70-kda Heat Shock Proteins And Their Associated Cofactors

我们的实验室对细胞中正常和病理蛋白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。我们继续研究Hsc70在网格蛋白介导的内吞作用，通过产生敲除Hsc70的合作伙伴、神经元特异性辅助素和无处不在表达的GAK的小鼠。这些伴侣蛋白将网格蛋白呈现给Hsc70，因此在将网格蛋白从包被网格蛋白的囊泡中剥离和在细胞质中陪伴网格蛋白中起主要作用。为了敲除GAK，我们必须制造条件敲除小鼠，因为传统的敲除是胚胎致死的。我们的GAK敲除研究表明，GAK在发育和成年小鼠中是必不可少的基因。对于神经元特异性辅助素，新生基因敲除小鼠死亡率较高，存活幼鼠体重普遍低于野生型幼鼠。存活下来的小鼠寿命正常，但它们存活下来可能只是因为GAK在存活的新生儿和成年抗氧化素敲除小鼠的大脑中上调了3倍。令人惊讶的是，正常水平的GAK不能弥补辅助素的缺失，这是由观察到的，在敲除小鼠的海马神经元中，网格蛋白介导的内吞作用被抑制。在原位和原代培养的神经细胞中，敲除突触上都存在网格蛋白包被的囊泡和空笼的数量增加。这些结果表明，辅助素在大脑突触囊泡的循环中具有特殊作用，这只能通过小鼠神经元中GAK水平的显著上调来补偿

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.