Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Covalent probe reveals Ubc13 interactome in the malaria parasite (#326)

Anna Truong 1 2 , Baiyi Quan 1 3 , Ruitian Hu 1 , Michael C Fitzgerald 1 4 , Emily R Derbyshire 1 5
  1. Chemistry, Duke University, Durham, North Carolina, United States
  2. Biochemistry & Pharmacology, University of Melbourne, Melbourne, Victoria, Australia
  3. Proteome Exploration Laboratory, California Institute of Technology, Pasadena, California, United States
  4. Biochemistry, Duke University, Durham, North Carolina, United States
  5. Molecular Genetics & Microbiology, Duke University, Durham, North Carolina, United States

Ubiquitination is an essential post-translational modification in the deadliest malaria parasite Plasmodium falciparum. The covalent attachment of ubiquitin, an 8-kDa protein that serves as a critical signaling molecule, to substrate proteins is mediated by an enzymatic cascade consisting of a ubiquitin-activating (E1), conjugating (E2), and ligase (E3) enzyme. Interestingly, deletion of the E2 PfUbc13 increases parasite sensitivity to the frontline antimalarial drug dihydroartemisinin. PfUbc13 is an essential gene and central mediator of Lys63-linked polyubiquitin (K63-Ub) chains, thus implicating this enzyme and specific modification in the activity of artemisinin-based combination therapies (ACTs). Drug resistance threatens the efficacy of ACTs, so chemically targeting PfUbc13 and elucidating its interactome is invaluable to further understanding the mechanism of ACTs and combatting malaria.

The compound NSC697923, covalent inhibitor of HsUbc13 via Michael addition, was identified as a covalent inhibitor of PfUbc13. Similar degrees of thermal destabilization were observed when recombinant HsUbc13 and PfUbc13 were treated with NSC697923, whereas no thermal shift was observed with the mutant protein PfUbc13-C86S. Using high-resolution mass spectrometry, the mass of the PfUbc13 covalently modified Cys86-containing tryptic peptide (929.4952 kDa) was detected. Furthermore, dose-dependent NSC697923 inhibition of Ub transfer from recombinant E1 PfUBA1 to PfUbc13 was observed in assays monitored by anti-Ub western blot. The Ki was determined to be 0.36 μM using a fluorescence polarization assay. The NSC697923 EC50 values against the parasite blood, liver, and sexual stages were determined to be 6.7, 7.8, <10 μM, respectively. Additionally, 64 putative PfUbc13 substrate proteins were identified by selective enrichment using K63-specific tandem Ub binding entities followed by differential bottom-up proteomic analysis. Overall, this work has identified the first-known chemical probe of PfUbc13 and provided insight into the PfUbc13 interactome, broadly informing future investigations into the role of PfUbc13 and K63-Ub in the mechanism of ACTs and furthering malaria drug discovery efforts.