70 KDA HEAT SHOCK PROTEINS AND THE HOMOLOGOUS UNCOATING ATPASE

我们实验室正在研究70 kDa的热休克蛋白，它可以作为 分子伴侣，也就是折叠和解开许多 活体内的过程。我们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 HSP70在脱膜过程中作用机理的详细研究 牛脑笼状蛋白包裹的囊泡和合成的笼状蛋白篮子。 我们先前发现，100 kDa的蛋白质辅因子需要 脱膜过程将会发生。我们现在已经演示了这一点 用不同类型组装蛋白HSP70制备的笼状蛋白篮子 总是导致脱膜的独特时间过程，包括快速 先是脱膜，然后是缓慢的稳定脱膜。我们 还证明了最初的脱涂层需要 100 kDa辅因子的存在与ATPase活性的最初爆发一样 它伴随着最初的脱涂层爆发。激活 在pH为6的条件下，在不脱涂层的情况下，通过网壳蛋白篮子生成HSP70 也需要辅因子的存在。因此，有一个高度的 HSP70、辅因子和网状蛋白篮子之间的特异性相互作用。 第二，我们发现我们发现的新的20 kDa组装蛋白 发现的是髓鞘碱性蛋白(MBP)，并已显示出 MBP诱导形成网状蛋白篮子的作用是特异的，并且不 与其他碱性蛋白质一起发生。MBP是目前为止最小的程序集 蛋白质尚未被发现，因此它可能有助于我们理解 在网状蛋白聚合中的组装蛋白。第三，我们是 ATPHSP70中聚合HSP70的性质研究 单体，但在ADP中它形成二聚体和三聚体。我们发现， 网状蛋白和多肽底物只与单体HSP70结合，而不与单体HSP70结合 聚合的热休克蛋白70。因此，蛋白质底物改变了平衡。 单体和聚合的HSP70之间向单体HSP70转变。论 另一方面，我们发现辅因子dna J的催化量 它与大肠杆菌中的HSP70相互作用，诱导HSP70聚合 变成非常大的聚合物。然而，这种影响只发生在ATP和 在ADP中则相反。我们还不知道dna j的这种影响是如何产生的。 与它向HSP70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 E-Coli中的HSP70，发现重组蛋白去包裹了笼状蛋白 并正在研究定点突变的效果 对该重组蛋白的生化性质的研究。

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.