Background
Deubiquitinating enzymes (DUBs) comprise a family of proteases responsible for cleaving the peptide or isopeptide bond between ubiquitin and its substrate proteins. Ubiquitin is essential for regulating diverse cellular functions by attaching to target proteins. The Hippo signaling pathway plays a crucial role in controlling tissue size, cell proliferation, and apoptosis. In a previous study, we discovered that YOD1 regulates the Hippo signaling pathway by deubiquitinating the neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4), an E3 ligase of large tumor suppressor kinase 1 (LATS1). Here, our
Conclusions
Our study revealed multiple cellular interactions between YOD1 and USP21. Moreover, our findings suggest that the combined activities of YOD1 and USP21 synergistically influence cell proliferation in A549 cells by regulating the Hippo signaling pathway.
Methods
We employed various bioinformatics tools (BioGRID, STRING, and Cytoscape) to identify novel potential substrates of YOD1. Furthermore, we used western blotting, co-immunoprecipitation (co-IP), glutathione S-transferase (GST) pull-down, immunocytochemistry (ICC) assays to investigate cellular interactions. To evaluate cell proliferation, we performed cell counting kit-8 (CCK-8), wound healing, colony forming, and flow cytometry assays using A549, HEK293T, and HeLa cells. Additionally, we assessed the expression levels of YAP and p-YAP in A549, HEK293T, and HeLa cells through western blotting.
Results
Our investigations revealed that YOD1 interacts with ubiquitin-specific proteases 21 (USP21), a DUB involved in the Hippo signaling pathway, and deubiquitinates the microtubule-affinity regulating kinase (MARK). Intriguingly, YOD1 and USP21 mutually deubiquitinate each other; while YOD1 regulates the protein stability of USP21, USP21 does not exert a regulatory effect on YOD1. Moreover, we observed the synergistic effect of YOD1 and USP21 on cell proliferation through the modulation of the Hippo signaling pathway. Conclusions: Our study revealed multiple cellular interactions between YOD1 and USP21. Moreover, our findings suggest that the combined activities of YOD1 and USP21 synergistically influence cell proliferation in A549 cells by regulating the Hippo signaling pathway.