Abstract
Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner. The technique involves tagging a protein of interest (POI) with a destabilizing domain (DD) specifically controlled by a small molecule. The successful construction of such fusion proteins may, however, be limited by functional interference of the DD epitope with electrostatic interactions required for full biological function of proteins. Another drawback of this approach is the remaining endogenous protein. Here, we combined the Cre-LoxP system with an advanced DD and generated a protein regulation system in which the loss of an endogenous protein, in our case the tumor suppressor PTEN, can be coupled directly with a conditionally fine-tunable DD-PTEN. This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models.
Highlights
Phosphatase and tensin homolog located on chromosome 10 (PTEN) is one of the most commonly mutated or deleted tumor suppressors in human cancer that is causally linked to autism spectrum disorder [1,2]
We proposed that in the absence of Shld1, FKBPÃ would be rapidly degraded by the ubiquitin proteasome system [13], resulting in the loss of FKBPÃ-PTEN fusion protein, whereas addition of Shld1 will stabilize the protein and inhibit PI3K/Akt signaling (Fig 1a)
Based on available knowledge regarding linker optimization [16,17], we composed a number of different peptide sequences with the aim of restoring PTEN activity in the FKBPÃ-linker-PTEN fusion protein (Fig 1c)
Summary
Phosphatase and tensin homolog located on chromosome 10 (PTEN) is one of the most commonly mutated or deleted tumor suppressors in human cancer that is causally linked to autism spectrum disorder [1,2]. Changes in PTEN expression level leads to aberrant cell cycle progression as well as to alterations in cell migration [3,4], amongst other cellular responses. It is still a considerable challenge to ascertain the minimum alteration in PTEN level or activity that can cause the onset of tumor formation [5] or neurological changes [6], or whether disease states can be ameliorated by reinstalling PTEN expression. We set out to exploit FKBP DD-technology to control PTEN protein function in a rapid, reversible and tunable manner. FKBP is a 12kDa FK506 binding protein, which is broadly expressed in various tissues and functions as a protein chaperone for newly synthesized polypeptides [8]. An engineered human FKBP has PLOS ONE | DOI:10.1371/journal.pone.0145783. An engineered human FKBP has PLOS ONE | DOI:10.1371/journal.pone.0145783 December 30, 2015
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