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，如阿尔茨海默病、帕金森病，其特征是蛋白质错误折叠、积累和聚集。蛋白质聚集体不会在非应激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，以及在聚合之前选择性地降解不正确折叠的多肽来抑制聚集体的形成。内质网已经进化出高度特异性的信号通路，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下生存所必需的。如果这种平衡被打乱，内质网应力反应就会被诱发；伴随着蛋白质合成的衰减，伴侣蛋白的表达出现了上调。这一过程被视为细胞保护作用。我们在睡眠剥夺中看到了这一点的证据：即使只有6小时的睡眠剥夺，我们也能看到内质网应激反应途径的诱导。虽然目前尚不清楚睡眠剥夺中哪些因素会诱发内质网应激样反应，但已知某些刺激如葡萄糖/能量降低、ca2 +水平扰动和活性氧（ROS）诱导内质网应激。我们也知道内质网中的蛋白质积累和钙稳态的紊乱会导致ROS的增加和NF - B的激活。显而易见的是，有几个因素可以诱导UPR，导致整体蛋白表达下降，如果应激严重，则氧化损伤，如果应激不适当缓解，可能会导致蛋白质聚集。这些蛋白质的变化是否在睡眠剥夺期间发生目前尚不清楚，将在本项目中进行评估。目前尚不清楚年龄对内质网应激反应和UPR的影响。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，这表明内质网应激反应可能受损，因为有缺陷的伴侣分子会影响伴侣系统。此外，由于这些伴侣是抗应激机制的关键参与者，因此可能会增加对细胞应激的脆弱性。因此，衰老的大脑对睡眠剥夺的这一方面的反应可能会发生变化。这一提议的总体假设是，睡眠剥夺导致内质网蛋白质的改变，这种变化的幅度与年龄有关。为了解决这一全球假设，我们有以下具体目标：1)我们提出睡眠剥夺导致内质网蛋白质表达减少和一部分蛋白质的氧化变化；2)我们假设睡眠剥夺的这种影响在老年动物中发生改变，特别是有更多证据表明氧化变化。我们将使用二维DIGE，以及氧化测定和质谱法，来识别内质网中因衰老和睡眠不足而改变的蛋白质。

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.