Abstract 5425: Human mitochondrial ClpXP protease degrades serine phosphorylated substrates
Bibliographic record
Abstract
Abstract ClpXP is an AAA+ protease located in mitochondria matrix. In human, ClpXP degrades damaged respiratory chain proteins and is essential for acute myeloid leukemia (AML) cell survival. This complex consists of the tetradecameric ClpP protease and regulatory particles ClpX. In bacteria, ClpXP targets substrates with a co-translationally added SsrA sequence. However, human lacks this tag and the degron for ClpXP remain unidentified. Notably, bacterial ClpXP homologues degrade substrates with phosphorylated arginine (pArg). We hypothesized that phosphorylated amino acids also facilitate substrate degradation by ClpXP in human mitochondria. Here, we demonstrate that ClpXP selectively degraded phosphorylated α-casein and tau proteins over dephosphorylated forms. Replacing ClpX with ONC201 or E. coli ClpA abolished phosphorylation selectivity, indicating ClpX imparts substrate specificity for phosphorylation. Next, we screened a panel of phosphorylated amino acids and peptides for their effect on 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. Free serine and phosphate also had no effect. Differential scanning fluorimetry showed that pSer stabilized ClpX, consistent with pSer binding to ClpX. We then used hydrogen/deuterium exchange mass spectrometry (HDX-MS) to reveal the ClpX conformational changes upon pSer containing peptides (Ala-pSer-Ala) binding. HDX analysis indicated the ClpX RKL loop as a putative binding site. Mutating this loop in ClpX (ClpXAAL) disrupted its interaction with pSer, resulting in loss of specificity for phosphorylated substrates. This suggests ClpXP selectively recruits phosphorylated protein for degradation through its RKL loop. In AML cells, ClpP knockdown impaired the respiratory chain and increased reactive oxygen species. Through mass spectrometry analysis of ClpXP interactome and their phosphorylation status upon ClpP inhibition, we identified potential ClpXP endogenous substrates SDHA and NDUFA4, components of the OXPHOS complex. Knockdown of ClpX or ClpP increased levels of pSer-SDHA and pSer-NDUFA4 without changing total protein levels. Recombinant ClpXP degraded pSer-SDHA in a dose-dependent manner, with degradation blocked by pSer peptides. ClpXP did not degrade total SDHA, consistent with its preference for phosphorylated substrates. 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, ClpXP degrades damaged mitochondrial proteins with serine phosphorylation, maintaining proper mitochondrial function. Citation Format: Yue Feng, Monica M. Goncalves, Yulia Jitkova, Alexander F. Keszei, Yongran Yan, Chaitra Sarathy, Jonathan St-Germain, Tristan M. Kenney, Matthew Tcheng, Vincent Trudel, Ross S. Mancini, Rahul Upadhyay, Rose Hurren, Marcela Gronda, Matthew Schultz, Kaylen Soriano, Kaitlin Lees, Neil C. Pomroy, S. Quinn W. Currie, Gilbert G. Privé, Mark A. Reed, Andrei K. Yudin, Linda Z. Penn, Cheryl H. Arrowsmith, Brian Raught, Mohammad Mazhab-Jafari, Siavash Vahidi, Aaron D. Schimmer. Human mitochondrial ClpXP protease degrades serine phosphorylated substrates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5425.
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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) | 0.002 | 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".