Regulation Of PIF1 Stability By Deubiquitination Enzymes OTUD5

Objective: In this study,we investigated the mechanism of ubiquitination enzyme OTUD5 on PIF1 protein.It is known that OTUD5 interacts with PIF1.It is unclear whether OTUD5 plays an important role in PIF1.So this topic will focus on regulation mechanism of OTUD5 on PIF1 was studied.To further explore as OTU family members OTUD5 on regulation of PIF1 whether there is an important function.Methods: Westernblotwere used to detect the changes of endogenous and exogenous PIF1 protein after overexpression of OTUD5.RNA interference was used to knock down OTUD5 to detect the changes of exogenous expressed PIF1 protein.Real time quantitative PCR(RT-PCR)was used to detect the effect of over expression or knockdown of OTUD5 on the level of PIF1 mRNA.The degradation rate of after PIF1 overexpression of OTUD5 was detected by half-life assay,and the regulation of OTUD5 on the stability of PIF1 protein was investigated.Finally,ubiquitination testwas used to detect the overexpression or knockdown of OTUD5 on ubiquitination of PIF1 protein,and the regulation of OTUD5 on the expression of PIF1 was investigated.Finally,ubiquitination test in vitro was used to detect the over ubiquitination of PIF1 full-length protein and PIF1 C truncated by OTUD5,and to determine the role of PINT domain in ubiquitination of OTUD5.Results: 1.Overexpression of OTUD5 increased the content of endogenous PIF1 and exogenous expressed PIF1 protein.2.Knock down OTUD5 decreased the exogenous expression of PIF1 protein content.3.Overexpression of OTUD5 increased the half-life of PIF1 protein,but overexpression or knockdown of OTUD5 had no effect on PIF1 mRNA.4.Overexpression of OTUD5 reduced the ubiquitination level of PIF1 protein,and knockdown of OTUD5 increased the ubiquitination level of PIF1.5.Deletion of the PINT domain inhibits the ubiquitination of OTUD5 by PIF1.Conclusion: OTUD5 binds to the PIF1 protein PINT domain,and dubiquitination of PIF1 increases the stability of PIF1 proteins.