Abstract 7053: Serine phosphorylation marks proteins for degradation by the mitochondrial matrix protease, ClpXP
Authors: Feng Yue , Goncalves Monica M. , Jitkova Yulia , Keszei Alexander , Sarathy Chaitra , Trudel Vincent , St-Germain Jonathan , Tcheng Matthew , Yan Yongran , Hurren Rose , Schultz Matthew , Raught Brian , Yudin Andrei , Mazhab-Jafari Mohammad , Vahidi Siavash , Schimmer Aaron D. 
Published: March 2024

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.