摘要
The endoplasmic reticulum (ER) serves as the entry point to the secretory system where nearly one-third of the cellular proteome must undergo synthesis, folding, and maturation events before being deployed. Proteins that fail to successfully navigate these processes and achieve their native conformation are detained by endoplasmic reticulum-associated degradation (ERAD), a quality-control mechanism responsible for targeting misfolded proteins for degradation by the cytosolic 26S proteasome4. Recent studies have demonstrated that treatment with the long chain acyl-CoA synthetase inhibitor triacsin c disrupts lipid droplet (LD) biogenesis and ERAD, suggesting a functional connection between the processes. However, whether LDs are involved in ERAD remains an outstanding question.
LDs are highly dynamic neutral lipid storage organelles that function as central hubs of lipid metabolism charged with storing lipids and maintaining energy homeostasis of the cell. The specific metabolic role of LDs is dictated by the cell type and the metabolic state of the cell, which can fluctuate in response to a number of cellular stimuli. LD functions are regulated by a complement of integral and peripheral proteins that associate with the bounding LD phospholipid monolayer. The ability to define a high-confidence LD proteome is paramount to understanding LD functions and dynamics. However, accurate analysis of the LD proteome composition has remained a challenge due to the presence of contaminating proteins in LD-enriched buoyant fractions.
In chapter one, we discuss the connection between protein and lipid regulatory systems within the ER and LDs, highlighting the importance of ERAD and lipophagy in maintaining cellular homeostasis. In chapter two, we use chemical and genetic approaches to disrupt LD biogenesis to explore a potential role for LDs in ERAD, ultimately providing evidence that LDs are dispensable for mammalian ERAD. Instead, our results suggest that triacsin c causes global alterations to the lipid landscape that disrupt ER proteostasis by interfering with the glycan trimming and dislocation steps of ERAD. Finally, in chapter three we develop a proximity labeling strategy that exploits LD-targeted APEX2 to biotinylate LD proteins in living cells. We apply this approach to two different cell types and are able to identify the vast majority of previously validated LD proteins, exclude common contaminating proteins, and identify the autophagy adaptor p62 as a mediator of hepatic lipophagy. Together these studies advance our understanding of the mechanisms that regulate lipid dynamics in the ER and LDs and their contribution towards maintaining cellular homeostasis.
摘要译文
内质网（ER）用作分泌系统的入口点，其中近三分之一的细胞蛋白质组必须在部署之前经历合成，折叠和成熟事件。通过内质网相关的降解（ERAD）释放未成功导航这些方法并达到其天然构象的蛋白质，其负责靶向靶向蛋白质的质量控制机制，用于通过细胞溶质26s蛋白酶体染色以降解。最近的研究表明，用长链酰基-CoA合成酶抑制剂三分子素C的处理破坏了脂液滴（LD）生物发生和ERAD，表明该方法之间的功能性连接。但是，LDS是否涉及ERAD仍然是一个突出的问题。 LDS是高度动态的中性脂质储存细胞器，其用作脂质代谢的中心毂，其带有储存脂质和维持细胞的能量稳态。 LDS的特定代谢作用由细胞类型和细胞的代谢状态决定，其可以响应于多种细胞刺激而波动。 LD函数通过与边界LD磷脂单层相关联的整体和外周蛋白的补体来调节。定义高信心LD蛋白质组的能力是理解LD功能和动态的最重要的。然而，由于在富集的浮力级分中存在污染蛋白质，对LD蛋白质组组合物的准确分析仍然存在挑战。在第一章中，我们讨论了ER和LDS内蛋白质和脂质调节系统之间的联系，突出了Erad和Pipophagy在维持细胞稳态中的重要性。在第二章中，我们使用化学和遗传方法来破坏LD生物发生，探讨ERAD中LDS的潜在作用，最终提供LDS对哺乳动物ERAD可分配的证据。相反，我们的结果表明，通过干扰Erad的甘草修剪和位错步骤，TriCsin C对脂质景观的全球变化引起了血液景观。最后，在第三章中，我们开发了一种接近标记策略，该策略将LD靶向的Apex2利用活细胞中的生物素化合物LD蛋白。我们将这种方法应用于两种不同的细胞类型，并且能够识别绝大多数先前验证的LD蛋白，排除常见的污染蛋白，并将自噬衔接子P62鉴定为肝脂肪的介质。这些研究共同推进了我们对调节ER和LDS中的脂质动态的机制以及它们对维持细胞稳态的贡献的理解。
Peterson, Clark W.. Mechanisms of Lipid Homeostasis in the Endoplasmic Reticulum and Lipid Droplets[D]. US: University of California, Berkeley, 2020