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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
 
Final Report Year 2024

Final Report Abstract

Proteins are marked with ubiquitin or the ubiquitin-like modifier FAT10 for degradation by the 26S proteasome. Until now, it was assumed that this protein degradation is mainly regulated by enzymes that mark these proteins with ubiquitin or FAT10. However, it has recently been shown that the activity of the 26S proteasome itself is also regulated by its phosphorylation or ubiquitylation, among other things. FAT10-mediated degradation is greatly accelerated by the protein NUB1. NUB1 and FAT10 together activate the 26S proteasome as potently as ubiquitin conjugates do, presumably because NUB1 binds to the same site of RPN1 on the 26S proteasome as the inhibitory USP14 and displaces it. In this project, we intended to characterize the role of NUB1 in 26S proteasome degradation, in the unfolded Protein response (UPR), in Alzheimer´s disease and in viral infection. We report that FAT10 and its interaction partner NUB1L facilitate the gate opening of the 20S proteasome in an ubiquitin- and USP14-independent manner. Furthermore, FAT10 is capable to activate all peptidolytic activities of the 26S proteasome, however, only together with NUB1L, by binding to the UBA domains of NUB1L and thereby interfering with NUB1L dimerization. The binding of FAT10 to NUB1L leads to an increased affinity of NUB1L for the sub-unit RPN1. In conclusion, the cooperation of FAT10 and NUB1L is a substrate-induced mechanism to activate the 26S proteasome. Ubiquitin-independent protein degradation via the 20S proteasome without the 19S regulato-ry particle has gained increasing attention over the last years. We found that FAT10 was rapidly degraded by purified 20S proteasomes in vitro, which was attributed to the weak folding of FAT10 and the N-terminally disordered tail. However, degradation of FAT10 in cellulo was strongly dependent on functional 26S proteasome. NUB1 modulates protein homeostasis through the targeted degradation of substrates via the proteasome system. In this study, we generated and in depth characterized the novel NUB1 knockout mouse strain. In contrast to a higher susceptibility upon BFA treatment in a human NUB1 knockout cell line we could not detect differences in various primary mouse cell lines. In an EAE model NUB1-deficient mice developed similar symptoms to wild-type mice. However, the pathogen-specific cytotoxic T cell response was strongly enhanced in NUB1-defiecient mice. Taken together, our data reveal an important in vivo function of NUB1 in the induction of a pathogen specific CTL response. Tauopathies, such as Alzheimer's disease, are marked by the pathological aggregation of tau, leading to neurodegeneration and cognitive decline. In this study, we explored the influ-ence of NUB1 on tau aggregation using a P301S tauopathy mouse model. Our findings demonstrate that NUB1 deficiency provides a protective effect against paralysis symptoms while we did not observe significant differences in the behavior of NUB1 knockout mice com-pared to wild-type mice. Notably, lysates from the brains of NUB1 wild-type mice exhibited higher seeding activity in a cellular tau reporter system. These results highlight the important role of NUB1 in modulating tau aggregation in neurodegenerative diseases.

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