Tir-like proteins seminar

8/15/2019 Tir-like proteins seminar

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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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Documents

Tir-like proteins seminar