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The ubiquitin-like protein PLIC-1 or ubiquilin 1 inhibits TLR3-Trif signaling.

Biswas N, Liu S, Ronni T, Aussenberg SE, Liu W, Fujita T, Wang T - PLoS ONE (2011)

Bottom Line: Interestingly, PLIC-1 had modest effect on TLR4-mediated signaling, but strongly suppressed the transcriptional activation of IFN-β promoter through the TLR3-Trif-dependent pathway.Finally, overexpression of PLIC-1 decreased Trif protein abundance in a Nocodazole-sensitive manner.Our results suggest that PLIC-1 is a novel inhibitor of the TLR3-Trif antiviral pathway by reducing the abundance of Trif.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT

Background: The innate immune responses to virus infection are initiated by either Toll-like receptors (TLR3/7/8/9) or cytoplasmic double-stranded RNA (dsRNA)-recognizing RNA helicases RIG-I and MDA5. To avoid causing injury to the host, these signaling pathways must be switched off in time by negative regulators.

Methodology/principal findings: Through yeast-two hybrid screening, we found that an ubiquitin-like protein named protein linking integrin-associated protein to cytoskeleton 1(PLIC-1 or Ubiquilin 1) interacted with the Toll/interleukin-1 receptor (TIR) domain of TLR4. Interestingly, PLIC-1 had modest effect on TLR4-mediated signaling, but strongly suppressed the transcriptional activation of IFN-β promoter through the TLR3-Trif-dependent pathway. Concomitantly, reduction of endogenous PLIC-1 by short-hairpin interfering RNA (shRNA) enhanced TLR3 activation both in luciferase reporter assays as well as in new castle disease virus (NDV) infected cells. An interaction between PLIC-1 and Trif was confirmed in co-immunoprecipitation (Co-IP) and GST-pull-down assays. Subsequent confocal microscopic analysis revealed that PLIC-1 and Trif colocalized with the autophagosome marker LC3 in punctate subcellular structures. Finally, overexpression of PLIC-1 decreased Trif protein abundance in a Nocodazole-sensitive manner.

Conclusions: Our results suggest that PLIC-1 is a novel inhibitor of the TLR3-Trif antiviral pathway by reducing the abundance of Trif.

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Related in: MedlinePlus

Interaction of PLIC-1 and Trif.A. 2 µg YFP-PLIC1or GFP and 2 µg Flag-Trif were cotransfected into 293T cells at 70% confluence in a 60-mm plate. 48 hours post-transfection, cell lysates were prepared and subjected to M2 anti-Flag affinity resin. After extensive wash with PBS, proteins were eluted by boiling the beads in 2× sample buffer and separated on a SDS-PAGE. Western blotting was performed using indicated antibodies. B. GST fusion proteins (around 2 µg) were mixed with cell lysates containing Flag-Trif for 2 hours and bound proteins were eluted from the GST column and analyzed for the presence of Flag-Trif. One tenth of the input protein was loaded. A Coomassie stained gel image was included showing the expression of each GST fusion protein.
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pone-0021153-g004: Interaction of PLIC-1 and Trif.A. 2 µg YFP-PLIC1or GFP and 2 µg Flag-Trif were cotransfected into 293T cells at 70% confluence in a 60-mm plate. 48 hours post-transfection, cell lysates were prepared and subjected to M2 anti-Flag affinity resin. After extensive wash with PBS, proteins were eluted by boiling the beads in 2× sample buffer and separated on a SDS-PAGE. Western blotting was performed using indicated antibodies. B. GST fusion proteins (around 2 µg) were mixed with cell lysates containing Flag-Trif for 2 hours and bound proteins were eluted from the GST column and analyzed for the presence of Flag-Trif. One tenth of the input protein was loaded. A Coomassie stained gel image was included showing the expression of each GST fusion protein.

Mentions: To explore the interaction between PLIC-1 and Trif, we first conducted Co-IP experiment. Fig. 4A showed that transiently expressed PLIC-1 was able to precipitate co-expressed Trif, but not the negative control protein. Moreover, in a separate experiment, we failed to pull down TLR3 and PLIC-1 together (data not shown). In order to determine which domain of PLIC-1 interacts with Trif, we produced GST fusion proteins containing PLIC-1 Uba domain alone, or PLIC-1 devoid of Uba or both Uba and Ubl domain and conducted a pull-down assay with Flag-Trif. It was found that the Uba domain of PLIC-1 alone is necessary and sufficient to bind Trif (Fig. 4B).


The ubiquitin-like protein PLIC-1 or ubiquilin 1 inhibits TLR3-Trif signaling.

Biswas N, Liu S, Ronni T, Aussenberg SE, Liu W, Fujita T, Wang T - PLoS ONE (2011)

Interaction of PLIC-1 and Trif.A. 2 µg YFP-PLIC1or GFP and 2 µg Flag-Trif were cotransfected into 293T cells at 70% confluence in a 60-mm plate. 48 hours post-transfection, cell lysates were prepared and subjected to M2 anti-Flag affinity resin. After extensive wash with PBS, proteins were eluted by boiling the beads in 2× sample buffer and separated on a SDS-PAGE. Western blotting was performed using indicated antibodies. B. GST fusion proteins (around 2 µg) were mixed with cell lysates containing Flag-Trif for 2 hours and bound proteins were eluted from the GST column and analyzed for the presence of Flag-Trif. One tenth of the input protein was loaded. A Coomassie stained gel image was included showing the expression of each GST fusion protein.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3117881&req=5

pone-0021153-g004: Interaction of PLIC-1 and Trif.A. 2 µg YFP-PLIC1or GFP and 2 µg Flag-Trif were cotransfected into 293T cells at 70% confluence in a 60-mm plate. 48 hours post-transfection, cell lysates were prepared and subjected to M2 anti-Flag affinity resin. After extensive wash with PBS, proteins were eluted by boiling the beads in 2× sample buffer and separated on a SDS-PAGE. Western blotting was performed using indicated antibodies. B. GST fusion proteins (around 2 µg) were mixed with cell lysates containing Flag-Trif for 2 hours and bound proteins were eluted from the GST column and analyzed for the presence of Flag-Trif. One tenth of the input protein was loaded. A Coomassie stained gel image was included showing the expression of each GST fusion protein.
Mentions: To explore the interaction between PLIC-1 and Trif, we first conducted Co-IP experiment. Fig. 4A showed that transiently expressed PLIC-1 was able to precipitate co-expressed Trif, but not the negative control protein. Moreover, in a separate experiment, we failed to pull down TLR3 and PLIC-1 together (data not shown). In order to determine which domain of PLIC-1 interacts with Trif, we produced GST fusion proteins containing PLIC-1 Uba domain alone, or PLIC-1 devoid of Uba or both Uba and Ubl domain and conducted a pull-down assay with Flag-Trif. It was found that the Uba domain of PLIC-1 alone is necessary and sufficient to bind Trif (Fig. 4B).

Bottom Line: Interestingly, PLIC-1 had modest effect on TLR4-mediated signaling, but strongly suppressed the transcriptional activation of IFN-β promoter through the TLR3-Trif-dependent pathway.Finally, overexpression of PLIC-1 decreased Trif protein abundance in a Nocodazole-sensitive manner.Our results suggest that PLIC-1 is a novel inhibitor of the TLR3-Trif antiviral pathway by reducing the abundance of Trif.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT

Background: The innate immune responses to virus infection are initiated by either Toll-like receptors (TLR3/7/8/9) or cytoplasmic double-stranded RNA (dsRNA)-recognizing RNA helicases RIG-I and MDA5. To avoid causing injury to the host, these signaling pathways must be switched off in time by negative regulators.

Methodology/principal findings: Through yeast-two hybrid screening, we found that an ubiquitin-like protein named protein linking integrin-associated protein to cytoskeleton 1(PLIC-1 or Ubiquilin 1) interacted with the Toll/interleukin-1 receptor (TIR) domain of TLR4. Interestingly, PLIC-1 had modest effect on TLR4-mediated signaling, but strongly suppressed the transcriptional activation of IFN-β promoter through the TLR3-Trif-dependent pathway. Concomitantly, reduction of endogenous PLIC-1 by short-hairpin interfering RNA (shRNA) enhanced TLR3 activation both in luciferase reporter assays as well as in new castle disease virus (NDV) infected cells. An interaction between PLIC-1 and Trif was confirmed in co-immunoprecipitation (Co-IP) and GST-pull-down assays. Subsequent confocal microscopic analysis revealed that PLIC-1 and Trif colocalized with the autophagosome marker LC3 in punctate subcellular structures. Finally, overexpression of PLIC-1 decreased Trif protein abundance in a Nocodazole-sensitive manner.

Conclusions: Our results suggest that PLIC-1 is a novel inhibitor of the TLR3-Trif antiviral pathway by reducing the abundance of Trif.

Show MeSH
Related in: MedlinePlus