Molecular bases for USP48 cis-activity regulation and hyperactivation by Cushing's disease-associated mutations

Ubiquitin-specific protease 48 (USP48) is a deubiquitinating enzyme involved in various pathophysiological processes, including DNA repair, tumorigenesis, and inflammation. Somatic mutations in USP48 (M415I/V) are associated with pituitary tumors in Cushings disease. These variants lead to increased USP48 enzyme activity and adrenocorticotropic hormone (ACTH) hypersecretion, a key feature of Cushings disease, indicating a direct link to the condition. Using biochemical experiments and in silico structural analyses, we identified Y414 in the catalytic USP domain as a critical residue for USP48 activity. In the wild-type USP domain, the Y414 side chain was oriented to closed, which led to catalytic triad misalignment and weak ubiquitin recognition. We also found that the C-terminal region (CTR) of USP48 is a cis-activating region. It interacts with the USP domain and ubiquitin tail, thereby facilitating the alignment of the catalytic triad and enhancing ubiquitin recognition ability. Additionally, genomic analyses of pituitary tumors in Japanese patients with Cushings disease (n=46) confirmed that USP48 M415I/V variants occurred at a frequency of 10%, which is consistent with previous reports. Our structural model suggests that M415I/V sterically interferes with and fixes the closed Y414 side chain to open, resulting in catalytic triad alignment and increased ubiquitin recognition ability. Furthermore, USP48 with M415I/V showed enhanced CTR-mediated activity, indicating that the open Y414 side chain and CTR can cause hyperactivation. This study provides a molecular basis for the novel cis-activation regulation of USP48 and its hyperactivation by Cushings disease-associated variants. Significance StatementThe regulatory mechanisms of DUB activity are not yet fully understood. This study identifies the CTR of USP48 as an activity-promoting region, a mechanism also present in USP7 and potentially other closely related DUBs such as USP40 and USP47. Despite the disease association of some DUBs, developing drugs to inhibit specific DUB members or variants remains challenging. The substitutions of USP48 M415 embedded in the USP domain to Ile/Val are associated with Cushings disease, which lacks adequate targeted therapeutics. This study reveals that these variants alter the orientation of Y414, leading to hyperactivation. These findings provide a molecular basis for developing USP48M415I/V-specific inhibitors for Cushings disease therapy.