Identification of Cysteines Involved in S-Nitrosylation, S-Glutathionylation, and Oxidation to Disulfides in Ryanodine Receptor Type 1
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Bibliographic record
Abstract
The skeletal muscle Ca(2+)-release channel (ryanodine receptor type 1 (RyR1)) is a redox sensor, susceptible to reversible S-nitrosylation, S-glutathionylation, and disulfide oxidation. So far, Cys-3635 remains the only cysteine residue identified as functionally relevant to the redox sensing properties of the channel. We demonstrate that expression of the C3635A-RyR1 mutant in RyR1-null myotubes alters the sensitivity of the ryanodine receptor to activation by voltage, indicating that Cys-3635 is involved in voltage-gated excitation-contraction coupling. However, H(2)O(2) treatment of C3635A-RyR1 channels or wild-type RyR1, following their expression in human embryonic kidney cells, enhances [(3)H]ryanodine binding to the same extent, suggesting that cysteines other than Cys-3635 are responsible for the oxidative enhancement of channel activity. Using a combination of Western blotting and sulfhydryl-directed fluorescent labeling, we found that two large regions of RyR1 (amino acids 1-2401 and 3120-4475), previously shown to be involved in disulfide bond formation, are also major sites of both S-nitrosylation and S-glutathionylation. Using selective isotopecoded affinity tag labeling of RyR1 and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, we identified, out of the 100 cysteines in each RyR1 subunit, 9 that are endogenously modified (Cys-36, Cys-315, Cys-811, Cys-906, Cys-1591, Cys-2326, Cys-2363, Cys-3193, and Cys-3635) and another 3 residues that were only modified with exogenous redox agents (Cys-253, Cys-1040, and Cys-1303). We also identified the types of redox modification each of these cysteines can undergo. In summary, we have identified a discrete subset of cysteines that are likely to be involved in the functional response of RyR1 to different redox modifications (S-nitrosylation, S-glutathionylation, and oxidation to disulfides).
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Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.001 |
| 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) | 0.000 | 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 it