Proteome-Wide Profiling of Cellular Targets Modified by Dopamine Metabolites Using a Bio-Orthogonally Functionalized...

Area of Science:

• Neuroscience
• Biochemistry
• Proteomics

Background:

• Selective death of midbrain dopaminergic neurons is a hallmark of Parkinson's disease (PD).
• Dysregulation of dopamine (DA) leads to neuronal stress and damage, potentially via conversion to reactive dopaquinone species (DQs).
• DA-protein adducts (DPAs) are implicated in neurotoxicity and PD pathogenesis, but their targets remain largely uncharacterized.

Purpose of the Study:

• To develop a novel chemical probe for characterizing DPAs and understanding their role in PD pathogenesis.
• To identify cellular proteins modified by DQs and the affected biological pathways in neuronal cells.

Main Methods:

• Developed a novel DA-mimetic probe, DA-yne, with a biorthogonal alkyne handle.
• Utilized DA-yne to label DPAs in fixed cells and lysates, followed by fluorescent visualization.
• Employed chemoproteomic enrichment and mass spectrometry to globally map DPA-modified proteins and their pathways in SH-SY5Y neuronal cells.

Main Results:

• DA-yne successfully visualized DPAs and enabled global mapping of proteins susceptible to DQ modification.
• Proteomic profiling revealed DPA formation affects diverse pathways, including ER stress, cytoskeletal instability, and proteotoxicity.
• Protein disulfide isomerase 3 (PDIA3), involved in ER stress, was identified as a DPA target and functionally inhibited by DA.

Conclusions:

• DA-yne provides new mechanistic insights into DA toxicity relevant to Parkinson's disease.
• Dysregulated dopamine can induce or exacerbate ER stress by modifying key proteins like PDIA3.
• The DA-yne probe is a valuable tool for studying quinone toxicity and its role in neurodegenerative diseases.