其具有含有靶向溶酶体降解的货物的腔内囊泡。这种不寻常的几何形状允许两套膜的并置，并且可能是这种几何形状在质膜和MVB中诱导朊病毒转化。 我实验室的另一个项目是研究酵母PSI+朊病毒的治疗，它是由错误折叠的Sup 35淀粉样蛋白聚集体形成的。与其他酵母朊病毒类似，PSI+通过灭活Hsp 104来治愈，这是切断朊病毒种子所必需的。令人惊讶的是，PSI+也通过Hsp 104的过表达被治愈，其机制尚不清楚，尽管许多不同的实验室已经研究了该过程。一种建议的通过Hsp 104过表达治愈的模型是由于种子在母细胞和子细胞之间的不对称分离。然而，我们通过使用流式细胞术根据年龄分离酵母菌排除了这种模型。然后，通过使用GFP标记的Sup 35的活细胞成像，我们观察到Hsp 104导致朊病毒种子的大小减小，这表明过量的Hsp 104可能正在从朊病毒纤维的末端去除分子。然而，我们发现，这种活性的热休克蛋白104，我们称之为修剪，是不足以治愈PSI+本身。此外，由修剪活性留下的淀粉样蛋白核心由过量的Hsp 104和其他分子伴侣呈递给蛋白酶体进行降解。这种通过过度表达治愈的机制已经证明了细胞摆脱朊病毒聚集体的新方法。 最后，我们还研究了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引起神经变性。我们首先发现，在酵母中，具有扩展的多聚谷氨酰胺重复序列的Htt片段的毒性取决于与Htt片段一起沿着存在于酵母中的朊病毒。我们发现，在polyQ重复区的C-末端具有聚脯氨酸区的HttQP 103在PSI+酵母中比在PIN+中具有更大的毒性，即使HttQP 103在PSI+和PIN+酵母中形成多个聚集体。此外，通过表达Sup 35的可溶性C末端片段，有效地挽救了由HttQP 103聚集体引起的毒性。这表明，在含有PSI+朊病毒的酵母中，HttQP 103的毒性主要是由于必需蛋白Sup 35的螯合。有趣的是，不含聚脯氨酸重复区的HttQ 103的表达在PSI+和PIN+酵母中都是有毒的，并且通过表达Sup 35的可溶性C-末端片段来挽救的效果很差。这显示了Htt毒性的复杂性质，其可能是由取决于细胞类型的不同蛋白质的螯合引起的。在哺乳动物细胞中的相关工作中，我们检查了具有不同长度polyQ重复区域的Htt片段的聚集，我们通过使用荧光相关光谱发现，非病理性和病理性Htt片段在胞质溶胶中形成可溶性寡聚体。因此，引起毒性的不是可溶性低聚物本身的存在，而是低聚物的性质决定了它们是否引起神经病理学，这可能是因为不同的低聚物螯合不同的蛋白质。

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.