Alcohol-induced alterations in protein acetylation: mechanisms and consequences

 描述(申请人提供)：每年有超过20,000人死于酒精性肝病，这是美国第十二大死因。因为肝脏是主要的部位 在酒精代谢中，它是最容易受到酒精损伤的器官。尽管酒精性肝病的进展在临床上已有很好的描述，但酒精性肝损伤的分子基础尚不清楚。我们的长期目标是了解导致酒精肝毒性的机制。这一建议是基于我们的发现，即乙醇处理的WIF-B细胞、来自乙醇喂养的大鼠的肝脏切片和肝脏中的微管更高度乙酰化和更稳定，并且增加微管乙酰化和稳定性可以通过损害基于微管的运动功能来解释酒精导致的蛋白质运输缺陷。这项建议旨在确定酒精诱导的微管乙酰化和稳定性如何直接导致脂肪变性和其他肝损伤。在这份提案中，我们提出了三个主要但相关的问题。我们的发现表明，在乙醇处理的WIF-B细胞中，动力蛋白/动力蛋白与停滞的跨细胞蛋白沿着乙酰化的微管共存，并且动力蛋白与微管结合得更紧密，这向我们表明，乙醇处理的细胞中运动处理能力的降低可以解释囊泡输送的障碍。我们将在目标1中提出的实验中验证这一假设。尽管我们之前的研究已经将微管超乙酰化与乙醇处理细胞中蛋白质运输和运动功能受损密切相关，但我们将使用目标2中描述的两种方法直接测试这一点。目标3将我们带向一个新的令人兴奋的方向。新的证据表明，微管和基于微管的马达是脂滴形成/降解和双向运动的重要调节因素。此外，在3T3-L1细胞中，乙酰化微管是脂肪形成所必需的。因此，我们认为乙酰化微管增强了乙醇诱导的脂肪变性。我们将按照目标3中的描述来测试这种可能性。通常，实验将在极化的肝脏WIF-B细胞中启动，并在可能的情况下在乙醇喂养的大鼠的肝脏中得到证实。我们将继续与迪恩·图马博士合作，并已获得其他几个人的支持，以帮助我们进行拟议的研究。我继续担任约翰·霍普金斯大学医学院细胞生物系的客座教授，确保成为霍普金斯显微镜设施的成员。我们的合作者广博的专业知识、获得高端资源的途径，再加上我们在肝细胞生物学方面的丰富专业知识，使我们非常适合进行这些重要的机械实验。这项研究还表明，调节肝细胞乙酰化状态可能是治疗脂肪变性和其他肝病症状的一种新的治疗策略。

 DESCRIPTION (provided by applicant): More than 20,000 people each year die of alcoholic liver disease, the twelfth largest cause of death in Americans. Because the liver is the major site of ethanol metabolism, it is the most susceptible organ to alcohol-induced injury. Although the progression of alcoholic liver disease is well-described clinically, the molecular basis for alcoho-induced liver injury is not understood. Our long-term goal is to understand the mechanisms that lead to alcohol-induced hepatotoxicity. This proposal is based on our findings that microtubules are more highly acetylated and more stable in ethanol-treated WIF-B cells, liver slices and livers from ethanol-fed rats, and that increased microtubule acetylation and stability can explain alcohol-induced defects in protein trafficking by impairing microtubule-based motor function. This proposal is aimed at identifying how alcohol-induced microtubule acetylation and stability directly contribute to steatosis and other liver injury. In this proposal, we ask three major, yet related, questions. Our findings that dynein/dynactin colocalizes with stalled transcytosing proteins along acetylated microtubules and that dynein binds microtubules more tightly in ethanol-treated WIF-B cells suggests to us that impaired vesicle delivery can be explained by decreased motor processivity in ethanol-treated cells. We will test that hypothesis in the experiments proposed in Aim 1. Although our previous studies have strongly correlated microtubule hyperacetylation with impaired protein trafficking and with impaired protein trafficking and motor function in ethanol-treated cells, we will test this directly using 2 approaches as described in Aim 2. Aim 3 takes us in a new and exciting direction. Emerging evidence implicates microtubules and microtubule-based motors as important regulators of lipid droplet formation/degradation and bidirectional motility. Furthermore, acetylated microtubules are required for adipogenesis in 3T3-l1 cells. Thus, we propose that acetylated microtubules enhance ethanol-induced steatosis. We will test that possibility as described in Aim 3. In general, experiments will be initiated in polarized, hepatic WIF-B cells and confirmed when possible in livers from ethanol-fed rats. We will continue our collaboration with Dr. Dean Tuma and have garnered the support of several others to help us perform the proposed studies. I continue my appointment as Visiting Faculty in the Department of Cell Biology at Johns Hopkins University School of Medicine securing membership to the Hopkins Microscope Facility. The expansive expertise of our collaborators, the access to high-end resources coupled with our considerable expertise in hepatic cell biology situate us perfectly to perform these important mechanistic experiments. This research also suggests that modulation of the hepatocyte acetylation state may be a novel therapeutic strategy for the treatment of steatosis and other symptoms of liver disease.