Rowe, D.C. et al. The myristoylation of TRIF-related adaptor molecule is essential for Toll-like receptor 4 signal transduction. Proc. Natl. Acad. Sci. USA 103, 6299-6304

Division of Infectious Disease and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 05/2006; 103(16):6299-304. DOI: 10.1073/pnas.0510041103
Source: PubMed


TRIF-related adaptor molecule (TRAM) is the fourth Toll/IL-1 resistance domain-containing adaptor to be described that participates in Toll-like receptor (TLR) signaling. TRAM functions exclusively in the TLR4 pathway. Here we show by confocal microscopy that TRAM is localized in the plasma membrane and the Golgi apparatus, where it colocalizes with TLR4. Membrane localization of TRAM is the result of myristoylation because mutation of a predicted myristoylation site in TRAM (TRAM-G2A) brought about dissociation of TRAM from the membrane and its relocation to the cytosol. Further, TRAM, but not TRAM-G2A, was radiolabeled with [3H]myristate in vivo. Unlike wild-type TRAM, overexpression of TRAM-G2A failed to elicit either IFN regulatory factor 3 or NF-kappaB signaling. Moreover, TRAM-G2A was unable to reconstitute LPS responses in bone marrow-derived macrophages from TRAM-deficient mice. These observations provide clear evidence that the myristoylation of TRAM targets it to the plasma membrane, where it is essential for LPS responses through the TLR4 signal transduction pathway, and suggest a hitherto unappreciated manner in which LPS responses can be regulated.

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    • "TRAM has previously been shown to localise to the plasma membrane and Golgi in resting cells but can also traffic independently of TLR4 to endosome membranes via a bipartite sorting motif [3]. TRAM is regulated by myristoylation, which is required for the adaptor molecule to be localized within plasma membrane and mutation of the myristoylation motif abolishes its functional activity [3], [24]. With this in mind, we examined whether expression of a myristoylation defective TRAM protein, TRAM-G2A, affected TLR7 mediated transcription factor activation. "
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    ABSTRACT: Toll-like receptor 7 (TLR7) plays a vital role in the immune response to ssRNA viruses such as human rhinovirus (HRV) and Influenza, against which there are currently no treatments or vaccines with long term efficacy available. Clearly, a more comprehensive understanding of the TLR7 signaling axis will contribute to its molecular targeting. TRIF related adaptor molecule (TRAM) plays a vital role in TLR4 signaling by recruiting TRIF to TLR4, followed by endosomal trafficking of the complex and initiation of IRF3 dependent type I interferon production as well as NF-κB dependent pro-inflammatory cytokine production. Towards understanding the molecular mechanisms that regulate TLR7 functionality, we found that TRAM-/- murine macrophages exhibited a transcriptional and translational impairment in TLR7 mediated RANTES, but not TNFα, production. Suppression of TRAM expression in human macrophages also resulted in an impairment in TLR7 mediated CCL5 and IFN-β, but not TNFα, gene induction. Furthermore, suppression of endogenous human TRAM expression in human macrophages significantly impaired RV16 induced CCL5 and IFNβ, but not TNFα gene induction. Additionally, TRAM-G2A dose-dependently inhibited TLR7 mediated activation of CCL5, IFNβ and IFNα reporter genes. TLR7-mediated phosphorylation and nuclear translocation of IRF3 was impaired in TRAM-/- cells. Finally, co-immunoprecipitation studies indicated that TRAM physically interacts with MyD88 upon TLR7 stimulation, but not under basal conditions. Our results clearly demonstrate that TRAM plays a, hitherto unappreciated, role in TLR7 signaling through a novel signaling axis containing, but not limited to, MyD88, TRAM and IRF3 towards the activation of anti-viral immunity.
    PLoS ONE 09/2014; 9(9):e107141. DOI:10.1371/journal.pone.0107141 · 3.23 Impact Factor
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    • "Contrary to other TLRs which are purely proatherogenic, TLR3 had an unexpected protective effect through vascular smooth muscle cell in arterial injury and atherosclerosis [21] [25]. All TLRs, except TLR3 which utilizes the TRIF-␤ dependent pathway, require the MyD88 dependent pathway to initiate downstream signaling [26]. TLR3 activation in circulation can elicit the production of IL-10 through IFN-␤ production which is a result of TLR3 dependent signaling [21] [27]. "
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    ABSTRACT: Background: Toll-like receptors, the most characterized innate immune receptors, have recently been demonstrated to play an important role in coronary atherosclerotic disease and diabetes mellitus (DM). TLR3 and TLR4 are known to act as anti-inflammatory and pro-inflammatory factors respectively in multi-factorial inflammatory disease states. However, there is less research about TLR3 and TLR4 expression in percutaneous transluminal coronary intervention (PCI) patients, particularly those with type 2 diabetes mellitus (DM2). Methods: We examined TLR3 and TLR4 expression and their downstream signaling pathway in PCI patients with (n=31) or without (n=32) DM2 compared with controls (n=35). Results: TLR3 and downstream anti-inflammatory factors (IRF-3, INF-β and IL-10) were significantly down-regulated in PCI patients with or without DM2 compared with controls, as determined by the quantification of both mRNA and protein. In contrast, TLR4 and downstream proinflammatory factors (MyD88 and TNF-α) were up-regulated in PCI patients with or without DM2 compared with controls. Conclusions: Patients undergoing PCI were shown to have a TLR-dependent pro-inflammatory state, mediated by a downregulation of TLR3 pathway, and upregulation of TLR4. This occurred in both with or without type 2 diabetes mellitus compared with controls in this research. The inflammatory imbalance observed in PCI patients was exacerbated in patients with DM2, consistent with a likely contribution of DM2 to the inflammatory state of coronary atherosclerotic disease, via impact on the innate immune response. This data supports the potential of TLRs as a novel therapeutic target in diabetics with coronary atherosclerotic disease.
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    • "For the TLR4 complex, phosphorylation of TLR4 and Mal has been suggested to be involved in the rearrangements [25], [26]. TRAM has been shown to be phosphorylated by Protein kinase C-ε (PKC-ε) upon stimulation by LPS, and the phosphorylation has been implicated in regulating the myristoylation state and thus the membrane targeting [10], [27]. The membrane targeting of another myristoylated protein, MARCKS, has been shown to be regulated by phosphorylation; MARCKS is released from the plasma membrane upon the PKC mediated phosphorylation of a serine near its myristoylation site [28]. "
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    ABSTRACT: MyD88, a Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR) or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the "sorting adaptor" for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.
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