Role of molecular chaperones in protein folding diseases

许多神经退行性疾病是由形成淀粉样沉积的细胞内或细胞外蛋白积累引起的。一般来说，淀粉样蛋白沉积可以穿过细胞膜，将有毒的淀粉样蛋白扩散到邻近的细胞。由于这种繁殖方式类似于传染性普恩病毒病中的播种方式，类似普恩病毒的传播似乎在许多神经退行性疾病中很常见。为了防止有毒淀粉样蛋白聚集体的积累，分子伴侣发挥解聚作用，但当它们不能保持质量控制时，就会形成蛋白质聚集体。我的研究处于这两个领域的十字路口；研究蛋白质聚集体的形成和繁殖，以及分子伴侣在酵母和哺乳动物细胞中的解聚。 酵母Pron需要分子伴侣Hsp104，Hsp104通过切断Pron种子在酵母分裂过程中繁殖Prion。去除淀粉样蛋白增殖构象的酵母蛋白通过使Hsp104失活而被治愈，但矛盾的是，酵母蛋白中的一种蛋白PSI+通过Hsp104的过表达而治愈。通过使用GFP标记的Sup35(形成PSI+Prion)的活细胞成像，我们发现Hsp104的过度表达实际上是由我们最近发现的一种名为Trimming的Hsp104活性引起的。修剪通过去除淀粉样蛋白种子末端的单体而不产生新种子来减小种子的大小，这与产生新种子的Hsp104的切断活性形成对比 在对酵母的第二项研究中，我们检查了亨廷丁(Htt)毒性的基础。HttPolyQ片段只在繁殖病毒的酵母菌中形成有毒聚集体。我们发现，不同的必需蛋白质被Htt聚集体隔离，这取决于Pron的不同。此外，Htt片段的氨基酸组成也影响了哪些蛋白质被隔离。未来的工作将包括过表达不同的Hsp104片段，以及不同酵母物种的Hsp104，以更好地了解Hsp104过表达导致PSI+固化的机制。HSP104解聚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的过度表达也被发现可以治愈一个普恩病毒，在这个案例中是URE3，我们正在研究这种治愈的机制。 我们还研究了蛋白质聚集和分子伴侣在哺乳动物细胞蛋白质解聚中的作用。我们对分子伴侣Hsc70在依赖于胞质蛋白运输中的作用的长期研究表明，Hsc70的主要功能是阻止胞浆中的胞质蛋白聚集。我们发现，这种活性依赖于两个与Hsc70和clathrin相互作用的J结构域蛋白，即普遍表达的GAK和神经元特异性的生长素。这些J-结构域蛋白对于细胞内所有依赖于笼状蛋白的运输都是必不可少的，当这些蛋白在小鼠体内被敲击时，就会导致发育和神经缺陷。通过表达GAK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状蛋白包裹的囊泡不可逆脱涂层所必需的。此外，我们发现这个结构域与帕金森氏病之间存在关联，我们现在正在使用GAK和生长素基因敲除小鼠来研究这一领域。 开展的另一个主要研究项目是研究Pron在哺乳动物细胞中的繁殖。PrPc到淀粉样蛋白PrPsc的转换已被证明发生在PrPc沿内体通路的交通中，但发生转换的间隔一直存在争议。通过在不同的时间点阻断PrPsc的细胞转运，我们发现PrPsc的胞内转换部位是多泡小体(MVB)。这一观察结果特别有趣，因为MVB具有不寻常的膜拓扑结构，这可能使PrPc和PrPsc分子之间以及细胞内和质膜上的反式相互作用成为可能。我们现在正在干扰其他的贩运途径，既是为了证实我们的结果，也是为了了解贩运的其他方面(例如，网状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.