tir-like proteins seminar
TRANSCRIPT
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Innate immunity:first line of defense against microbes
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Three arms for innate immune detectionof pathogens
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Suzuki et al , Trends in Immunology , 2002
Toll-like receptor signaling pathway
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TIR 3D fold belongsto the flavodoxin-like
/ familythat includes
bacterial proteins
Crystal structure of human Toll-likereceptor 2 TIR domain
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Model for TIR-TIR signaling interaction:homotypic via E-helix and heterotypic via BB loop
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Sequence alignment between prokaryotic & eukaryotic TIRs
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Genome location of TIR-like genes:pathogenicity islands of infectious bacteria
(1) TIR-like gene; (20) integrase gene; (12) type IV secretion pilin protein precursor gene
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aims
3D structure of a prokaryote TIR domain Comparison with a eukaryote TIR receptor
TIR signaling: prokar eukar complex Homo- vs. hetero-typic interactions
Function of TIR-like domain in bacteria
Prevent host cell inflammation signals (virulent factor)
Prokaryote programmed cell death
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Prokaryotic TIRs Status Bacillus subtilis Cloned Brucella abortus Cloned, no expression Burkholderia fungorum Cloned, no expression
Escherichia coli CFT073 Cloned, toxic, refolded, unstable Paracoccus denitrificans Cloned, expressed, purified, >20mg/ml, xtal trialsSalmonella enteriditis Cloned, toxicStaphylococus aureus Cloned, no expression
Prokaryote TIR-like proteinsexpression trials in E. coli
P. denitrificans TIR-like protein ( Pd TLP )
could only be expressed in high quantity
using the Rosetta(DE3)-pLysS E. coli strain.
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Limited proteolysis analysis of Pd TLP
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Mass spectrometry identification of Pd TLP digestion fragments
Full length
C-terminalfragment
(S 146 -A-M-K-P)
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158kDa 44kDa 17kDa
Gel filtration chromatograms for Pd TLP
Full length(33 kDa)
C-terminalTIR-like(17 kDa)
N-terminalCoiled-coil(16 kDa)
Dimer
Monomer
Oligomer
TIR-likeCoiled-coilpI 5.7pI 10.2 Full length pI 9.6
Leu145 aa299aa1
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Differential scanning calorimetry profiles for Pd TLP full length, coiled-coil & TIR-like domains
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Circular dichroism spectrum of the coiled-coil region of Pd TLP
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1H-15 N 2D HSQC NMR spectrum of Pd TLPTIR-like domain
( p p
m )
(ppm)
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Salmonel la Paracoccus Bruc ella
Prokaryote TIR-like proteins suppress NFkB activation induced by MyD88
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In vitro interaction between MyD88and Pd TLP TIR domains
GSTGST-mMyD88-TIR
PdTLP-TIR
GSTGST-mMyD88-TIR
hTLR2-TIR
GSTGST-hTLR2-TIR
mMyD88-TIR
GSTGST-hTLR2-TIR
PdTLP-TIR
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Model for virulent action by TIR-like mimicry healthy
cellinfected
cell
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Striking a balance through host mimicry :
Salmonella SopE ( GEF ) & SptP ( GAP ) virulent factors
Stebbins, C. Nature 412:701-5 (2001)
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Drug ability of the TLR pathway
Beutler, B. Nature 430:257-263 (2004)
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BB loop mimic as inhibitor of prot-prot inter:a low molecular weight pyrrolidine interacts with MyD88 TIR
Davis, CN, Rebek, J & Bartfai, B. PNAS 103:2953-2958 (2006)
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Acknowle dgements
Ray Low
Genaro Pimienta
Takashi Mukasa
Robert C. Liddington
John C. Reed
Adam Godzik
Group Collaborators
Funding: R21AI065602 (NIAID-NIH )
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Second life avatar:
jamie pascal
See you in the metaverse
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