Zhao, Zhou2024-08-272024-08-272024http://hdl.handle.net/2003/4265910.17877/DE290R-24495Ubiquitin as a macromolecule-based modification has been validated as the regulator of protein stability. Ubiquitin signals can be significantly diversified by forming polymerized chains as well as conjugating with some structurally similar modifiers (Ubiquitin-like proteins, Ubls). Therefore, cellular events can be modulated in a more complicated way through these modifications, which are reversible by specialized proteases called deubiquitinases (DUBs) and Ubiquitin-like proteases (ULPs). Some of these enzymes show specificity toward different modifiers, while some prefer to cleave linkage-specific polyubiquitin chains. To study the modifier specificity and linkage specificity, high quality chemical biology tools are the cornerstones. Isopeptide-linked fluorescence polarization substrates are powerful tools for analyzing modifier specificity of DUBs/ULPs quantitatively. Since Ub/Ubl-based substrates can be sensitive to harsh reaction and purification conditions, a native semisynthetic method was developed based on recombinantly expressed proteins which were further functionalized with fluorophores and purified in aqueous buffer to ensure the homogeneity of substrates. Six substrates were prepared to assemble an assay panel for characterizing several DUBs/ULPs. USP16 and USP36 were unprecedently identified as the first proteases with triple modifier specificity. Studies on linkage specificity were enabled by the development of novel diUb probes with internal warheads, which are based on alkyl bromide. Alkyl-bromide-based probes were successfully used for studying SnVTD, which is a Lys6-specific DUB. The complex structure was solved to reveal a unique recognition and activation mechanism. The probes were also applied for capturing two linkage-specific E3s. Collectively, a high-quality fluorescence polarization assay platform was established and used for exploring modifier specificity of DUBs/ULPs. Novel diUb probes were developed to capture DUBs with linkage specificity. These tools will facilitate the understanding of DUBs/ULPs.enDeubiquitinaseUbiquitin-like proteasesProtein chemistry570540PhDThesisProteineUbiquitin