Identification Of Global Substrates Of Ubiquitin-specific Proteases 14 And Studies On Its Biological Functions

Ubiquitin-specific protease 14(USP14) is one of the ubiquitin-specific processing enzymes/ubiquitin-specific proteases(UBPs/USPs), and it is the only one of the USPs to interact with 26 S proteasome. USP14 may influence several cellular physiology and pathology such as signaling pathways, neurogenesis and tumorigenesis, since it could stabilize substrates via trimming ubiquitin chains from them. Nevertheless, the substrates of USP14 remain largely unknown, which hinders the understanding of the important role of USP14 in biological process.In this study, we finished a system-wide screening of USP14 substrates and regulatory network for the first time. We carried out quantitative proteomics analysis and protein immunoprecipitation combined with mass spectrometry analysis(IP-MS). Via these methods, we identified 530 potential interacted proteins and we found 8 potential substrates among these proteins. They are FASN, CKB, ACADM, GNAS, IDH2, ABLIM1, MYO1 E and CTSB, most of these proteins are related to energy metabolism and cell fate.Besides, we identified 12178 ubiquitination sites by ubiquitination peptide enrichment, and we found that some ubiquitination sites of CKB, FASN, UBB and MYO1 E showed an upward tendency, these results further suggest that these proteins may be the substrates of USP14. We also found some additional potential substrates of USP14 according to this result in combination with protein immunoprecipitation data. Our results could expand our understanding of the role USP14 played in many biological processes and its molecular mechanism.