Protein Modification by 3,4-Dihydroxyphenylacetaldehyde

描述(由申请人提供) 多巴胺(DA)经酶催化脱氨生成3，4-二羟基苯乙醛(DOPAL)，DOPAL主要通过线粒体醛脱氢酶(ALDH2)代谢为酸，也可通过醛还原酶(ALR)代谢为醇。最近的工作表明，氧化应激和脂质过氧化产物，如4-羟基壬烯醛(4HNE)，是帕金森病(PD)的致病因素。4HNE先前被证明可以使ALDH2失活，因此被预测会损害DOPAL的代谢。本申请中提供的初步数据表明，4HNE通过人ALDH2和大鼠脑ALR抑制DOPAL的代谢。此外，还发现DOPAL可以修饰多肽胺，生成席夫碱产物。泛素依赖的蛋白酶体受损被认为是PD发病的一个因素，DOPAL可能通过影响赖氨酸的泛素结合来抑制蛋白酶体的降解。根据已有的研究和初步数据，推测4HNE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，并损害蛋白酶体的降解。为了验证这一假设，我们将提出三个具体目标。特异性目标1将评估4HNE是否在体外抑制DOPAL的代谢。用DA和4HNE处理突触体，并测定DA、DOPAL和DA-酸的水平。在特定目标2中的实验将评估在体外产生的DOPAL是否会修饰蛋白质。突触体将与[14C]DA和不同浓度的4HNE孵育。利用基于蛋白质组学的二维凝胶电泳和质谱学方法，可以可视化和鉴定由[14C]DOPAL加合的蛋白质。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。模型蛋白将被DOPAL加合，并与突触体蛋白酶体孵育，并通过Western blotting监测底物蛋白的降解。本申请中提出的研究将作为未来解决DOPAL修饰蛋白质的功能后果的工作/研究建议的基础，例如，确定DOPAL是否损害DA的生物合成和抑制DA囊泡转运蛋白。由于DOPAL在结构上类似于DA和活性电泳体，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.