HNE damage of adenine nucleotide translocase in ethanol-mediated neuron apoptosis

乙醇介导的神经元凋亡中腺嘌呤核苷酸转位酶的 HNE 损伤

• 批准号: 7934507
• 负责人: GEORGE I HENDERSON
• 金额: $ 21.46万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-20 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7934507
• 关键词: 4 hydroxynonenal; Address; Adenine Nucleotide Translocase; Aldehydes; Amino Acids; Apoptosis; Apoptotic; Area; Binding; Brain; Cell Death; Cell Membrane Permeability; Cerebrum; Cessation of life; Clinical; Complex; Cyclophilins; Data; Development; Ethanol; Event; Experimental Models; Extravasation; Family; Induction of Apoptosis; Inner mitochondrial membrane; Intervention; Lead; Literature; Mass Spectrum Analysis; Mediating; Mitochondria; Modification; Necrosis; Neurons; Oxidative Stress; Permeability; Play; Rattus; Regulation; Role; Side; Sulfhydryl Compounds; Treatment Protocols; adduct; alcohol response; alcohol sensitivity; antiporter; clinically relevant; crosslink; cyclophilin D; fetal; insight; member; mitochondrial permeability transition pore; neuron apoptosis; novel; pro-apoptotic protein; protein folding; public health relevance; research study; response

DESCRIPTION (provided by applicant): The overall hypothesis of this exploratory proposal is that a key mechanism underlying ethanol-mediated opening of the mitochondrial permeability transition pore complex and possibly apoptosis activation, is perturbation of adenine nucleotide translocase (ANT) function by 4- hydroxynonenal (HNE) adduct formation. Key responses to HNE are, as previously documented, inhibition of ANT activity, activation of the mitochondrial permeability transition pore, and ultimate release of pro-apoptotic proteins. It is proposed that this occurs as a multifactorial event which includes direct modification of specific amino acid residues of ANT and its impaired interactions with cyclophilin D (CyP-D). Significance: This proposal address a new and novel, mitochondrially-mediated, fundamental mechanism by which ethanol may elicit apoptotic death of neurons. Neuron survival control points such as this are prime targets for the ultimate development of new clinical interventions. Specific Aim 1 will address the hypothesis that modifications of adenine nucleotide translocase by 4- hydroxynonenal adduct formation is a mechanism underlying ethanol-related effects on the mitochondrial permeability transition pore complex and ultimately on induction of apoptosis. Specific Aim 2 will address the hypothesis that direct modifications of cyclophilin-D by 4- hydroxynonenal adduct formation alter critical interactions with adenine nucleotide translocase and that this is one mechanism underlying ethanol-related effects on the mitochondrial permeability transition pore complex. Experimental Approaches: The experimental approaches will utilize Mass Spectrometry to determine specific amino acid residues on ANT and CyP-D that are modified and point-mutational manipulations to establish the functional significance of these ethanol-related modifications. Studies will also establish effects of these specific ANT and CyP-D modifications on CyP-D binding to ANT, on permeability transition regulation, and on sensitivity to ethanol-mediated neuron death. The experimental model will be cultured fetal rat cerebral cortical neurons exposed to clinically relevant ethanol treatment regimens. PUBLIC HEALTH RELEVANCE: Exposure of cultured fetal rat cerebral cortical neurons to ethanol rapidly elicits oxidative stress and increased formation of 4-hydroxynonenal (HNE) in mitochondria, which is followed by mitochondrial leakage of pro-apoptotic proteins and apoptotic death. This exploratory proposal addresses the hypothesis that a mechanism underlying ethanol-mediated opening of the permeability transition pore complex (PTPC) is connected to HNE modifications of the pore components, adenine nucleotide translocase (ANT) and cyclophilin D (CyP-D). Thus, the proposed studies will provide insight into basic mechanisms underlying mitotoxic responses to ethanol as well as fundamental mechanisms of PTPC function.

描述（由申请人提供）：该探索性提案的总体假设是，乙醇介导的线粒体通透性转换孔复合物打开以及可能的细胞凋亡激活的关键机制是通过 4-羟基壬烯醛（HNE）加合物形成扰动腺嘌呤核苷酸转位酶（ANT）功能。如前所述，对 HNE 的关键反应是抑制 ANT 活性、激活线粒体通透性转换孔以及最终释放促凋亡蛋白。据推测，这是一个多因素事件，其中包括 ANT 特定氨基酸残基的直接修饰及其与亲环蛋白 D (CyP-D) 的相互作用受损。意义：该提案提出了一种新的、新颖的、线粒体介导的基本机制，乙醇可以通过该机制引起神经元凋亡。诸如此类的神经元存活控制点是最终开发新的临床干预措施的主要目标。具体目标 1 将提出以下假设：通过 4-羟基壬烯醛加合物形成对腺嘌呤核苷酸转位酶的修饰是乙醇相关影响线粒体通透性转换孔复合物并最终诱导细胞凋亡的潜在机制。具体目标 2 将解决以下假设：通过 4-羟基壬烯醛加合物形成对亲环蛋白-D 的直接修饰改变了与腺嘌呤核苷酸转位酶的关键相互作用，并且这是乙醇相关效应对线粒体通透性过渡孔复合物的潜在机制之一。实验方法：实验方法将利用质谱法来确定 ANT 和 CyP-D 上经过修饰的特定氨基酸残基，并通过点突变操作来确定这些乙醇相关修饰的功能意义。研究还将确定这些特定 ANT 和 CyP-D 修饰对 CyP-D 与 ANT 结合、通透性转变调节以及对乙醇介导的神经元死亡敏感性的影响。该实验模型将培养暴露于临床相关乙醇治疗方案的胎鼠大脑皮层神经元。 公共健康相关性：培养的胎鼠大脑皮层神经元暴露于乙醇会迅速引发氧化应激并增加线粒体中 4-羟基壬烯醛 (HNE) 的形成，随后导致促凋亡蛋白线粒体渗漏和细胞凋亡。该探索性提议提出了这样的假设：乙醇介导的渗透性过渡孔复合物 (PTPC) 打开的机制与孔成分、腺嘌呤核苷酸转位酶 (ANT) 和亲环蛋白 D (CyP-D) 的 HNE 修饰有关。因此，拟议的研究将深入了解乙醇有丝毒性反应的基本机制以及 PTPC 功能的基本机制。