Biochemical, molecular and cellular studies on negative regulators of TLR-signalling

Abstract

Toll-like receptors (TLRs) recognise pathogenic microorganisms through conserved pathogen associated molecular patterns, which activates the innate immune response. TLR signalling is mediated by cytoplasmic adaptor proteins via Toll/interleukin-1 receptor (TIR) domains. Sterile α- and armadillo-motif- containing protein (SARM) is the fifth TLR adaptor protein identified in humans and has been described as a negative regulator of the innate immune response. Several pathogenic bacteria are also known to express proteins with TIR- domains, which are believed to be involved in disruption of TLR signalling. This raises the question of whether SARM functions in a similar manner, as phylogenetic studies have shown that SARM is closely related to bacterial proteins. In this project, functional characterisation of SARM and a bacterial TIR domain protein from Bacillus anthracis (BaTdp) have been performed using both recombinantly expressed and purified proteins, as well as cellular assays. The TIR domains of both SARM and BaTdp were found to form heterotypic TIR-TIR interactions with multiple human TLR adaptors, including Myeloid differentiation factor 88 (MyD88). SARM and MyD88 both localised to mitochondria when overexpressed in mammalian cells, and SARM overexpression was associated with a reduction of TLR2-, TLR4- and MyD88- induced cytokine activation. A single amino acid residue in the SARM BB-loop motif, G601, was also identified as being critical for SARM’s anti-inflammatory effect. A short peptide derived from this motif was able to target MyD88 in vitro and slightly reduce TLR4-mediated cytokine activation. Overexpression of BaTdp in mammalian cells had no significant effect on TLR-mediated cytokine activation. Instead, the protein targeted microtubular networks in the cell and BaTdp expression was associated with a significant increase in cellular autophagy activity. The findings further enhance our understanding of the underlying mechanisms by which SARM suppress the innate immune response, and also describe previously unknown functions of BaTdp.