Regulation of hepatic lysine N-acetylation by cysteine proximity due to alcohol toxicity

项目总结 Ruth L.Kirschstein NRSA F31奖学金的目标是发展申请者在 酒精诱导的翻译后修饰(PTMS)的发病机制。酒精性肝 疾病(ALD)是全世界可预防的死亡的主要原因，每年造成约5.9%的死亡 一年了，但缺乏预防性或再生性疗法。肝是酒精解毒的主要部位 产生对肝脏生物化学产生负面影响的产品。酒精新陈代谢的一个主要后果是 赖氨酸乙酰化的诱导，一种代谢诱导的PTM。一个特征不佳的因素是干扰 到半胱氨酸蛋白质组中所涉及的半胱氨酸硫醇网络，这对酶的活性至关重要，蛋白质 结构和信号。早期ALD模型已被应用于蛋白质组学研究，以更好地理解 酒精代谢的下游影响，并阐明扰乱的途径。使用为期6周的利伯-德卡利 (LD)模型中，我们使用一种新的基于点击化学的方法来量化肝脏蛋白质组中的硫醇氧化还原变化。 NHPLC-MS/MS联用分析。在相同的模型中，我们研究了长期饮酒对肝脏的影响 赖氨酸N-乙酰化通过乙酰IP定量分析，然后nHPLC-MS/MS，简要介绍这些研究 结果表明，酒精摄入诱导半胱氨酸蛋白质组整体减少，赖氨酸乙酰化增加。 最近的研究已经确定了赖氨酸和半胱氨酸氨基酸之间的生物物理空间联系 来指导互动。当半胱氨酸残基在15？以内时，赖氨酸乙酰化被放大，称为 半胱氨酸-赖氨酸对(CysLys)。我们的工作表明，酒精诱导的N-乙酰化增加是部分原因 到更具活性的半胱氨酸硫醇的乙酰化，然后转移到附近的赖氨酸，得到S？N 乙酰转移反应。拟议的培训计划将通过以下方式在慢性学习障碍模型中调查这种关系 整合乙酰和硫醇氧化还原分析以评估病理上相关的蛋白质靶标。因此， F31应用的中心假设是蛋白质CysLys对是赖氨酸增加的一种机制 乙酰化和半胱氨酸在酒精中毒时的氧化还原敏感性，并在ALD的病理中起作用。特定的 目标1将表征受Lys乙酰化和Cys氧化还原变化影响的肝脏蛋白质组特征 慢性酒精代谢的多层次综合分析。具体目标2将定义监管 肌萎缩侧索硬化症关键蛋白中S的N-乙酰转移机制。这些目标将是 采用多学科方法进行询问，将提供对赖氨酸乙酰化的透彻了解 以及通过调节蛋白质结构的硫醇氧化还原信号。完成这些具体目标将支持 申请成为酒精引起的代谢紊乱领域的独立研究人员。

PROJECT SUMMARY The goal of this Ruth L. Kirschstein NRSA F31 fellowship is to develop the expertise of the applicant in pathogenic mechanisms of alcohol-induced post-translational modifications (PTMs). Alcohol-associated liver disease (ALD) is a leading cause of preventable mortality worldwide causing approximately 5.9% of deaths every year, yet lacks preventative or regenerative therapies. The liver is the primary site of alcohol detoxification generating products that negatively impact hepatic biochemistry. A major consequence of alcohol metabolism is the induction of lysine acetylation, a metabolically induced PTM. One poorly characterized factor is the disruption to the network of cysteine thiols involved in the cysteine proteome, which is crucial to enzyme activity, protein structure, and signaling. Models of early-stage ALD have been applied to proteomic studies to better understand the downstream effects of alcohol metabolism and elucidate perturbed pathways. Using a 6-week Lieber-DeCarli (LD) model, we quantified thiol redox changes in the hepatic proteome employing a novel click-chemistry-based nHPLC-MS/MS assay. With the same model, we examined the impact of chronic alcohol consumption on hepatic lysine N-acetylation through quantitative analysis via acetyl-IP followed by nHPLC-MS/MS. Briefly, these studies identified that alcohol feeding induced an overall reduced cysteine proteome and increased lysine acetylation. Recent studies have identified a biophysical spatial association between lysine and cysteine amino acids leading to direct interactions. Lysine acetylation is amplified when cysteine residues are within 15 Å, referred to as a cysteine-lysine pair (CysLys). Our work suggests the alcohol-induced increase in N-acetylation is partially due to the acetylation of the more reactive cysteine thiol followed by transfer to a nearby lysine, resulting in an SàN acetyl transfer reaction. The proposed training plan will investigate this relationship in a chronic LD model by integrating acetyl- and thiol redox OMIC analyses to evaluate pathologically relevant protein targets. Therefore, the central hypothesis of this F31 application is that protein CysLys pairs are a mechanism for increased lysine acetylation and cysteine redox sensitivity during alcohol toxicity and contributes to the pathology of ALD. Specific aim 1 will characterize hepatic proteomic signatures impacted by Lys acetylation and Cys redox changes due to chronic alcohol metabolism using a multi-layered integrated analysis. Specific aim 2 will define regulatory mechanisms of SàN acetyl transfer in specific proteins critical to the progression of ALD. These aims will be interrogated utilizing a multidisciplinary approach that will provide a thorough understanding of lysine acetylation and thiol redox signaling by regulating protein structure. Completion of these specific aims will support the applicant in becoming an independent researcher in the field of alcohol-induced metabolic dysregulation.