Identification and Functional Characterization of Phosphorylation Sites on GTP cyclohydrolase I
Bibliographic record
Abstract
GTP cyclohydrolase I (GCH‐1) is the rate‐limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases (NOS) and aromatic amino acid hydrolases. In this study, we aim to identify specific phosphorylation sites on GCH‐1 and characterize their function. Rat GCH‐1 with FLAG tag was subcloned into a Tet‐On plasmid which was stably expressed in Flp‐in 293 cells. Mass spectrometry studies revealed GCH‐1 was phosphorylated in vivo at serine(S) 167 and threonine (T) 231. Sequence analysis of GCH‐1 protein showed 8 potential phosphorylation sites [S51, S72, T85, T91, T103, S130, S167 and T231]. Functional studies demonstrated that GCH‐1 activity and BH4 levels were significantly decreased in cells expressing phospho‐defective alanine (A) mutants S72 A, T85A, T91A, T103A or S130A while they were increased in cells expressing the T231A mutant. Furthermore, GCH1 activity, and BH4 levels were increased in cells expressing the phospho‐mimic glutamic acid (E)/aspartic acid (D) mutants S72E, T85E, T91E, T103D or T130D, while they were decreased in cells expressing the T231D mutant. Interestingly, cells expressing the S51E, but not S51A had increased cellular BH4 production, while cells expressing S167A or S167E mutant did not affect BH4 synthesis. In summary, our study suggests that GCH‐1 is positively and negatively regulated by multiple phosphorylation sites.
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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".