An affinity-directed phosphatase, AdPhosphatase, system for targeted protein dephosphorylation

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Signaling

Background:

• Reversible protein phosphorylation is crucial for cellular signaling, with dysregulation implicated in numerous diseases.
• Controlling phosphorylation at the substrate level is complex due to kinases phosphorylating multiple substrates and substrates being targeted by multiple kinases.

Purpose of the Study:

• To develop a novel system, AdPhosphatase, for targeted dephosphorylation of specific phospho-substrates.
• To demonstrate the specificity and efficacy of AdPhosphatase in altering protein phospho-status and function.

Main Methods:

• Engineered AdPhosphatase by conjugating catalytic subunits of Protein Phosphatase 1 or 2A to an anti-GFP nanobody.
• Utilized CRISPR-Cas9 to knock in GFP-tags into target phospho-proteins (FAM83D, ULK1).
• Assessed AdPhosphatase-mediated dephosphorylation of tagged proteins.

Main Results:

• AdPhosphatase system achieved highly specific dephosphorylation of GFP-tagged FAM83D and ULK1.
• Demonstrated successful redirection of protein phosphatases to neo-substrates via nanobody-mediated proximity.
• Showcased the ability to alter the phospho-status and function of target proteins.

Conclusions:

• The AdPhosphatase system provides a precise method for targeted protein dephosphorylation.
• This technology represents a new modality for potential therapeutic interventions and drug discovery, particularly in diseases linked to aberrant phosphorylation.