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 作为分子伴侣，也就是参与了ATP依赖的 蛋白质的折叠和展开，蛋白质的形成和溶解 蛋白质复合体，以及蛋白质跨膜转移。 在许多这样的过程中，DNAJ类蛋白质似乎是 参与；这类蛋白质作为辅助因子参与hsc70类 蛋白质显然是向hsc70“呈递”蛋白质底物。然而， 这一陈述的机制尚不清楚。我们已经拿了几个 了解这些蛋白质作用机制的方法。 首先，我们正在进行详细的机制调查。 Hsc70在牛脑蛋白包被小泡脱膜过程中的作用。在……里面 在进行这项研究时，我们发现蛋白质辅因子，即生长素， 是脱膜过程发生所必需的，而粘附素起作用 催化诱导网蛋白篮子与ATP中的hsc70结合。 因此，生长素的作用看起来像是DNAJ同系物，实际上 它的羧基末端有一个DNAJ区。我们也在研究 DNAJ同系物对hsc70的直接作用。我们的结果表明， 几个DNAJ同源物通过以下方式引起ATP水解酶的初始爆发 Hsc70和hsc70在三磷酸腺苷存在下的可逆聚合。 这可能代表民政署署长将一份hsc70提交给另一份 不存在底物的条件。我们目前正在 研究生长素是否与hsc70和hsc70直接相互作用 使其在没有网状蛋白篮子的情况下发生聚合。我们是 还研究了生长素在不寻常的观察中的作用 HSC70只支持在其活性之前进行一轮去涂层 以一种未知的方式强烈抑制。除了这些研究外， 在去包被的情况下，我们表达了一些重组hsc70突变体。 它将ATP或将ATP和ADP绑定几个数量级 比正常的hsc70更弱。它们也不再分解三磷酸腺苷。这些 突变对无核苷酸的特性几乎没有影响 Hsc70；突变的hsc70仍然结合了cathrin triskelion和 各种多肽底物。然而，与之形成对比的是 强结合的ATP和ADP，弱结合的核苷酸没有作用 关于网状蛋白和多肽与hsc70结合的研究。此外，两者 DNaJ同系物的聚合和依赖于生长素的脱膜 网状蛋白被突变完全抑制，这表明 Hsc70涉及DNAJ同系物的作用需要强结合的ATP和 也可能是三磷酸腺苷的水解。我们目前正在将hsc70 突变体、改变的生长素和DNAJ同源物进入哺乳动物细胞以 确定它们在体内对hsc70作用的影响。

Our laboratory is studying the 70-kDa class of heat shock proteins 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 the DnaJ class of proteins appears to be involved; this class of proteins acts as cofactors to the hsc70 class of proteins apparently "presenting" protein substrates to hsc70. However, the mechanism of this presentation is unknown. 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 hsc70 in uncoating bovine brain clathrin-coated vesicles. In carrying out this study we discovered that the protein cofactor, auxilin, is required for the uncoating process to occur, and that auxilin acts catalytically to induce clathrin baskets to bind to hsc70 in ATP. Therefore, auxilin appears to be acting like a DnaJ homolog and indeed it has a DnaJ region at its carboxyl-terminal end. We are also studying the direct effect of DnaJ homologs on hsc70. Our results show that several DnaJ homologs cause both an initial burst of ATP hydrolysis by hsc70 and reversible polymerization of the hsc70 in the presence of ATP. This may represent presentation of one hsc70 to another by the DnaJ under conditions where substrates are not present. We are currently investigating whether auxilin also directly interacts with hsc70 and causes its polymerization in the absence of clathrin baskets. We are also investigating the role of auxilin in the unusual observation that hsc70 supports only one round of uncoating before its activity is strongly inhibited in an unknown manner. In addition to these studies on uncoating, we have expressed a number of recombinant hsc70 mutants which bind either ATP or both ATP and ADP several orders of magnitude more weakly than normal hsc70. They also no longer hydrolyze ATP. These mutations had almost no effect on the properties of nucleotide-free hsc70; the mutated hsc70s still bound both clathrin triskelions and various peptide substrates. However, in contrast to the effects of strongly bound ATP and ADP, the weakly bound nucleotides had no effect on the binding of clathrin and peptides to hsc70. Furthermore, both polymerization by DnaJ homologs and auxilin dependent uncoating of clathrin were completely inhibited by the mutations suggesting that actions of hsc70 involving DnaJ homologs require strongly bound ATP and possibly ATP hydrolysis as well. We are currently transfecting hsc70 mutants, altered auxilin, and DnaJ homologs into mammalian cells to determine their effect on the actions of hsc70 in vivo.