Synergy of lipolysis and lipophagy in alcoholic liver disease

项目摘要/摘要 我的长期目标是发现与酒精性肝脏有关的新的细胞机制 疾病(ALD)是一种进行性疾病，可导致致命的不可逆转阶段。其中之一 酒精性脂肪变性是酒精性脂肪变性的早期和可逆的先兆，在这种脂肪变性中，大量的甘油三酯和 富含胆固醇的脂滴(LDs)积聚在肝细胞内，肝细胞是主要的功能细胞类型 肝脏的。虽然LDS被认为是ALD进展的中心，但细胞 酒精破坏这些细胞器的机制很差。 明白了。为了解决这一重要的知识差距，这项提议将定义协同效应 在脂滴分解代谢的两个看似不同的过程之间：脂解作用，它利用 CAMP途径激活和募集胞浆脂肪酶到LD表面，以及脂噬作用，这是 利用膜转运事件，导致溶酶体脂肪酶分解LD。 初步数据表明，脂解和吞脂机制针对离散的、基于大小的LD 亚群，小的LDs被多囊泡体直接吞噬 (MVBS)通过晚期内体途径进行分解代谢。因此，这一点的中心假设 建议是，Etoh破坏了脂肪分解作用于大LD的顺序机制，以 缩小它们的大小，以便通过内体微脂吞噬直接摄取。在目标1中，我将确定 胞浆脂肪酶在防止ALD进展中的作用。在目标2中，我将确定 Etoh在一个连续的“脂解-脂噬”途径上的作用，并定义了 MVB/内体促进小LDS的微脂吞噬作用。在目标3中，我将整合我以前的 具备这一新知识的专业知识和培训，以定义新的cAMP信号机制 支持脂肪分解和吞脂作用，抗击酒精性脂肪变性。异常强大的研究 梅奥诊所消化科和肝科的环境非常适合这样做。 训练。为了实现这些目标，我将接受慢性病啮齿动物模型的实践培训 酒精消费、比较蛋白质组学和生物信息学。这项新培训 补充了我目前在细胞信号、显微镜和生物化学方面的技能，并提供了一种 多学科工具箱，全面评估肝脏中的脂质分解代谢。建议数 研究将整合我在毕业时在cAMP/PKA信号机制方面的专业知识 我在学校接受了关于脂滴生物学和酒精性脂肪变性的博士后培训。 在我的导师Mark McNiven博士的帮助下，以及一个强大的研究团队，其中包括两个 合作者和四名咨询委员会成员，我将拥有所有的专业知识和培训 需要成功实现这些目标并过渡到独立研究 位置。从拟议的研究中获得的结果将提供一种机制 对酒精性脂肪肝脂滴分解代谢的认识。重要的是，这些研究将 提供已发表的研究手稿和初步数据，以支持未来的R01 求婚。

Project Summary/Abstract My long-term objective is to discover new cellular mechanisms that contribute to alcoholic liver disease (ALD), a progressive disease leading to non-reversible stages that can be fatal. One of the early and reversible precursors of ALD is alcoholic steatosis, in which large triglyceride and cholesterol-rich lipid droplets (LDs) accumulate within hepatocytes, the main functional cell type of the liver. While LDs are believed to be central to the progression of ALD, the cellular mechanisms whereby alcohol disrupts the breakdown of these organelles are poorly understood. To address this important gap in knowledge, this proposal will define the synergy between two seemingly-distinct processes of lipid droplet catabolism: lipolysis, which utilizes the cAMP pathway to activate and recruit cytosolic lipases to the LD surface, and lipophagy, which utilizes membrane trafficking events that lead to LD breakdown by lysosomal lipases. Preliminary data suggest that lipolysis and lipophagy machinery target discrete, size-based LD subpopulations, and that small LDs are targeted for direct engulfment by multivesicular bodies (MVBs) for catabolism through the late endosomal pathway. Thus, the central hypothesis of this proposal is that EtOH disrupts a sequential mechanism whereby lipolysis acts on large LDs to reduce their size for direct uptake by endosomal microlipophagy. In Aim 1, I will determine the role of cytosolic lipases in protecting against ALD progression. In Aim 2, I will determine effect of EtOH on a sequential “lipolysis-to-lipophagy” pathway and define the mechanisms by which MVB/endosomes facilitate microlipophagy of small LDs. In Aim 3, I will integrate my previous expertise with this new knowledge and training to define novel cAMP signaling mechanisms that support lipolysis and lipophagy to combat alcoholic steatosis. The exceptionally strong research environment within Mayo Clinic’s Division of Gastroenterology and Hepatology is ideal for this training. To accomplish these aims, I will receive hands-on training in rodent models of chronic alcohol consumption, comparative proteomics, and bioinformatics. This new training complements my current skillset in cell signaling, microscopy, and biochemistry, and provides a multidisciplinary toolbox to comprehensively assess lipid catabolism in the liver. The proposed research will integrate my expertise in cAMP/PKA signaling mechanisms from my graduate school training with my postdoctoral expertise in lipid droplet biology and alcoholic steatosis. With the help of my mentor, Dr. Mark McNiven, along with a strong research team including two collaborators and four advisory committee members, I will have all the expertise and training needed to successfully accomplish these aims and transition to an independent research position. The results gained from the proposed research will provide a mechanistic understanding of lipid droplet catabolism in alcoholic fatty liver. Importantly, these studies will provide published research manuscripts and preliminary data in support of a future R01 proposal.