Biomedical subjects

Brian G Monks

Publications and source records attributed to Brian G Monks.

9 recordsLinked to original sources

PubMed2006

MD-2 expression is not required for cell surface targeting of Toll-like receptor 4 (TLR4).

The cell surface receptor complex formed by TLR4 and myeloid differentiation 2 (MD-2) is engaged when cells are exposed to LPS. Recent studies suggested that surface localization of functional mouse TLR4 (mTLR4) depends on the simultaneous expression of MD-2. As we did not observe a similar requirement, we conducted a comparative study of human TLR4 and mTLR4 surface expression in immune cells derived from the MD-2 knockout mouse and LPS-responsive cell lines and in cells that ectopically express TLR4. Our results indicate that in the human and mouse models, neither TLR4 function nor TLR4 surface targeting requires MD-2 coexpression. Accordingly, we report on one human cell line, which constitutively expresses functional TLR4 on the cell surface in the absence of MD-2 expression.

Animals↗

PubMed2006

MD-2.

Toll-like receptors (TLRs) are a small family of type-I glycoproteins that bind to and are activated by conserved non-self molecular signatures carried by microorganisms. Toll-like receptor 4 is triggered by most lipopolysaccharides (LPS). LPS is a complex amphipathic saccharolipidic glycan derived from Gram-negative bacteria. Unique among TLRs, TLR4 activity and interaction with its natural ligand(s) strictly depends on the presence of the extracellular adaptor MD-2. MD-2 is a small secreted glycoprotein that binds with cytokine-like affinities to both the hydrophobic portion of LPS and to the extracellular domain of TLR4. The interaction between MD-2 and LPS induces a triggering event on TLR4, which involves the molecular rearrangement of the receptor complex and its homotypic aggregation. In silico analysis suggests that MD-2 and MD-1 are paralogs derived from a common predecessor at the level of early vertebrates. In this review, we summarize the current state of knowledge concerning MD-2.

Amino Acid Sequence↗

PubMed2006

The myristoylation of TRIF-related adaptor molecule is essential for Toll-like receptor 4 signal transduction.

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.

Adaptor Proteins, Vesicular Transport↗

PubMed2005

Pharmacological inhibition of endotoxin responses is achieved by targeting the TLR4 coreceptor, MD-2.

The detection of Gram-negative LPS depends upon the proper function of the TLR4-MD-2 receptor complex in immune cells. TLR4 is the signal transduction component of the LPS receptor, whereas MD-2 is the endotoxin-binding unit. MD-2 appears to activate TLR4 when bound to TLR4 and ligated by LPS. Only the monomeric form of MD-2 was found to bind LPS and only monomeric MD-2 interacts with TLR4. Monomeric MD-2 binds TLR4 with an apparent Kd of 12 nM; this binding avidity was unaltered in the presence of endotoxin. E5564, an LPS antagonist, appears to inhibit cellular activation by competitively preventing the binding of LPS to MD-2. Depletion of endogenous soluble MD-2 from human serum, with an immobilized TLR4 fusion protein, abrogated TLR4-mediated LPS responses. By determining the concentration of added-back MD-2 that restored normal LPS responsiveness, the concentration of MD-2 was estimated to be approximately 50 nM. Similarly, purified TLR4-Fc fusion protein, when added to the supernatants of TLR4-expressing cells in culture, inhibited the interaction of MD-2 with TLR4, thus preventing LPS stimulation. The ability to inhibit the effects of LPS as a result of the binding of TLR4-Fc or E5564 to MD-2 highlights MD-2 as the logical target for drug therapies designed to pharmacologically intervene against endotoxin-induced disease.

Cell Line↗

PubMed2005

The RNA helicase Lgp2 inhibits TLR-independent sensing of viral replication by retinoic acid-inducible gene-I.

The paramyxovirus Sendai (SV), is a well-established inducer of IFN-alphabeta gene expression. In this study we show that SV induces IFN-alphabeta gene expression normally in cells from mice with targeted deletions of the Toll-IL-1 resistance domain containing adapters MyD88, Mal, Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF), and TRIF-related adaptor molecule TLR3, or the E3 ubiquitin ligase, TNFR-associated factor 6. This TLR-independent induction of IFN-alphabeta after SV infection is replication dependent and mediated by the RNA helicase, retinoic acid-inducible gene-I (RIG-I) and not the related family member, melanoma differentiation-associated gene 5. Furthermore, we characterize a RIG-I-like RNA helicase, Lgp2. In contrast to RIG-I or melanoma differentiation-associated gene 5, Lgp2 lacks signaling caspase recruitment and activation domains. Overexpression of Lgp2 inhibits SV and Newcastle disease virus signaling to IFN-stimulated regulatory element- and NF-kappaB-dependent pathways. Importantly, Lgp2 does not prevent TLR3 signaling. Like RIG-I, Lgp2 binds double-stranded, but not single-stranded, RNA. Quantitative PCR analysis demonstrates that Lgp2 is present in unstimulated cells at a lower level than RIG-I, although both helicases are induced to similar levels after virus infection. We propose that Lgp2 acts as a negative feedback regulator of antiviral signaling by sequestering dsRNA from RIG-I.

Adaptor Proteins, Signal Transducing↗

PubMed2004

TLR9 signals after translocating from the ER to CpG DNA in the lysosome.

Microbial DNA sequences containing unmethylated CpG dinucleotides activate Toll-like receptor 9 (TLR9). We have found that TLR9 is localized to the endoplasmic reticulum (ER) of dendritic cells (DCs) and macrophages. Because there is no precedent for immune receptor signaling in the ER, we investigated how TLR9 is activated. We show that CpG DNA binds directly to TLR9 in ligand-binding studies. CpG DNA moves into early endosomes and is subsequently transported to a tubular lysosomal compartment. Concurrent with the movement of CpG DNA in cells, TLR9 redistributes from the ER to CpG DNA-containing structures, which also accumulate MyD88. Our data indicate a previously unknown mechanism of cellular activation involving the recruitment of TLR9 from the ER to sites of CpG DNA uptake, where signal transduction is initiated.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Lysines 128 and 132 enable lipopolysaccharide binding to MD-2, leading to Toll-like receptor-4 aggregation and signal transduction.

Three cell-surface proteins have been recognized as components of the mammalian signaling receptor for bacterial lipopolysaccharide (LPS): CD14, Toll-like receptor-4 (TLR4), and MD-2. Biochemical and visual studies shown here demonstrate that the role of CD14 in signal transduction is to enhance LPS binding to MD-2, although its expression is not essential for cellular activation. These studies clarify how MD-2 functions: we found that MD-2 enables TLR4 binding to LPS and allows the formation of stable receptor complexes. MD-2 must be bound to TLR4 on the cell surface before binding can occur. Consequently, TLR4 clusters into receptosomes (many of which are massive) that recruit intracellular toll/IL-1/resistance domain-containing adapter proteins within minutes, thus initiating signal transduction. TLR4 activation correlates with the ability of MD-2 to bind LPS, as MD-2 mutants that still bind TLR4, but are impaired in the ability to bind LPS, conferred a greatly blunted LPS response. These findings help clarify the earliest events of TLR4 triggering by LPS and identify MD-2 as an attractive target for pharmacological intervention in endotoxin-mediated diseases.

Amino Acid Sequence↗

PubMed2002

Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction.

Mammalian responses to LPS require the expression of Toll-like receptor 4 (TLR4), CD14, and MD-2. We expressed fluorescent TLR4 in cell lines and found that TLR4 densely localized to the surface and the Golgi. Similar distributions were observed in human monocytes. Confocal imaging revealed rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and the plasma membrane. Fluorescent LPS followed these trafficking pathways in CD14-positive cells. The TLR4- adapter protein, MyD88, translocated to the cell surface upon LPS exposure, and cross-linking of surface TLR4 with antibody induced signaling. Golgi-associated TLR4 expression was disrupted by brefeldin A, yet LPS signaling was preserved. We conclude that LPS signaling may be initiated by surface aggregation of TLR4 and is not dependent upon LPS trafficking to the Golgi.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Involvement of toll-like receptor (TLR) 2 and TLR4 in cell activation by mannuronic acid polymers.

The alginate capsule produced by the human pathogen Pseudomonas aeruginosa is composed mainly of mannuronic acid polymers (poly-M) that have immunostimulating properties. Poly-M shares with lipopolysaccharide the ability to stimulate cytokine production from human monocytes in a CD14-dependent manner. In the present study we examined the role of Toll-like receptor (TLR) 2 and TLR4 in responses to poly-M. Blocking antibodies to TLR2 and TLR4 partly inhibited tumor necrosis factor production induced by poly-M in human monocytes, and further inhibition was obtained by combining the antibodies. By transiently transfecting HEK293 cells, we found that membrane CD14 together with either TLR2 or TLR4/MD-2 could mediate activation by poly-M. Transfection of HEK293 cells with TLR2 and fluorescently labeled TLR4 followed by co-patching of TLR2 with an antibody revealed no association of these molecules on the plasma membrane. However, macrophages from the Tlr4 mutant C3H/HeJ mice and TLR4 knockout mice were completely non-responsive to poly-M, whereas the tumor necrosis factor release from TLR2 knockout macrophages was half of that seen with wild type cells. Taken together the results suggest that both TLR2 and TLR4 are involved in cell activation by poly-M and that TLR4 may be required in primary murine macrophages.

Animals↗