Immune and Inflammatory Responses

Injury and Infection

NFκ B and StressKinases

Cytokines

Other Cell Types Leukocytes

Wound Healing etc Adaptive Immunity

Innate Response( eg , , )pathogens mechanical heat shock

The TIR domain Family of Immediate-EarlyHost Defence Proteins

Extracellular

Cytoplasmic

TIR DomainIg DomainDeath DomainLeucine RichRepeatTlr1Tlr2Tlr3Tlr4Tlr5Tlr6Tlr7Tlr8Tlr9Tlr10N-proteinRRP1L6RRP5B15RA46RA52RIL1RIIST2IL1RIAcPIL18AcPIL18RIL1RrPMyD88

sIL1RIIsST2sIL1RIMyD88Human TollsTolls 1-8Plant Pathogen Resistance Proteins

Human Cytokine ReceptorsViral GeneProductsDrosophila

Schematic of TIR signalling

aa

*?PRISMTollipMyD88

IRAK 1/2/3TRAF6TAB/TAK1IKKComplex IIP-1IRAK 1/2/3

Small GTPasesMKKKsMKKsMKsJNKp38ERKScaffoldsScaffolds

ScaffoldsRel FamilyTranscription FactorsUbProteosome

Jun FamilyTranscription Factors Scaffolds???

EXTRACELLULAR SIGNAL

a

MechanicalTemperatureNutrientsChemical StressHormones

DivisionlocomotionSecretionApoptosisDifferentiation

Cells as computational devices

• Contains 1 copy of the genome• Contains ca 1010- 1011 protein molecules in a volume of ca 1 picolitre• Contains ca 104 different proteins present at 104 - 107 copies/cell• The proteins plus small molecules and ions form a spatially distributed, dissipative computational network capable of robust self regulation within a narrow range of physical environments.• The network is non-linear- binary (?)• The network is embodied as a semi-solid state device - most reactions do not occur in free solution

The Computational Network is Built from Noisy

Components Nuclear Concentration of NFκB Measured by Confocal Imaging of Anti-relA Labelled Fibroblasts

Protein concentration in cells is controlled in part by rate of gene transcription

transcription is stochastic

The concentration of any given protein must therefore vary between different cells in clonal population

Since most if not all proteins participate in the control network, this will in turn affect control of gene expression differentially in each individual cell

aa

Time(min)10301030103010301030103010301030100 2010201020Relative Fluorescence

IL-1 ( )M0251020306090

10-1010-810-12

Theoretical Analysis suggests that the Segment Polarity network in Flies is Binary

Random Graph Representation of Signal Transduction Network

Nodes = ReactantsEdges = Reactions

Implementation 1. Chemical kinetics -ODEs2. Cellular Automata3. Software agents

Basic considerations1. Edges/nodes2. Edge/node distribution3. Rules4. Bit depth5. Word length

Input

Promoter

Signal Transduction Pathways in a Network ?

Input

Perturbation of Internal State of Network Can Mimic Agonist

Example Mechanisms

1. Titre out inhibitor

2. Increase concentration aboveKd for activator

aaaa

Experimental strategycDNAlibrarypoolsof200-500independentcoloniesofbacteriaplasmidDNApreparation

1. transfection of mammalian cells2. analysisby DualLuciferaseassay3. pool-breakdown

REPORTERCONSTRUCTS:IL8-lucTK-luc(internal control)

00.10.20.30.40.50.60.70.80.900.20.40.60.8IL-8 luc

Positive pools are detectable by both screen strategies

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 0.2 0.4 0.6 0.8

TK

-RL

IL8-luc

5

25

45

65

85

105

125

145

50 550 1050 1550 2050

bri

gh

tness

area

negative pool

5

25

4 5

65

85

105

125

145

50 550 1 05 0 15 50 2050

positive pool

area

Dual-Luciferase assay

EGFP assay

Clone ID Gene name Orientation Clone ID

Proposed mech. of action

2E3 N10/NUR77 Sense Inhibition of survival signals

3E1 RelA Sense Induction of NFκB responsive genes

4D8 hTrb-1(hTrb-2, hTrb-3) Sense (3’ UTR) Inhibition of AP-1 activation

2B2

2G1

19-6.2

23-5.8

IFN Sense Induces TRAIL expression which activates

Both apoptosis and NFκB

19-3.7 Prothymosin 4 Antisense (full) Thymosin beta is an anti-inflammatory protein

83-3.8 Ferritin Sense Redox effect on NFκB

91-2.8 Mitochondrial NADPH Oxidase

Antisense Redox

53-1.7 Homer1B Sense 3’ UTR Scaffold

126 Genomic clone + est

Summary of clones isolated from 3% of library

Adaptation of Mammalian Expression Screen to High throughput Robotic Platform

aa

cDNATransformE. ColiLibrary of 3x 106independanttransformants

96 pinhigh speedpicking headLibrary in traysca 3,000colonies per tray2 copies ofgridded library106 clones eachin bar coded384 well plates1 clone per well

Ligate ExpressionVectorMEGAPIX

Construct20,833 poolsof 48 fromone copy andmake Minprepsin bar coded96 well platesAssay bymammaliantransfection

Q-BOT

Cherry pick source wellsfor positive pools frommaster libraryIdentify source wellby rows and columns

working copyBackup copy

VERIFY ACTIVITYSEQUENCE INSERTStore at -80

Output from High Throughput Screen showing Candidate Positive Pools - each Pool from 48 Library Wells

1. 1/1000 clones regulate pIL82. pIL8 has 4 TF sites3. 1/4000 per site4. 3,000,000 clones in library5. Each mRNA is represented by

ca 10 clones6. 200 unique cDNAs per site7. The signal transduction network

is highly overlapping.8. Few if any components will

be unique to a gene , class of genes or agonist

9. Specifity is likely to be a higher order property - ie “words” rather than letters.

10. What is the word length ?

Conclusions/Speculations

1. Signal Transduction networks are likely binary2. Data suggest ca 200 elements of network “control”

a given transcription factor in the IL-8 promoter3. Since each element is in turn coded by a gene ----4. Many elements must be used generically 5. The notion that there are dedicated/specfic pathways

and pathway components cannot be correct6. Programs that are specific may use generic elements

by combining them into longer “words”.