Tandem Bioorthogonal Labeling Uncovers Endogenous Cotranslationally <i>O</i>-GlcNAc Modified Nascent Proteins
Bibliographic record
Abstract
Hundreds of nuclear, cytoplasmic, and mitochondrial proteins within multicellular eukaryotes have hydroxyl groups of specific serine and threonine residues modified by the monosaccharide N -acetylglucosamine (GlcNAc). This modification, known as O -GlcNAc, has emerged as a central regulator of both cell physiology and human health. A key emerging function of O -GlcNAc appears to be to regulate cellular protein homeostasis. We previously showed, using overexpressed model proteins, that O -GlcNAc modification can occur cotranslationally and that this process prevents premature degradation of such nascent polypeptide chains. Here, we use tandem metabolic engineering strategies to label endogenously occurring nascent polypeptide chains within cells using O -propargyl-puromycin (OPP) and target the specific subset of nascent chains that are cotranslationally glycosylated with O -GlcNAc by metabolic saccharide engineering using tetra- O -acetyl-2- N -azidoacetyl-2-deoxy- d -galactopyranose (Ac 4 GalNAz). Using various combinations of sequential chemoselective ligation strategies, we go on to tag these analytes with a series of labels, allowing us to define conditions that enable their robust labeling. Two-step enrichment of these glycosylated nascent chains, combined with shotgun proteomics, allows us to identify a set of endogenous cotranslationally O -GlcNAc modified proteins. Using alternative targeted methods, we examine three of these identified proteins and further validate their cotranslational O -GlcNAcylation. These findings detail strategies to enable isolation and identification of extremely low abundance endogenous analytes present within complex protein mixtures. Moreover, this work opens the way to studies directed at understanding the roles of O -GlcNAc and other cotranslational protein modifications and should stimulate an improved understanding of the role of O -GlcNAc in cytoplasmic protein quality control and proteostasis.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
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 | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
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".