The Deubiquitinating Enzyme USP1 is Auto-Ubiquitinated and Destabilized by ML323 in Colorectal Cancer Cells
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.
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