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的目标。Kirschstein NRSA F31奖学金旨在培养申请人在以下方面的专业知识： 酒精诱导的翻译后修饰（PTM）的致病机制。酒精相关性肝 疾病（ALD）是全球可预防死亡的主要原因，每年造成约5.9%的死亡。 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有负面影响的产物。酒精代谢的一个主要后果是 赖氨酸乙酰化的诱导，代谢诱导的PTM。一个不好描述的因素是 对于半胱氨酸蛋白质组中涉及的半胱氨酸巯基网络，这对酶活性至关重要， 结构和信号。早期ALD模型已被应用于蛋白质组学研究，以更好地了解 酒精代谢的下游影响，并阐明扰动途径。使用为期6周的利伯-迪卡里 (LD)模型，我们定量巯基氧化还原变化的肝蛋白质组采用一种新的点击化学为基础的 nHPLC-MS/MS测定。用同样的模型，我们研究了长期饮酒对肝脏的影响。 通过乙酰基-IP随后nHPLC-MS/MS的定量分析进行赖氨酸N-乙酰化。简言之，这些研究 确定了酒精喂养诱导整体减少半胱氨酸蛋白质组和增加赖氨酸乙酰化。 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关联， to direct直接interactions互动.当半胱氨酸残基在15个碱基内时，赖氨酸乙酰化被放大，称为乙酰化。 半胱氨酸-赖氨酸对（CysLys）。我们的工作表明，酒精诱导的N-乙酰化增加部分是由于 反应性更强的半胱氨酸巯基的乙酰化，然后转移到附近的赖氨酸，导致SàN 乙酰基转移反应拟议的培训计划将通过以下方式在慢性LD模型中研究这种关系： 整合乙酰基和巯基氧化还原OMIC分析，以评估病理相关的蛋白质靶标。因此，我们认为， 该F31应用的中心假设是蛋白质CysLys对是增加赖氨酸的机制 乙酰化和半胱氨酸氧化还原敏感性在酒精毒性和有助于ALD的病理。具体 目的1将表征受Lys乙酰化和Cys氧化还原变化影响的肝蛋白质组特征， 慢性酒精代谢使用多层综合分析。具体目标2将定义监管 对ALD进展至关重要的特定蛋白质中的SàN乙酰转移机制。这些目标将是 利用多学科方法进行询问，这将提供对赖氨酸乙酰化的透彻理解。 和巯基氧化还原信号通过调节蛋白质结构。完成这些具体目标将有助于 申请人成为酒精诱导的代谢失调领域的独立研究人员。