RESUMO
It is known that SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) mediates autophagy through its E3 ubiquitin ligase activity, but the ubiquitinated substrates of SMURF1 need to be further explored. In this paper, the interacting proteins of SMURF1 in THP-1 cells were captured and identified by co-immunoprecipitation (Co-IP) combined with mass spectrometry. It was found that SMURF1 could physically bind to 222 proteins in THP-1 cells, and Adenosine deaminase acting on RNA 1 (ADAR1) had a higher peptide binding score. SMURF1 overexpression vectors were constructed and transfected into HEK-293T cells, then Co-IP and Western blotting assays verified the interaction between exogenous SMURF1 and endogenous ADAR1. qRT-PCR and Western blotting assays were carried out after transfecting SMURF1 overexpression vectors in HEK-293T cells, which identified that overexpression of SMURF1 attenuated the protein levels of ADAR1 (P<0. 05). However, there was no significant difference in the mRNA level of ADAR1. HEK-293T cells with normal and overexpressing SMURF1 were treated with cycloheximide (CHX), respectively, and Western blotting assays showed a shortened half-life of ADAR1 after overexpression of SMURF1 (P < 0. 05). Furthermore, overexpression of SMURF1 increased the polyubiquitination level of ADAR1 as detected by Co-IP and Western blot (P<0. 05). After the proteasome inhibitor (MG132) treatment, the Western blotting assay was performed to demonstrate that the negative regulatory effect of SMURF1 on ADAR1 was weakened after the proteasome degradation pathway was attenuated (P<0. 05). This study shows that SMURF1 interacts with ADAR1, catalyzes the polyubiquitination of ADAR1 and mediates its degradation through the proteasome pathway, which provides a theoretical basis for exploring the various biological functions of SMURF1 by affecting the stability of ADAR1.