Use of CellPermeable Peptides to

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Use of Cell-Permeable Peptides to Dissect
Toll-Like Receptor Signaling Matthew J. Fenton,
Ph.D. Asthma, Allergy, and Inflammation
Branch National Institute of Allergy and
Infectious Diseases NIH, Bethesda
MD fentonm_at_niaid.nih.gov
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Toll-like receptor (TLR) structure
Bell et al., Trends Immunol. 24 528-33, 2003
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TLR proteins, their ligands, and their adaptor
proteins
Bulendran and Ahmed, Cell 124 849-863,
2006 (TIRAP MAL)
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TLR adaptor proteins and signaling pathways
ONeill and Bowie, 2007 (TIRAP MAL)
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IL-1, IL-6, TNF, IP-10
ONeill and Bowie, 2007 (TIRAP MAL)
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IFN-a/b, RANTES
ONeill and Bowie, 2007
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TLR adaptor proteins contain a conserved TIR
domain
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Crystal structure of the TIR domain
Xu et al., Nature 408 111-115, 2000
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Conservation of the BB-loop domain
Xu et al., Nature 408 111-115, 2000
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• Questions
• How do the different adapter proteins interact
  with TLR4?
• How do the different adapter proteins interact
  with each other?
• Can exogenous peptides containing conserved TIR
  sequences act as competitive inhibitors?
• How can these exogenous peptides be transported
  into the cells?

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A Protein Transduction Domain (PTD) from the
Drosophila antennapedia protein transports
cargo peptides into cells
Kabouridis, 2003
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Cell-permeable BB-loop peptide (BP) sequences
Antennapedia PTD BB
loop TLR1/6-BP RQIKIWFQNRRMKWKKLHERNFVPGKSIVE TLR
2-BP RQIKIWFQNRRMKWKKLHKRDFVPGKWIID TLR4-BP RQIKIW
FQNRRMKWKKLHYRDFIPGVAIAA MyD88-BP RQIKIWFQNRRMKWKK
VSDRDVLPGTCVWS TRIF-BP RQIKIWFQNRRMKWKKFCEEFQVPGRG
ELH TIRAP-BP RQIKIWFQNRRMKWKKLQLRDAAPGGAIVS TRAM-B
P RQIKIWFQNRRMKWKKIVFAEMPCGRLHLQ
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A TIRAP/Mal-BP inhibits LPS-induced IL-6 and
IP-10 gene expression
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Effect of BPs on cytokine induction by LPS
40 mM peptide
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Effect of BPs on the LPS-stimulated activation of
MAP kinases and STAT-1
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Effect of BPs on dimerization of IRF-3 and
expression of IRF-1
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Comparative effects of BPs on TLR2- vs
TLR4-mediated activation of MAP kinases and IL-1
gene expression
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Use of TLR BPs on MyD88-dependent and
-independent signaling pathways
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• Conclusions (1)
• Exogenous BB-loop peptides can block LPS-induced
  cytokine gene expression, MAPK activation, and
  IRF activation in a dose-dependent manner
• The BB-loops of TIR-containing adaptor proteins
  are unable to significantly block cytokine gene
  expression and MAPK activation induced by the
  TLR2 ligand P3C
• Only the BB-loop of TLR2 can block P3C-induced
  cytokine gene expression and MAPK activation

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• Conclusions (2)
• The BB-loops of TLR2 and TLR4 can block both
  MyD88-dependent and independent signaling
  induced by LPS
• None of the BB-loops peptides tested
  could block activation by the TLR3 ligand poly
  IC
• Although most TLR signal transduction events
  appear to be dependent on the BB-loop, the
  BB-loops of the TLRs and the adapter proteins
  appear to perform distinct functions

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The TLR4 signalsome
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The TLR4 signalsome
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Acknowledgements Stefanie N. Vogel (Univ. of
Maryland) Vladimir U. Toschakov (Univ. of
Maryland) Subhendu Basu (Univ. of
Maryland) Dagmar Schilling (Paul-Ehrlich-Institu
te)