70-kda Heat Shock Proteins And Their Associated Cofactors

我们的实验室对细胞中正常和病理性蛋白质复合物的形成和溶解感兴趣，重点是分子伴侣在这一过程中的作用。 我们继续我们的研究HSC 70在网格蛋白介导的内吞作用的作用，产生敲除小鼠的HSC 70 cochaperones，神经元特异性生长素和普遍表达的GAK。 这些分子伴侣将网格蛋白呈递给Hsc 70，因此在从网格蛋白包被的囊泡中揭开网格蛋白和在胞质溶胶中陪伴网格蛋白中具有主要作用。 为了敲除GAK，我们必须制造条件性敲除小鼠，因为常规敲除是胚胎致死的。 我们的GAK基因敲除研究表明，GAK是一个重要的基因在发育和成年小鼠。 至于神经元特异性生长素，新生敲除小鼠的死亡率相对较高，存活的幼崽通常比野生型幼崽体重低。 存活的小鼠有正常的寿命，但它们可能只是因为GAK在存活的新生和成年生长素基因敲除小鼠的大脑中上调了3倍。 令人惊讶的是，GAK的正常水平不能补偿生长素的缺乏，如通过观察所示，网格蛋白介导的内吞作用在敲除小鼠的海马神经元中被抑制。 在原位和原代神经元培养物中，在敲除突触处存在数量增加的网格蛋白包被的囊泡和空笼。 这些结果表明，生长素在大脑中回收突触囊泡中具有专门的作用，这只能通过小鼠神经元中GAK水平的显著上调来补偿 小鼠胚胎成纤维细胞（MEF）来源于该条件性GAK敲除小鼠，并且通过使用表达Cre重组酶的腺病毒破坏MEF中的GAK。 敲除GAK，完全阻断MEFs中网格蛋白介导的内吞作用。 此外，MEFs的网格蛋白组织被完全破坏，从质膜和trans-Golgi网络的网格蛋白的损失。 为了补偿网格蛋白介导的内吞作用的损失，液相摄取显著增加。 有趣的是，在没有GAK的情况下，细胞分裂被阻止，但MEFs仍然可以存活数周。 这些结果表明，GAK的不存在以及反过来Hsc 70在胞质溶胶中的网格蛋白的伴侣作用的缺乏对细胞中的多个系统具有深远的影响。 除了网格蛋白介导的内吞作用，我们还研究了朊病毒的繁殖，朊病毒是一种感染性蛋白质，可以在酵母和哺乳动物细胞中错误折叠成淀粉样蛋白构象。 在酵母中，分子伴侣Hsp 104调节包括PSI+在内的几种酵母朊病毒的遗传，PSI+是翻译终止因子Sup 35 p的朊病毒形式。 通过活细胞成像，我们发现Hsp 104不仅切断朊病毒种子，而且修剪或减小种子的大小。 我们发现，当Hsp 104被胍中生长的酵母灭活时，种子变得更小，因为Hsp 104仍然能够修剪它们。 然而，它不能切断它们，种子数量减少，朊病毒固化发生。 然而，当通过表达显性负性Hsp 104突变体使Hsp 104完全失活时，Hsp 104不再修剪或切断种子，因此随着熟化的发生，种子的大小增加，数量减少。 因此，当酵母分裂时，Hsp 104既修剪种子以保持它们的稳态大小，又切断它们以保持种子的稳态数量。

Our laboratory is interested in the formation and dissolution of both normal and pathological protein complexes in the cell with an emphasis on the role of molecular chaperones in this process. We have continued our studies on the role of Hsc70 in clathrin-mediated endocytosis by generating knockout mice of the HSC70 cochaperones, neuronal-specific auxilin and ubiquitously expressed GAK. These chaperones present clathrin to Hsc70 and therefore have a major role in uncoating clathrin from clathrin coated vesicles and chaperoning clathrin in the cytosol. To knock-out GAK, we had to make a conditional knock-out mouse since the conventional knock-out was embryonic lethal. Our GAK knockout studies showed that GAK was an essential gene during development and in adult mice. As for neuronal-specific auxilin, the newborn knockout mice had relatively high mortality and surviving pups generally had a lower body weight than the wild-type pups. The surviving mice had a normal life-span, but they probably survived only because GAK was up-regulated as much as 3-fold in the brains of both surviving neonate and adult auxilin knockout mice. Surprisingly, normal levels of GAK could not compensate for the absence of auxilin as shown by the observation that clathrin-mediated endocytosis was inhibited in the hippocampal neurons of the knockout mice. An increased number of clathrin-coated vesicles and empty cages were present at the knockout synapses both in situ and in primary neuronal cultures. These results show that auxilin has a specialized role in recycling synaptic vesicles in the brain, which can only be compensated for by marked up-regulation of GAK levels in mouse neurons Mouse embryonic fibroblasts (MEFs) were derived from this conditional GAK knockout mouse and GAK was disrupted in the MEFs by using adenovirus expressing Cre recombinase. Knocking-out GAK, completely blocked clathrin-mediated endocytosis in the MEFs. In addition, the clathrin organization of the MEFs was totally disrupted with loss of clathrin from both the plasma membrane and trans-Golgi network. To compensate for the loss of clathrin-mediated endocytosis, there was a marked increase in fluid phase uptake. Interestingly, in the absence of GAK, cell division was arrested, but the MEFs were still viable for several weeks. These results show that the absence of GAK and in turn the lack of chaperoning of clathrin in the cytosol by Hsc70 had a profound effect on multiple systems in the cell. In addition to our research on clathrin-mediated endocytosis, we also studied the propagation of prions, infective proteins that can misfold into an amyloid conformation both in yeast and mammalian cells. In yeast, the molecular chaperone, Hsp104, regulates the inheritance of several yeast prions including PSI+, which is the prion form of the translation termination factor Sup35p. By using live cell imaging, we found that Hsp104 not only severs prion seeds, but trims or reduces the size of the seeds. We found that when Hsp104 was inactivated by growing yeast in guanidine, the seeds became smaller because Hsp104 was still able to trim them. However, it could not sever them and the number of seeds decreases and prion curing occurred. However, when Hsp104 was completely inactivated by expressing a dominant negative Hsp104 mutant, Hsp104 no longer trimmed or severed the seeds and therefore the seeds both increased in size and decreased in number as curing occurred. Therefore, Hsp104 both trims the seed to maintain their steady-state size and severs them to maintain the steady-state number of seeds as the yeast divides.