Structural and biochemical studies on ANKRD9 as a novel substrate receptor for ubiquitin ligase complex

Abstract

The ubiquitin-proteasome system (UPS) regulates various cellular pathways, such as signaling, proliferation and apoptosis by modulating degradation of target proteins through multi-step catalyzed reaction, which consist of three disparate enzymes. E1

ubiquitin activating enzyme, E2

ubiquitin conjugating enzyme, and E3

ubiquitin ligase complex. Among E3 ligase complexes, Cullin-Ring finger E3 ubiquitin ligase (CRL) family has been extensively studied. CRL is composed of Cullin scaffold protein, RING box protein, an adaptor protein and a substrate receptor protein, and the substrate receptor confers substrate specificity to CRL by recruiting target proteins to the E3 ubiquitin ligase complex. Here, I identified ankyrin repeat domain 9 (ANKRD9), as a novel substrate receptor for CRL. Using quantitative proteomic methodologies in conjunction with immunoblotting and in-vitro binding assays, I showed that ANKRD9 forms a complex with components of Cul5 E3 ubiquitin ligase complex, including EloB, EloC and Cul5. Interestingly, ANKRD9 contains unique SOCS box from that of other substrate receptor proteins in that the BC box within SOCS box of ANKRD9 is not conserved. Furthermore, I identified Inosine Monophosphate Dehydrogenase (IMPDH) as a substrate for ANKRD9. IMPHD is the rate-limiting enzyme in de novo biosynthesis of guanine nucleotide, implicated for cellular proliferation and cancer. In addition, overexpressing ANKRD9 reduces and downregulating ANKRD9 increases IMPDH protein level. Taken together, our results suggest that ANRKD9 functions as a novel substrate receptor for CRL and regulates IMPDH level through proteasomal degradation.