New technology detects protein loss by turning ‘signal weakening’ into ‘signal amplification’

A technology has been developed that detects protein loss by turning it into “signal amplification” rather than “signal weakening.” Researchers at Stanford University introduced a technology called “ratio-based transcriptional activation” (RTA) for highly sensitive detection of protein degradation inside cells.

According to the international journal Cell on the 1st (local time), a team led by Professor Steven Banik at Stanford University announced RTA, a technology that converts protein degradation into a programmable, amplified gene-expression signal.

Protein degradation is a key mechanism by which cells maintain homeostasis. Existing methods, such as immunoblotting and fluorescent reporters, directly link a decrease in the target protein to a weaker signal, resulting in low sensitivity and making large-scale screening difficult.

RTA was inspired by the NF-κB signaling pathway. Under normal conditions, NF-κB is held in the cytoplasm by the inhibitory protein IκB. When IκB is degraded, NF-κB enters the nucleus and activates transcription of target genes.

The researchers engineered a system to reproduce this “inhibitor degradation→transcription factor release” mechanism. They used the target protein as a degradable inhibitor, fused it to the inhibitory domain of a transcription factor, and adjusted the ratio of the transcription factor to the fused inhibitor.

When the target protein is degraded, inhibition is lifted, allowing the transcription factor to activate a reporter gene and amplify the output signal. By changing the signal from “weakening due to loss” to “amplification of loss,” the system greatly increases detection sensitivity and overcomes limitations imposed by target protein abundance.

The RTA circuit is highly modular and versatile. Using a triple orthogonal circuit, the researchers simultaneously detected the downregulation of multiple proteins in a single cell and generated a range of outputs, including fluorescent protein expression, cell viability and barcode sequencing.

The technology is not limited to a particular degradation pathway and can detect both the ubiquitin-proteasome and lysosomal pathways. The researchers also applied RTA to detect oncogenic proteins and identify new molecular glue degraders.

However, the technology is still at the proof-of-concept stage. The researchers said that overexpression of fusion proteins could interfere with the function of endogenous proteins, and that the long-term stability and off-target effects of in vivo applications require further evaluation.