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蛋白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。我们继续研究Hsc70在网格蛋白介导的内吞作用，通过产生敲除Hsc70的合作伙伴、神经元特异性辅助素和无处不在表达的GAK的小鼠。具体来说，我们想要确定敲除GAK的效果，并确定GAK的结构域，这些结构域对其伴侣蛋白的功能至关重要。使用条件GAK敲除小鼠获得的小鼠胚胎成纤维细胞（mef），用表达Cre重组酶的腺病毒破坏GAK。敲除GAK，完全阻断了mef中网格蛋白介导的内吞作用，破坏了质膜和反式高尔基网络上的网格蛋白组织。此外，gak缺失的mef在G1/S检查点被阻止，但仍然经历了多轮中心体复制。有趣的是，所有这些表型都被截断的GAK分子所拯救，该分子仅由网格蛋白结合结构域和j结构域组成。此外，这个截断的GAK片段，作为转基因表达，能够挽救当GAK从小鼠的特定组织中被敲除时观察到的致命性。这些结果表明，GAK的网格蛋白结合结构域和j结构域，而不是GAK的激酶和pten样结构域，是其协同伴侣功能所必需的。

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.