Characterization of interaction and ubiquitination of phosphoenolpyruvate carboxykinase by E3 ligase UBR5
Bibliographic record
Abstract
Phosphoenolpyruvate carboxykinase (PEPCK1) is ubiquitinated by E3 ubiquitin ligase UBR5, which was thought to be facilitated by the acetylation of Lys70, Lys71 and Lys594 in PEPCK1. Here, we made a series of UBR5 HECT domain truncation variants and, through pull-down assay, showed that the N-terminal lobe of the UBR5 HECT domain is largely responsible for interacting with PEPCK1. We mutated all three lysine residues thought to be acetylated in PEPCK1 but were surprised to observe no loss of binding to UBR5 HECT domain. Furthermore, two PEPCK1 truncation variants (74-622 aa and 10-560 aa) lacking these lysine residues were still able to bind with UBR5 and ubiquitinated in HEK293T cells. To discover the ubiquitination site(s) of PEPCK1, which is currently unknown, the Lys residues of PEPCK1 were mutated to Ala and the ubiquitination level of the PEPCK1 mutants was assessed. Results revealed at least two ubiquitination sites (Lys243 and Lys342), which represent the first time that ubiquitination sites of PEPCK1 have been identified. Our pull-down experiments further show that the lack of ubiquitination of PEPCK1 Lys243Ala and Lys342Ala mutants is not due to their binding to UBR5, which remained unchanged. Taken together, our work has provided new insights into UBR5 mediated ubiquitination of PEPCK1.
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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 | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 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".