HomeInVivo Biosystems BlogView from the BenchHow do I get rid of this pesky protein? The Hows and Whys of Protein Degradation

How do I get rid of this pesky protein? The Hows and Whys of Protein Degradation

Being able to understand how a protein acts in specific tissues and times can be key for the general understanding of a protein’s function.  There are methods to do this at the genomic level such as the Cre/Lox system or RNAi. However, often a more rapid and direct approach is needed. This can be achieved through the degradation of the target protein. There are a few methods to achieve this degradation but they can be summarized in 3 steps.

  1. Tag your protein of interest at the endogenous locus using CRISPR/Cas9.
  2. Express a recognition and degradation targeting protein (RAD protein) using tissue-specific/condition-specific promoter . 
  3. The RAD protein binds to the tag on the protein of interest and targets the protein of interest for degradation.

Controlled protein degradation of a target can be a powerful tool for scientists as they look to understand the role of a protein at different points of development (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233056/).

Additionally, valuable tools can be built using this technology to precisely control protein expression at key developmental stages (https://doi.org/10.1534/g3.118.200278). As this technique becomes more widespread, there is abundant potential for new tools and discoveries through controlled protein degradation.

Image made created with BioRender.Com

Applications for protein degradation: 

  • Avoiding maternal contribution when studying essential genes during embryonic development (10.1091/mbc.E18-06-0347).
  • Isoform specific depletion to understand the roles of different isoforms.
  • Identifying tissue-specific protein functions (eLife 2020;9:e53603).

There are multiple strategies for inducing degradation. Here I will contrast 2 commonly used methods.

GFP-nanobody system

This method uses a single-domain antibody fragment known as a nanobody for tag recognition. These are much smaller than traditional antibodies and with only one chain can easily be encoded for expression in the genome. This nanobody is anti-GFP and GFP is the tag. The nanobody is fused to a component of E3 ubiquitin ligase complex. When this fusion protein is expressed it will bind to the GFP tag and ubiquitinate the target protein which will then be degraded by the proteosome.

 Advantages:

  • Protein degradation can be monitored using GFP
  • Can use existing GFP-tagged strains
  • Spatial and temporal control of degradation depending on the GFP-nanobody expression

 Disadvantages:

  • GFP tag can interfere with native protein function
  • Can’t be used in the germline

Auxin degradation system

This method has been adapted from the auxin system in Arabidopsis, involved in plant hormone regulation, for use in C. elegans. Target proteins are tagged with a 44 amino acid degron sequence. This is recognized by the F-box protein TIR1, which is a part of an E3 ubiquitin ligase complex called the SCF complex. However, TIR1 only binds to the degron sequence in the presence of auxin. Auxin is a plant hormone with easy uptake into the worm and egg, but with no effect on wild-type C. elegans growth, development, or behavior. The Auxin degradation system provides spatial control of degradation by expressing the TIR1 under tissue specific proteins, and provides temporal control of degradation with the application of auxin at a specific time. 

Advantages:

  • Small tag
  • Spatial and temporal control
  • Can be used in the germline

Disadvantages:

  • Degron tag can interfere with native protein function
  • Second method needed to validate protein degradation (antibody staining)

When using these methods, it is important to monitor the degradation of the target protein. Different levels and rates of depletion have been observed depending on the protein abundance, localization, and accessibility.   Both of these methods have the advantage that a single tagged protein can be combined with multiple different tissue-specific expression patterns of the RAD protein for precise evaluation of the effect of the target protein depletion. 

Degradation tagging of proteins is a powerful genetic tool that is only beginning to be fully utilized to uncover protein function.  It’s a good tool to keep in mind when digging deeper into the genetic pathways that make our favorite worm tick.

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