Role of molecular chaperones in protein folding diseases

许多神经退行性疾病是由形成淀粉样蛋白沉积物的细胞内或细胞外蛋白质的积累引起的。一般来说，淀粉样蛋白沉积物可以穿过细胞膜，将有毒的淀粉样蛋白扩散到邻近的细胞。由于这种传播模式类似于传染性朊病毒疾病中发生的播种，因此朊病毒样传播似乎在许多神经退行性疾病中很常见。为了防止有毒的淀粉样蛋白聚集体的积累，分子伴侣的功能是使它们解聚，但当它们不能保持质量控制时，就会形成蛋白质聚集体。我的研究是在这两个领域的十字路口;研究蛋白质聚集体的形成和繁殖，以及酵母和哺乳动物细胞中分子伴侣的解聚。 酵母朊病毒需要分子伴侣HSP 104，HSP 104通过切断朊病毒种子在分裂的酵母中繁殖朊病毒。酵母朊病毒通过Hsp 104的失活而被治愈，其摆脱了淀粉样蛋白繁殖构象，但矛盾的是，酵母朊病毒之一PSI+通过Hsp 104的过表达而被治愈。通过使用GFP标记的Sup 35（形成PSI+朊病毒）的活细胞成像，我们发现Hsp 104过表达的治愈实际上是由我们最近发现的称为修剪的Hsp 104活性引起的。与产生新种子的Hsp 104的切断活性相反，修剪通过从淀粉样蛋白种子的末端去除单体而不产生新种子来减小种子的大小 在酵母的第二项研究中，我们研究了亨廷顿蛋白（Htt）毒性的基础。HttpolyQ片段仅在繁殖朊病毒的酵母中形成毒性聚集体。我们发现，根据朊病毒，不同的必需蛋白质被Htt聚集体隔离。此外，Htt片段的氨基酸组成也影响哪些蛋白被隔离。未来的工作将包括过表达不同的Hsp 104片段以及来自不同酵母物种的Hsp 104，以更好地理解通过Hsp 104过表达治疗PSI+的机制。Hsp 104去活化淀粉样蛋白的能力有可能成为治疗人类淀粉样蛋白疾病的治疗工具。有趣的是，Htt的过表达也被发现可以治愈朊病毒，在这种情况下是URE 3，我们正在研究治愈的机制。 我们还研究了哺乳动物细胞中蛋白质聚集和分子伴侣在蛋白质解聚中的作用。我们对分子伴侣Hsc 70在网格蛋白依赖性运输中的作用的长期研究表明，Hsc 70的主要功能是防止网格蛋白在胞质溶胶中聚集。我们发现这种活性依赖于两个J结构域蛋白，它们与Hsc 70和网格蛋白、普遍表达的GAK和神经元特异性生长素相互作用。这些J结构域蛋白对于细胞中所有网格蛋白依赖性运输是必不可少的，当这些蛋白质在小鼠中被敲除时，这会导致发育和神经缺陷。通过表达GAK的片段，我们发现GAK的pten样结构域不是必需的，这是令人惊讶的，因为有人提出该结构域是网格蛋白包被的囊泡不可逆去包被所必需的。此外，我们发现这个结构域与帕金森病之间存在关联，我们现在正在使用我们的GAK和生长素敲除小鼠进行研究。 开展的另一个主要研究项目是研究朊病毒在哺乳动物细胞中的繁殖。PrPc向淀粉样蛋白PrPsc的转化已被证明发生在PrPc沿着内体途径沿着运输时，但发生转化的隔室一直存在争议。通过阻断PrPsc的细胞运输，我们发现朊病毒转化的细胞内位点是多泡体（MVB）。这一观察结果特别令人感兴趣，因为MVB具有不寻常的膜拓扑结构，可能使PrPc和PrPsc分子之间的反式相互作用在细胞内以及在质膜上。我们现在正在扰乱其他贩运途径，以确认我们的结果，并了解贩运的其他方面（例如网格蛋白介导的内吞作用）如何影响PrPsc的繁殖。

Many neurodegenerative diseases are caused by the accumulation of intracellular or extracellular proteins that form amyloid deposits. In general, amyloid deposits can cross cell membranes to spread the toxic amyloids to neighboring cells. Since this mode of propagation is similar to the seeding that occurs in infectious prion disease, prion-like transmission appears to be common to many neurodegenerative diseases. To protect against the accumulation of toxic amyloid aggregates, molecular chaperones function to disaggregate them, but when they do not maintain quality control, protein aggregates are formed. My research is at the crossroads of these two areas; examining the formation and propagation of protein aggregates and their disaggregation by molecular chaperones in yeast and mammalian cells. Yeast prions require the molecular chaperone, Hsp104, which propagates prion in dividing yeast by severing the prion seeds. Yeast prions are cured that is rid of the amyloid propagating conformation, by inactivation of Hsp104, but paradoxically, one of the yeast prions, PSI+, is cured by overexpression of Hsp104. By using live cell imaging of GFP-labeled Sup35, which forms PSI+ prion, we found that curing by Hsp104 overexpression was actually caused by an activity of Hsp104 that we recently uncovered termed trimming. Trimming reduces the size of the seeds by removing monomers from the ends of the amyloid seeds without creating new seeds, in contrast to the severing activity of Hsp104 that creates new seeds In a second study in yeast, we examined the basis of huntingtin (Htt) toxicity. HttpolyQ fragments form toxic aggregates only in yeast propagating a prion. We found that depending on the prion, different essential proteins are sequestered by the Htt aggregates. In addition the amino acid composition of the Htt fragment also affected which proteins are sequestered. Future work will include overexpressing different Hsp104 fragments and also Hsp104 from different yeast species to better understand the mechanism of PSI+ curing by Hsp104 overexpression. The ability of Hsp104 to depolymerize amyloids has potential as a therapeutic tool to treat amyloid diseases in man. Interestingly, overexpression of Htt has also been found to cure a prion, in this case URE3, and we are investigating the mechanism of the curing. We have also studied both protein aggregation and the role of molecular chaperones in protein disaggregation in mammalian cells. Our long-term study of the role of the molecular chaperone, Hsc70, in clathrin-dependent trafficking has shown that the major function of Hsc70 is to prevent aggregation of clathrin in the cytosol. We discovered that this activity is dependent on two J-domain proteins that interact with Hsc70 and clathrin, the ubiquitously expressed GAK and the neuronal-specific auxilin. These J-domain proteins are essential for all clathrin-dependent trafficking in the cell and, when these proteins were knocked in the mouse, this caused both developmental and neurological defects. By expressing fragments of GAK, we found its pten-like domain of GAK is not essential, which is surprising, since it was proposed that this domain is required for irreversible uncoating of clathrin coated vesicles. Furthermore, we found that there was an association between this domain and Parkinsons disease, an area that we are now pursuing by using our GAK and auxilin knockout mice. Another major research project carried out was studying prion propagation in mammalian cells. The conversion of the PrPc to the amyloid PrPsc has been shown to occur as PrPc traffics along the endosomal pathway, but the compartment where conversion takes place has been controversial. By blocking the cellular trafficking of PrPsc at various points, we found that the intracellular site of prion conversion is the multivesicular body (MVB). This observation is of particular interest because the MVB has an unusual membrane topology that might enable trans-interaction between PrPc and PrPsc molecules within the cell as well as at the plasma membrane. We are now perturbing other trafficking pathways both to confirm our results and to understand how other aspects of trafficking (e.g. clathrin-mediated endocytosis) affect PrPsc propagation.