PDF《LPS stimulation》

ARTICLE nature communications | 3:707 | DOI: 10.

1038/ncomms1706 | www.nature.com/naturecommunications ?

2012 Macmillan Publishers Limited. All rights reserved. Received

23 Jun

2011 | Accepted

25 Jan

2012 | Published

28 Feb

2012 DOI: 10.1038/ncomms1706 Toll-like receptor

4 is an innate immune receptor responsible for the recognition of the Gram-negative cell wall component lipopolysaccharide. Here we show that transmembrane emp24 domain-containing protein

7 (TMED7) inhibits MyD88-independent toll-like receptor

4 signalling. TMED7 overexpression inhibits the ability of TRAM, an adaptor utilized by toll- like receptor 4, or lipopolysaccharide to activate the interferon regulatory factor 3-signalling pathway, whereas TMED7 knockdown enhances production of the cytokine, RANTES, following lipopolysaccharide stimulation. Upon lipopolysaccharide stimulation, TMED7 co-localizes with TRAM and toll-like receptor

4 in late endosomes where it encounters the negative regulator of TRAM, TAG. The TMED7 sequence is found in TAG because of a read-through from the tmed7 gene into the ticam2 gene. TMED7 is essential for TAG-mediated disruption of the TRAM/ TRIF complex and the degradation of toll-like receptor 4. A TMED homologue, logjam, has a negative role in the Toll and IMD pathways in Drosophila melanogaster;

therefore, TMEDs may have a conserved role in the regulation of innate immunity.

1 Trinity Biomedical Sciences Institute, School of Biochemistry and Immunology, Trinity College Dublin, Pearse Street, Dublin 2, Ireland.

2 Institute of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, N-7491, Trondheim. Correspondence and requests for materials should be addressed to A.F.M. (email: [email protected]). The GOLD domain-containing protein TMED7 inhibits TLR4 signalling from the endosome upon LPS stimulation Sarah L. Doyle1, Harald Husebye2, Dympna J. Connolly1, Terje Espevik2, Luke A.J. O'

Neill1 &

Anne F. McGettrick1 ARTICLE nature communications | DOI: 10.1038/ncomms1706 nature communications | 3:707 | DOI: 10.1038/ncomms1706 | www.nature.com/naturecommunications ?

2012 Macmillan Publishers Limited. All rights reserved. T oll-like receptors (TLRs) are type

1 transmembrane receptors involved in the detection of invading pathogens. They possess a ligand-binding domain of leucine-rich repeats and a sig- nalling Toll-IL-1 receptor (TIR) domain, which interacts with TIR domain containing adaptor molecules. TLR4 senses lipopolysaccha- ride (LPS) from Gram-negative bacteria1 and launches a complex immune response. TLR4 is also reported to sense endogenous mol- ecules produced during tissue injury, provoking inflammation2,3. Excessive host responses towards LPS have been shown to contribute to such diseases as Gram-negative sepsis, rheumatoid arthritis and atherosclerosis4. It is therefore essential that LPS-induced responses are tightly regulated. TLR4 activates two separate signalling path- ways, the MyD88-dependent and MyD88-independent pathways. The TIR-domain containing adaptors Mal and MyD88 facilitate the activation of the MyD88-dependent pathway at the plasma mem- brane, resulting in the activation of the pro-inflammatory tran- scription factor nuclear factor (NF)-κB. TIR-domain-containing adaptor-inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM) facilitate the MyD88-independent pathway, leading to interferon regulatory factor

3 (IRF3) activation, which induces the expression of genes containing the interferon-sensitive response element (ISRE) in their promoter, such as those encoding interferon β (IFN β) and CCL5 (RANTES)5. Recent studies have revealed an important role for intracellular trafficking in the TLR ligand recognition and in the regulation of their downstream signalling pathways. Wang et al.6 revealed that Rab10 acts as a positive regulator of TLR4 signalling by promoting transport of TLR4 from the Golgi to the plasma membrane. TLR4 and TRAM traffic from the plasma membrane to the early endo- some upon LPS stimulation, and it is from these endosomes that the MyD88-independent pathway signals7. Rab7b acts as a negative regulator of the TLR4 signalling pathway by promoting the move- ment of TLR4 into late endosomes for degradation8C10. Husebye et al.11 showed that Rab11a is essential for the trafficking of TLR4 into the Rab11a?+? endocytic recycling compartment (ERC) and onto Escherichia coli-containing phagosomes. Rab11a knockdown reduced IRF3 signalling following E. coli (or LPS) stimulation, with little or no effect on NF-κB signalling. Thetransmembraneemp24domain-containingprotein(TMED)/ p24 family are involved in the vesicular trafficking of proteins and are therefore functionally similar to the Rab family. Ten TMED genes are present in mammals and they are separated into four dif- ferent subfamilies, α, β, δ and γ12. TMED proteins are highly con- served in eukaryotes13 and exist as monomers, dimers, oligomers and hetero-oligomers14,15. They possess a GOLD domain, which is a β-strand-rich domain found in several proteins involved in Golgi dynamics, as well as intracellular protein trafficking14,16. Here we report on a TMED family member, TMED7, which neg- atively regulates TLR4 signalling. We have previously reported that the TMED7 transcript occurs in a protein we named TRAM adap- tor molecule with GOLD domain (TAG), owing to a read-through from the gene encoding tmed7 into the ticam2 gene on chromosome 59,17,18. We now report that TMED7, like TAG9, acts as an inhibi- tor of the MyD88-independent TLR4 signalling pathway. TMED7 resides mainly in the Golgi and endosomal structures in resting cells. It co-localizes with TRAM intracellularly, but not TLR4, and is not present on the plasma membrane. Upon LPS stimulation, TMED7 trafficks into the maturing endosomal network along with TRAM and TLR4. Its GOLD domain appears to project into the cytosol where it can interact with TRAM and TAG, providing an explanation for why TAG has TMED7 in its sequence allowing for a homotypic interaction between the GOLD domains of TAG and TMED7. We have found that the disruption of the TRIF/TRAM complex by TAG requires TMED7, and that TMED7 targets TLR4 for degradation, resulting in the consequent cessation of TLR4 sig- nalling. Our study therefore reveals a role for TMED7, as a critical inhibitor of TLR4 signalling, and implicates the TMED family, alongside the Rab family, as important regulators in the control of innate immune signalling. Results ExpressionprofileofTMED7. It was originally reported that Tmed7 and Ticam2 (Tram) were two separate genes on chromosome 5;