70 KDA HEAT SHOCK PROTEINS AND THEIR ASSOCIATED COFACTORS

我们实验室正在研究70 kDa的热休克蛋白 (Hsp70和Hsc70)作为分子伴侣，即 参与依赖于ATP的蛋白质的折叠和去折叠， 蛋白质复合体的形成和溶解，以及移位 跨膜的蛋白质。在许多这样的过程中， DNAJ类蛋白作为Hsc70蛋白的辅助因子， 显然是向Hsc70“呈递”蛋白质底物。其中一个 Hsc70作用需要DNAJ同源基因的过程是 去包被网状蛋白的囊泡。在研究这一机制时 Hsc70在脱包覆牛脑蛋白囊泡中的作用 发现蛋白质辅因子，即生长素，是 脱膜过程发生了，我们也证明了阿维菌素是一种DNAJ 同源同源。比较生长素与其他DNAJ的直接相互作用 与Hsc70同源，我们发现，正如我们与其他DNAJ所显示的那样 同系物，生长素增加了ATP水解步骤的速度和 导致Hsc70在三磷酸腺苷存在下的可逆聚合。这 后一种效应可能代表DNAJ将一个Hsc70呈现给另一个 在不存在底物的条件下的同系物。然而， 我们还发现，虽然大多数DNAJ同源物与Hsc70结合很弱 在三磷酸腺苷中，生长素结合得更强。我们目前正在 研究哪部分的生长素是造成这种强烈的 与Hsc70结合。我们还在调查哪些比例的粘菌素和 其他DNAJ同源物阻止它们与ADP中的Hsc70相互作用。 在我们对脱膜反应的研究中，我们还发现， 在一轮脱涂层后，某种类型的铝土素络合物， 组装蛋白和可溶性的笼状蛋白三离子形成强烈的 抑制Hsc70进一步脱涂层。我们目前正在调查 这种复合体的性质，可能在生理上作为一种重要的 不在胞浆中聚合的成核复合体 Hsc70的存在，但促进神经中包被的凹陷的形成 执行快速内吞作用的细胞。最后，我们发现， 生长素在CoS细胞和HeLa细胞中的过表达抑制 这些细胞的内吞作用。此效果不依赖于 Hsc70与粘附素的相互作用，因为它也与粘附素一起发生 DNAJ区突变，使其不再与Hsc70相互作用 体外培养。此外，对内吞作用的抑制作用甚至更强。 大脑特有的组装蛋白AP-180。与这些形成对比的是 结果，有报道称，组装蛋白的添加， AP-2对通透性细胞有促进内吞作用的作用。 因此，我们的数据表明，大脑特异的过度表达 正常情况下不表达这些蛋白的细胞中的组装蛋白 可能会抑制内吞作用。

Our laboratory is studying the 70-kDa class of heat shock proteins (Hsp70s and Hsc70s) which act as molecular chaperones, that is, are involved in the ATP-dependent folding and unfolding of proteins, the formation and dissolution of protein complexes, and the translocation of proteins across membranes. In many of these processes members of the DnaJ class of proteins act as cofactors to the Hsc70 proteins, apparently "presenting" protein substrates to Hsc70. One of the processes where a DnaJ homolog is required for Hsc70 action is the uncoating of clathrin-coated vesicles. In studying the mechanism of action of Hsc70 in uncoating bovine brain clathrin-coated vesicles, we discovered that the protein cofactor, auxilin, is required for the uncoating process to occur, and we also showed that auxilin is a DnaJ homolog. In comparing the direct interaction of auxilin and other DnaJ homologs with Hsc70, we found that, as we showed with other DnaJ homologs, auxilin increases the rate of the ATP hydrolysis step and causes reversible polymerization of Hsc70 in the presence of ATP. This latter effect may represent presentation of one Hsc70 to another by DnaJ homologs under conditions where substrates are not present. However, we also found that, while most DnaJ homologs bind quite weakly to Hsc70 in ATP, auxilin binds much more strongly. We are currently investigating what portion of auxilin is responsible for this strong binding to Hsc70. We are also investigating what portion of auxilin and other DnaJ homologs prevents them from interacting with Hsc70 in ADP. In our studies on the uncoating reaction, we have also found that, following one round of uncoating, some type of complex of auxilin, assembly proteins, and soluble clathrin triskelions forms which strongly inhibits further uncoating by Hsc70. We are currently investigating the nature of this complex which may be important physiologically as a nucleating complex that does not polymerize in the cytosol in the presence of Hsc70 but facilitates formation of coated pits in nerve cells which carry out rapid endocytosis. Finally, we have found that overexpression of auxilin in either Cos cells or HeLa cells inhibits endocytosis by these cells. This effect does not depend on the interaction of Hsc70 with auxilin because it also occurs with auxilin mutated in the DnaJ region so that it no longer interacts with Hsc70 in vitro. In addition, an even stronger inhibition of endocytosis occurs with the brain-specific assembly protein, AP-180. In contrast to these results, it has been reported that the addition of the assembly protein, AP-2, to permeabilized cells, increases the rate of endocytosis. Therefore, our data suggest that overexpression of brain-specific assembly proteins in cells which normally do not express these proteins may inhibit endocytosis.