USE1 is a bispecific conjugating enzyme for ubiquitin and FAT10, which FAT10ylates itself in cis

Abstract

The ubiquitin-like modifi er FAT10 targets proteins for degradation by the proteasome and is activated by the E1 enzyme UBA6. In this study, we identify the UBA6-specifi c E2 enzyme (USE1) as an interaction partner of FAT10. Activated FAT10 can be transferred from UBA6 onto USE1 in vitro , and endogenous USE1 and FAT10 can be coimmunoprecipitated from intact cells. Small interfering RNA-mediated downregulation of USE1 mRNA resulted in a strong reduction of FAT10 conjugate formation under endogenous conditions, suggesting that USE1 is a major E2 enzyme in the FAT10 conjugation cascade. Interestingly, USE1 is not only the fi rst E2 enzyme but also the fi rst known substrate of FAT10 conjugation, as it was effi ciently auto-FAT10ylated in cis but not in trans .

The ubiquitin-like modifier FAT10 in cancer development

“…On the one hand FAT10 may act as a regulator of NF-B signaling and on the other hand FAT10 expression itself is regulated by NF-B. As already pointed out, FAT10 mRNA and protein expression is highly and synergistically inducible in all cell types by the pro-inflammatory cytokines IFN-␥ and TNF-␣ (Aichem et al, 2010;Raasi et al, 1999;Ren et al, 2011) or by a combination of TNF-␣ and IL-6 as shown in HepG2 cells (Choi et al, 2014). In HCT116 cells it was shown, that TNF-␣ signaling via the TNF-␣ receptor 1 (TNFR1) induced FAT10 expression, a receptor mainly found on tumor and stromal cells where it mediates the activation of NF-B (Balkwill, 2009;Ren et al, 2011).…”

“…Upon IFN-␥/TNF-␣ mediated induction of FAT10 in the human embryonic kidney cell line HEK293, the FAT10 mRNA was detectable already after 4-5 h, however, FAT10 protein was prominently detected in western blots only 24 h after cytokine stimulation with the highly sensitive monoclonal antibody 4F1 (Aichem et al, , 2010. Nevertheless, upon immunoprecipitation of FAT10 and its modified substrates with mAb 4F1, combined with a subsequent western blot analysis with a polyclonal FAT10 specific antibody, bulk FAT10 conjugates of a large range of sizes are detectable, similar to ubiquitin, ISG15 or SUMO conjugates.…”

“…Ebstein and colleagues (Ebstein et al, 2012) showed that FAT10 could enhance the MHC class I presentation of the human cytomegalovirus (HCMV)-derived antigen pp65 in HeLa cells by about two-fold as compared to untagged pp65 and Schliehe and colleagues (Schliehe et al, 2012) showed that the degradation rate as well as the presentation of specific lymphocyte choriomeningitis virus (LCMV) epitopes on MHC class I molecules was enhanced when FAT10 was fused to the N-terminus of the LCMV nucleoprotein. Apart from its expression in mTECs FAT10 is also constitutively expressed in mature B cells and dendritic cells (Bates et al, 1997) and its expression is highly and synergistically inducible on mRNA and protein level in numerous tissues and cell types by the pro-inflammatory cytokines IFN␥ and TNF␣ (Aichem et al, 2010;Raasi et al, 1999). A recent publication showed, that a high induction of FAT10 expression is also achieved by a synergistic treatment of HepG2 cells with TNF-␣ in combination with IL-6 (Choi et al, 2014).…”

FAT10 and NUB1L bind to the VWA domain of Rpn10 and Rpn1 to enable proteasome-mediated proteolysis

“…For induction of endogenous FAT10 expression, HEK293 cells were transfected with control or hRpn10-specific siRNA and after 24 h stimulated with 200 U ml − 1 IFN-γ and 400 U ml − 1 TNFα for extra 24 h (both from Peprotech). Cells were lysed and subjected to immunoprecipitation using a FAT10 specific mAb (4F1) 23 for immunoprecipitation and a FAT10-reactive polyclonal antibody 21 for western blot analysis. hRpn10 knockdown.…”

“…For negative controls (lane 1-3), 20 µg of His-peptide (Abcam) was added. After 2 h of binding at 4 °C, the resin was washed three times with NET-TON buffer 23 , two times with NET-T buffer 23 and once with 20 mM Tris buffer (pH 7.8). Bound proteins were analysed by western blotting.…”