Serine and Threonine Phosphorylation Marks Proteins for Degradation By Clpxp
Authors: Feng Yue , Jitkova Yulia , Soor Harjeet , Trudel Suzanne , Lees Kaitlin , Soriano Kaylen , Vahidi Siavash , Yudin Andrei , Schimmer Aaron D 
Published: November 2021

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 a subset of AML and inhibiting or hyperactivating it kills leukemic cells and stem cells in vitro and in vivo. Yet, it is unknown how the mitochondrial ClpXP recognizes proteins for degradation.
In Bacillus subtilis, the bacterial ClpXP homologue recognizes proteins tagged with phospho-arginine for degradation. To determine if phosphorylated amino acids influence mitochondrial 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 phospho-tyrosine (pTyr) 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, RRApSVA, and ApTA peptides inhibited ClpXP enzyme activity, while the non-phosphorylated version (ASA, RRASVA, and ATA) had 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, κ-casein with low levels of phosphorylation and dephosphorylated α-casein were not cleaved by ClpXP.
As ClpX 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. 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 bound ClpX and none of the phosphorylated amino acids bound ClpP. Similarly, ApSA, RRApSVA, and ApTA peptides bound ClpX, while the non-phosphorylated ASA, RRASVA, and ATA did not bind ClpX.
Previously we showed that ClpP interacted with respiratory chain complex II subunit SDHA, and ClpP knockdown in AML cells impaired respiratory chain complex II activity and reactive oxygen species increased. Therefore, we tested how ClpXP knockdown impacts levels of phospho-serine SDHA. Using shRNA, we knocked down ClpP and ClpX individually in OCI-AML2 cells. After target knockdown, we pulled down pSer proteins and then probed for SDHA. Knockdown of both ClpP and ClpX increased the abundance of serine phosphorylated SDHA comparing to control, suggesting that ClpXP degrades serine phosphorylated SDHA.
Finally, as a chemical approach, we generated small molecules that mimic pSer and demonstrated that they inhibited ClpXP-mediated degradation of FITC-casein and bound ClpX with a potency similar to pSer.
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 AML.

Disclosures
Schimmer: Takeda Pharmaceuticals: Consultancy, Research Funding; Medivir AB: Research Funding; Otsuka Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; UHN: Patents & Royalties.