The role of Pirh2 C-terminal residues and ubiquitin lysine chains in ubiquitination mechanism.
Bibliographic record
Abstract
11110 Background: Pirh2, a p53 inducible gene, is proposed to be a main regulator of p53 family proteins fine tuning the DNA damage response by acting as an E3 ligase. A negative feedback loop between Pirh2 and p53 exists where under unstressed conditions, Pirh2 induces p53 ubiquitination followed by proteosomal degradation. In case of cellular stress when the activity of tumor suppressors is essential, Pirh2 is self-ubiquitinated releasing p53 continuous repression. Interestingly among all E3 ligases, Pirh2 is the only one to be over-expressed in a wide range of human tumors. This over-expression indicates a disruption in the self-ubiquitination mechanism or a defect in the degradation mechanism post ubiquitination. Based on that, we aimed to analyze Pirh2 ubiquitination mechanism through mapping Pirh2 domains to reveal the essential residues for this process and the ubiquitin chains utilized. Methods: Pirh2 constructs deleting major residues in the three domains were designed, and also ubiquitin mutant constructs were designed through single/multiple mutations at specific positions where lysine residues are mutated to arginine. Using these constructs and in comparison to WT proteins, we tested Pirh2 in-vitro self and p53 ubiquitination activity. Results: Regarding Pirh2, we were able to reveal that residues 240-250 of the c-terminal along with the ring domain are essential for self-ubiquitination.K63R and K48R ubiquitin did not affect Pirh2 self-ubiquitination minimizing the impact of ubiquitin mutations on Pirh2 self-ubiquitination. However, KO, which had all lysine residues mutated to arginine, showed total inhibition of Pirh2 self-ubiquitination confirming the importance of lysine residues. Interestingly, Pirh2 self-ubiquitination reaction showed no difference in the presence or absence of p53 proteins. Concerning Pirh2-p53 ubiquitination, K48 was found to be critical for E3 ubiquitin ligase activity because K48R and not K36R showed defective ubiquitination. All results were confirmed by quantifying ubiquitin. Conclusions: Our data added knowledge to the Pirh2 self-ubiquitination mechanism that can resolve the constant overexpression of Pirh2 proteins hence maximizing p53 response to DNA damage.
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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 | 0.001 |
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".