Regulatory Landscapes of USP8: Understanding Complex Deubiquitination Control Systems in Cancer Progression

• Applicant: Privatdozent Dr. Klaus-Peter Knobeloch
• Subject Area: Biochemistry ; General Genetics and Functional Genome Biology ; Structural Biology ; Cell Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 566971408

Posttranslational modification by ubiquitin regulates a wide range of biological processes, counterbalanced by deubiquitinating enzymes (DUBs). Ubiquitin-specific proteases (USPs) form the largest subgroup of DUBs and often contain multiple domains that enable complex interactions with various signaling pathways via protein-protein interactions (PPIs). Despite their significance, the interconnected regulatory networks of DUBs remain poorly understood. USP8, a multidomain DUB, plays a pivotal role in regulating receptor tyrosine kinase stability and early endosomal trafficking. Phosphorylation at a specific serine residue (hUSP8S718 or mUSP8S680) is crucial for binding to 14-3-3 proteins. Mutations in this binding motif (BM) lead to the proteolytic cleavage of USP8, generating a truncated form (USP8tc) with enhanced deubiquitinase activity. These mutations have been identified in ~35% of ACTH-releasing pituitary adenomas associated with Cushing's Disease (CD), where the increased activity of USP8tc stabilizes EGFR. Similar mutations have also been found in patients with chronic lymphocytic leukemia, further emphasizing the critical role of USP8-14-3-3 interaction in preventing tumorigenesis. Despite the importance of USP8-14-3-3 binding, its regulation and physiological role remain unclear, and structural-functional relationships have been minimally explored. To address these gaps, we have developed a mouse model for conditional and constitutive inactivation of USP8-14-3-3 interaction, identified ubiquitin-associated (UBA) domain-containing kinases that mediate USP8 14-3-3 BM phosphorylation, created a specific anti-phospho-hUSP8S718/mUSP8S680 antibody, and produced recombinant USP8 and USP8tc proteins. Additionally, we have used structural prediction models to explore USP8 interactions with 14-3-3, ubiquitin, and associated kinases, generating new hypotheses for their regulation. In an interdisciplinary approach combining structural biology, mouse genetics, and molecular biology, we aim to: (1) Analyze the regulation of USP8 activity by S680 phosphorylation on the structural level and its physiological role in vivo. (2) Evaluate the role of newly identified hUSP8S718 kinases and define underlying structure-function relationships. (3) Define potential alterations in substrate recognition upon USP8 regulation. (4) Characterize molecular mechanisms of USP8 substrate preferences and inhibition by small molecules. We anticipate that these findings will uncover novel insights into tumor pathogenesis and deepen our understanding of the complex regulation of deubiquitination. This could pave the way for new therapeutic strategies targeting USP8 in cancer.

• DFG Programme: Research Grants
• International Connection: South Korea
• Partner Organisation: National Research Foundation of Korea, NRF
• Cooperation Partner: Professor Donghyuk Shin, Ph.D.