70 KDA HEAT SHOCK PROTEINS AND THEIR ASSOCIATED COFACTORS

我们的实验室正在研究70 kDa的热休克蛋白， 作为分子伴侣，也就是说，参与ATP依赖的 蛋白质的折叠和展开， 蛋白质复合物和蛋白质跨膜转运。 在许多这些过程中，DnaJ类蛋白似乎是 参与;这类蛋白质作为辅助因子的hsc 70类， 蛋白质显然将蛋白质底物“呈递”给HSC 70。 然而，在这方面， 这种表现的机制是未知的。 我们采取了多项 了解这些蛋白质的作用机制的方法。 首先，我们正在进行一个详细的调查机制， HSC 70在未包被的牛脑网格蛋白包被的囊泡中的作用。 在 进行这项研究，我们发现蛋白质辅因子，生长素， 是脱膜过程发生所必需的，而生长素 催化诱导网格蛋白篮结合ATP中的hsc 70。 因此，生长素似乎表现得像DnaJ同系物， 其羧基末端具有DnaJ区。 我们也在研究 DnaJ同源物对hsc 70的直接作用。 我们的结果显示 几种DnaJ同源物引起ATP水解的初始爆发， hsc 70和hsc 70在ATP存在下的可逆聚合。 这可以表示在细胞内DnaJ将一个hsc 70呈递给另一个hsc 70。 不存在底物的条件。 我们目前正在 研究生长素是否也直接与HSC 70相互作用， 在不存在网格蛋白篮的情况下引起其聚合。 我们 同时研究生长素在不寻常的观察中的作用， hsc 70在其活性被破坏之前仅支持一轮去涂层。 以一种未知的方式强烈抑制。 除了这些研究 在未包被的情况下，我们表达了许多重组hsc 70突变体。 其结合ATP或ATP和ADP几个数量级 比正常HSC 70弱。 它们也不再水解ATP。 这些 突变对无核苷酸的性质几乎没有影响， hsc 70;突变的hsc 70仍然结合网格蛋白三骨架， 各种肽底物。 然而，与 强结合ATP和ADP的核苷酸，弱结合的核苷酸没有影响 对网格蛋白和肽与HSC 70结合的影响。 此外，双方 聚合通过DnaJ同系物和生长素依赖性的未包衣的 网格蛋白被突变完全抑制，表明 涉及DnaJ同系物的hsc 70的作用需要强烈结合的ATP， 也可能是ATP水解。 我们目前正在测试HSC 70 突变体、改变的生长素和DnaJ同源物导入哺乳动物细胞， 测定它们对HSC 70在体内的作用的影响。

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.