Aining protein (VCP)/p97 at the endoplasmic reticulum (ER) via the ER-associated protein degradation (ERAD) pathway (Claessen et al., 2010; Ernst et al., 2009). We locate that YOD1 straight associates with TRAF6 and competes with p62 for TRAF6 binding and activation. YOD1 is released from TRAF6 upon IL-1 stimulation and YOD1 depletion enhances canonical NF-kB activation. These benefits define YOD1 as novel damaging regulator of TRAF6/p62-triggered IL-1 signaling.ResultsYOD1 associates with all the C-terminal MATH domain of TRAFTo recognize regulators of TRAF6, we searched for interaction partners by yeast-two-hybrid (Y2H). We discovered a novel interaction of YOD1 with full length TRAF6, but not with a fragment comprising the catalytic RING-Zinc Finger1 (Z1) domain (Figure 1A; Figure 1–figure supplement 1A). Initially, mammalian YOD1 and its yeast homolog OTU1 have been identified as co-factor in the AAAATPase p97/Cdc48 (Rumpf and Jentsch, 2006; Ernst et al., 2009), which was also confirmed within the Y2H (Figure 1A). Apart from the binding to YOD1, TRAF6 self-associated and bound for the E2 enzyme UBC13 as well as the ubiquitin editing enzyme A20, but not to p97 (Figure 1–figure supplement 1B). To get information around the selectivity of YOD1/TRAF6 interaction, we checked binding of YOD1 to a panel of E3 ligases at the same time as other proteins involved in regulatory ubiquitination by Y2H (Figure 1–figure supplement 1C). Regardless of some weak interaction with cIAP2 and SHARPIN, within this panel YOD1 was pretty selectively binding to p97 and TRAF6. Protein expression was determined by Western Blotting and functionality of some proteins in yeast was confirmed by verifying identified interactions of selected constructs (Figure 1–figure supplement 1D and E). Of note, despite the fact that expression of some E3 ligases was barely detectable, the interaction with identified partners (e.CD150/SLAMF1, Mouse (HEK293, His) g. HOIP with OTULIN or cIAP1/2 with UBC13) was readily detectable within the growth assay. To confirm that TRAF6 and YOD1 are straight associating and to narrow down the YOD1 binding web page on TRAF6, we expressed and purified recombinant YOD1 and TRAF6 proteins and performed pull-down (PD) experiments (Figure 1B and Figure 1–figure supplement 2A). Clearly, YOD1 was binding towards the C-terminal MATH (346 -504) but to not the N-terminal RING-Z1 (50sirtuininhibitor59) domain of TRAF6.IL-1 beta Protein supplier Moreover, we confirmed binding of recombinant YOD1 to p97 by GST-PD (Figure 1–figure supplement 2B). Within a triple PD experiment we neither identified that YOD1-p97 association was prevented by TRAF6 nor that p97 did influence interaction of YOD1 to the TRAF6 MATH domain, revealing that each proteins can bind to YOD1 independently (Figure 1–figure supplement 2C).PMID:24576999 To investigate whether YOD1 and TRAF6 also interact in cells, we co-expressed full length FLAGYOD1 and HA-TRAF6 in HEK293 cells. Co-immunoprecipitations (IP) employing anti-HA or anti-FLAG antibodies confirmed the interaction of TRAF6 and YOD1 (Figure 1C ). Congruent using the Y2H and PD results, YOD1 bound for the C-terminal MATH domain, but not to the N-terminal RING-Z1-Z4 region of TRAF6 (Figure 1D). Around the side of YOD1, the N-terminal UBX domain was critical to mediate TRAF6 interaction (Figure 1E, scheme Figure 1F). Sequence comparison indicated theSchimmack et al. eLife 2017;6:e22416. DOI: ten.7554/eLife.2 ofResearch articleCell BiologyFLAG-YODAAD mock TRAF6 TRAF6 50-159 p97 +HIS -HISBInput GST GST-YOD1 HIS-TRAF6 346-504 HIS-TRAF6 346-504 GST-YOD1 + + + + + GST-PD + + +CFLAG-YOD1 HA-TRAF6.
