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Genetic code expansion reveals site-specific lactylation in living cells reshapes protein function

ABSTRACT: Genetic code expansion reveals site-specific lactylation in living cells reshapes protein function

ORGANISM(S): Homo Sapiens

SUBMITTER: Hui Ye

PROVIDER: PXD048127 | iProX | Tue Dec 26 00:00:00 GMT 2023

REPOSITORIES: iProX

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Genetic code expansion reveals site-specific lactylation in living cells reshapes protein function

Project description:Still in its infancy, the functions of lactylation remain elusive. To address this, we established a comprehensive workflow for lactylation studies that integrates the discovery of lactylation sites with proteomics, the expression of site-specifically lactylated proteins in living cells via genetic code expansion (GCE), and the evaluation of the resulting biological consequences. Specifically, we developed a wet-and-dry-lab combined proteomics strategy, and identified highly conserved lactylation at ALDOA-K147. Driven by its potential biological significance, we site-specifically expressed this lactylated ALDOA in mammalian cells and interrogated the biological changes. We discovered that it not only inhibited enzyme activity but also elicited gain-of-function effects——it dramatically reshaped the functionality of ALDOA by improving stability, enhancing nuclear translocation and affecting gene expression. Further, we demonstrated broad applicability of this workflow to study distinct histone lactylation sites. Together, we anticipate its wide uses in elucidating causative links between site-specific lactylation and target-centric or cell-wide changes.

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Genetic code expansion reveals aminoacylated lysine ubiquitination mediated by UBE2W

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Chimeric design of Pyrrolysyl-tRNA synthetase/tRNA pairs and canonical synthetase/tRNA pairs for genetic code expansion

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Engineering unnatural cells with a 21st amino acid as a living epigenetic sensor

Project description:Living animals rely extensively on posttranslational modifications (PTMs) to regulate protein activity, stability, subcellular localization, and protein protein interactions. These modifications are tightly controlled by the balance of writer and eraser enzymes, which add or remove PTMs on proteins. Current strategies to measure writer and eraser activities in living animals largely depend on invasive methods such as single cell sequencing, quantitative mass spectrometry, or activity based probes, which often lack cell or tissue specificity. In this study, we report the development of autonomous cells, both prokaryotic and eukaryotic, with the ability to biosynthesize and genetically encode acetyllysine using genetic code expansion technology. These engineered living sensors with a site specific acetyllysine modification can be transplanted into living animals, enabling real time monitoring of PTM dynamics in living cells and their visualization within animal models. We further demonstrate the utility of these cells in tracking deacetylase activity and assessing the effects of deacetylase inhibitors on PTM dynamics in living animals.

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