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

酒精毒性导致的半胱氨酸接近对肝脏赖氨酸 N-乙酰化的调节

• 批准号: 10752320
• 负责人: Courtney McGinnis
• 金额: $ 3.72万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752320
• 关键词: Acetaldehyde; Acetates; Acetylation; Advanced Development; Affect; Age; Alcohol consumption; Alcoholic Liver Diseases; Alcoholic liver damage; Alcohols; Amines; Amino Acids; Analytical Biochemistry; Binding; Biochemical; Biochemistry; Biological Assay; Biological Markers; Biophysics; Carbon; Cell Respiration; Cessation of life; Charge; Chemistry; Chronic; Classification; Clinical; Consumption; Cysteine; Data; Data Set; Development; Drug Metabolic Detoxication; Early Diagnosis; Enzymes; Equilibrium; Ethanol Metabolism; Ethanol toxicity; Evaluation; Fellowship; Goals; Grant; Hepatic; Homeostasis; Individual; Iron; Lead; Liver; Liver diseases; Lysine; Mediating; Mentors; Mentorship; Metabolic; Metals; Mitochondria; Modeling; Modification; Mus; National Center for Advancing Translational Sciences; National Institute on Alcohol Abuse and Alcoholism; National Research Service Awards; Nicotinamide adenine dinucleotide; Outcome; Oxidation-Reduction; Pathogenicity; Pathologic; Pathology; Pathway interactions; Patients; Post-Translational Protein Processing; Preventive therapy; Proteins; Proteome; Proteomics; Reaction; Recombinants; Regulation; Research; Research Personnel; Role; Signal Transduction; Sirtuins; Site; Stress; Structure; Sulfhydryl Compounds; Supportive care; Therapeutic; Therapeutic Intervention; Training; United States National Institutes of Health; Work; Zinc; alcohol consequences; alcohol effect; alcohol exposure; bench to bedside; chronic alcohol ingestion; clinically relevant; detection method; enzyme activity; feeding; hepatoprotective; improved; in vitro activity; in vivo; insight; interdisciplinary approach; liver injury; mouse model; new therapeutic target; novel; oxidation; prevent; preventable death; protein function; protein structure; proteomic signature; regenerative therapy; repaired; response; success; therapeutic target

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.

项目摘要 这个Ruth L. Kirschstein NRSA F31奖学金的目标是发展申请人的专业知识 酒精引起的翻译后修饰（PTM）的致病机制。酒精相关的肝脏 疾病（ALD）是全球可预防死亡率的主要原因，每次死亡约5.9％ 一年，但缺乏预防或再生疗法。肝脏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。酒精代谢的主要结果是 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破坏 到参与半胱氨酸蛋白质组的半胱氨酸硫醇网络，这对于酶活性至关重要 结构和信号传导。早期ALD的模型已应用于蛋白质组学研究以更好地理解 酒精代谢和阐明扰动途径的下游影响。使用6周的Lieber-Decarli （LD）模型，我们使用新颖的基于点击化学的肝蛋白质组中的硫醇氧化还原变化量化 NHPLC-MS/MS分析。通过相同的模型，我们检查了慢性饮酒对肝的影响 赖氨酸N-乙酰基通过定量分析通过乙酰IP，然后是NHPLC-MS/MS。简而言之，这些研究 发现酒精喂养诱导了整体降低半胱氨酸蛋白质组并增加赖氨酸乙酰化。 最近的研究确定了赖氨酸和半胱氨酸氨基酸领先的生物物理空间关联 指导互动。当半胱氨酸保留在15Å以内时，赖氨酸乙酰化会扩增，称为 半胱氨酸赖氨酸对（Cyslys）。我们的工作表明，酒精引起的N-乙酰化的增加部分是由于 乙酰化更具反应性半胱氨酸硫醇，然后转移到附近的赖氨酸中，导致Sàn 乙酰转移反应。拟议的培训计划将在慢性LD模型中调查这种关系 整合乙酰基和硫醇氧化还原氧杂质分析以评估病理相关的蛋白质靶标。所以， 该F31应用的中心假设是蛋白质cyslys对是增加歌词的机制 酒精毒性期间的乙酰化和半胱氨酸氧化还原敏感性有助于ALD的病理。具体的 AIM 1将表征受LYS乙酰化影响和CYS氧化还原影响的肝蛋白质组学特征 使用多层综合分析的慢性酒精代谢。具体目标2将定义监管 特异性蛋白质中Sàn乙酰基转移的机理对ALD的进展至关重要。这些目标将是 使用多学科方法进行询问，该方法将提供对赖氨酸乙酰化的透彻理解 通过控制蛋白质结构，和硫醇氧化还原信号传导。这些特定目标的完成将支持 申请人成为酒精诱导的代谢失调领域的独立研究人员。