Characterization of lysine demethylase KDM5 family: Substrate specificity and identification of potential novel non-histone substrates
Bibliographic record
Abstract
A major regulatory influence over cell biology is lysine methylation and demethylation within histone proteins.The KDM5/JARID1 sub-family are 2oxoglutarate and Fe(II)-dependent lysine-specific histone demethylases that are characterized by their Jumonji catalytic domains.This enzyme family is known to facilitate the removal of tri-/di-methyl modifications from lysine 4 of histone H3 (i.e., H3-K4me3/2), a mark associated with active gene expression.As a result, studies to date have revolved around KDM5's influence on disease through their ability to regulate H3-K4me2/3.Recently, evidence has demonstrated that KDM5's may influence disease beyond H3-K4 demethylation, making it critical to further investigate KDM5 demethylation of non-histone proteins.In efforts to help identify potential non-histone substrates for the KDM5 family, we developed a library of 180 permutated peptide substrates (PPS), with sequences that are systematically altered from the WT H3-K4me3 sequence.From this library, we characterized recombinant KDM5A/B/C/D substrate preference.Subsequently we developed recognition motifs for each KDM5 demethylase and used them to predict potential substrates for KDM5A/B/C/D.Demethylation activity was then profiled to generate a list of high/medium/low-ranking substrates for further in vitro validation for each of KDM5A/B/C/D.Through this approach, we analyzed prediction success rate and identified 66 high-ranking substrates in which KDM5 demethylases displayed significant in vitro activity towards.We further shown the ability to monitor changes in cellular methylation in a handful of the 66 high ranking candidate substrates in response to KDM5 inhibition.Specifically, we iii focused validation efforts on a high-ranking KDM5A novel substrate: p53-K370me3.We demonstrated significant recombinant KDM5A1-588ΔAP and KDM5A1-801 activity towards the p53-K370me3 substrate in vitro.We then monitored KDM5A-mediated demethylation of the p53-K370me3/2 substrate in HCT 116 cells using a combination of wild-type KDM5A and inactive-mutant KDM5AH483A overexpression plasmids, along with immunoblotting, (co-) immunoprecipitation and mass spectrometry analysis.Furthermore, we have shown that KDM5A expression influences the established p53-53BP1 interaction.Finally, we identified a novel p53-TAF5 interaction dominated through the p53-K370me3 state and how KDM5A activity might affect this interaction.Ultimately, we have provided the first evidence of a KDM5 demethylase targeting a nonhistone substrate for demethylation, via the novel KDM5A demethylation of the p53-K370me3 substrate.
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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".