Development and Characterization of a Novel Protein Stability Probe
Bibliographic record
Abstract
As a regulator of gene transcription, (MYC) modulates cell proliferation, growth, and metabolism. The deregulation of c-MYC (MYC) drives over 50% of cancers, making it a promising therapeutic target. Unfortunately, traditional approaches to target MYC have been unsuccessful. MYC turnover is tightly regulated, with a half-life of just 30 minutes in non-transformed cells, however only one pathway has been well-characterized as regulating MYC stability. Current strategies to measure protein half-life are low-throughput or prone to false readouts, and have hampered the discovery of novel regulatory pathways for MYC that could be targeted therapeutically. To overcome this barrier, we developed a protein stability probe, consisting of c-MYC (MYC) fused to Venus fluorescent protein (MYC-Venus), and a high-content confocal screening pipeline to identify regulators of MYC using automated image analysis. This probe enables protein half-life to be scored as a function of fluorescence intensity and distribution. The MYC-Venus probe was piloted by screening a kinase inhibitor library to identify known and novel kinases that regulate MYC stability. The Venus probe was also validated with another short half-life protein, MCL-1. This validated stability probe was expanded to a cell system that models MYC-driven human breast cancer, in order to further characterize the functionality of the MYC-Venus stability probe. A 438-compound library was screened, and compounds that either increase or decrease MYC-Venus levels with minimal cytotoxicity were identified. Compounds that increased MYC-Venus levels can be attributed to an artefact of the CMV promoter used to express MYC-Venus, demonstrating that the Venus probe can also detect changes in protein levels. While compounds that decreased MYC-Venus levels were also identified, none modulated MYC stability at the timepoints tested. The utility of the MYC-Venus probe was further demonstrated by adapting an existing assay to quantify changes in protein stability through confocal imaging, increasing the number of compounds that can be validated for future screens. The development and characterization of the Venus stability probe lays the foundation for future work to identify novel regulators of short half-life proteins, a field that has been hampered by a lack of screening tools.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".