Age and Sleep Effects on ER Protein Function

描述(申请人提供)：许多与衰老相关的疾病，如阿尔茨海默氏症和帕金森氏症，都以蛋白质错误折叠、堆积和聚集为特征。蛋白质聚集体不会在未受压力的细胞中积累的事实，部分是由于细胞“质量控制”机制的存在。内质网(ER)就是这样一个质量控制系统。它通过确保转录和翻译的保真度，通过陪伴新生或未折叠的蛋白质，以及通过在错误折叠的多肽可以聚集之前选择性地降解它们，来抑制聚集的形成。内质网进化出了高度特异的信号通路，以确保其蛋白质折叠能力不会被淹没。这些途径统称为未折叠蛋白反应(UPR)，是细胞在内质网应激中生存所必需的。如果这种平衡被破坏，内质网应激反应就会被诱导；伴随着蛋白质合成的减弱，伴侣蛋白会上调。这一过程被视为具有细胞保护作用。我们在睡眠剥夺中看到了这一点的证据：在短短6小时的睡眠剥夺中，我们看到了内质网应激反应通路的诱导。虽然目前还不清楚睡眠剥夺中的哪些因素会引发内质网应激样反应，但已知某些刺激，如葡萄糖/能量降低、钙水平的扰动和活性氧自由基(ROS)等，都会诱导内质网应激。众所周知，内质网中蛋白质的积累和钙稳态的紊乱导致ROS增加和核因子？B的激活。显而易见的是，几个因素可以诱导UPR，导致整体蛋白质表达减少，如果压力很严重，如果压力没有得到适当的缓解，氧化损伤和可能的蛋白质聚集。这些蛋白质的变化是否发生在睡眠剥夺期间目前尚不清楚，将在这个项目中进行评估。年龄对内质网应激反应和UPR的影响尚不清楚。随着年龄的增长，内质网伴侣在小鼠肝脏中的氧化程度越来越高，这表明可能存在受损的内质网应激反应，因为有缺陷的伴侣分子将影响伴侣系统。此外，由于这些伴侣是抗应激机制的关键参与者，因此很可能会增加对细胞应激的脆弱性。因此，老化的大脑对睡眠不足的这一方面的反应可能会发生变化。这一建议的总体假设是，睡眠不足会导致内质网中蛋白质的变化，并且这种变化的幅度与年龄有关。为了解决这一全球假说，我们有以下具体目标：1)我们认为睡眠剥夺会导致内质网蛋白质表达减少，并导致蛋白质子集的氧化改变；2)我们假设睡眠剥夺的这种影响在老年动物中发生了改变，尤其是更多的氧化改变的证据。我们将使用2-D DGE，以及氧化分析和质谱分析，来识别内质网中因衰老和睡眠剥夺而改变的蛋白质。

DESCRIPTION (provided by applicant): Many aging related diseases such as Alzheimer's, and Parkinson's are characterized by protein misfolding, accumulation and aggregation. The fact that protein aggregates do not accumulate in unstressed cells is due in part to the existence of cellular "quality control" machinery. One such quality control system is the endoplasmic reticulum (ER). It suppresses the formation of aggregates by ensuring fidelity of transcription and translation, by chaperoning nascent or unfolded proteins, and by selectively degrading improperly folded polypeptides before they can aggregate. The ER has evolved highly specific signaling pathways to ensure that its protein folding capacity is not overwhelmed. These pathways collectively called the unfolded protein response (UPR) are required if the cell is to survive the ER stress. If this equilibrium is disturbed the ER stress response is induced; there is an up regulation of chaperones with attenuation of protein synthesis. This process is seen as cytoprotective. We see evidence of this during sleep deprivation: with as little as 6 hours of sleep deprivation we see an induction of the ER stress response pathway. While it is not clear what in sleep deprivation would induce an ER stress-like response, it is known that certain stimuli such as reduced glucose/energy, perturbations in Ca 2+ levels, and reactive oxygen species (ROS) induce ER stress. It is also known that protein accumulation in the ER, and disturbances in calcium homeostasis lead to an increase in ROS and activation of NF?B. What is evident is that several factors can induce the UPR, resulting in decreased global protein expression, and if the stress is severe, oxidative damage and possibly aggregation of proteins if the stress is not suitably relieved. Whether these changes in protein occur during sleep deprivation is currently unknown and will be assessed in this project. It is not known what the effect of age is on the ER stress response and the UPR. ER chaperones are increasingly oxidized in mouse liver with age suggesting that there may be an impaired ER stress response, as defective chaperone molecules will impact on the chaperoning system. Further, there is likely to be increased vulnerability to cellular stress as these chaperones are key participants in an anti-stress mechanism. Thus, there are likely to be alterations in the aging brain to this aspect of the response to sleep deprivation. The overall hypothesis of this proposal is that sleep deprivation leads to alterations in proteins in the endoplasmic reticulum and that the magnitude of this change is age-dependent. To address this global hypothesis, we have the following specific aims: 1) We propose that sleep deprivation results in decreased protein expression in the ER and oxidative change to a subset of proteins and 2) We hypothesize that this effect of sleep deprivation is altered in older animals with in particular more evidence of oxidative change. We will use 2-D DIGE, as well as oxidation assays and mass spectrometry, to identify proteins that are altered by aging and sleep deprivation in the ER.