The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells
Xin Xu1, Xiao Mei1, Kunkun Han2, Guanting Wu3, Ruili Li1, Yili Yang11Center for Self-Propelled Nanotechnologies, College of Biotechnology, Suzhou Industrial Park Institute of Services Outsourcing Suzhou, Jiangsu, P. R. China, 2China Regional Research Center, International Centre for Genetic Engineering and Biotechnology, Taizhou, Jiangsu, P. R. China, 3Department of General Surgery, The Affiliated Jiangsu Shengze Hospital of Nanjing Medical University, Suzhou, Jiangsu, P. R. China,
Objectives: Our previous study indicated that USP1 inhibitor ML323 downregulated USP1 in colorectal cancer (CRC) cells, but the specific mechanism was still unknown. Methods: CRC cells were lysed for immunoblotting to detect protein expressions. Quantitative real-time PCR was performed to examine mRNA levels. Cycloheximide chase assays were carried out to evaluate the half-life of USP1. Coimmunoprecipitation was used to analyze the polyubiquitination of USP1. Results: USP1 protein stability was enhanced by the proteasome inhibitor MG132 in CRC cells. The wild-type USP1 was upregulated by MG132, but not its catalytic mutant. Additionally, the polyubiquitination of USP1 was enhanced by MG132 as well, which indicated USP1 was degraded through the ubiquitin-proteasome pathway. Meanwhile, we confirmed ML323 downregulated USP1 expression in CRC cells, and cycloheximide chase assay also revealed ML323 reduced USP1 protein stability. Further results showed ML323-induced USP1 downregulation and destabilization were abolished by MG132. Moreover, USP1 protein destabilization was not reversed by the caspase inhibitor Z-VAD, which further suggested ML323-induced USP1 downregulation was not dependent on the effects of cell death in CRC cells. Conclusion: Our results showed USP1 was auto-ubiquitinated, and ML323 destabilized USP1 through the ubiquitinproteasome pathway in CRC cells, providing a theoretical basis for anti-CRC drugs’ development targeting USP1. Keywords: Colorectal cancer, USP1, ML323
Cite This Article
Xu X, Mei X, Han K, Wu G, Li R, Yang Y. The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells. EJMO. 2023; 7(2): 174-179
Corresponding Author: Yili Yang