[Show abstract][Hide abstract] ABSTRACT: The adapter protein MyD88 adapter-like (Mal), encoded by TIR-domain containing adapter protein (Tirap) (MIM 606252), is the most polymorphic of the five adapter proteins involved in Toll-like receptor signaling, harboring eight
non-synonymous single nucleotide polymorphisms in its coding region. We screened reported mutations of Mal for activity in
reporter assays to test the hypothesis that variants of Mal existed with altered signaling potential. A TIR domain variant,
Mal D96N (rs8177400), was found to be inactive. In reconstituted cell lines, Mal D96N acted as a hypomorphic mutation, with
impaired cytokine production and NF-κB activation upon lipopolysaccharide or PAM2CSK4 stimulation. Moreover, co-immunoprecipitation studies revealed that Mal D96N is unable to interact with MyD88, a prerequisite
for downstream signaling to occur. Computer modeling data suggested that residue 96 resides in the MyD88 binding site, further
supporting these findings. Genotyping of Mal D96N in three different cohorts suggested that it is a rare mutation. We, thus,
describe a rare variant in Mal that exerts its effect via its inability to bind MyD88.
[Show abstract][Hide abstract] ABSTRACT: Developing T cells encounter peripheral self-antigens in the thymus in order to delete autoreactive clones. It is now known
that the autoimmune regulator protein (AIRE), which is expressed in thymic medullary epithelial cells, plays a key role in
regulating the thymic transcription of these peripheral tissue-specific antigens. Mutations in the AIRE gene are associated
with a severe multiorgan autoimmune syndrome (APECED), and autoimmune reactivities are manifest in AIRE-deficient mice. Functional
AIRE protein is expressed as distinct nuclear puncta, although no structural basis existed to explain their relevance to disease.
In addressing the cell biologic basis for APECED, we made the unexpected discovery that an AIRE mutation hot spot lies in
a caspase recruitment domain. Combined homology modeling and in vitro data now show how APECED mutations influence the activity of this transcriptional regulator. We also provide novel in vivo evidence for AIRE's association with a global transcription cofactor, which may underlie AIRE's focal, genome-wide, alteration
of the transcriptome.
Full-text · Article · Feb 2008 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The Toll-like receptor 4 (TLR4) is a class I transmembrane receptor expressed on the surface of immune system cells. TLR4 is activated by exposure to lipopolysaccharides derived from the outer membrane of Gram negative bacteria and forms part of the innate immune response in mammals. Like other class 1 receptors, TLR4 is activated by ligand induced dimerization, and recent studies suggest that this causes concerted conformational changes in the receptor leading to self association of the cytoplasmic Toll/Interleukin 1 receptor (TIR) signalling domain. This homodimerization event is proposed to provide a new scaffold that is able to bind downstream signalling adaptor proteins. TLR4 uses two different sets of adaptors; TRAM and TRIF, and Mal and MyD88. These adaptor pairs couple two distinct signalling pathways leading to the activation of interferon response factor 3 (IRF-3) and nuclear factor kappaB (NFkappaB) respectively. In this paper we have generated a structural model of the TLR4 TIR dimer and used molecular docking to probe for potential sites of interaction between the receptor homodimer and the adaptor molecules. Remarkably, both the Mal and TRAM adaptors are strongly predicted to bind at two symmetry-related sites at the homodimer interface. This model of TLR4 activation is supported by extensive functional studies involving site directed mutagenesis, inhibition by cell permeable peptides and stable protein phosphorylation of receptor and adaptor TIR domains. Our results also suggest a molecular mechanism for two recent findings, the caspase 1 dependence of Mal signalling and the protective effects conferred by the Mal polymorphism Ser180Leu.