Deciphering the biological functions of F-box proteins through the use of Parallel Adaptor Capture (PAC) proteomics.

Abstract

The timely and selective proteasomal degradation of proteins is important for the maintenance of proper cellular processes. Prior to proteasomal degradation, proteins destined for degradation are polyubiquitiylated by ubiquitin ligases (E3s). The SKP1-CUL1-F-box protein (SCF) complex is a member of the cullin-RING ligase (CRL) superfamily of modular, multi-protein E3s, in which the F-box protein (FBP) acts as a substrate specificity factor for the recruitment of substrates to the SCF complex. Many of the 69 human FBPs remain uncharacterized. From our current knowledge of FBPs, we know that they regulate a myriad and diverse set of cellular processes and their misregulation is associated with diseases, including cancer. Though many approaches exist for the identification of SCF substrates and/or interactors, most existing genetic and quantitative proteomic methods are not capable of adaptor identification, while interaction proteomic approaches are generally performed in a low throughput manner. To facilitate our characterization of FBPs, we have developed a novel, facile proteomic approach for the identification of interactors, including substrates, of FBPs. In this method, FBPs, which together with SKP1 form the adaptor subunit of the SCF complex, are individually expressed in cells. These cells are subsequently exposed to 3 conditions: left untreated, treated with MLN4924 (neddylation inhibitor) or treated with Bortezomib (proteasome inhibitor). After treatment, cells are lysed, and the lysates are affinity-purified and processed in parallel for proteomic analysis. This approach, which we have named Parallel Adaptor Capture (PAC) proteomics, was successfully applied for the characterization of three novel FBP/substrate interactions: FBXW11 (β-TrCP2)/DEPTOR (Appendix 1), FBXL17/BACH1 (Chapter 2) and FBXO22/KDM4A (Chapter 3). Besides the characterization of these FBPs, PAC proteomics was performed on 19 leucine-rich repeats containing FBPs, the FBXLs, identifying 230 high confidence interacting proteins, including known regulatory proteins and substrates. Deciphering the biological context and significance of these interactions will allow us to understand the importance of FBXLs in the cellular processes in which they regulate. Since CRLs require neddylation to efficiently ubiquitylate their substrates and most CRL substrates are degraded by the proteasome, PAC proteomics, in principle, can also be applied to other CRL adaptors.