Degradation of Endoplasmic Reticulum Chaperones by Chaperone-mediated Autophagy

描述（由申请人提供）：自噬是细胞质量控制的主要机制，因为它负责消除溶酶体中改变的细胞内成分。自噬也有助于维持细胞蛋白质和细胞器的稳态。在这些细胞器中，内质网（ER）在蛋白质生物合成中起着核心作用，并经历巨大的变化以适应细胞对特定蛋白质的需求。内质网中的伴侣蛋白对于保证许多新合成蛋白的正确折叠是至关重要的。这些伴侣的紧密合成调节确保细胞在各种应激和非应激条件下存活。因此，这些伴侣蛋白合成的增加是内质网中蛋白质折叠受损的第一个细胞反应之一，并且导致其上调的机制现在已经得到了很好的表征。然而，相对而言，人们对这些伴侣蛋白的正常转换或它们在应激诱导的翻译上调后的最终命运知之甚少。我们最近发现一种选择性的自噬，被称为伴侣介导的自噬（CMA），可能在内质网伴侣的周转和应激后正常内质网稳态的恢复中发挥核心作用。本项目的目的是阐明内质网伴侣降解的机制，特别强调伴侣介导的自噬（CMA）在其转换中的作用。我们将：1)研究正常基础条件下特异性内质网伴侣蛋白（GRP94， BiP和calreticulin）的降解机制；2)确定这些降解机制是否会在两种应激条件下（内质网应激和饥饿）发生变化；3)研究之前描述的CMA活性随年龄的下降是否会影响内质网伴侣的降解，并可能导致衰老过程中细胞对内质网应激的反应不佳。为此，我们将使用我们实验室开发的生化和基于图像的分析来跟踪CMA对溶酶体中ER伴侣的降解。此外，在培养细胞和啮齿类动物的不同组织中，使用CMA关键成分的遗传操作，我们将确定ER伴侣蛋白正常转换失败的后果。公共卫生相关性：细胞对内质网应激的适应不足已被确定为越来越多的破坏性人类疾病的发病基础，如神经退行性疾病、代谢性疾病如糖尿病和严重肝病等。因此，更好地了解细胞对内质网应激反应的分子机制可能会导致调节这种反应并防止其失败的新方法。

DESCRIPTION (provided by applicant): Autophagy is a major mechanism for cellular quality control, as it is responsible for the elimination of altered intracellular components in lysosomes. Autophagy also contributes to the maintenance of cellular protein and organelle homeostasis. Among these organelles, the endoplasmic reticulum (ER) plays a central role in protein biosynthesis and undergoes dramatic changes to accommodate the cellular demands of particular proteins. Chaperones resident in the ER are critical to assure proper folding of many newly synthesized proteins. Tight synthetic regulation of these chaperones ensures cell survival under a variety of both stress and non-stress conditions. Thus, increased synthesis of these chaperones is one of the first cellular responses to compromised protein folding in the ER, and the mechanisms leading to their upregulation are now well-characterized. However, comparatively little is known about the normal turnover of these chaperones or their eventual fate following their stress-induced translational upregulation. We have recently found that a selective type of autophagy, known as chaperone-mediated autophagy (CMA), may play a central role in the turnover of ER chaperones and in the recovery of normal ER homeostasis after stress. The purpose of this project is to elucidate the mechanism(s) of degradation of ER chaperones with special emphasis on the role of chaperone mediated autophagy (CMA) in their turnover. We will: 1) Examine the mechanisms for degradation of specific ER chaperones (GRP94, BiP and calreticulin) under normal basal conditions; 2) Determine if these mechanisms of degradation change in response to two stress conditions (ER stress and starvation), and 3) examine whether the previously described decline in CMA activity with age affects the degradation of ER chaperones and could contribute to the poor cellular response to ER stress in aging. For this purpose we will use both biochemical and image-based assays developed in our laboratory to track the degradation of ER chaperones in lysosomes by CMA. Furthermore, using genetic manipulations of critical components of CMA, both in cells in culture and in different tissues in rodents we will determine the consequences of failure in proper turnover of ER chaperones. Public Health Relevance: Inadequate cellular adaptation to ER stress has been identified as the pathogenetic basis of a growing list of devastating human disorders such as neurodegenerative disorders, metabolic diseases such as diabetes and severe liver diseases, among others. Consequently, a better understanding of the molecular mechanisms involved in the cellular response to ER stress could lead to novel approaches to modulate this response and to prevent its failure.