Design and Construction of a Protein Degradation Switch

  • Type:
    Conference Presentation
  • Conference Type:
    AIChE Annual Meeting
  • Presentation Date:
    November 7, 2010
  • Duration:
    30 minutes
  • Skill Level:
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Targeted protein degradation in eukaryotic cells requires recruitment of the target protein to proteasome which is usually triggered by ubiquitination of the target protein. It has recently been shown that ubiquitination is not necessary and localization to the proteasome is sufficient for degradation [1]. One intriguing way to bypass the ubiquitination step is artificial induction of target protein and proteasome dimerization. In this method, both the target protein and the proteasome are fused to particular protein domains and upon addition of a small molecule that can simultaneously bind to both domains, dimerization and consequently localization of the target protein to the proteasome occur. To implement this idea, we took advantage of a naturally occurring dimerizing molecule, rapamycin. We fused the Rpn10 subunit of Saccharomyces cerevisiae's proteasome with Fpr1, and on the other hand we fused the target protein, such as Ura3, with the Fpr1-rapamycin binding domain of Tor1. Upon adding rapamycin to the cell culture medium, Fpr1 and Tor1 domains come together and hence the target protein is localized to the proteasome and degrades. We have shown previously that the above approach is applicable for certain target proteins [2]. However, its efficiency varies substantially across different proteins and the method remains ineffective for proteins that are inherently difficult to degrade. To overcome this limitation, we propose to utilize multiple binding modules to speed up the degradation process. After confirmation from simulation studies that increasing the number of binding modules would lead to faster degradation, we constructed a series of differently tagged proteins to test our hypothesis. To study systematically the effect of the number of binding modules, we made use of a mutated version of Tor1 that cannot bind to rapamycin. Triple tags with different combinations of wild-type and mutated Tor1 domains (eight combinations in total) were constructed and fused to Ura3. The temporal profile of Ura3 in response to rapamycin has been measured in various constructs and the half-life of the Ura3 protein has been estimated accordingly. This work highlights the design-orientated approach of synthetic biology in engineering molecular tools for switch-like and tunable control of protein degradation. [1] Stankunas K, Crabtree GR, Exploiting protein destruction for constructive use, PNAS, 2007, 104(28): 11511-11512. [2] Janse DM, Crosas B, Finley D, Church GM, Localization to the proteasome is sufficient for degradation, J Biol Chem, 2004, 279(20): 21415-21420.

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