70-kda Heat Shock Proteins And Their Associated Cofactor

我们的实验室对细胞中正常和病理蛋白质复合体的形成和溶解感兴趣，并强调分子伴侣在这一过程中的作用。特别是，我们正在研究普遍存在的分子伴侣Hsc70和J-结构域辅助因子蛋白，它们可以诱导特定的底物与Hsc70结合。在我们以前的工作中，我们研究了Hsc70在网状蛋白介导的内吞作用中的作用，特别是它从网状蛋白包裹的小泡中解离网状蛋白的能力。我们首先发现，去涂层不仅需要Hsc70，还需要100 kDa神经特异的J结构域蛋白Axlin或Axin的非神经元同源物，150 kDa的蛋白GAK类似于Axin，但还包含一个N-末端的激酶结构域。然后我们发现，在体内，包被网状蛋白的凹坑是动态的结构，并且在网状蛋白介导的内吞作用中，网状蛋白和其他包被网状蛋白的成分，包括网状蛋白适配器蛋白AP2交换。类似地，跨高尔基体网络上的网状蛋白和网状蛋白接头蛋白AP1与胞浆中的游离网状蛋白和AP1交换。根据我们的数据，我们得出结论，在以分子筛包被的凹陷形式发生的分子筛的结构重排中，分子筛交换是必需的。然后，我们使用通透性细胞表明，Hsc70不仅在包被包络蛋白的小泡萌发后解离包被包被的小泡，而且在质膜上包被的小凹或TGN上包被包被的包被的芽的内陷过程中发生的包被的小泡在TGN上内陷的过程中，Hsc70也是必需的。 在过去的一年里，我们使用全内反射显微镜来确定在网状蛋白包被的凹陷过程中，GAK结合相对于动力蛋白和网状蛋白结合的时间。在动力素短暂地募集到网状蛋白斑点之后，大量的GAK被瞬时募集。然后，随着凹坑从质膜内陷，Gak和clathrin从消失场中消失，最后这些蛋白质从荧光发射场中消失，可能是因为Hsc70将clathrin从萌发的小泡中去掉。GAK的募集依赖于其PTEN样结构域，我们发现该结构域与磷脂结合。这表明与磷脂的相互作用对于GAK和Hsc70的募集是必不可少的，但仅有Hsc70的募集可能不足以导致不可逆转的clathrin脱涂层。当肌动蛋白解聚抑制包被网孔的萌发时，包被网孔上有GAK的反复闪烁，但没有出现断裂和不可逆脱包现象。因此，不可逆的网状蛋白脱壳可能需要GAK的萌发和同步补充。 在过去的一年里，在动物水平的进一步研究中，我们继续对生长素和GAK基因敲除小鼠的研究。我们之前已经确定，在胚胎第10天GAK被有条件地敲除神经细胞的小鼠表现出大脑的主要发育缺陷，并在出生后不久死亡。我们现在发现，当GAK有条件地从肝脏或皮肤细胞中剔除时，小鼠也会在出生后不久死亡。对肝脏和皮肤的组织分析都显示出严重的发育缺陷，这些缺陷发生在组织中，就像在大脑中发生的那样。此外，当成年动物被他莫昔芬有条件地敲除GAK时，小鼠在治疗后的几天内死亡。因此，GAK对于成年动物的生存和发育都是必需的。

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. In particular we are studying the ubiquitous molecular chaperone Hsc70 and the J-domain cofactor proteins that induce specific substrates to bind to Hsc70. In our previous work we have studied the role of Hsc70 in clathrin-mediated endocytosis, in particular its ability to dissociate clathrin from clathrin-coated vesicles. We first discovered that uncoating not only requires Hsc70 but also the 100 kDa nerve-specific J-domain protein auxilin or the non-neuronal homolog of auxilin, the 150 kDa protein GAK that is similar to auxilin but also contains an N-terminal kinase domain. We then showed that in vivo clathrin-coated pits are dynamic structures and both clathrin and other components of clathrin-coated pits including the clathrin adaptor protein AP2 exchange during clathrin-mediated endocytosis. Similarly, clathrin and the clathrin adaptor protein AP1 on the trans-Golgi network exchanges with free clathrin and AP1 in the cytosol. From our data we concluded that clathrin exchange is required for the structural rearrangement of clathrin that occurs as clathrin-coated pits invaginate. We then showed using permeabilized cells that Hsc70 not only dissociates clathrin after clathrin-coated vesicles bud off but is also required for the clathrin exchange that occurs during invagination of clathrin-coated pits on the plasma membrane or clathrin-coated buds on the TGN. During the past year we used total internal reflectance microscopy to determine the timing of GAK binding relative to dynamin and clathrin binding during invagination of clathrin-coated pits. Following transient recruitment of dynamin to the clathrin puncta, large amounts of GAK are transiently recruited. GAK and clathrin then disappear from the evanescent field as the pit invaginates from the plasma membrane and finally these proteins disappear from the epifluorescence field, probably as the clathrin is uncoated from the budded vesicles by Hsc70. The recruitment of GAK is dependent on its PTEN-like domain, which we found binds to phospholipids. This suggests that interaction with phospholipids is essential for recruitment of GAK and, in turn, Hsc70, but Hsc70 recruitment alone might not be sufficient to induce irreversible clathrin uncoating. When budding of clathrin-coated pits was inhibited by actin depolymerization, there was repeated flashing of GAK on the clathrin-coated pit but neither scission nor irreversible uncoating occur. Therefore, budding as well as synchronous recruitment of GAK might be required for irreversible clathrin uncoating. In further studies at the animal level during the past year, we continued our studies on the auxilin and GAK knock-out mice. We had previously determined that mice in which GAK is conditionally knocked-out of neuronal cells at embryonic day ten show major developmental defects in the brain and die shortly after birth. We have now found that the mice also die shortly after birth when GAK is conditionally knocked-out of liver or skin cells. Histological analysis of both the liver and the skin show profound developmental defects these in tissues just as occurred in the brain. Furthermore, when the adult animal is treated with tamoxifen to conditionally knock-out the GAK, the mice die within a few days of the treatment. Therefore GAK is required for viability of the adult animal as well as in development.