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Peli1 facilitates TRIF-dependent Toll-like receptor signaling and proinflammatory cytokine production.

Chang M, Jin W, Sun SC - Nat. Immunol. (2009)

Bottom Line: Peli1 was required for TLR3-induced activation of IkappaB kinase (IKK) and its 'downstream' target, transcription factor NF-kappaB, but was dispensable for IKK-NF-kappaB activation induced by several other TLRs and the interleukin 1 (IL-1) receptor.Notably, Peli1 bound to and ubiquitinated RIP1, a signaling molecule that mediates IKK activation induced by the TLR3 and TLR4 adaptor TRIF.Our findings suggest that Peli1 is a ubiquitin ligase needed for the transmission of TRIF-dependent TLR signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.

ABSTRACT
Toll-like receptors (TLRs) are pivotal in innate immunity and inflammation. Here we show that genetic deficiency in Peli1, an E3 ubiquitin ligase, attenuated the induction of proinflammatory cytokines by ligands of TLR3 and TLR4 and rendered mice resistant to septic shock. Peli1 was required for TLR3-induced activation of IkappaB kinase (IKK) and its 'downstream' target, transcription factor NF-kappaB, but was dispensable for IKK-NF-kappaB activation induced by several other TLRs and the interleukin 1 (IL-1) receptor. Notably, Peli1 bound to and ubiquitinated RIP1, a signaling molecule that mediates IKK activation induced by the TLR3 and TLR4 adaptor TRIF. Our findings suggest that Peli1 is a ubiquitin ligase needed for the transmission of TRIF-dependent TLR signals.

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Peli1 is essential for IKK-NF-κB activation by TLR3(a) Primary Peli1+/+ and Peli1−/− MEFs were unstimulated (NT) or stimulated with poly(I:C) (50 μg/ml) or LPS (1 μg/ml). Nuclear extracts were subjected to EMSA for detection of NF-κB and NF-Y. (b) MEFs were treated as indicated and subjected to kinase assay (KA) by isolating IKK holoenzyme by IP using anti-IKKγ and using GST-IκBα(1-54) as substrate (upper). Kinase assay membrane was subjected to IB using anti-IKKα (lower). (c,d) Peli1+/+ and Peli1−/− B cells were stimulated as indicated and subjected to EMSA (c) and kinase assays (d). (e) Peli1+/+ and Peli1−/− splenocytes were stimulated as indicated and subjected to kinase assays as described in b. (f) Primary Peli1+/+ and Peli1−/− MEFs were stimulated as in A. IKKε was isolated by IP and subjected to kinase assays using GST-IRF3 as substrate. Data are representative of 3 or more independent experiments.
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Figure 4: Peli1 is essential for IKK-NF-κB activation by TLR3(a) Primary Peli1+/+ and Peli1−/− MEFs were unstimulated (NT) or stimulated with poly(I:C) (50 μg/ml) or LPS (1 μg/ml). Nuclear extracts were subjected to EMSA for detection of NF-κB and NF-Y. (b) MEFs were treated as indicated and subjected to kinase assay (KA) by isolating IKK holoenzyme by IP using anti-IKKγ and using GST-IκBα(1-54) as substrate (upper). Kinase assay membrane was subjected to IB using anti-IKKα (lower). (c,d) Peli1+/+ and Peli1−/− B cells were stimulated as indicated and subjected to EMSA (c) and kinase assays (d). (e) Peli1+/+ and Peli1−/− splenocytes were stimulated as indicated and subjected to kinase assays as described in b. (f) Primary Peli1+/+ and Peli1−/− MEFs were stimulated as in A. IKKε was isolated by IP and subjected to kinase assays using GST-IRF3 as substrate. Data are representative of 3 or more independent experiments.

Mentions: A central signaling event involved in TLR-mediated gene induction is activation of IKK and its downstream transcription factor NF-κB 2. To investigate the function of Peli1 in this process, we examined the effect of Peli1 deficiency on TLR-stimulated activation of IKK and NF-κB using different cell types. Consistent with the gene induction studies (Fig. 2,3), poly(I:C)-stimulated NF-κB activation was severely attenuated in Peli1−/− MEFs (Fig. 4a). The Peli1 deficient MEFs also showed a partial inhibition of NF-κB activation by LPS (Fig. 4a). Parallel kinase assays revealed that Peli1 deficiency largely impaired poly(I:C)-stimulated activation of IKK and also partially inhibited the LPS-stimulated activation of IKK (Fig. 4b). Since TLR4 activates IKK through both MyD88- and TRIF-dependent pathways, whereas TLR3 activates IKK exclusively through TRIF 2, these results suggest that Peli1 plays a non-redundant role in promoting IKK activation in the TRIF-dependent TLR pathway.


Peli1 facilitates TRIF-dependent Toll-like receptor signaling and proinflammatory cytokine production.

Chang M, Jin W, Sun SC - Nat. Immunol. (2009)

Peli1 is essential for IKK-NF-κB activation by TLR3(a) Primary Peli1+/+ and Peli1−/− MEFs were unstimulated (NT) or stimulated with poly(I:C) (50 μg/ml) or LPS (1 μg/ml). Nuclear extracts were subjected to EMSA for detection of NF-κB and NF-Y. (b) MEFs were treated as indicated and subjected to kinase assay (KA) by isolating IKK holoenzyme by IP using anti-IKKγ and using GST-IκBα(1-54) as substrate (upper). Kinase assay membrane was subjected to IB using anti-IKKα (lower). (c,d) Peli1+/+ and Peli1−/− B cells were stimulated as indicated and subjected to EMSA (c) and kinase assays (d). (e) Peli1+/+ and Peli1−/− splenocytes were stimulated as indicated and subjected to kinase assays as described in b. (f) Primary Peli1+/+ and Peli1−/− MEFs were stimulated as in A. IKKε was isolated by IP and subjected to kinase assays using GST-IRF3 as substrate. Data are representative of 3 or more independent experiments.
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Figure 4: Peli1 is essential for IKK-NF-κB activation by TLR3(a) Primary Peli1+/+ and Peli1−/− MEFs were unstimulated (NT) or stimulated with poly(I:C) (50 μg/ml) or LPS (1 μg/ml). Nuclear extracts were subjected to EMSA for detection of NF-κB and NF-Y. (b) MEFs were treated as indicated and subjected to kinase assay (KA) by isolating IKK holoenzyme by IP using anti-IKKγ and using GST-IκBα(1-54) as substrate (upper). Kinase assay membrane was subjected to IB using anti-IKKα (lower). (c,d) Peli1+/+ and Peli1−/− B cells were stimulated as indicated and subjected to EMSA (c) and kinase assays (d). (e) Peli1+/+ and Peli1−/− splenocytes were stimulated as indicated and subjected to kinase assays as described in b. (f) Primary Peli1+/+ and Peli1−/− MEFs were stimulated as in A. IKKε was isolated by IP and subjected to kinase assays using GST-IRF3 as substrate. Data are representative of 3 or more independent experiments.
Mentions: A central signaling event involved in TLR-mediated gene induction is activation of IKK and its downstream transcription factor NF-κB 2. To investigate the function of Peli1 in this process, we examined the effect of Peli1 deficiency on TLR-stimulated activation of IKK and NF-κB using different cell types. Consistent with the gene induction studies (Fig. 2,3), poly(I:C)-stimulated NF-κB activation was severely attenuated in Peli1−/− MEFs (Fig. 4a). The Peli1 deficient MEFs also showed a partial inhibition of NF-κB activation by LPS (Fig. 4a). Parallel kinase assays revealed that Peli1 deficiency largely impaired poly(I:C)-stimulated activation of IKK and also partially inhibited the LPS-stimulated activation of IKK (Fig. 4b). Since TLR4 activates IKK through both MyD88- and TRIF-dependent pathways, whereas TLR3 activates IKK exclusively through TRIF 2, these results suggest that Peli1 plays a non-redundant role in promoting IKK activation in the TRIF-dependent TLR pathway.

Bottom Line: Peli1 was required for TLR3-induced activation of IkappaB kinase (IKK) and its 'downstream' target, transcription factor NF-kappaB, but was dispensable for IKK-NF-kappaB activation induced by several other TLRs and the interleukin 1 (IL-1) receptor.Notably, Peli1 bound to and ubiquitinated RIP1, a signaling molecule that mediates IKK activation induced by the TLR3 and TLR4 adaptor TRIF.Our findings suggest that Peli1 is a ubiquitin ligase needed for the transmission of TRIF-dependent TLR signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.

ABSTRACT
Toll-like receptors (TLRs) are pivotal in innate immunity and inflammation. Here we show that genetic deficiency in Peli1, an E3 ubiquitin ligase, attenuated the induction of proinflammatory cytokines by ligands of TLR3 and TLR4 and rendered mice resistant to septic shock. Peli1 was required for TLR3-induced activation of IkappaB kinase (IKK) and its 'downstream' target, transcription factor NF-kappaB, but was dispensable for IKK-NF-kappaB activation induced by several other TLRs and the interleukin 1 (IL-1) receptor. Notably, Peli1 bound to and ubiquitinated RIP1, a signaling molecule that mediates IKK activation induced by the TLR3 and TLR4 adaptor TRIF. Our findings suggest that Peli1 is a ubiquitin ligase needed for the transmission of TRIF-dependent TLR signals.

Show MeSH
Related in: MedlinePlus