Abstract 4834: Mitochondrial ClpXP degrades serine phosphorylated protein aggregates

Abstract ClpXP is a serine protease localized to the mitochondrial matrix and maintains mitochondrial proteostasis by degrading damaged/aggregated respiratory chain complex proteins. Both inhibition and hyperactivation of ClpP lead to impaired OXPHOS function and kill leukemic cells in vitro and in vivo. Yet, the marks that tag proteins for degradation by ClpXP remain unknown. The bacterial homologue of ClpXP degrades damaged proteins with arginine phosphorylation. Therefore, we assessed how phosphorylation affects ClpXP protease activity. Recombinant ClpXP was incubated with its unnatural substrate, FITC-tagged casein, in the presence of increasing concentrations of phospho-serine (pSer), phospho-threonine (pThr), phospho-arginine (pArg), or phospho-tyrosine (pTyr). In a dose-dependent manner, pSer and pThr free amino acids and short peptides inhibited casein degradation by ClpXP. In contrast, free Ser, Thr, phosphate, pArg and pTyr did not inhibit ClpXP protease activity. In addition, we characterized ClpP peptidase activity and ClpX ATPase activity using the Ac-Trp-Leu-Ala-AMC (Ac-WLA-AMC) peptidase activity assay and the Malachite Green ATPase assay. Neither pSer nor pThr inhibited ClpX ATPase activity or ClpP peptidase activity. Thus, pSer and pThr only blocked ClpXP from degrading full-length protein without altering the enzymatic activity of the complex. Next, we characterized pSer and pThr binding to ClpXP by thermal shift binding assay. pSer and pThr free amino acids and short peptides bound to ClpX but not ClpP. All the dephosphorylated counterparts did not bind to ClpX. Therefore, pSer and pThr interact with ClpX outside the ATPase site. cyroEM studies of ClpX and pSer peptides indicated a putative pSer docking site in the central pore of ClpX. Previously, we identified OXPHOS complex II subunit SDHA as a potential ClpXP endogenous substrate. Therefore, we used SDHA as a model to assess whether serine phosphorylation influenced ClpXP protein degradation in intact cells. Using pSer immunoprecipitation, we observed increased serine phosphorylated SDHA in AML cells after ClpP and ClpX knockdown. The increased serine phosphorylated SDHA was specifically found in the insoluble fraction of the mitochondrial proteins. Supporting that serine phosphorylated SDHA represents aggregated protein, treatment of cells with ROS-inducing antimycin or 42°C heat shock also increased levels of serine phosphorylated SDHA in the fraction of insoluble mitochondrial proteins. Finally, adding recombinant ClpXP to mitochondrial lysates specifically degraded SDHA with serine phosphorylation. In summary, mitochondrial ClpXP degrades aggregated mitochondrial proteins marked by serine phosphorylated. Thus, this work identifies new degradation machinery for mitochondrial protease, advances the function of mitochondrial ClpXP, and highlights a new therapeutic strategy to target this protease. Citation Format: Yue Feng, Yulia Jitkova, Alexander Keszei, Jonathan St-Germain, Yongran Yan, Brian Raught, Mohammad Mazhab-Jafari, Aaron Schimmer. Mitochondrial ClpXP degrades serine phosphorylated protein aggregates. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4834.