Abstract
Background:
Parkinson's disease (PD), a chronic and severe neurodegenerative disease, is pathologically characterized by the selective loss of nigrostriatal dopaminergic neurons. Dopamine (DA), the neurotransmitter produced by dopaminergic neurons, and its metabolites can covalently modify proteins, and dysregulation of this process has been implicated in neuronal loss in PD. However, much remains unknown about the protein targets.
Methods:
In the present work, we designed and synthesized a dopamine probe (DA-P) to screen and identify the potential protein targets of DA using activity-based protein profiling (ABPP) technology in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In situ pull-down assays, cellular thermal shift assays (CETSAs) and immunofluorescence were performed to confirm the DA modifications on these hits. To investigate the effects of DA modifications, we measured the enzymatic activities of these target proteins, evaluated glycolytic stress and mitochondrial respiration by Seahorse tests, and systematically analyzed the changes in metabolites with unbiased LC-MS/MS-based non-targeted metabolomics profiling.
Results:
We successfully identified three glycolytic proteins, aldolase A, α-enolase and pyruvate kinase M2 (PKM2), as the binding partners of DA. DA bound to Glu166 of α-enolase, Cys49 and Cys424 of PKM2, and Lys230 of aldolase A, inhibiting the enzymatic activities of α-enolase and PKM2 and thereby impairing ATP synthesis, resulting in mitochondrial dysfunction.
Conclusions:
Recent research has revealed that enhancing glycolysis can offer protection against PD. The present study identified that the glycolytic pathway is vulnerable to disruption by DA, suggesting a promising avenue for potential therapeutic interventions. Safeguarding glycolysis against DA-related disruption could be a potential therapeutic intervention for PD.
Keywords:
Dopamine (DA); Glycolysis; Mitochondrial dysfunction; Parkinson’s disease (PD).