Protein Modification by 3,4-Dihydroxyphenylacetaldehyde

描述（由申请人提供） 多巴胺（DA）经过酶催化的脱氨作用产生3，4-二羟基苯乙醛（DOPAL），其主要通过线粒体醛脱氢酶（ALDH 2）代谢为酸，但也通过醛还原酶（ALR）代谢为醇。最近的工作涉及氧化应激和脂质过氧化产物，例如，4-羟基壬烯醛（4 HNE），作为帕金森病（PD）的因子。4 HNE先前被证明可抑制ALDH 2，因此预计会损害DOPAL代谢。本申请中提供的初步数据表明，4 HNE通过人ALDH 2和大鼠脑ALR抑制DOPAL的代谢。此外，发现DOPAL修饰肽胺，产生席夫碱产物。泛素依赖性蛋白酶体的损伤已被认为是PD发病机制中的一个因素，DOPAL可能通过损伤赖氨酸的DOPAL修饰引起的泛素缀合来抑制蛋白质的蛋白酶体降解。基于先前的研究和本文提供的初步数据，假设4 HNE可以抑制DOPAL代谢，导致儿茶酚胺醛水平异常，产生DOPAL-蛋白加合物和蛋白酶体降解受损。为了检验这一假设，将讨论三个具体目标。具体目标1将评价4 HNE是否在体外抑制DOPAL代谢。将用DA和4 HNE处理突触体，并测量DA、DOPAL和DA酸的水平。具体目标2中的实验将评估体外产生的DOPAL是否修饰蛋白质。将突触体与[14 C]DA和不同浓度的4 HNE一起孵育。使用基于蛋白质组学的方法与2-D凝胶电泳和质谱，[14 C]DOPAL加合的蛋白质将被可视化和鉴定。具体目标3将确定蛋白质的DOPAL修饰损害蛋白酶体降解的程度。模型蛋白将通过DOPAL加合并与突触体蛋白酶体孵育，并通过Western印迹监测底物蛋白的降解。本申请中提出的研究将作为未来工作/研究建议的基础，解决DOPAL修饰蛋白质的功能后果，例如，确定DOPAL是否损害DA生物合成并抑制DA囊泡转运蛋白。由于DOPAL在结构上类似于DA和反应性亲电体，因此它可能是DA结合蛋白的自杀配体，未来的工作将解决这个问题。

DESCRIPTION (provided by applicant) Dopamine (DA) undergoes enzyme-catalyzed deamination to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is metabolized primarily to an acid via mitochondrial aldehyde dehydrogenase (ALDH2) but also to an alcohol by aldehyde reductase (ALR). Recent work implicates oxidative stress and lipid peroxidation products, e.g., 4-hydroxynonenal (4HNE), as factors in Parkinson's Disease (PD). 4HNE was previously shown to inactivate ALDH2 and is therefore predicted to impair DOPAL metabolism. Preliminary data presented in this application demonstrate that 4HNE inhibits metabolism of DOPAL via human ALDH2 and rat brain ALR. Furthermore, DOPAL was found to modify peptide amines, yielding a Schiff base product. Impairment of the ubiquitin-dependent proteasome has been implicated as a factor in PD pathogenesis, and DOPAL may inhibit proteasomal degradation of proteins by impairing ubiquitin conjugation due to DOPAL modification of Lys. Based on previous studies and preliminary data presented here, it is hypothesized that 4HNE can inhibit DOPAL metabolism resulting in aberrant levels of the catecholamine aldehyde, yielding DOPAL-protein adducts and impaired proteasomal degradation. To test this hypothesis, three specific aims will be addressed. Specific Aim 1 will evaluate whether 4HNE inhibits DOPAL metabolism in vitro. Synaptosomes will be treated with DA and 4HNE and levels of DA, DOPAL and DA-acid measured. Experiments in Specific Aim 2 will assess if DOPAL generated in vitro modifies proteins. Synaptosomes will be incubated with [14C]DA and varying concentrations of 4HNE. Using a proteomics based approach with 2-D gel electrophoresis and mass spectrometry, proteins adducted by [14C]DOPAL will be visualized and identified. Specific Aim 3 will establish the extent to which DOPAL-modification of proteins impairs proteasomal degradation. Model proteins will be adducted by DOPAL and incubated with synaptosomal proteasome, and degradation of substrate protein monitored via Western blotting. The studies proposed in this application will serve as a foundation for future work/research proposals addressing the functional consequences of protein modification by DOPAL, e.g., determining whether DOPAL impairs DA biosynthesis and inhibits the DA vesicular transporter. Because DOPAL is structurally analogous to DA and a reactive electrophile, it may be a suicide ligand for DA binding proteins, and future work will address this issue.