Rapid cell type-specific nascent proteome labeling in Drosophila

Area of Science:

• Molecular Biology
• Neuroscience
• Biochemistry

Background:

• Controlled gene expression relies on regulated protein synthesis, often cell type-specific.
• Existing methods for measuring nascent protein synthesis in vivo are cumbersome.
• O-propargyl-puromycin (OPP) labels nascent proteins but lacks cell specificity.

Purpose of the Study:

• To develop a method for cell type-specific in vivo labeling of nascent proteins.
• To investigate age-related changes in protein synthesis in the *Drosophila* brain.

Main Methods:

• Developed phenylacetyl-OPP (PhAc-OPP), a puromycin analog with an enzyme-labile blocking group.
• Utilized cell type-specific expression of penicillin G acylase (PGA) to enable targeted PhAc-OPP incorporation.
• Applied PGA-dependent OPP incorporation (PoPPI) for imaging and affinity purification of nascent proteins in intact *Drosophila* brains.

Main Results:

• Achieved robust, cell-specific labeling of nascent proteins in targeted *Drosophila* brain cells within 2 hours.
• Demonstrated the method's ability to capture in vivo protein synthesis states quantitatively.
• Revealed a significant age-related decline in neuronal protein synthesis in the fly brain.

Conclusions:

• PGA-dependent OPP incorporation (PoPPI) enables rapid, cell-specific visualization and identification of nascent proteins in vivo.
• The method is suitable for studying protein synthesis under various physiological and pathological conditions.
• PoPPI provides new insights into age-related changes in neuronal protein synthesis.