Lipotoxic Protective Response of the Endoplasmic Reticulum

内质网的脂毒性保护反应

• 批准号: 10706013
• 负责人: ROBERT V FARESE
• 金额: $ 19.19万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10706013
• 关键词: Lipotoxic; Protective; Response; Endoplasmic; Reticulum

Lipid homeostasis is crucially important for cells, and disruptions in this balance can lead to diseases of lipid overload, such as obesity-related disorders. Lipid homeostasis is mainly coordinated in the endoplasmic reticulum (ER), the largest membrane system in cells. The ER is a well-recognized location for synthesis of membrane and secreted proteins, processes that are safeguarded by the “unfolded protein response” (UPR). Recent studies suggest that a complementary and interrelated pathway, which we term the “lipotoxic protective response” (LPR), acts in parallel to maintain ER lipid homeostasis. In contrast to the UPR, however, knowledge of the molecular aspects of the LPR remain rudimentary. Here we describe studies to overcome this knowledge gap through investigations through our recent identification of the mammalian FIT2 protein as a key guardian of ER lipid homeostasis. FIT2 has been a mysterious ER protein, of crucial importance for cell health and function, that was implicated in ER homeostasis and lipid metabolism but lacked an identified function. After years of effort and a biochemical tour de force, we have discovered that FIT2 is an acyl-CoA diphosphatase enzyme that catabolizes fatty acyl-CoAs, the activated forms of fatty acids, in the ER. Our preliminary data indicate that this activity may be localized on the luminal leaflet and is crucially important for cell health and viability. Absence of FIT2 triggers ER stress and, in a mouse model we have generated, liver injury. Our findings break new ground and raise many important questions about FIT2 and the LPR. Here we propose to answer these questions by using an interdisciplinary approach. Aim 1 will determine mechanisms of the LPR at the molecular level by deciphering the biochemical mechanism for FIT2’s catabolism of fatty acyl-CoA in the ER. We will combine biochemical and structural biology approaches to answer: What is the enzyme’s catalytic mechanism? Is FIT2 active towards ER luminal substrates? Is its activity regulated by lipid metabolites (e.g., diacylglycerol, DAG)? What is the fate of the metabolites generated by FIT2 activity? Aim 2 will determine mechanisms of the LPR at the cellular level by elucidating how FIT2 and the LPR are integrated with other ER stress protection pathways. Specifically, we will address: How does FIT2 mechanistically trigger the UPR? Does the mammalian UPR pathway depend on FIT2 activity? Can the interdependency of the FIT2/LPR and the UPR be exploited for therapeutic purposes in cancer, for example those with high demands on ER function, such hepatocellular carcinoma and multiple myeloma? Aim 3 will determine mechanisms of the LPR at the physiological level by determining how FIT2 maintains lipid homeostasis in mammalian liver. We have generated mice lacking FIT2 in hepatocytes, and preliminary studies indicate these mice have ER stress, increases in hepatocyte lipid storage, defects in lipid secretion, and signs of liver disease. We will mechanistically dissect this phenotype to elucidate how FIT2 is required in cellular lipid metabolism. Completion of these aims will reveal how a fast, enzymatic mechanism functions in the LPR to protect cells and maintain lipid homeostasis. It will also shed light on the pathogenesis of human disease, such as the rare human disorder of FIT2 deficiency and more common metabolic diseases involving ER lipid metabolism, with implications for both liver steatosis and cancers that rely heavily of the ER (such as liver cancer and multiple myeloma).

脂质平衡对细胞至关重要，这种平衡的破坏会导致脂质过载的疾病， 例如与肥胖相关的疾病。脂类平衡主要协调在内质网中，内质网是最大的 细胞膜系统。内质网是膜和分泌蛋白质合成的公认位置， 受“未折叠蛋白反应”(UPR)保护的过程。最近的研究表明，一种互补的 而相互关联的途径，我们称之为“脂毒保护性反应”(LPR)，平行地作用于维持内质网脂肪 动态平衡。然而，与UPR相反，对LPR的分子方面的知识仍然是初级的。这里 我们描述了通过调查来克服这种知识差距的研究，通过我们最近发现的 哺乳动物FIT2蛋白作为内质网脂质稳态的关键守护者。FIT2是一种神秘的ER蛋白，对 对细胞健康和功能的重要性，这与内质网稳态和脂肪代谢有关，但缺乏 已确定的功能。经过多年的努力和生化考察，我们发现FIT2是一种酰基辅酶A 内质网中分解脂肪酰基-COAS的二磷酸酶，脂肪酰基-COAS是脂肪酸的活化形式。我们的初步数据 表明这种活性可能定位在管腔小叶上，并且对细胞健康和活性至关重要。 在我们建立的小鼠模型中，FIT2的缺失会触发内质网应激，并导致肝脏损伤。我们的发现开辟了新天地 并提出了关于FIT2和LPR的许多重要问题。在这里，我们建议通过使用 跨学科方法。目标1将通过破译LPR的分子水平来确定LPR的机制 内质网中脂肪酰辅酶A FIT2‘S分解代谢的生化机制我们将把生化和结构结合起来 生物学方法回答：这种酶的催化机制是什么？FIT2是否对内质网管腔底物起作用？ 它的活性是否受脂代谢产物(如二酰甘油，DAG)的调节？产生的代谢物的命运是什么？ FIT2活动？AIM 2将通过阐明FIT2和LPR如何在细胞水平上确定LPR的机制 与其他内质网应激保护途径整合。具体地说，我们将解决：FIT2是如何机械触发的 普遍定期审议？哺乳动物的UPR通路依赖于FIT2的活性吗？FIT2/LPR和 UPR被用于癌症的治疗目的，例如那些对ER功能有高要求的癌症，如 肝细胞癌和多发性骨髓瘤？目标3将在生理水平上确定LPR的机制 通过确定FIT2如何维持哺乳动物肝脏中的脂类平衡。我们已经产生了缺乏FIT2的小鼠 肝细胞，初步研究表明这些小鼠有内质网应激，肝细胞脂质储存增加， 脂肪分泌和肝病的征兆。我们将机械地解剖这种表型，以阐明FIT2是如何 细胞脂代谢所必需的。这些目标的完成将揭示快速、酶机制如何在 LPR具有保护细胞、维持脂质平衡的作用。它还将阐明人类疾病的发病机制，如 作为罕见的人类FIT2缺乏症和更常见的涉及ER脂代谢的代谢性疾病， 对肝脏脂肪变性和严重依赖ER的癌症(如肝癌和多发性骨髓瘤)都有影响。