Sensitive detection of proteasomal activation using the Deg-On mammalian synthetic gene circuit.
The ubiquitin proteasome system (UPS) has emerged as a drug target for diverse diseases characterized by altered proteostasis, but pharmacological agents that enhance UPS activity have been challenging to establish. Here we report the Deg-On system, a genetic inverter that translates proteasomal degradation of the transcriptional regulator TetR into a fluorescent signal, thereby linking UPS activity to an easily detectable output, which can be tuned using tetracycline. We demonstrate that this circuit responds to modulation of UPS activity in cell culture arising from the inhibitor MG-132 and activator PA28γ. Guided by predictive modelling, we enhanced the circuit's signal sensitivity and dynamic range by introducing a feedback loop that enables self-amplification of TetR. By linking UPS activity to a simple and tunable fluorescence output, these genetic inverters will enable a variety of applications, including screening for UPS activating molecules and selecting for mammalian cells with different levels of proteasome activity.
Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas 77005, USA.
Wenting
W
Zhao
Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas 77005, USA.
Matthew
M
Bonem
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, USA.
Claire
C
McWhite
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, USA.
Jonathan J
JJ
Silberg
1] Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas 77005, USA [2] Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, USA [3] Department of Bioengineering, Rice University, Houston, Texas 77005, USA.
Laura
L
Segatori
D029968:Escherichia coli Proteins; D053263:Gene Regulatory Networks; D005813:Genes, Synthetic; D006367:HeLa Cells; D006801:Humans; D007976:Leupeptins; D046988:Proteasome Endopeptidase Complex; D011500:Protein Synthesis Inhibitors; D049490:Systems Biology; D013752:Tetracycline; D043743:Ubiquitin-Protein Ligase Complexes
D029968:Escherichia coli Proteins; D007976:Leupeptins; D011500:Protein Synthesis Inhibitors; C470187:TetR(B) protein, E coli; D043743:Ubiquitin-Protein Ligase Complexes; D046988:Proteasome Endopeptidase Complex; D013752:Tetracycline; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde