E3-independent Monoubiquitination Of Annexin A1

As one kind of conventional protein post-translational modifications(PTMs),ubiquitination has been shown to be involved in a variety of non-proteosomal processes including membrane trafficking,signal transduction,transcription,DNA repair and protein translation.Conventional ubiquitination,in the first step,E1 activates Ub,which is then transferred on an E2 Ub-conjugating enzyme that finally cooperates with an E3 Ub ligase to attach Ub to a lysine residue on the substrate.In 2007 year,Daniela Hoeller and other scientists demonstrated that multiple ubiquitin binding domain containing proteins(UBD-containing proteins)can be monoubiquitinated in vitro independently of the activity of E3 ligases,showed that UBDs can directly recruit Ub-loaded E2 enzymes and named this kind of ubiquitination after the E3 independent of nonconventional ubiquitination.After Daniela Hoeller reported,scientists identified the E3 independent of nonconventional ubiquitination be involved in variety processes including Protein activity regulation,transcription,DNA repair and found this kind of ubiquitination prevail among vital activities.Annexins are traditionally thought of as calcium-dependent phospholipid-binding proteins,but recent work suggests a more complex set of functions.More than a thousand proteins of the annexin superfamily have been identified in major eukaryotic phyla,but annexins are absent from yeasts and prokaryotes.Annexin 1(Anx A1)is the first characterized member of the annexin family in 1970 s and has been researched from that moment.Anx A1 has been identified that can contribute in variety processes including cell proliferation,differentiation,apoptosis,anti-inflammatory.The faction of Anx A1 in may processes is dependent on its protein level.In head and neck cancers,the protein level of Anx A1 has been associated with advanced stage of the disease,metastasis,and differentiation status and could be an effective differentiation marker for the detection of epithelial dysplasia and histopathological grading of head and neck squamous cell carcinomas.Although the expression level of Anx A1 is so important,the study of protein degradation of Anx A1 was rarely.Using database and protein-protein interacting experiment,we found that the protein of Ub-conjugating enzyme Ubc H5 family proteins interact with Anx A1.This direct interaction were confirmed by Coimmunoprecipitation and pulldown assay in vivo and in vitro.GFP Co-IP assay suggest that Anx A1 N-terminal domain is important for Ubc H5 family protein binding.Anx A1 can be monoubiquitin modified in cell when overexpression Ubc H5 family proteins.Using pure protein ubiquitination in vitro,we found that Ub can be transferred to Anx A1 by Ubc H5 family protein directly and independent E3 ubiquitin ligase.Alexander Sorkin reported that the E3 independent of nonconventional ubiquitination with two models.First model is that a UBD-containing protein binds to an E2-conjugating enzyme by means of the interaction of the UBD with ubiquitin linked to E2 via a thioester bond,and then the ubiquitin is transferred to an ?-amino group of the lysine residue in the UBD-containing protein.The other model is that A UBD-containing protein binds to an E2-conjugating enzyme by means of the interaction of the UBD with ubiquitin linked to E2 via a thioester bond,and then The ubiquitin moiety of the UBD-containing protein can interact either with the UBD of the same substrate molecule or the UBD of another protein.Different with two kinds of reported models of E3 independent ubiquitination,Anx A1 interacts with E2 Ub-conjucting enzyme but not ubiquitin and has not ubiquitin binding domain.We identified that Anx A1 interacts Ubc H5 family proteins and then transfers Ub that linked to Ubc H5 family proteins via a thioester bond.The interaction of Anx A1 and Ubc H5 family proteins is dependent on the calcium concentration.Both high or low concentration of calcium are adverse to the interaction.We infer that 8 of calcium binding sites of Anx A1 have different adhesions and different sites binding calcium change the structure of Anx A1 protein diversely.The structure of Anx A1 dependent on the calcium concentration may provide a foundation for its functional diversity in cells.The calcium concentration not only regulates the interaction,but also regulated the ubiquitination of Anx A1 by Ubc H5 family proteins.This result also confirmed that the ubiquitination of Anx A1 is depend on the interaction of Ubc H5 family proteins.When overexpression Ubc H5 family protein in cells,the content of Anx A1 protein has an obvious reducing.By restrain protein translation,we confirmed that overexpression of Ubc H5 family proteins can degrade the Anx A1 protein.This degradation is depending on lysosomal degradation pathway but not proteasome degradation pathway.This degradation may be due to the effect of the ubiquitination of the Ubc H5 family proteins on the subcellular localization of Anx A1,which leads to its localization in the lysosome for degradation.This degradation of Anx A1 may play an important role in cell proliferation,apoptosis,inflammatory response and cell carcinogenesis.In summary,we demonstrated in vitro and in cells that the Ubc H5 family proteins can directly interact with the membrane coupling protein Anx A1 in a calcium concentration-dependent manner.By interacting with Anx A1,the Ubc H5 family proteins can directly ubiquitinate Anx A1 through a novel nonconventional ubiquitination modification model that is not resistant to E3 ubiquitin ligase.This monoubiquitination of Anx A1 regulates the localization of Anx A1 to lysosomes for degradation,thereby reducing the protein content of Anx A1 in cells.