The Ubiquitin-Like Protein PLIC-1 or Ubiquilin 1 Inhibits TLR3-Trif Signaling

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. © 2011 Biswas et al

Regulation of interferon-inducible antiviral gene expression in human cells

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Common Interaction Surfaces of the Toll-Like Receptor 4 Cytoplasmic Domain Stimulate Multiple Nuclear Targets

Toll-like receptor 4 (TLR4) mediates the host response to lipopolysaccharide (LPS) by promoting the activation of pro- and anti-inflammatory cytokine genes. To activate each gene, numerous signal transduction pathways are required. The adaptor proteins MyD88 and TIRAP contribute to the activation of several and possibly all pathways via direct interactions with TLR4's Toll/interleukin-1 receptor (IL-1R) (TIR) domain. However, additional adaptors that are required for the activation of specific subsets of pathways may exist, which could contribute to the differential regulation of target genes. Furthermore, it remains unknown whether direct interactions that have been reported between TIR domains and other proteins are required for TLR4 signaling. To address these issues, we systematically mutated the TLR4 TIR domain in the context of a CD4/TLR4 fusion protein. Several exposed residues defining at least two structural surfaces were required in macrophages for activation of the proinflammatory IL-12 p40 and anti-inflammatory IL-10 promoters, as well as promoters dependent on individual transcription factors. Interestingly, the same residues were required by all promoters tested, suggesting that the signaling pathways diverge downstream of the adaptors. The mutant phenotypes provide a framework for future studies of TLR4 signaling, as the interaction supported by each critical surface residue will need to be defined

A common mechanism for mitotic inactivation of C2H2 zinc finger DNA-binding domains

Many nuclear proteins are inactivated during mitotic entry, presumably as a prerequisite to chromatin condensation and cell division. C2H2 zinc fingers define the largest transcription factor family in the human proteome. The linker separating finger motifs is highly conserved and resembles TGEKP in more than 5000 occurrences. However, the reason for this conservation is not fully understood. We demonstrate that all three linkers in the DNA-binding domain of Ikaros are phosphorylated during mitosis. Phosphomimetic substitutions abolished DNA-binding and pericentromeric localization. A linker within Sp1 was also phosphorylated, suggesting that linker phosphorylation provides a global mechanism for inactivation of the C2H2 family

The Ubiquitin-Like Protein PLIC-1 or Ubiquilin 1 Inhibits TLR3-Trif Signaling

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

Reduction of PLIC-1 level enhanced TLR3-Trif-mediated signaling.

<p><b>A</b>. Knocking down PLIC-1 by shRNA. 0.1 µg of HA-PLIC-1 was transfected with 0.1,1,2,4 µg of indicated shRNA constructs in 293T cells. Western blot was performed to detect HA-PLIC-1. <b>B</b>. shRNA 1602, 733, the scramble construct, and LMP (targeting mouse PLIC-1) were expressed in 293T cells. Knockdown of endogenous PLIC-1 was verified by western blotting using an anti-PLIC-1 antibody. <b>C</b>. Indicated combinations of constructs were transfected into 293T cells. 48 hours following transfection, luciferase activity was determined and normalized against renilla luciferase. <b>D</b>. Reduction of PLIC-1 by shRNA 1602 enhanced the activation of IFN-β promoter activity by poly I∶C stimulation. 293T cells were transfected with indicated plasmids and further stimulated with poly I∶C (40 µg/ml) for 24 hours prior to lysis. Notice that transfection with shRNA construct itself did not activate the IFN-β promoter.</p

PLIC-1 blocked TLR3-induced production of IFN-α during NDV infection.

<p>The parental human lung cancer cell line A549 or the A549 stably expressing shRNA #1602 and 733 (specifically target hPLIC-1) or the negative control shRNA (LMP) were stimulated with poly I∶C (100 µg/ml) for 24 hours and then infected by NDV-GFP (MOI 1). 12 hours post-infection, images were taken to visualize cells that had been productively infected by the green virus. Recombinant IFN-α was added as a positive control to suppress NDV infection. Percentages of positive infection were calculated by averaging the number of green cells over that of blue cells (DAPI staining of nuclei) in 4 fields. The numbers were indicated in the upper right corner of each merged image.</p

Overexpression of PLIC-1 decreased Trif abundance in a Nocodazole-sensitive manner.

<p><b>A</b>. 293T cells were transfected with indicated plasmids. 48 hours post-transfection, lysates were prepared and subjected to western blot analysis. HCV NS3/4A was known to cleave Trif and was included as a positive control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021153#pone.0021153-Li1" target="_blank">[8]</a>. However, it only marginally reduced the level of Trif. PLIC-1 has no effect on the expression of a protein named Flap, which was included as a negative control here. <b>B</b>. 293T cells were transfected with 0.2 µg Flag-Trif and indicated amount of HA-PLIC-1 plasmid for 24 hours. Cells were then treated with 30 µM Nocodazole or MG132 (50 µM) for 6 hours prior to cell lysis. Western blotting was performed to quantitate the level of Flag-Trif, HA-PLIC-1. A non-specific band was indicated as the loading control.</p

PLIC-1 and Trif co-localized with LC3.

<p>The human hepatoma cell line Huh7.5.1 cells were seeded on glass cover slip and then transfected with 0.1 µg of YFP-PLIC-1 and RFP-LC3 (<b>A</b>), or 0.1 µg YFP-Trif and RFP-LC3 in HEK (<b>B</b>) or Huh7.5.1 (<b>C</b>) cells in a 24-well plate format. 24 hours post-infection, cells were fixed and imaged using a Zeiss Meta LSM510 microscope. In all figures, co-localization was indicated as yellow dots in the merged images.</p