70 KDA HEAT SHOCK PROTEINS AND THE HOMOLOGOUS UNCOATING ATPASE

我们的实验室正在研究70 kDa的热休克蛋白， 分子伴侣，即折叠和展开蛋白质在许多 在体内的过程。 我们已经采取了几种方法来理解 这些蛋白质的作用机制。 首先，我们正在进行一项 详细研究了HSP 70在脱膜中的作用机制 牛脑网格蛋白包被的囊泡和合成网格蛋白篮。 我们以前发现，一个100 kDa的蛋白质辅因子是必需的， 去涂层过程发生。 我们已经证明，对于 用各种类型的组装蛋白、HSP 70制备的网格蛋白篮 总是会导致一个独特的时间过程，由快速的 脱包衣初始爆发，随后是缓慢的稳态脱包衣。 我们 也证明了脱涂层的初始爆发需要 100 kDa辅因子的存在以及ATP酶活性的初始爆发 这伴随着最初的爆脱涂层。 的激活 在pH 6下通过网格蛋白篮测定hsp 70，其中未发生去包衣 还需要辅因子的存在。 因此， 热休克蛋白70、辅因子和网格蛋白篮之间的特异性相互作用。 第二，我们发现，新的20 kDa组装蛋白，我们 髓鞘碱性蛋白（MBP）的发现，并已表明， MBP诱导形成网格蛋白篮是特异性的， 与其他碱性蛋白质结合。 到目前为止，MBP是最小的组装 蛋白质，因此它可以帮助我们了解 组装蛋白质的网格蛋白聚合。 第三，我们是 研究聚合的hsp 70的性质;在ATP hsp 70中， 但在ADP中形成二聚体和三聚体。 我们发现 网格蛋白和肽底物仅与单体HSP 70结合，而不与 聚合HSP 70。 因此蛋白质底物改变了平衡 在单体和聚合的hsp 70之间朝向单体hsp 70。 上 另一方面，我们发现催化量的dnaJ，一种辅因子， 在大肠杆菌中与热休克蛋白70相互作用，诱导热休克蛋白70聚合 变成非常大的聚合物。 然而，这种效应只发生在ATP和 在ADP中逆转。 我们还不知道dnaJ的这种作用是如何产生的 这与其向HSP 70呈递各种底物的报道有关。 最后，我们成功地表达了大量的人类压力， hsp 70在大肠杆菌中的表达，已经发现重组蛋白使网格蛋白脱膜， 并正在研究定点突变的效果 这种重组蛋白的生化特性。

Our laboratory is studying the 70-kDa heat shock proteins which act as molecular chaperones, that is, fold and unfold proteins in numerous processes in vivo. We have taken several approaches to understanding the mechanism of action of these proteins. First, we are carrying out a detailed investigation of the mechanism of action of hsp70 in uncoating bovine brain clathrin-coated vesicles and synthetic clathrin baskets. We previously discovered that a 100 kDa protein cofactor is required for the uncoating process to occur. We have now demonstrated that for clathrin baskets prepared with various types of assembly proteins, hsp70 always cause a distinctive time course of uncoating consisting of a rapid initial burst of uncoating followed by slow steady-state uncoating. We have also demonstrated that the initial burst of uncoating requires the presence of 100 kDa cofactor as does the initial burst of ATPase activity which accompanies the initial burst of uncoating. The activation of hsp70 by clathrin baskets at pH 6 where uncoating does not take place also requires the presence of cofactor. Therefore there is a highly specific interaction between hsp70, cofactor and clathrin baskets. Second, we have found that the new 20 kDa assembly protein which we discovered is myelin basic protein (MBP) and have shown that the ability of MBP to induce formation of clathrin baskets is specific and does not occur with other basic proteins. MBP is, by far, the smallest assembly protein yet discovered and therefore it may help us understand the role of assembly proteins in clathrin polymerization. Third, we are investigating the properties of polymerized hsp70; in ATP hsp70 is monomeric but in ADP it forms dimers and trimers. We have found that clathrin and peptide substrates bind only to monomeric hsp70 and not to polymerized hsp70. Therefore protein substrates shift the equilibrium between monomeric and polymerized hsp70 toward monomeric hsp70. On the other hand, we have found that catalytic amounts of dnaJ, a cofactor which interacts with hsp70 in E Coli, induces the polymerization of hsp70 into very large polymers. However, this effect only occurs in ATP and is reversed in ADP. We do not yet understand how this effect of dnaJ is related to reports that it presents various substrates to hsp70. Finally, we have succeeded in expressing large amounts of human stress hsp70 in E Coli, have found that the recombinant protein uncoats clathrin coated vesicles and are studying the effect of site-directed mutagenesis on the biochemical properties of this recombinant protein.