Protein Reactivity of 3,4-Dihydroxyphenylacetaldehyde, a Toxic Dopamine Metabolite, Is Dependent on Both the Aldehyde and the Catechol

Abstract

Dopamine (DA) has been implicated as an endogenous neurotoxin to explain the selective neurodegeneration as observed for Parkinson's disease (PD). However, previous work demonstrated 3,4-dihydroxyphenylacetaldehyde (DOPAL) to be more toxic than DA. DOPAL is generated as a part of DA catabolism via the activity of monoamine oxidase and the mechanism of DOPAL toxicity is proposed to involve protein modification. Previous studies have demonstrated protein reactivity via the aldehyde moiety; however, DOPAL contains two reactive functional groups (catechol and aldehyde) both with the potential for protein adduction. The goal of this work was to determine whether protein modification by DOPAL occurs via a thiol-reactive quinone generated from oxidation of the catechol, which is known to occur for DA, or if the aldehyde forms adducts with amine nucleophiles. To accomplish this objective, the reactivity of DOPAL towards N-acetyl-lysine (NAL), N-acetyl-cysteine (NAC) and two model proteins was determined. In addition, several DOPAL analogues were obtained and used for comparison of reactivity. Results demonstrate that at pH 7.4 and 37°C, the order of DOPAL reactivity is NAL ≫ NAC and the product of NAL and DOPAL is stable in the absence of reducing agent. Moreover, DOPAL will react with model proteins, but in the presence of amine-selective modifiers citraconic anhydride and 2-iminothiolane hydrochloride, the reactivity of DOPAL towards the proteins is diminished. In addition, DOPAL-mediated protein cross-linking is observed when a model protein or a protein mixture (i.e. mitochondria lysate) are treated with DOPAL at concentrations of 5-100 μM. Protein cross-linking was diminished in the presence of ascorbate, suggesting the involvement of a quinone in DOPAL-mediated protein modification. These data indicate DOPAL to be highly reactive towards protein nucleophiles with the potential for protein cross-linking.

Oxidation of 3,4-Dihydroxyphenylacetaldehyde, a Toxic Dopaminergic Metabolite, to a Semiquinone Radical and an ortho-Quinone

“…Purity was 97-99% as determined by HPLC analysis as described previously (18,24). Synthesized DOPAL was used without further purification.…”

“…SDS-PAGE Protein Cross-linking-Oxidized or non-oxidized DOPAL (50 M) was reacted with glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 0.3 mg/ml) in 50 mM NaH 2 PO 4 , pH 7.4, at 37°C for 4 h and then analyzed via SDS-PAGE (4% stacking, 10% resolving gels, and 9 g of protein loaded/well) and densitometry (NIH ImageJ software) as described previously (18). DOPAL oxidized by various methods (1.0 eq of NaIO 4 , 36 units/ml tyrosinase, and 2.0 eq of K 3 Fe(CN) 6 ) was added to the protein at specific time points (10 or 60 s following oxidation).…”

“…100-l aliquots were removed at various time points, treated with perchloric acid (6 l), centrifuged (10,000 ϫ g, 5 min), and analyzed quantitatively with an Agilent 1200 series capillary system HPLC with photodiode array detector. The concentration of DOPAL (non-oxidized) was determined by the calibration curve method described previously (18,24). To estimate the concentrations of oxidized DOPAL in each aliquot, a calibration curve (not shown) was constructed using 50 M DOPAL oxidized by various concentrations of NaIO 4 .…”

“…Work from our laboratory suggests that protein reactivity associated with DOPAL is partially mediated via catechol oxidation (18). Similarly, oxidation of DOPAL to a radical could help explain its high toxicity.…”

“…Physiological levels of DOPAL in the substantia nigra are ϳ2 M; levels as low as 6 M can exert significant toxicity (16). Reactivity with proteins, presumably via Schiff base formation, is an important mechanism of toxicity for DOPAL (17,18). Protein modification can result in inhibition of enzyme activity and loss of function for cellular proteins.…”