Induced TRIM21 ISGylation By IFN-? Enhances P62 Ubiquitination To Prevent Its Autophagosome Targeting

ISG15 was firstly reported as an ubiquitin-like protein.Like ubiquitin,it can be covalently conjugated to target proteins,known as ISGylation.Similar to ubiquitination,ISGylation also requires corresponding E1 ISG15 activating enzyme,E2 ISG15 coupling enzyme,and E3 ISG15 ligase.So far,only one E1 ISG15 activating enzyme(UBE1L),one E2 ISG15 coupling enzyme(UBCH8),and three E3 ISG15 ligases(HHARI,EFP,and HERC5)have been identified.In the past decades,hundreds of ISGylated proteins have been identified by proteomic analysis.However,most of them have not been verified by molecular and biochemical experiments.Furthermore,little is known about the function of ISGylation in regulating the target proteins that have been validated.Therefore,it is important to uncover new substrates of ISGylation that can be covalently modified by ISG15,and to further study the detailed mechanism by which ISGylation regulating the target proteins.Our study demonstrated that TRIM21 can be ISGylated in HEK293 cells when TRIM21 was co-expressed together with ISG15 conjugating system,or in A549 cells upon the stimulation of IFN-? for 48 h.ISGylation promoted the K63-linkage specific E3 ubiquitin ligase activity,and thus enhancing the levels of K63-linked ubiquitination on itself and its substrate p62.That was why the self-oligomerization of p62 was prevented,as well as its targeting to autophagosome.In summary,it is the first time to identify TRIM21 as a substrate of ISGylation,and the enzyme activity of TRIM21 was afftected by ISGlation.ISGylated TRIM21 prevents p62 localizing to autophagosome by enhancing its K63-linked ubiquitination.