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Regulation of Toll-like receptor signaling by NDP52-mediated selective autophagy is normally inactivated by A20.

Inomata M, Niida S, Shibata K, Into T - Cell. Mol. Life Sci. (2011)

Bottom Line: However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II.NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation.Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Microbiology, Division of Oral Infections and Health Sciences, Asahi University School of Dentistry, Hozumi 1851, Mizuho, Gifu 501-0296, Japan.

ABSTRACT
Toll-like receptor (TLR) signaling is linked to autophagy that facilitates elimination of intracellular pathogens. However, it is largely unknown whether autophagy controls TLR signaling. Here, we report that poly(I:C) stimulation induces selective autophagic degradation of the TLR adaptor molecule TRIF and the signaling molecule TRAF6, which is revealed by gene silencing of the ubiquitin-editing enzyme A20. This type of autophagy induced formation of autophagosomes and could be suppressed by an autophagy inhibitor and lysosomal inhibitors. However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II. Through screening of TRIF-interacting 'autophagy receptors' in human cells, we identified that NDP52 mediated the selective autophagic degradation of TRIF and TRAF6 but not TRAF3. NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation. Intriguingly, only under the condition of A20 silencing, NDP52 could effectively suppress poly(I:C)-induced proinflammatory gene expression. Thus, this study clarifies a selective autophagic mechanism mediated by NDP52 that works downstream of TRIF-TRAF6. Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling.

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NDP52 promotes aggregation of TRAF6. a, c, e, g, i, k HeLa cells were transfected with Flag-TRAF6 and HA-NDP52 for 24 h (a, c, e). HeLa cells were transfected with control siRNA or A20 siRNA for 24 h. Then cells were further transfected with HA-NDP52 and Flag-TRAF6 for 18 h (g, i, k). Cells were fixed and stained with anti-Flag mouse monoclonal antibody and Alexa564-conjugated anti-mouse IgG antibody and then with anti-NDP52 rabbit polyclonal antibody and Alexa488-conjugated anti-rabbit IgG antibody (a, g), with anti-TRAF6 rabbit polyclonal antibody and Alexa488-conjugated anti-rabbit IgG antibody and then with anti-FK2 mouse monoclonal antibody and Alexa564-conjugated anti-mouse IgG antibody (c, i) or with anti-Flag mouse monoclonal antibody and Alexa564-conjugated anti-mouse IgG antibody and then with anti-Rab7 rabbit monoclonal antibody and Alexa488-conjugated anti-rabbit IgG antibody (e, k). Arrows autolysosome-like vesicles. Scale bar 10 μm. b, d, f, h, j, l Quantification of the percentage of cells containing TRAF6 aggregates (b, h), polyUb aggregates (d, j), and Rab7-positive vesicles (f, l) were performed by counting Flag-TRAF6-positive cells on five different images of microscopic fields including at least 30 cells. Results, shown as the mean ± SD (n = 5), are representative of three independent experiments. *P < 0.01
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Fig7: NDP52 promotes aggregation of TRAF6. a, c, e, g, i, k HeLa cells were transfected with Flag-TRAF6 and HA-NDP52 for 24 h (a, c, e). HeLa cells were transfected with control siRNA or A20 siRNA for 24 h. Then cells were further transfected with HA-NDP52 and Flag-TRAF6 for 18 h (g, i, k). Cells were fixed and stained with anti-Flag mouse monoclonal antibody and Alexa564-conjugated anti-mouse IgG antibody and then with anti-NDP52 rabbit polyclonal antibody and Alexa488-conjugated anti-rabbit IgG antibody (a, g), with anti-TRAF6 rabbit polyclonal antibody and Alexa488-conjugated anti-rabbit IgG antibody and then with anti-FK2 mouse monoclonal antibody and Alexa564-conjugated anti-mouse IgG antibody (c, i) or with anti-Flag mouse monoclonal antibody and Alexa564-conjugated anti-mouse IgG antibody and then with anti-Rab7 rabbit monoclonal antibody and Alexa488-conjugated anti-rabbit IgG antibody (e, k). Arrows autolysosome-like vesicles. Scale bar 10 μm. b, d, f, h, j, l Quantification of the percentage of cells containing TRAF6 aggregates (b, h), polyUb aggregates (d, j), and Rab7-positive vesicles (f, l) were performed by counting Flag-TRAF6-positive cells on five different images of microscopic fields including at least 30 cells. Results, shown as the mean ± SD (n = 5), are representative of three independent experiments. *P < 0.01

Mentions: Autophagy receptors are known to mediate selective aggregation of targets, and this process is required for selective removal of them by autophagy [21]. We therefore tested whether NDP52 is associated with TRAF6 aggregation. Transfected TRAF6 hardly formed aggregates in HeLa cells and was diffusedly present in the cytoplasm (Fig. 7a, upper, and b). However, cotransfection of TRAF6 with NDP52 was found to be markedly increased in aggregated TRAF6 in the cytoplasm, and the TRAF6 aggregates were colocalized with NDP52 (Fig. 7a, middle, and b). NDP52-induced TRAF6 aggregates were detectable as proteins with polyUb (Fig. 7c, middle and lower, and d), although TRAF6 itself could sufficiently increase protein aggregates with polyUb even in the absence of NDP52 transfection (Fig. 7c, upper, and d). In some cells, formation of autolysosome-like vacuoles was observed (Fig. 7a, lower, and e, lower), and such vacuoles were partly colocalized with the autolysosomal marker Rab7 (Fig. 7e, lower). Rab7-positive vacuoles hardly colocalized with TRAF6 aggregations (Fig.  7e), suggesting that TRAF6 may be rapidly degraded in Rab7-positive vacuoles. Transfection of TRAF6 with NDP52 did not significantly increase the formation of Rab7-positive vacuoles (Fig. 7f). We next tested whether A20 silencing affects these observations. In A20-silenced HeLa cells, NDP52-induced TRAF6 aggregates were clearly increased (Fig. 7g, h). Aggregates of polyUb proteins were also increased and TRAF6 aggregates were colocalized with polyUb of proteins (Fig. 7i, j). Under the condition of A20 silencing, autolysosome-like vacuoles were increased and partly colocalized with Rab7 (Fig. 7k, l). These results collectively suggest that, after recruitment to TRAF6, NDP52 participates in the aggregation of the TRAF6 conjugated with polyubiquitin chains. This process may lead to degradation of TRAF6 in autolysosome although such a function of NDP52 is restricted by A20.Fig. 7


Regulation of Toll-like receptor signaling by NDP52-mediated selective autophagy is normally inactivated by A20.

Inomata M, Niida S, Shibata K, Into T - Cell. Mol. Life Sci. (2011)

NDP52 promotes aggregation of TRAF6. a, c, e, g, i, k HeLa cells were transfected with Flag-TRAF6 and HA-NDP52 for 24 h (a, c, e). HeLa cells were transfected with control siRNA or A20 siRNA for 24 h. Then cells were further transfected with HA-NDP52 and Flag-TRAF6 for 18 h (g, i, k). Cells were fixed and stained with anti-Flag mouse monoclonal antibody and Alexa564-conjugated anti-mouse IgG antibody and then with anti-NDP52 rabbit polyclonal antibody and Alexa488-conjugated anti-rabbit IgG antibody (a, g), with anti-TRAF6 rabbit polyclonal antibody and Alexa488-conjugated anti-rabbit IgG antibody and then with anti-FK2 mouse monoclonal antibody and Alexa564-conjugated anti-mouse IgG antibody (c, i) or with anti-Flag mouse monoclonal antibody and Alexa564-conjugated anti-mouse IgG antibody and then with anti-Rab7 rabbit monoclonal antibody and Alexa488-conjugated anti-rabbit IgG antibody (e, k). Arrows autolysosome-like vesicles. Scale bar 10 μm. b, d, f, h, j, l Quantification of the percentage of cells containing TRAF6 aggregates (b, h), polyUb aggregates (d, j), and Rab7-positive vesicles (f, l) were performed by counting Flag-TRAF6-positive cells on five different images of microscopic fields including at least 30 cells. Results, shown as the mean ± SD (n = 5), are representative of three independent experiments. *P < 0.01
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Related In: Results  -  Collection

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Fig7: NDP52 promotes aggregation of TRAF6. a, c, e, g, i, k HeLa cells were transfected with Flag-TRAF6 and HA-NDP52 for 24 h (a, c, e). HeLa cells were transfected with control siRNA or A20 siRNA for 24 h. Then cells were further transfected with HA-NDP52 and Flag-TRAF6 for 18 h (g, i, k). Cells were fixed and stained with anti-Flag mouse monoclonal antibody and Alexa564-conjugated anti-mouse IgG antibody and then with anti-NDP52 rabbit polyclonal antibody and Alexa488-conjugated anti-rabbit IgG antibody (a, g), with anti-TRAF6 rabbit polyclonal antibody and Alexa488-conjugated anti-rabbit IgG antibody and then with anti-FK2 mouse monoclonal antibody and Alexa564-conjugated anti-mouse IgG antibody (c, i) or with anti-Flag mouse monoclonal antibody and Alexa564-conjugated anti-mouse IgG antibody and then with anti-Rab7 rabbit monoclonal antibody and Alexa488-conjugated anti-rabbit IgG antibody (e, k). Arrows autolysosome-like vesicles. Scale bar 10 μm. b, d, f, h, j, l Quantification of the percentage of cells containing TRAF6 aggregates (b, h), polyUb aggregates (d, j), and Rab7-positive vesicles (f, l) were performed by counting Flag-TRAF6-positive cells on five different images of microscopic fields including at least 30 cells. Results, shown as the mean ± SD (n = 5), are representative of three independent experiments. *P < 0.01
Mentions: Autophagy receptors are known to mediate selective aggregation of targets, and this process is required for selective removal of them by autophagy [21]. We therefore tested whether NDP52 is associated with TRAF6 aggregation. Transfected TRAF6 hardly formed aggregates in HeLa cells and was diffusedly present in the cytoplasm (Fig. 7a, upper, and b). However, cotransfection of TRAF6 with NDP52 was found to be markedly increased in aggregated TRAF6 in the cytoplasm, and the TRAF6 aggregates were colocalized with NDP52 (Fig. 7a, middle, and b). NDP52-induced TRAF6 aggregates were detectable as proteins with polyUb (Fig. 7c, middle and lower, and d), although TRAF6 itself could sufficiently increase protein aggregates with polyUb even in the absence of NDP52 transfection (Fig. 7c, upper, and d). In some cells, formation of autolysosome-like vacuoles was observed (Fig. 7a, lower, and e, lower), and such vacuoles were partly colocalized with the autolysosomal marker Rab7 (Fig. 7e, lower). Rab7-positive vacuoles hardly colocalized with TRAF6 aggregations (Fig.  7e), suggesting that TRAF6 may be rapidly degraded in Rab7-positive vacuoles. Transfection of TRAF6 with NDP52 did not significantly increase the formation of Rab7-positive vacuoles (Fig. 7f). We next tested whether A20 silencing affects these observations. In A20-silenced HeLa cells, NDP52-induced TRAF6 aggregates were clearly increased (Fig. 7g, h). Aggregates of polyUb proteins were also increased and TRAF6 aggregates were colocalized with polyUb of proteins (Fig. 7i, j). Under the condition of A20 silencing, autolysosome-like vacuoles were increased and partly colocalized with Rab7 (Fig. 7k, l). These results collectively suggest that, after recruitment to TRAF6, NDP52 participates in the aggregation of the TRAF6 conjugated with polyubiquitin chains. This process may lead to degradation of TRAF6 in autolysosome although such a function of NDP52 is restricted by A20.Fig. 7

Bottom Line: However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II.NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation.Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Microbiology, Division of Oral Infections and Health Sciences, Asahi University School of Dentistry, Hozumi 1851, Mizuho, Gifu 501-0296, Japan.

ABSTRACT
Toll-like receptor (TLR) signaling is linked to autophagy that facilitates elimination of intracellular pathogens. However, it is largely unknown whether autophagy controls TLR signaling. Here, we report that poly(I:C) stimulation induces selective autophagic degradation of the TLR adaptor molecule TRIF and the signaling molecule TRAF6, which is revealed by gene silencing of the ubiquitin-editing enzyme A20. This type of autophagy induced formation of autophagosomes and could be suppressed by an autophagy inhibitor and lysosomal inhibitors. However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II. Through screening of TRIF-interacting 'autophagy receptors' in human cells, we identified that NDP52 mediated the selective autophagic degradation of TRIF and TRAF6 but not TRAF3. NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation. Intriguingly, only under the condition of A20 silencing, NDP52 could effectively suppress poly(I:C)-induced proinflammatory gene expression. Thus, this study clarifies a selective autophagic mechanism mediated by NDP52 that works downstream of TRIF-TRAF6. Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling.

Show MeSH
Related in: MedlinePlus