Ubiquitin signaling for protein degradation

Division Of Basic Sciences - Nci

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Abstract

The 26S proteasome performs regulated proteolysis in eukaryotes and has emerged as a major therapeutic target. Protein degradation occurs within a 20S catalytic core particle (CP) that can be capped at either end by a 19S regulatory particle (RP). Proteasome substrates are distinguished by post-translational modification with ubiquitin chains, which bind directly to receptor sites in the RP. A major goal of our section is to understand how the proteasome recognizes substrates and becomes committed to substrate proteolysis. We aim to use this knowledge to design new methods that target specific components of the ubiquitin-proteasome pathway. In collaboration with extramural researchers, we established Rpn1 and Rpn13 as substrate receptors in the proteasome that can directly interact with ubiquitin or ubiquitin-like domains of shuttle factors that also bind to ubiquitin by ubiquitin-associated domains. We used NMR spectroscopy to solve the structure of the three major substrate receptor sites in the RP (Rpn1, Rpn10 and Rpn13) complexed with ubiquitin chains. This work is being extended to evaluate the specific functional roles of Rpn1, Rpn10 and Rpn13, both by biophysical methods and cell biology techniques. The Rpn13-binding site in proteasomes is at the extreme C-terminal end of Rpn2 and the Rpn2-binding surface of Rpn13 has emerged as a therapeutic target, particularly for hematological cancers. Rpn13-binding molecules induce apoptosis and prevent tumor growth in myeloma and ovarian cancer xenograft models. We solved the structure of hRpn13 complexed with its proteasome binding site in subunit hRpn2/PSMD1 and used this structure to conduct an integrated virtual and biophysical screen for small molecule binders of hRpn13. This effort identified a new hRpn13-targeting scaffold and lead compound, which we named XL5. We solved the structure of XL5-complexed hRpn13, which informed on how to generate a bifunctional PROTAC molecule for hRpn13 targeting. Our hRpn13 PROTAC contains XL5 for hRpn13 binding, which is linked to a small molecule that binds to an E3 ligase, allowing it to direct ubiquitination machinery to induce hRpn13 ubiquitination and in turn, its degradation. Compared to unmodified XL5, its fusion with E3 ligase warheads, particularly VHL, yielded higher efficacy at inducing apoptosis and restricting growth of multiple myeloma cell lines, and with gene editing, we found the effects are hRpn13-dependent. The hRpn13 PROTAC further revealed the presence of a cleaved hRpn13 product that contains an intact Pru domain but lacks its UCHL5-binding DEUBAD domain. This hRpn13 truncated product (hRpn13Pru) cannot bind UCHL5 and has lost regulatory restrictions imposed by an inter-domain DEUBAD:Pru interaction. hRpn13Pru is degraded preferentially over hRpn13 full-length protein in multiple myeloma cells by the PROTAC because of the lost DEUBAD domain, which in the full-length protein restricts access to the Pru and recruits UCHL5 for hRpn13 deubiquitination. We are also further defining proteasome structure at atomic level resolution. As part of this effort, we discovered a binding site for E6AP/UBE3A at the C-terminal end of hRpn10, the first known binding site for a ubiquitination enzyme. We named this domain RAZUL based on its binding to the E6AP AZUL domain. We solved the structure of the RAZUL:AZUL protein complex to discover that RAZUL adopts a helical structure upon binding to E6AP to form a 4-helix bundle with two AZUL helices. Altogether, this project is providing fundamental information on how the proteasome recognizes ubiquitinated substrates at atomic level resolution, as well as the therapeutic potential of targeting sites in the proteasome RP.