Anti-UCHL5 monoclonal antibody Made to order

Background: Ubiquitin-proteasome system (UPS) is integral to cell survival by maintaining protein homeostasis, and its dysfunction has been linked to cancer and several other human diseases. Through counteracting ubiquitination, deubiquitinases (DUBs) can either positively or negatively regulate UPS function, thereby representing attractive targets of cancer therapies. Previous studies have shown that metal complexes can inhibit tumor growth through targeting the UPS; however, novel metal complexes with higher specificity for cancer therapy arc still lacking. Methods: We synthesized a new gold(l) complex, Au(PPh3)PT. The inhibitory activity of Au(PPh3)PT on the UPS and the growth of multiple cancer cell types were tested in vitro, ex vivo, and in vivo. Furthermore, we compared the efficacy of Au(PPh3)PT with other metal compounds in inhibition of UPS function and tumor growth. Findings: Here we report that (i) a new gold(I) complex-pyrithione, i.e., Au(PPh3)PT, induced apoptosis in two lung cancer cell lines A549 and NCI-H1299; (ii) Au(PPh3)PT severely impaired UPS proteolytic function; (iii) Au(PPh3)PT selectively inhibited 19S proteasome-associated DUBS (UCHL5 and USP14) and other nonproteasomal DUBs with minimal effects on the function of 20S proteasome; (iv) Au(PP113)PT induced apoptosis in cancer cells from acute myeloid leukemia patients; (v) Au(PP113)PT effectively suppressed the growth of lung adenocarcinoma xenografts in nude mice; and (vi) Au (PP113)PT elicited less cytotoxicity in normal cells than several other metal compounds. Interpretation: Together, this study discovers a new gold(l) complex to be an effective inhibitor of the DUBs and a potential anti-cancer drug. Fund: The National High Technology Research and Development Program of China, the project of Guangdong Province Natural Science Foundation, the projects from Foundation for Higher Education of Guangdong, the project from Guangzhou Medical University fur Doctor Scientists, the Medical Scientific Research Foundation of Guangdong Province, and the Guangzhou Key Medical Discipline Construction Project Fund, (C) 2018 The Authors. Published by Elsevier B.V.

USP14 is a cysteine protease deubiquitinase associated with the proteasome and plays important catalytic and allosteric roles in proteasomal degradation. USP14 inhibition has been considered a therapeutic strategy for accelerating degradation of aggregation-prone proteins in neurodegenerative diseases and for inhibiting proteasome function to induce apoptotic cell death in cancers. Here we studied the effects of USP14 inhibition in mammalian cells using small molecule inhibitors and an inactive USP14 mutant C114A. Neither the inhibitors nor USP14 C114A showed consistent or significant effects on the level of TDP-43, tau or a-synuclein in HEK293T cells. However, USP14 C114A led to a robust accumulation of ubiquitinated proteins, which were isolated by ubiquitin immunoprecipitation and identified by mass spectrometry. Among these proteins we confirmed that ubiquitinated beta-catenin accumulated in the cells expressing USP14 C114A with immunoblotting and immunoprecipitation experiments. The proteasome binding domain of USP14 C114A is required for its effect on ubiquitinated proteins. UCHL5 is the other cysteine protease deubiquitinase associated with the proteasome. Interestingly, the inactive mutant of UCHL5 C88A also caused an accumulation of ubiquitinated proteins in HEK293T cells but did not affect beta-catenin, demonstrating USP14 but not UCHL5 has a specific effect on beta-catenin. We used ubiquitin immunoprecipitation and mass spectrometry to identify the accumulated ubiquitinated proteins in UCHL5 C88A expressing cells which are mostly distinct from those identified in USP14 C114A expressing cells. Among the identified proteins are well established proteasome substrates and proteasome subunits. Besides beta-catenin, we also verified with immunoblotting that UCHL5 C88A inhibits its own deubiquitination and USP14 C114A inhibits deubiquitination of two proteasomal subunits PSMC1 and PSMD4. Together our data suggest that USP14 and UCHL5 can deubiquitinate distinct substrates at the proteasome and regulate the ubiquitination of the proteasome itself which is tightly linked to its function.