70-kda Heat Shock Proteins and Associated Cofactors

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RNA干扰来耗尽GAK的细胞，这些研究以及对通透性细胞的进一步研究表明，Hsc70和GAK不仅通过解离笼蛋白而引起交换，还通过陪伴它并促进其与凹坑的重新结合来引起交换。令人惊讶的是，这些研究还表明，Hsc70直接招募与细胞膜和跨高尔基体网络的结合，而不依赖于它对clathrin的影响。在过去的一年里，在动物水平的进一步研究中，我们继续对我们的生长素和GAK基因敲除小鼠进行研究。我们现在可以肯定的是，阿司匹林基因敲除的小鼠减少了活产、较小的初始出生体重和较低的产仔数。此外，我们发现，当大脑中GAK的产生高度上调时，这些影响会得到改善，这种影响显然是在一些小鼠身上自然发生的。由于GAK和生长素通常在大脑中的含量几乎相等，因此似乎需要相对大量的GAK来克服大脑中的生长素的损失。类似地，我们发现GAK在第10天被有条件地敲除神经细胞的小鼠表现出高度异常的大脑发育，并在出生后不久死亡，尽管这些细胞中存在生长素。因此，我们发现生长素和GAK是不可互换的。相反，每一个都在神经细胞中扮演着重要而独立的角色。 在过去的一年里，我们还开始了用绿色荧光蛋白标记的酵母和哺乳动物蛋白的研究，以研究它们的运输和聚集。特别是，在我们对酵母Pron的研究中，我们使用了荧光光漂白来跟踪融合到GFP上的酵母Pron蛋白Sup35p的聚集。令人惊讶的是，与目前的教条相反，我们发现，在分子伴侣Hsp104被灭活后，不需要细胞分裂来稀释剩余的Sup35p普恩。相反，即使在未分裂的细胞中也会发生聚集的Sup35p的丢失，这可能是因为灭活的Hsp104不能持续促进新的Sup35p普恩的形成。我们目前正在调查这种意想不到的现象是否也发生在其他酵母蛋白上。

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 RNA interference to deplete cells of GAK and these studies along with further studies on permeabilized cells showed that Hsc70 and GAK not only cause exchange by dissociating clathrin but also by chaperoning it and facilitating its rebinding to pits. Surprisingly, these studies also showed that Hsc70 directly recruits binding of clathrin adaptors to both the plasma membrane and the trans-Golgi network independent of its effects on clathrin. In further studies at the animal level during the past year, we continued our studies on our auxilin and GAK knock-out mice. We are now certain that auxilin knock-out mice have decreased live births, smaller initial birth weights, and lower litter sizes. Furthermore, we find that these effects are ameliorated when GAK production is highly up-regulated in the brain, an effect that apparently occurs naturally in some of the mice. Since GAK and auxilin are normally present in nearly equal amounts in the brain, it appears that relatively large amounts of GAK are needed to overcome loss of auxilin from the brain. Similarly, we have found that mice in which GAK is conditionally knocked-out of neuronal cells at day 10 pc show highly abnormal brain development and die shortly after birth despite the presence of auxilin in these cells. Therefore we find that auxilin and GAK are not interchangeable. Rather each plays an important and independent role in nerve cells. During the past year we also began to work on both yeast and mammalian prion proteins labeled with GFP to study their trafficking and aggregation. In particular in our studies on yeast prion we used fluorescence photobleaching to follow aggregation of the yeast prion protein Sup35p fused to GFP. Surprisingly, in contrast to current dogma, we found that, after the molecular chaperone Hsp104 is inactivated, which is known to prevent prion propagation, cell division is not needed to dilute out the remaining Sup35p prion. Rather, loss of aggregated Sup35p occurs even in non-dividing cells, perhaps because inactivated Hsp104 does not continuously facilitate formation of new Sup35p prion. We are currently investigating whether this unexpected phenomenon also occurs with other yeast prions.