Modulation of TLR Signaling by Multiple MyD88-Interacting Partners Including Leucine-Rich Repeat Fli-I-Interacting Proteins

Department of Biochemistry and Biophysics.
The Journal of Immunology (Impact Factor: 4.92). 04/2009; 182(6):3450-60. DOI: 10.4049/jimmunol.0802260
Source: PubMed


Emerging evidences suggest TLR-mediated signaling is tightly regulated by a specific chain of intracellular protein-protein interactions, some of which are yet to be identified. Previously we utilized a dual-tagging quantitative proteomics approach to uncover MyD88 interactions in LPS-stimulated cells and described the function of Fliih, a leucine-rich repeat (LRR) protein that negatively regulates NF-kappaB activity. Here we characterize two distinct LRR-binding MyD88 interactors, LRRFIP2 and Flap-1, and found that both are positive regulators of NF-kappaB activity. Upon LPS stimulation, LRRFIP2 was also found to positively regulate cytokine production in macrophages, suggesting a functional role in TLR4-mediated inflammatory response. Furthermore, we observed that immediately following LPS stimulation both LRRFIP2 and Flap-1 compete with Fliih for interacting with MyD88 to activate the signaling. By using a novel multiplex quantitative proteomic approach, we found that at endogenous levels these positive and negative regulators interact with MyD88 in a timely and orderly manner to differentially mediate the NF-kappaB activity through the course of signaling from initiation to prolongation, and to repression. Based on these data, we describe a mechanistic model in which selective modulation of TLR signaling is achieved by temporal and dynamic interactions of MyD88 with its regulators.

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Available from: Ling Xie, Jan 14, 2014
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    • "With regards to the mouse isoforms, three different proteins have been reported, two of which, Fli-I leucine-rich repeat associated protein 1 (Flap-1) and Lrrfip1, have been studied. The first one has been described as interacting with Fli-I and myeloid differentiation factor 88 (Myd88) and to positively regulate TLR immune response and Nuclear Factor-KappaB (NF-κB) activation through Myd88 interaction (Liu and Yin, 1998, Reed et al., 1998, Dai et al., 2009, Bagashev et al., 2010). On the other hand, Lrrfip1 has been described as an intracellular sensor of pathogens that regulates the innate immune response via a β-catenin-dependent pathway (Yang et al., 2010). "
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    ABSTRACT: Lrrfip1 is an up-regulated protein after cerebral ischaemia whose precise role in the brain both in healthy and ischaemic conditions is unclear. Different Lrrfip1 isoforms with distinct roles have been reported in human and mouse species. The present study aimed to analyse the Lrrfip1 transcriptional variants expressed in rat cortex, to characterise their expression patterns and subcellular location after ischaemia, and to define their putative role in the brain. Five transcripts were identified and three of them (Lrrfip1, CRA_g and CRA_a' (Flap-1)) were analysed by qPCR. All the transcripts were up-regulated and showed differential expression patterns after in-vivo and in-vitro ischaemia models. The main isoform, Lrrfip1, was found to be up-regulated from the acute to the late phases of ischaemia in the cytoplasm of neurons and astrocytes of the peri-infarct area. This study demonstrates that Lrrfip1 activates β-catenin, Akt, and mTOR proteins in astrocytes and positively regulates the expression of the glutamate transporter GLT-1. Our findings point to Lrrfip1 as a key brain protein that regulates pro-survival pathways and proteins and encourages further studies to elucidate its role in cerebral ischaemia as a potential target to prevent brain damage and promote functional recovery after stroke.
    Neuroscience 03/2014; 268. DOI:10.1016/j.neuroscience.2014.02.051 · 3.36 Impact Factor
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    • "Endotoxin tolerance phenomenon has consistently been linked with particular regulatory events including the deficient recruitment of the adapter MyD88 to TLR4 and consequently reduction in TLR4-MyD88 complex formation , impairment of IRAK-1 activity, and defects in the activation of NF-kB (Medvedev et al., 2002). Of interest, individual signal proteins such as LRRFIP2, Flap-1, and Fliih, all of which interact with MyD88, were found to play an opposing regulatory role in TLR signaling (Wang et al., 2006; Dai et al., 2009). However, the induction of negative regulators of TLR by Natterins with a consequent interference with inside-out in integrin signaling remains elusive. "
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    ABSTRACT: Here we evaluated whether Natterins affect the leukocyte-endothelial cell interaction, hampering leukocyte mobilization and extravasation. Leukocyte-endothelial cell interactions were evaluated in venules of mouse cremaster muscle using intravital microscopy. We reported that low doses of Natterins interfere with the cell capturing, inhibiting the interaction of blood neutrophils with the post-capillary venules induced by the TLR4 agonist LPS, or the chemokine KC. Using endotoxemic mice challenged with LPS, we confirmed that Natterins reduce neutrophil accumulation in the peritoneum exudates. The rolling of leukocytes induced by KC or LPS was not impaired in Natterins-treated TLR2, MyD88 deficient or TLR4 mutant mice, indicating that TLR2- or TLR4-MyD88-mediated signals are required for the anti-inflammatory effect of Natterins. The inhibitory effect was not influenced by endogenous regulators of inflammation such as IL-10, corticosteroids, the HO-1 or the antagonist of the receptor of IL-1, nor by the disruption of their proteolytic activity. However, it was completely dependent on the activation of serine/threonine phosphatases and the PI3K signaling pathway, but independent on increased proteasome activity. This work started asking how the main toxins in the T nattereri venom contributes for the deficient influx of inflammatory leukocytes, which consequently drive to the delayed inflammatory reaction finalization in injured tissue; and finished demonstrating that Natterins can control the leukocyte-endothelial wall interactions in a mechanism dependent on negative signals derived from TLR2-TLR4/Myd88 signaling cascade. Interestingly, we confirmed that the antagonist effect of Natterins is mediated by the activation of serine/threonine phosphatases and by the key signaling PI3K molecule
    Toxicon 01/2014; 87(1):54. · 2.49 Impact Factor
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    • "The LRR region of Flii shares 29% sequence identity and 42% similarity to TLR4 [20]. Through its interaction with MyD88, it has been suggested that Flii can modulate inflammation by suppressing TLR4-MyD88-mediated activation of NF-í µí¼…B [21]. Conversely, a reduction in the Flii level may enhance activation of NF-í µí¼…B and increase cytokine secretion [20]. "
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    ABSTRACT: Impaired wound healing and ulceration represent a serious complication of both type 1 and type 2 diabetes. Cytoskeletal protein Flightless I (Flii) is an important inhibitor of wound repair, and reduced Flii gene expression in fibroblasts increased migration, proliferation, and adhesion. As such it has the ability to influence all phases of wound healing including inflammation, remodelling and angiogenesis. Flii has the potential to modulate inflammation through its interaction with MyD88 which it an adaptor protein for TLR4. To assess the effect of Flii on the inflammatory response of diabetic wounds, we used a murine model of streptozocin-induced diabetes and Flii genetic mice. Increased levels of Flii were detected in Flii transgenic murine wounds resulting in impaired healing which was exacerbated when diabetes was induced. When Flii levels were reduced in diabetic wounds of Flii-deficient mice, healing was improved and decreased levels of TLR4 were observed. In contrast, increasing the level of Flii in diabetic mouse wounds led to increased TLR4 and NF- κ B production. Treatment of murine diabetic wounds with neutralising antibodies to Flii led to an improvement in healing with decreased expression of TLR4. Decreasing the level of Flii in diabetic wounds may therefore reduce the inflammatory response and improve healing.
    02/2013; 2013(3):389792. DOI:10.1155/2013/389792
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