70-kda Heat Shock Proteins And Their Associated Cofactors

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一过程中的作用。特别是，我们正在研究无处不在的分子伴侣Hsc70和诱导特定底物与Hsc70结合的j结构域辅因子蛋白。在我们之前的工作中，我们研究了Hsc70在网格蛋白介导的内吞作用中的作用，特别是它从网格蛋白包被的囊泡中分离网格蛋白的能力。我们首先发现，脱膜不仅需要Hsc70，还需要100 kDa的神经特异性j结构域蛋白auxilin或auxilin的非神经元同源物，150 kDa的蛋白GAK，与auxilin相似，但也含有n端激酶结构域。然后我们发现，在体内，网格蛋白包被的坑是动态结构，并且网格蛋白和网格蛋白包被的坑的其他成分，包括网格蛋白介导的内吞过程中网格蛋白接头蛋白AP2的交换。同样，反式高尔基网络上的网格蛋白和网格蛋白接头蛋白AP1与胞浆中的自由网格蛋白和AP1交换。从我们的数据中我们得出结论，网格蛋白交换是网格蛋白结构重排所必需的，当网格蛋白包覆的凹坑发生内陷时。然后，我们通过渗透细胞证明，Hsc70不仅在网格蛋白包被的囊泡脱落后解离网格蛋白，而且在质膜上网格蛋白包被的凹坑或TGN上网格蛋白包被的芽内陷时发生的网格蛋白交换也需要Hsc70。

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. In particular we are studying the ubiquitous molecular chaperone Hsc70 and the J-domain cofactor proteins that induce specific substrates to bind to Hsc70. In our previous work we have studied the role of Hsc70 in clathrin-mediated endocytosis, in particular its ability to dissociate clathrin from clathrin-coated vesicles. We first discovered that uncoating not only requires Hsc70 but also the 100 kDa nerve-specific J-domain protein auxilin or the non-neuronal homolog of auxilin, the 150 kDa protein GAK that is similar to auxilin but also contains an N-terminal kinase domain. We then showed that in vivo clathrin-coated pits are dynamic structures and both clathrin and other components of clathrin-coated pits including the clathrin adaptor protein AP2 exchange during clathrin-mediated endocytosis. Similarly, clathrin and the clathrin adaptor protein AP1 on the trans-Golgi network exchanges with free clathrin and AP1 in the cytosol. From our data we concluded that clathrin exchange is required for the structural rearrangement of clathrin that occurs as clathrin-coated pits invaginate. We then showed using permeabilized cells that Hsc70 not only dissociates clathrin after clathrin-coated vesicles bud off but is also required for the clathrin exchange that occurs during invagination of clathrin-coated pits on the plasma membrane or clathrin-coated buds on the TGN. During the past year our laboratory has characterized mouse embryonic fibroblasts derived from GAK K/O mice. When we examined transferrin uptake in the cre-treated MEFs we found that transferrin uptake was almost completely blocked. Consistent with these results, the transferrin receptor had a diffusive appearance over the plasma membrane with no internalized pool of transferrin visible. When we then examined the status of the CCPs in the cell, we found that there were very few clathrin puncta on the plasma membrane, while the AP-2 had an aberrant distribution, appearing clustered, along with epsin, eps15 and dynamin. When the MEFs were transfected with GFP-clathrin, the few remaining clathrin puncta on the plasma membrane showed no clathrin exchange. Therefore, these results show that deletion of GAK causes a profound disruption in clathrin-mediated endocytosis, which is most likely the cause of the lethality that we observe in developing and mature mice in which GAK has been knocked out in specific tissues. We have also carried out studies on primary neurons cultured from auxilin knock-out mice. Western blots showed that these neurons had a marked increase in clathrin and in agreement with these results, light microscopy showed an increase in clathrin structures in the cell. More specifically, electron microscopy studies showed a marked increase in clathrin-coated vesicles in the cells. These results are consistent with auxilin being required for rapid uncoating of clathrin-coated vesicles in the neurons; apparently even in the presence of GAK, auxilin is required for this process. Apparently, in the absence of auxilin, the cell compensates for the clathrin trapped in clathrin-coated vesicles by increasing the total amount of clathrin in the cell. In a related study, on the mechanism of formation of clathrin-coated pits in cells, we have investigated the interaction of clathrin with GGAs, a class of monomeric clathrin adaptors involved in the sorting of cargo at the trans-Golgi network of eukaryotic cells. Previous studies have shown that GGAs interact with clathrin both in solution and in the cell, but it has not yet been shown whether they assemble clathrin. We find that GGA1 promoted assembly of clathrin with complete assembly achieved when one GGA1 molecule is bound per heavy chain. In the presence of excess GGA1, we obtained the unusual stoichiometry of five GGA1s per heavy chain, and even at this stoichiometry the binding was not saturated. The assembled structures were mostly baskets, but approximately 10% of the structures were tubular with an average length of 180 +/- 40 nm and width of approximately 50 nm. From these results we conclude that the clathrin adaptor GGA1 is a clathrin assembly protein unique in its ability to polymerize clathrin into tubules. Finally, we have studied the effect of normal cellular prion protein (PrP(C)) on abnormal protein aggregation by transfecting huntingtin fragments (Htt) into SN56 neuronal-derived cells depleted of PrP(C) by RNA interference. PrP(C) depletion caused an increase in both the number of cells containing granules and the number of apoptotic cells. Consistent with the increase in Htt aggregation, PrP(C) depletion caused an decrease in proteasome activity and a decrease in the activities of cellular defense enzymes compared with control cells whereas reactive oxygen species (ROS) increased more than threefold. Therefore, PrP(C) may protect against Htt toxicity in neuronal cells by increasing cellular defense proteins, decreasing ROS and increasing proteasome activity thereby increasing Htt degradation. Depletion of endogenous PrP(C) in non-neuronal Caco-2 and HT-29 cells did not affect ROS levels or proteasome activity suggesting that only in neuronal cells does PrP(C) confer protection against Htt toxicity. The protective effect of PrP(C) was further evident in that overexpression of mouse PrP(C) in SN56 cells transfected with Htt caused a decrease in both the number of cells with Htt granules and the number of apoptotic cells, whereas there was no effect of PrP(C) expression in non-neuronal NIH3T3 or CHO cells. Finally, in chronically scrapie (PrP(Sc))-infected cells, ROS increased more than twofold while proteasome activity was decreased compared to control cells. Although this could be a direct effect of PrP(Sc), it is also possible that, since PrP(C) specifically prevents pathological protein aggregation in neuronal cells, partial loss of PrP(C) itself increases PrP(Sc) aggregation.