NLRP11 attenuates Toll-like receptor signalling by targeting TRAF6 for degradation via the ubiquitin ligase RNF19A
Authors: Wei Zhao
Meng Lin
Jiayu Ou
Jun Cui
Chenglei Wu
Zexiong Su
Rong-Fu Wang
Journal: Nature Communications (2017)
Discipline: Multidisciplinary
Keywords: TRAF6; NLRP11; RNF19A; TLR signalling; degradation
Affiliations: Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, People's Republic of China., Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, People's Republic of China., Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, People's Republic of China. ., Collaborative Innovation Center of Cancer Medicine, Sun Yat-sen University, Guangzhou, Guangdong, 510080, People's Republic of China. ., Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX, 77030, USA. ., Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY, 10065, USA. ., Institute of Biosciences and Technology, College of Medicine, Texas A & M University, Houston, TX, 77030, USA. .

Abstract:  The adaptor protein TRAF6 has a central function in Toll-like receptor (TLR) signalling, yet the molecular mechanisms controlling its activity and stability are unclear. Here we show that NLRP11, a primate specific gene, inhibits TLR signalling by targeting TRAF6 for degradation. NLRP11 recruits the ubiquitin ligase RNF19A to catalyze K48-linked ubiquitination of TRAF6 at multiple sites, thereby leading to the degradation of TRAF6. Furthermore, deficiency in either NLRP11 or RNF19A abrogates K48-linked ubiquitination and degradation of TRAF6, which promotes activation of NF-κB and MAPK signalling and increases the production of proinflammatory cytokines. Therefore, our findings identify NLRP11 as a conserved negative regulator of TLR signalling in primate cells and reveal a mechanism by which the NLRP11-RNF19A axis targets TRAF6 for degradation. 

Retracted: No

PubPeer comments:
 In Fig.2b and 3a, the expression pattern is way different for control lane. ![file] , 
 In their Embo Reports paper, they claimed that NLRP11, a Nod-like receptor, is induced by type I IFN and translocates to mitochondria to interact with MAVS upon viral infection, however in this paper they said NLRP11 recruits RNF19A to degrade TRAF6 in the absence of MAVS, this does not make any sense. ![file] ![file] , 
 In Wt panel, LPS treatment will promote degradation of TRAF6, but it did not show any degradation, but increase TRAF6 expression, this does not make any sense. ![file] , 
 To add to the confusion in this article: Figure 6f: The loading control (Actin) contains 12 lanes, but the rest of the blots contain 10 lanes. This is also seen in the Supplementary Figure 11 as a support for the western blot in Figure 1f. So called  Full-lenght uncropped western blots are presented in the Supplementary figures for all the Western blots. Without a loading control clearly seen on the film, this data is not a proof that the individual blots are retrieved from the same run same gel. The problem is clearly seen in Supplementary Figure 6 and Figure 9 (support for Figure 1g and 4e, respectively). The blots seem to be randomly taken and can be any western blot the authors may have available. Furthermore, many of the Western blots look artificial. I have included an example from Supplementary Figure 13, where it looks like the Western immunoblot bands have been pasted on a background, which appear very different from the background around the bands. ![file] ![file] , 
 #4 ![file] Thanks for raising this concern. In Figure 6F The loading control contains two more lanes for miss cutting. Now, we provide the original figures and you can find HA-TRAF6 and b-actin come from the same film. Because NLRP11 is 118 kDa and b-actin is 45 kDa, to get a better signal we run NLRP11 on 8% gel and transfer for 2.5 h and b-actin on 10% gel and transfer for 1.5 h. Thatu2019s why these blots are from different membranes. ![file] For all the immunoblots, we collected the data from Bio-rad ChemiDoc, and have the original scn files, and here we provided the merged figure of Supplementary Figure 13 with protein marker. Best regards Chenglei Wu , 
 #1u00a0 In EMBO Reports, we reported that NLRP11 degrades TRAF6 via MAVS after virus infection, and in Nature Communications, we demonstrated that NLRP11 recruits RNF19A to degrade TRAF6 after LPS treatment. First of all, we did not test whether MAVS is involved for TRAF6 degradation by LPS treatment in NC paper, and did not make a conclusion that NLRP11 recruits RNF19A to degrade TRAF6 in the absence of MAVS, as the questioner claimed. Secondary, these two works demonstrate the function of NLRP11 in two different pathways, which are activated by different stimuli to transmit signals through different receptors and adapters. Upon binding to viral RNA, RIG-I interacts with MAVS and recruits TRAF3 and TRAF6 to form a large signalosome, which in turn activates downstream signaling pathway. However, upon LPS treatment, TLR4 associates with IRAK family and recruits TRAF6 to activate downstream signaling pathway, which does not require MAVS. Thus, MAVS mainly participates in RLR signaling pathway but not TLR signaling pathway. It is reasonable that NLRP11 degrades TRAF6 via MAVS under viral infection ,while it degrades TRAF6 with the help of other adaptor proteins under LPS treatment.There are numerous examples in immunology that one protein have multiple functions by targeting different molecular targets. #2 Figure 2b and Figure 3a used different types of cells. We used stable cell lines (insert control vector or NLRP11 plasmid) in Figure 2b and transfected control or NLRP11 siRNA into THP1 cells in Figure 3a. Thus each one should compare to its own control, because even the condition of LPS treatment is different among these two experiments. For siRNA transfection, it will wait at least 24 h before LPS treatment, while for THP1-EV cells, LPS could directly add to the medium. Thus the expression pattern may have some time delay under experimental conditions. #3 We estimate that the questioner may have misunderstood this data in our article. In our paper, we showed many times that LPS treatment does not significant promote degradation of TRAF6 in WT cells (Fig 4e,f,g, Fig 5b,d,e), which is contrary to what the questioner put forward. In our experimental system, we only observed dramatic TRAF6 degradation by NLRP11 overexpression in THP-1 cells. In Figure 5i, we showed the ubiquitination of TRAF6 by pulldown of endogenous TRAF6 from WT and NLRP11 KO cells after LPS treatment. From our data, we can find that although we pulled down more TRAF6 in NLRP11 KO THP-1 cells, the K48-linked ubiquitination of TFAF6 was decreased in NLRP11 KO THP-1 cells, which is consistent with our conclusion. , 
 #1 In EMBO Reports, we reported that NLRP11 degrades TRAF6 via MAVS **after virus infection**, and in Nature Communications, we demonstrated that NLRP11 recruits RNF19A to degrade TRAF6 **after LPS treatment**. **First of all, we did not test whether MAVS is involved for TRAF6 degradation by LPS treatment in NC paper, and did not make a conclusion that NLRP11 recruits RNF19A to degrade TRAF6 in the absence of MAVS, as the questioner claimed**. Secondary, these two works demonstrate the function of NLRP11 in **two different pathways, which are activated by different stimuli to transmit signals through different receptors and adapters**. Upon binding to viral RNA, RIG-I interacts with MAVS and recruits TRAF3 and TRAF6 to form a large signalosome, which in turn activates downstream signaling pathway. However, upon LPS treatment, TLR4 associates with IRAK family and recruits TRAF6 to activate downstream signaling pathway, which does not require MAVS. Thus, MAVS mainly participates in RLR signaling pathway but not TLR signaling pathway. It is reasonable that NLRP11 degrades TRAF6 via MAVS under viral infection ,while it degrades TRAF6 with the help of other adaptor proteins under LPS treatment.There are numerous examples in immunology that one protein have multiple functions by targeting different molecular targets. , 
 #2 Figure 2b and Figure 3a used different types of cells. We used stable cell lines (insert control vector or NLRP11 plasmid) in Figure 2b and transfected control or NLRP11 siRNA into THP1 cells in Figure 3a. Thus each one should compare to its own control, because even the condition of LPS treatment is different among these two experiments. For siRNA transfection, it will wait at least 24 h before LPS treatment, while for THP1-EV cells, LPS could directly add to the medium. Thus the expression pattern may have some time delay under experimental conditions. , 
 #3 We estimate that the questioner may have misunderstood this data in our article. In our paper, we showed many times that LPS treatment does not significant promote degradation of TRAF6 in WT cells (Fig 4e,f,g, Fig 5b,d,e), which is contrary to what the questioner put forward. In our experimental system, we only observed dramatic TRAF6 degradation by NLRP11 overexpression in THP-1 cells. In Figure 5i, we showed the ubiquitination of TRAF6 by pulldown of endogenous TRAF6 from WT and NLRP11 KO cells after LPS treatment. From our data, we can find that although we pulled down more TRAF6 in NLRP11 KO THP-1 cells, the K48-linked ubiquitination of TFAF6 was decreased in NLRP11 KO THP-1 cells, which is consistent with our conclusion. ,