70-kda Heat Shock Proteins And Their Associated Cofactors

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一过程中的作用。 我们继续我们的研究内吞作用，检查是什么驱动组装的网格蛋白涂层坑。 我们以前表明，在网格蛋白介导的内吞作用，网格蛋白和AP 2的网格蛋白包被的坑交换与自由网格蛋白和AP 2在细胞中。 为了更好地理解网格蛋白和AP 2交换的机制，在无细胞系统中检查这些过程。 我们发现，Hsc 70不仅需要解离的网格蛋白，但，此外，通过结合到解离的网格蛋白，它也促进了网格蛋白的重新绑定的坑。 至于AP 2的交换，我们发现它是由AP 2的β链的磷酸化和去磷酸化的循环引起的，前者引起AP 2的解离，后者引起AP 2的重新结合。 最后，我们发现，网格蛋白包被的小坑能够通过添加胞质溶胶再生提供PIP 2和AP 2的小核都存在于质膜上。 相反，不可逆的溶解坑发生后，完全解离的AP 2或PIP 2水平的降低。 除了网格蛋白介导的内吞作用的研究外，我们还专注于朊病毒的繁殖，朊病毒是具有淀粉样构象的感染性蛋白质。 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。 通过使用活细胞成像，我们发现非朊病毒形式的Sup 35 p-GFP在PSI-细胞中扩散，而朊病毒形式的Sup 35 p-GFP在PSI+细胞中聚集。 为了测试这些病灶是否是负责维持朊病毒表型的实际种子或繁殖体，我们检查了当使用显性负性Hsp 104突变体通过灭活Hsp 104治愈酵母时，酵母细胞中病灶的数量是否与PSI+治愈的程度相关。 接种后的细胞成像显示，具有病灶的细胞数量与PSI+表型的治愈之间存在直接相关性。 此外，我们重新检查了胍添加后发生的荧光变化，这也通过灭活Hsp 104来治愈PSI+酵母。 与我们以前的研究一致，我们发现在胍的存在下，酵母中没有可见的仍然是PSI+的病灶。 然而，我们现在发现，在应激处理后，这些细胞中的病灶变得可见，并且显示出具有病灶的细胞的百分比与固化程度之间的直接相关性。 因此，使用显性失活的Hsp 104突变体或胍处理灭活Hsp 104来治愈细胞产生的数据与抑制Hsp 104防止种子或繁殖体复制的模型一致，并且细胞分裂是稀释剩余种子或繁殖体以治愈朊病毒表型的酵母所需的。 我们也一直在研究小鼠神经细胞系中朊病毒蛋白的运输，包括天然形式和淀粉样形式的瘙痒症蛋白。 使用RNA干扰使细胞耗尽网格蛋白以确定网格蛋白介导的内吞作用是否是朊病毒蛋白内化所需的。 我们的研究结果表明，组成和铜刺激的朊病毒的内化发生通过网格蛋白的独立，动力蛋白依赖的途径。 最后，为了了解羊瘙痒病的传播，我们一直在研究羊瘙痒病形式的朊病毒蛋白通过内吞途径进入溶酶体的运输

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 this process. We have continued our studies on endocytosis, examining what drives the assembly of the clathrin-coated pit. We previously showed that during, clathrin-mediated endocytosis, both clathrin and AP2 on clathrin-coated pits exchanged with free clathrin and AP2 in cells. To better understand the mechanism of clathrin and AP2 exchange, these processes were examined in a cell-free system. We found that Hsc70 was not only required for dissociation of clathrin but, in addition, by binding to the dissociated clathrin, it also facilitated the rebinding of clathrin to the pits. As for the exchange of AP2, we found that it was caused by a cycle of phosphorylation and dephosphorylation of the beta chain of AP2 with the former causing dissociation and the latter rebinding of AP2. Finally, we found that clathrin-coated pits were able to be regenerated by addition of cytosol provided that both PIP2 and a small nucleus of AP2 were present on the plasma membrane. In contrast, irreversible dissolution of the pits occurred upon complete dissociation of AP2 or a decrease in the PIP2 level. Aside from our research on clathrin-mediated endocytosis, we have focused on the propagation of prions, which are infective proteins having an amyloid conformation. 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. By using live cell imaging, we found that Sup35p-GFP in the non-prion form was diffuse in psi- cells, while Sup35p-GFP in prion-form was aggregated in PSI+ cells. To test whether these foci are the actual seeds or propagons, which are responsible for maintaining the prion phenotype, we examined whether the number of foci in yeast cells correlate with the extent of curing of PSI+ when yeast are cured by inactivation of Hsp104 using a dominant negative Hsp104 mutant. Imaging of the cells followed by plating showed there is a direct correlation between the number of cells with foci and the curing of the PSI+ phenotype. In addition, we re-examined the fluorescence changes that occur upon guanidine addition, which also cure PSI+ yeast by inactivating Hsp104. In agreement with our previous studies, we found in the presence of guanidine, there are no visible foci in yeast that are still PSI+. However, we now find that upon stress treatments, the foci become visible in these cells and show a direct correlation between the percentage of cells having foci and the extent of curing. Therefore, curing cells either using the dominant negative Hsp104 mutant or guanidine treatment to inactivated Hsp104 yield data that are consistent with a model in which inhibiting Hsp104 prevents the seeds or propagons from replicating and cell division is required for dilution of the remaining seeds or propagons to cure yeast of the prion phenotype. We also have been examining the trafficking of prion protein, both the native form and the amyloid form of the protein called scrapie, in mouse neuronal cell lines. Cells were depleted of clathrin using RNA interference to determine whether clathrin-mediated endocytosis was required for the internalization of prion protein. Our results showed that both the constitutive and copper-stimulated internalization of prion occurred via a clathrin-independent, dynamin-dependent pathway. Finally, to understand scrapie propagation, we have been examining the trafficking of the scrapie form of the prion protein through the endocytic pathway to the lysosome