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。我们正在采取的一种方法来理解 这些蛋白质的作用机制是对其进行详细的 热休克蛋白70在脱皮牛体中作用机制的研究 脑部网状蛋白包裹的囊泡和合成的网状蛋白篮子。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。我们现在已经证明了这一点 辅因子是生长素，它是神经细胞中存在的一种次要组装蛋白。 我们对粘菌素的研究表明，它具有有限的区域，这些区域 与DNAJ区域同源，DNAJ是一种已显示的蛋白质辅助因子 参与将底物呈递给E.线圈和HSP70。 高等有机体。我们的数据表明，粘液素可能是 网状蛋白篮子转到热休克蛋白70。相比之下，我们也证明了这一点 根据早期的发现，无论是笼状蛋白轻链还是末端 需要网状蛋白结构域才能去除涂层。这是一个重大的变化 在目前的观点中，HSP70的脱涂层机制。在另一个国家 对去涂层的研究，我们先前发现HSP70导致了最初的 先是爆裂脱膜，然后是缓慢的稳定脱膜。我们现在发现 这种脱壳的时间过程只发生在组装蛋白质的时候 都存在；对于纯络合菊酯篮子，去涂层的时间进程是 线性提示组装蛋白以某种方式参与了 显著降低了稳态脱膜率。我们还有 直接研究DNAJ对HSP70的影响。我们之前发现， DNAJ引起HSP70的聚合。我们现在发现这个 聚合反应发生得比活化要快得多 ATPase活性表明，ATP在聚合后发生了水解。我们的 数据表明，当DNAJ发生聚合和ATP水解时 将一种HSP70呈现给另一种，而不是将底物呈现给 热休克蛋白70。最后，我们之前表达了大量的人类压力 HSP70在大肠杆菌中的表达，发现该重组蛋白可脱壳 包被网状蛋白的囊泡。我们现在已经修改了其中一个关键残留物 在参与三磷酸腺苷结合的ATP结合位点D10 通过镁离子与活性中心结合。我们发现这一修改 将ATP和ADP的结合常数降低约两个数量级 震级。此外，在ATP或ADP结合的情况下，该突变体 酶与无核苷酸的热休克蛋白70具有几乎相同的性质 这表明，通过影响核苷酸结合的能力 热休克蛋白70，D10突变，实际上，转换构象 即使在ATP或ADP仍然存在的情况下，酶也不同于无核苷酸的酶 捆绑在它身上。

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. One approach we are taking to understanding the mechanism of action of these proteins is to carry 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 this cofactor is auxilin, a minor assembly protein present in neuronal cells. Our studies on auxilin suggest that it has limited regions which are homologous to regions of DnaJ, a protein cofactor which has been shown to be involved in presenting substrates to hsp70 both in E. coil and higher organisms. Our data suggests that auxilin may be presenting clathrin baskets to hsp70. We have also demonstrated that, in contrast to earlier findings, neither the clathrin light chains nor the terminal domains of clathrin are required for uncoating. This is a major change in the current view of the mechanism of uncoating by hsp70. In another study on uncoating, we previously found that hsp70 causes an initial burst of uncoating followed by slow steady-state uncoating. We now find that this time course of uncoating only occurs when assembly proteins are present; with pure clathrin baskets the time course of uncoating is linear suggesting that in some way assembly proteins are involved in markedly decreasing the rate of steady-state uncoating. We have also directly studied the effect of DnaJ on hsp70. We previously found that DnaJ causes polymerization of hsp70. We have now found that this polymerization occurs much more rapidly than the activation of the ATPase activity showing that ATP hydrolysis follows polymerization. Our data suggests that polymerization and ATP hydrolysis may occur when DnaJ presents one hsp70 to another rather than presenting a substrate to hsp70. Finally, we previously expressed large amounts of human stress hsp70 in E. coli and found that this recombinant protein uncoats clathrin coated vesicles. We have now modified one of the key residues at the ATP binding site, D10, which is involved in the binding of ATP to the active site through Mg. We have found that this modification reduces the binding constants of both ATP and ADP about two orders of magnitude. Furthermore, with either ATP or ADP bound, this mutant enzyme has almost the same properties as nucleotide-free hsp70 suggesting that, by affecting the ability of nucleotide to bind to hsp70, the D10 mutation, in effect, converts the conformation of the enzyme to that of nucleotide-free enzyme even when ATP or ADP is still bound to it.