Role of molecular chaperones in endocytosis and protein folding diseases

我们的实验室对细胞中正常和病理性蛋白质复合物的形成和溶解感兴趣，重点是分子伴侣在这些过程中的作用。 我们继续我们的研究HSC 70在网格蛋白介导的内吞作用的作用，产生敲除小鼠的HSC 70 cochaperones，神经元特异性生长素和普遍表达的GAK。 具体地说，我们想确定敲除GAK的效果，并确定GAK的结构域对其辅伴侣功能至关重要。 使用来自条件性GAK敲除小鼠的小鼠胚胎成纤维细胞（MEF），通过使用表达Cre重组酶的腺病毒破坏GAK。 敲除GAK，完全阻断了MEFs中网格蛋白介导的内吞作用，并破坏了质膜上和高尔基体网络上的网格蛋白组织。 此外，GAK缺失的MEFs在G1/S检查点被捕获，但仍经历多轮中心体复制。 有趣的是，所有这些表型都用仅由网格蛋白结合结构域和J结构域组成的截短GAK分子拯救。 此外，该截短的GAK片段，其作为转基因表达，能够挽救当GAK从小鼠的特定组织中敲除时观察到的致死性。 这些结果表明，网格蛋白结合和J-结构域的GAK，而不是激酶和pTen-like结构域的GAK，是必不可少的辅伴侣功能。 此外，我们还研究了朊病毒的繁殖，朊病毒是一种感染性蛋白质，在哺乳动物和酵母细胞中都可以错误折叠成淀粉样蛋白构象。 在哺乳动物细胞中，慢性感染的羊瘙痒病细胞，具有错误折叠的淀粉样蛋白形式的朊病毒，已经被证明可以被许多不同的药物治愈，这些药物已经被报道具有不同的靶点。 然而，羊痒病的免疫染色显示，使用所有这些不同的抗朊病毒药物，不再发现局部化的羊痒病在整个内体运输途径中循环，而是全部定位在lamp-1阳性内体中。 这些结果表明，这些药物清除羊瘙痒病的机制是将羊瘙痒病隔离在一个死端隔室中，这反过来又阻止了正确折叠的朊病毒蛋白转化为错误折叠的淀粉样蛋白构象。 我们对朊病毒的研究也扩展到酵母，酵母是研究朊病毒繁殖的简单模型系统。 在酵母中，分子伴侣Hsp 104调节包括PSI+在内的几种酵母朊病毒的遗传，PSI+是翻译终止因子Sup 35 p的朊病毒形式。 Hsp 104的失活和过表达都能治愈PSI+朊病毒的酵母菌。 通过使用活细胞成像，我们现在已经发现分子伴侣Hsp 104具有两种活性。 它不仅切断朊病毒种子，而且通过从淀粉样蛋白纤维末端去除Sup 35分子来修剪或减小种子的大小。 这些释放的Sup 35分子不会使新种子成核，因此，修剪不会增加朊病毒种子的数量。 相反，切断雌鹿种子会增加新种子的数量。 我们现在还发现，修整活性对于通过Hsp 104的过表达治愈正朊病毒以及在繁殖PSI正酵母中维持朊病毒种子的稳态大小是重要的。

Our laboratory is interested in the formation and dissolution of both normal and pathological protein complexes in the cell with an emphasis on the role of molecular chaperones in these processes. We have continued our studies on the role of Hsc70 in clathrin-mediated endocytosis by generating knockout mice of the HSC70 cochaperones, neuronal-specific auxilin and ubiquitously expressed GAK. Specifically, we wanted to determine the effect of knocking out GAK and also determine the domains of GAK that are essential for its cochaperone function. Using mouse embryonic fibroblasts (MEFs) derived from the conditional GAK knockout mouse, GAK was disrupted by using adenovirus expressing Cre recombinase. Knocking-out GAK, completely blocked clathrin-mediated endocytosis in the MEFs and disrupted the clathrin organization on the plasma membrane and at the trans-Golgi network. Moreover, the GAK-depleted MEFs were arrested at the G1/S checkpoint, but still underwent multiple rounds of centrosome duplication. Interestingly, all of these phenotypes were rescued with a truncated GAK molecule consisting of just the clathrin binding domain and J-domain. Furthermore, this truncated GAK fragment, which was expressed as a transgene, was able to rescue the lethality observed when GAK was knocked out of specific tissues in mice. These results show that the clathrin-binding and J-domains of GAK, but not the kinase and pTen-like domains of GAK, are essential for its cochaperone function. In addition, we also studied the propagation of prions, infective proteins that can misfold into an amyloid conformation both in mammalian and yeast cells. In mammalian cells, chronically infected scrapie cells, having the misfolded amyloid form of prion, have been shown to be cured by a number of different drugs that have been reported to have different target sites. However, immunostaining of scrapie showed that with all these different anti-prion drugs, the scrapie localized was no longer found cycling throughout the endosomal trafficking pathway, but instead was all localized in lamp-1 positive endosomes. These results indicate that the mechanism by which these drugs clear scrapie is to sequester scrapie in a dead-end compartment, which, in turn, prevents the conversion of the properly folded prion protein to the misfolded amyloid conformation. Our research on prion has also been extended to yeast, which is a simple model system to study prion propagation. In yeast, the molecular chaperone, Hsp104, regulates the inheritance of several yeast prions including PSI+, which is the prion form of the translation termination factor Sup35p. Both inactivation and overexpression of Hsp104 cures yeast of the PSI plus prion. By using live cell imaging, we have now found that the molecular chaperone, Hsp104, has two activities. It not only severs prion seeds, but also trims or reduces the size of the seeds by removing Sup35 molecules from the end of the amyloid fiber. These released Sup35 molecules do not nucleate new seeds and therefore, trimming does not increase the number of prion seeds. In contrast, severing the does seeds increase the number of new seeds. We have also now found that trimming activity is important for curing of plus prion by overexpression of Hsp104, as well as maintaining the steady-state size of the prion seeds in propagating PSI plus yeast.