All living cells are constantly producing new proteins to replace the older ones or those that have become damaged. Furthermore, the stability of some proteins must be controlled temporally during the cell cycle or normal cellular function is disrupted. Thus, eukaryotic cells have a massive regulatory system for controlling the stability of proteins. Unwanted proteins are covalently modified by poly-ubiquitin chains, targeting them to the proteasome where they are recycled. Ubiquitin chains are synthesized by a large family of enzymes named E3s. E3s act as a scaffold recruiting both the protein substrate as well as a charged E2 enzyme that is pre-loaded with an activated ubiquitin. In mammalian cells, ubiquitin-mediated proteolysis is controlled by some 600 E3s that each serves unique roles in important biological functions such as apoptosis, NF?B signaling, circadian rhythms, perception of metabolites, and cell division. During the process of ubiquitylation, ubiquitin serves two major roles: (1) it serves as a donor where an E2 enzyme transfers the ubiquitin from its active site to a protein substrate bound to an E3; and (2) it serves as an acceptor when substrate modified ubiquitin attacks an E2~ubiquitin. Consecutive ubiquitins in the poly-ubiquitin chain are covalently linked between 1 of 7 possible lysine residues on an acceptor ubiquitin and the C-terminus of the donor. The identity of the lysine residue that tethers ubiquitins in the chain is o critical importance since only some ubiquitin lysines such as Lys 48 can promote protein degradation. The E2 enzyme Cdc34 catalyzes poly-ubiquitin chain formation through its interactions with donor and acceptor ubiquitins. Cdc34 functions with the largest family of E3s, the Cullin-RING ligases, and thus its impact on the cell is considerable. Furthermore, inhibitors of Cdc34 have recently been developed indicating that Cdc34 is potentially druggable. Cdc34 generates poly-ubiquitin chains with Lys 48 specificity which promotes the degradation of the substrate. During catalysis, Cdc34 must place the donor ubiquitin C-terminus and Lys 48 on the acceptor ubiquitin within proximity of its active site. However, the interface between Cdc34 and acceptor ubiquitin is unknown, and though the interface between Cdc34 and donor ubiquitin has been characterized, there is reason to believe that E3 may also play a role in stabilizing the donor ubiquitin's conformation. The molecular details of the interactions between Cdc34 and ubiquitin will be uncovered using a combination of biochemical assays and cutting edge molecular modeling.
Cdc34 functions with the majority of the cullin-RING ligases (CRLs), the largest family of ubiquitin ligases in human cells. The multi-purpose drug thalidomide is known to target a CRL, and a potent inhibitor of CRLs named MLN4924 is currently under investigation in clinical trials as a therapy for cancer. More recently, a direct inhibitor of Cdc34 function (CC0651) was developed and stops cancer proliferation in tissue culture. Given these prior successes in drug discovery, the work proposed in this grant is likely to help in the development of future therapies against cancer and other human pathologies.
| Hill, Spencer; Hill, Connor; Kleiger, Gary (2018) Using In Vitro Ubiquitylation Assays to Estimate the Affinities of Ubiquitin-Conjugating Enzymes for Their Ubiquitin Ligase Partners. Methods Mol Biol 1844:39-58 |
| Hill, Spencer; Kleiger, Gary (2017) Self-regulating ubiquitin ligases. EMBO J 36:392-393 |
| Ibarra, Rebeca; Sandoval, Daniella; Fredrickson, Eric K et al. (2016) The San1 Ubiquitin Ligase Functions Preferentially with Ubiquitin-conjugating Enzyme Ubc1 during Protein Quality Control. J Biol Chem 291:18778-90 |
| Hill, Spencer; Harrison, Joseph S; Lewis, Steven M et al. (2016) Mechanism of Lysine 48 Selectivity during Polyubiquitin Chain Formation by the Ube2R1/2 Ubiquitin-Conjugating Enzyme. Mol Cell Biol 36:1720-32 |
