deGradFP-Targeted Protein Degradation via GFP protein

Trypanosoma brucei, an evolutionarily divergent eukaryote, causes devastating diseases like African trypanosomiasis. Studying its biology is critical for understanding eukaryotic evolution and developing therapies. However, functional genomics in T. brucei has been hindered by the lack of efficient tools to deplete stable proteins. Traditional methods like RNAi and CRISPR are slow or ineffective for proteins with low turnover. degradFP, a protein degradation system previously used in model organisms, to T. brucei.

degradFP combines the substrate-recognition domain of an SCF E3 ubiquitin ligase with VhhGFP4, a nanobody that binds GFP derivatives. Upon induction with doxycycline, degradFP recruits GFP/YFP-tagged proteins to the ubiquitin-proteasome pathway, leading to their rapid degradation.

degradFP's key advantage is its speed, bypassing the lag time inherent to RNAi or gene knockout methods. Its reliance on endogenous GFP tagging-a routine technique in T. brucei-makes it widely applicable. However, the system's success depends on GFP accessibility and functionality of the tagged protein. For instance, if GFP is sterically hindered or the fusion disrupts protein activity, degradFP may fail.

By enabling rapid protein depletion, it accelerates phenotypic analysis of essential genes, particularly those involved in cell division, trafficking, and virulence. Future work could explore nanobodies against other epitopes (e.g., HA or Myc tags) to broaden applicability. Testing degradFP in bloodstream-form parasites and optimizing F-box proteins for different life stages will further enhance its utility. Ultimately, this tool may expedite drug target validation and anti-parasitic drug discovery.

By combining precision with speed, this system opens new avenues for dissecting T. brucei biology and combating neglected tropical diseases. As the tool evolves, it could become a cornerstone of functional genomics in non-model eukaryotes.

Reference

• Ishii M, Akiyoshi B. Targeted protein degradation using deGradFP in Trypanosoma brucei. Wellcome Open Res. 2022 Oct 19;7:175. doi: 10.12688/wellcomeopenres.17964.2. PMID: 35865221; PMCID: PMC9277568.