Spatiotemporal-resolved protein networks profiling with photoactivation dependent proximity labeling

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Existing proximity labeling methods using activated esters or phenoxy radicals have limitations in reactivity and specificity.
• Activated esters exhibit broad labeling radii, while phenoxy radicals can disrupt cellular redox balance.
• There is a need for precise, spatiotemporally controlled proximity labeling techniques in living cells.

Purpose of the Study:

• To develop and validate a novel photoactivation-dependent proximity labeling (PDPL) method.
• To compare PDPL's performance against existing methods like TurboID for proteomic coverage and specificity.
• To apply PDPL for identifying novel interactors and substrates of disease-relevant proteins, such as BRD4 and Parkin.

Main Methods:

• Genetically fused the photosensitizer protein miniSOG to a protein of interest.
• Utilized blue light irradiation to trigger singlet oxygen generation for spatiotemporally resolved labeling.
• Employed aniline probes to label histidine residues, enabling mapping of proximal proteins.
• Performed side-by-side comparisons with TurboID and applied PDPL to BRD4 and Parkin.

Main Results:

• PDPL demonstrated high fidelity in mapping organelle-specific proteomes.
• PDPL achieved more specific and deeper proteomic coverage compared to TurboID.
• PDPL identified previously unknown interactors for BRD4 and Parkin.
• Two novel Parkin substrates, Ssu72 and SNW1, were identified via overexpression screening, mediated by the ubiquitination-proteasome pathway.

Conclusions:

• Photoactivation-dependent proximity labeling (PDPL) is a powerful tool for precise and efficient proteomic analysis in living cells.
• PDPL overcomes limitations of previous methods, offering enhanced specificity and depth in protein interaction mapping.
• The identification of new substrates for Parkin highlights PDPL's utility in disease-related biological discovery.