Alcohol-induced changes in protein acetylation: mechanisms and consequences

总结 仅在美国，每年就有超过22，000人死于酒精性肝病，估计高达3.3 全球每年有100万人死亡（占全球死亡总数的5.9%）。所以很明显，酒精性肝病是一种主要的 生物医学健康问题。由于肝脏是乙醇代谢的主要场所，因此它是最重要的 易受酒精损伤的器官。虽然酒精性肝病的进展是很好的描述 在临床上，酒精诱导的肝损伤的分子基础尚不清楚。这一建议扩大了我们的 发现微管在乙醇处理的WIF-B细胞，肝切片， 来自乙醇喂养的大鼠/小鼠的肝脏-以及我们的初步数据-也在人类肝脏组织中。我们进一步 表明微管的超乙酰化直接解释了酒精诱导的蛋白质运输和脂质的缺陷， 液滴动力学在这个建议中，我们将测试乙醇诱导的蛋白质修饰的广泛假设， 差异性地破坏基于微管的蛋白质/细胞器运动，导致过氧化物酶体功能障碍 并促进酒精性脂肪变性我们还将检验补充热量 限制模拟物保护免受伤害。我们发现微管乙酰化和乙醛 内收损伤蛋白质运输的程度相似，表明两种修饰都有助于受损的 在乙醇处理的细胞中观察到运动性。我们将研究如何微管修饰（和修饰的 其他蛋白质）差异地影响Aim 1中的蛋白质和细胞器动力学。在目标2中，我们扩大了我们的研究 改变细胞器运动性和过氧化物酶体动力学。尽管它们在调节氧化应激和 脂肪酸代谢，过氧化物酶体在其对酒精诱导的 脂肪变性新出现的证据表明，微管和相关的马达是重要的调节器， 过氧化物酶体的动力学，并通过扩展，它们的功能-我们将在酒精的背景下探索的关系- 诱发脂肪变性。目标3把我们带到一个令人兴奋的方向，我们扩大了我们的研究与亚精胺对 它对肝纤维化的保护作用。亚精胺和羟基柠檬酸盐（热量限制模拟物）诱导 蛋白质脱乙酰化（包括微管脱乙酰化）通过不同的机制。因此，在目标3中，我们提出 这种增强的蛋白质去乙酰化将抵消酒精诱导的整体蛋白质乙酰化（和酒精- 诱导的微管依赖性蛋白运输）以赋予肝保护。我们进一步建议， 亚精胺促进细胞保护性自噬，从而降低积累的脂滴水平， 功能失调的线粒体和过氧化物酶体。一般而言，研究将在极化的肝脏WIF-B中启动 细胞，在来自乙醇/高脂肪饮食喂养的小鼠的肝脏中确认，并且在可能的情况下，在人体组织中确认。我们 已经获得了其他几个人的支持，为该项目提供他们的专业知识。我们可以继续访问 位于约翰霍普金斯基础生物医学研究所附近的成像设备， 霍普金斯地理信息中心允许我们获得许多服务和资源。我们丰富的专业知识 合作者，获得高端资源，加上我们在肝细胞生物学方面的丰富专业知识， 我们非常荣幸能够完成这些重要的机械和翻译研究。

SUMMARY More than 22,000 people each year die of alcoholic liver disease in the US alone with estimates as high as 3.3 million deaths each year globally (5.9% of all global deaths). So clearly, alcoholic liver disease is a major biomedical health concern world-wide. 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 alcohol-induced liver injury is not understood. This proposal expands on our findings that microtubules are more highly acetylated and more stable in ethanol-treated WIF-B cells, liver slices, livers from ethanol-fed rats/mice - and from our preliminary data - also in human liver tissue. We have further shown that microtubule hyperacetylation directly explains alcohol-induced defects in protein trafficking and lipid droplet dynamics. In this proposal, we will test the broad hypothesis that ethanol-induced protein modification differentially disrupts microtubule-based protein/organelle motility that leads to peroxisome dysfunction and promotes alcoholic steatosis. We will also test the hypothesis that supplementation with caloric restriction mimetics protects against injury. Our findings that both microtubule acetylation and acetaldehyde adduction impair protein trafficking to similar extents suggest both modifications contribute to the impaired motility observed in ethanol-treated cells. We will examine how microtubule modifications (and modifications on other proteins) differentially impact protein and organelle dynamics in Aim 1. In Aim 2, we expand our studies on altered organelle motility to peroxisome dynamics. Despite their known role in regulating oxidative stress and fatty acid metabolism, peroxisomes are under-studied in their contribution to the progression of alcohol-induced steatosis. Emerging evidence indicates that microtubules and associated motors are important regulators of peroxisomal dynamics, and by extension, their function – a relationship we will explore in the context of alcohol- induced steatosis. Aim 3 takes us in an exciting direction where we expand on our studies with spermidine on its hepatoprotective effects against fibrosis. Spermidine and hydroxycitrate (caloric restriction mimetics) induce protein deacetylation (including microtubule deacetylation) by different mechanisms. Thus, in Aim 3 we propose that this enhanced protein deacetylation will counteract alcohol-induced global protein acetylation (and alcohol- induced microtubule-dependent protein trafficking) to confer hepatoprotection. We further propose that spermidine promotes cytoprotective autophagy thereby decreasing the levels of accumulated lipid droplets and dysfunctional mitochondria and peroxisomes. In general, studies will be initiated in polarized, hepatic WIF-B cells, confirmed in livers from ethanol/high fat diet-fed mice, and where possible, confirmed in human tissue. We have garnered the support of several others to provide their expertise to the project. We have continued access to the Imaging Facility located at nearby Johns Hopkins Institute of Basic Biomedical Studies and are members of the Hopkins GI Center allowing us access to many services and resources. 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 and translational studies.