Post-translational modifications (PTMs) regulate protein structure, function, and localization, influencing key processes like gene transcription and metabolism.
These chemical marks are written and erased by enzymes that, when dysregulated, can drive disease.
Targeting such enzymes has great therapeutic potential, but observing their activity inside living cells has remained a challenge.
Static Assays to Living Sensors
Conventional methods, such as mass spectrometry or activity-based probes, reveal enzyme activity only after cells are disrupted.
Genetic code expansion (GCE) enables scientists to insert noncanonical amino acids (ncAAs) like AcK into proteins at specific sites, but this typically requires feeding cells synthetic amino acids that are poorly absorbed.
The new study overcomes this barrier by creating autonomous cells capable of producing AcK internally without external supplementation.
Researchers identified RDW23166.1, a free-lysine acetyltransferase that uses acetyl-CoA for acetylation. When combined with GCE machinery, this enzyme allowed both bacterial and mammalian cells to biosynthesize and incorporate AcK into proteins.
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Building Live Enzyme Reporters
The team paired these engineered cells with fluorescent (EGFP-K85AcK) and bioluminescent (Fluc-K529AcK) reporters to monitor sirtuin deacetylase activity in real time.
The fluorescent sensor detected activity across multiple sirtuins (SIRT1/2/3/5), while the bioluminescent version was SIRT1-focused (SIRT1»SIRT2) and enabled real-time imaging of SIRT1 activity in living mice.
These autonomous cells produced AcK-modified proteins at multiple defined sites, yielding stronger and more uniform incorporation than methods relying on external amino acid feeding.
Testing in Live Tumor Models
Using HCT116 colon cancer cells expressing the SIRT1 sensor, the researchers assessed how inhibitors affected enzyme activity and tumor growth in mice.
The SIRT1 inhibitor EX-527 sharply reduced the bioluminescent signal but did not shrink tumors, whereas Inauhzin lowered both the signal and tumor size, likely through off-target, p53-dependent effects.
These findings demonstrate that blocking an enzyme’s activity doesn’t always translate to therapeutic benefit.
A Platform for Real-Time Biology
This cell-based system is a powerful tool for studying post-translational modifications and evaluating drug responses in their native biological contexts.
Its light-based readouts could streamline drug screening and potentially be applied to other modifications or organoid models.
By eliminating the need for externally supplied AcK, the technology enables dynamic observation of natural protein regulation in living cells, providing scientists with a window into the molecular changes that drive health and disease.
Journal Reference
Hu, Y. et al. (2025). Engineering unnatural cells with a 21st amino acid as a living epigenetic sensor. Nature Communications, 16(1), 1-17. DOI: 10.1038/s41467-025-64448-1
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