Abstract 2407: Serine and threonine phosphorylation marks proteins for degradation by ClpXP
Bibliographic record
Abstract
Abstract The mitochondrial serine protease ClpXP regulates the integrity of the respiratory chain by degrading damaged and/or misfolded proteins. This protease is over-expressed in multiple malignancies and inhibiting or hyperactivating it kills malignant cells in vitro and in vivo. In Bacillus subtilis, the bacterial ClpXP homologue recognizes proteins tagged with phosphor-arginine for degradation. Yet, it is unknown how the mitochondrial ClpXP recognizes proteins for degradation. To determine how phosphorylated amino acids influence ClpXP-mediated protein degradation, we incubated recombinant ClpXP with its unnatural substrate FITC-casein and increasing concentrations of phospho-serine (pSer), phospho-threonine (pThr), phospho-arginine (pArg), or phosphor-tryrosine (pTry) in a cell-free assay and measured release of fluorogenic FITC. In a dose-dependent manner, pSer and pThr free amino acids inhibited casein cleavage by ClpXP while pTyr, pArg, and the dephosphorylated amino acids had no effect on ClpXP activity. Likewise, ApSA and ApTA peptides inhibited ClpXP enzyme activity, while the non-phosphorylated version (ASA and ATA) and no effect. Next, we tested whether the phosphorylation state of full length proteins would influence their degradation by ClpXP. Using gel-based cell-free assays, the phosphorylation enriched α-casein and β-casein were degraded by recombinant ClpXP. In contrast, the κ-casein with low phosphorylation level and dephosphorylated α-casein were not cleaved by ClpXP. In addition, we treated α-casein with lambda-phosphatases to dephosphorylate the protein and confirmed the release of free phosphate by the Malachite green phosphate assay. Phosphatase treated α-casein was resistant to ClpXP cleavage. As ClpXP is an AAA ATPase, we asked if pSer and pThr acted on the ATPase of the enzyme. pSer and pThr did not inhibit the ATPase activity of ClpX, as measured by the malachite green ATPase assay. We also measured the effect of pSer and pThr on the peptidase activity of ClpP alone without its regulatory subunit ClpX. Neither pSer or pThr inhibited ClpP peptidase activity. We investigated if pSer and pThr could bind to ClpX. Using thermal shift binding assays, we demonstrated that pSer and pThr but not pTyr and pArg bind ClpX and none of the phosphorylated amino acids bind ClpP. Finally, we characterized the total and phopho- mitochondrial proteome of T-REX HEK293 wild type and ClpP knockout cells by mass spectrometry. We identified 402 unique mitochondrial proteins and 38 phosphorylated mitochondrial proteins in wild type cells. 20 phosphorylated mitochondrial proteins increased in the ClpP knockout cells compared to wild type with a cut-off of 2 fold. In summary, we discovered that ClpX binds pSer and pThr and phosphorylation of these amino acids mark proteins for degradation by the ClpXP mitochondrial protease. This work highlights a new strategy to develop inhibitors of ClpXP for the treatment of cancer. Citation Format: Yue Feng, Yulia Jitkova, Aaron D. Schimmer. Serine and threonine phosphorylation marks proteins for degradation by ClpXP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2407.
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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) | 0.001 | 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 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 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".