Intracellular Toll-like receptor recruitment and cleavage in endosomal/lysosomal organelles

Methods Enzymol. 2014:535:141-7. doi: 10.1016/B978-0-12-397925-4.00009-2.

Abstract

Microbial pathogens are recognized through multiple, distinct receptors such as intracellular Toll-like receptors (TLRs 3, 7, 8, 9, and 13) which reside in the endosomes and lysosomes. TLRs are sensitive to chloroquine, a lysomotropic agent that neutralizes acidic compartments indicating a role for endo/lysosomal proteases for their signaling. Indeed, upon stimulation, full-length TLR7 and 9 are cleaved into a C-terminal fragment and this processing is highly dependent on a cysteine protease named asparagine endopeptidase (AEP) in dendritic cells. A recruitment and a boost in AEP activity, which was induced shortly after TLR7 and 9 stimulation, are shown to promote TLR7 and 9 cleavage and correlate with an increased acidification in endosomes and lysosomes. Moreover, mutating a putative AEP cleavage site in TLR7 or 9 strongly decreases their signaling in DCs, suggesting perhaps a direct cleavage of TLR7 and 9 by AEP. These results demonstrate that TLR7 and 9 require a proteolytic cleavage for their signaling and identified a key endocytic protease playing a critical role in this process.

Keywords: Dendritic cells; Intracellular TLRs; MHCII; MyD88; Processing; Proteases.

MeSH terms

  • Animals
  • Cell Fractionation
  • Cells, Cultured
  • Dendritic Cells / metabolism
  • Endosomes / enzymology*
  • Endosomes / metabolism
  • Enzyme Assays
  • Lysosomes / enzymology*
  • Lysosomes / metabolism
  • Mice
  • Peptide Hydrolases / isolation & purification
  • Peptide Hydrolases / metabolism
  • Primary Cell Culture
  • Protein Transport
  • Proteolysis
  • Signal Transduction
  • Toll-Like Receptors / metabolism*

Substances

  • Toll-Like Receptors
  • Peptide Hydrolases