Regulation of 26S proteasome activity and coping with protein aggregation diseases by NUB1

• Applicant: Professor Dr. Michael Basler, since 10/2022
• Subject Area: Biochemistry ; Immunology
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 423436224

Proteins are targeted for degradation by the 26S proteasome by attachment of ubiquitin chains or the ubiquitin-like modifier FAT10. Until recently it was assumed that proteasomal degradation was mainly regulated by enzymatic linkage of ubiquitin or FAT10 to their substrates. However, it is emerging now that the activity of the 26S proteasome itself is regulated e.g. by phosphorylation or ubiquitylation. Moreover, ubiquitylated substrates bind the inhibitory proteasome-associated deubiquitylating enzyme USP14 and by this means conformationally activate the 26S proteasome. FAT10-mediated proteasomal degradation is strongly accelerated by NEDD8 ultimate buster 1 (NUB1). Remarkably, we have now found that NUB1 and FAT10 together but not alone activated the 26S proteasome in vitro as potently as ubiquitin conjugates. Moreover, we showed that NUB1 binds to the same site in the 26S proteasome subunit RPN1 as USP14 and that NUB1 replaced USP14 from the 26S proteasome. In this project we will investigate the mechanism and functional consequences of 26S proteasome activation by NUB1. We will determine whether the ubiquitin-like (UBL) domain or the ubiquitin associated (UBA) domains of NUB1 and/or the N- or C-terminal UBL domains of FAT10 are required for this activation. We will test if NUB1 competes with USP14 for 26S proteasome binding, whether NUB1 directly binds to USP14 and whether USP14 is required for NUB1/FAT10 mediated proteasome activation. As NUB1 also binds to loosely folded proteins other than FAT10 we will test, whether these will likewise cooperate with NUB1 to activate the proteasome. Importantly, we will investigate if NUB1/FAT10 activate the degradation of ubiquitin conjugates by the 26S proteasome. By cryo electron microscopy we will investigate if NUB1/FAT10 conformationally activate the 26S proteasome and whether this activation relies on 20S proteasome gate opening. Using the NUB1-/- mouse, which we have recently generated, we will test whether cells from this mouse are hypersensitive to protein misfolding-inducing drugs as we have shown in NUB1-deficient HeLa cells. We will pair the NUB1-/- mouse with transgenic mouse models of Parkinson’s and Huntington’s disease to examine whether a lack of NUB1 exacerbates disease symptoms and protein aggregate formation in neurons. Virally infected or tumor cells require higher proteasome activity. Hence we will infect NUB1-/- mice with lymphocytic choriomeningitis virus and measure virus replication, the cytotoxic T cell response and the unfolded protein response to infection. Finally, we will determine whether NUB1-/- mice are more resistant to colon carcinoma formation chemically induced by AOM/DSS or by breeding with APCMin/+ mice. Taken together, this project has the potential to identify a new mechanism of 26S proteasome activation and further establish NUB1 and/or FAT10 as pharmacological targets for the therapy of neurological protein aggregation diseases and cancer.

• DFG Programme: Research Grants
• Ehemaliger Antragsteller: Professor Dr. Marcus Groettrup, until 9/2022 (†)