Abstract 7053: Serine phosphorylation marks proteins for degradation by the mitochondrial matrix protease, ClpXP
Bibliographic record
Abstract
Abstract ClpXP is a conserved protein degradation system. In human mitochondria, ClpXP degrades damaged respiratory chain proteins and is vital for the survival of acute myeloid leukemia (AML) cells. This system consists of the barrel-like protease ClpP, capped by regulatory particles (RP) ClpX. In bacteria, ClpXP targets substrates with a co-translationally added SsrA sequence. However, in humans, the SsrA tag is absent, and the degradation markers for ClpXP remain unidentified. Notably, bacterial ClpXP homologues degrade substrates with phosphorylated arginine (pArg). We hypothesized that phosphorylated amino acids also mark substrates for ClpXP degradation in human mitochondria. We showed that ClpXP preferentially degraded phosphorylated α-casein substrate over dephosphorylated α-casein. In contrast, ClpP activated by ONC201, without ClpX, degraded phosphorylated and dephosphorylated α-casein with equal efficiency. Next, we screened a panel of phosphorylated amino acids and peptides for their ability to inhibit ClpXP-mediated degradation of α-casein. Phosphorylated serine (pSer) amino acids and peptides inhibited ClpXP protease activity, while phosphorylated tyrosine (pTyr) or pArg did not. Likewise, Ser and free phosphate did not inhibit ClpXP protease activity. Thermal shift assays showed that pSer amino acids and peptides bind to ClpX, not ClpP. Hydrogen/deuterium exchange mass spectrometry revealed that the addition of Ala-pSer-Ala (ApSA) peptide, but not ASA peptide induced conformational changes in the negatively-charged N terminus of ClpX, thus revealing a putative binding site for ApSA. Earlier, we established that ClpP interacts with the respiratory chain complex II subunit SDHA. Knocking down of ClpP in AML cells affected the respiratory chain and increased reactive oxygen species. Therefore, we tested how ClpXP knockdown impacts levels of phospho-serine SDHA (pSer-SDHA) in intact cells. Using shRNA, we knocked down ClpP and ClpX individually in OCI-AML2 cells. After target knockdown, we pulled down pSer proteins and probed for SDHA. Knockdown of both ClpP and ClpX increased the abundance of pSer-SDHA compared to control. We subsequently added recombinant ClpXP protein to total mitochondrial lysates and the pSer immunoprecipitated fraction from OCI-AML2 cells. This led to decreased levels of pSer-SDHA but did not alter levels of total SDHA, consistent with its preference for pSer-marked substrates suggesting that ClpXP degrades pSer-SDHA. Further analysis showed elevated pSer-SDHA levels only in the insoluble mitochondrial protein fraction after ClpX knockdown, suggesting these might be damaged proteins. In conclusion, our findings demonstrate that serine phosphorylation promotes the degradation of damaged mitochondrial proteins by ClpXP protease. Citation Format: Yue Feng, Monica M. Goncalves, Yulia Jitkova, Alexander Keszei, Chaitra Sarathy, Vincent Trudel, Jonathan St-Germain, Matthew Tcheng, Yongran Yan, Rose Hurren, Matthew Schultz, Brian Raught, Andrei Yudin, Mohammad Mazhab-Jafari, Siavash Vahidi, Aaron D. Schimmer. Serine phosphorylation marks proteins for degradation by the mitochondrial matrix protease, ClpXP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7053.
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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 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 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".