? ? 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. ? ? ?
