Advanced Bioinformatics Lecture 5: Signal transduction and simulation ZHU FENG Innovative Drug Research Centre in CQU 创新药物研究与生物信息学实验室

1.Components in signal transduction 2.Growth factor and receptor 3.RTK signal transduction 4.Constructing a pathway model 5.Signaling oncogene & therapeutics Table of Content 2

Signal Transduction is the process by which a cell converts an extracellular signal into a response. Involved in: (1) Cell-cell communication; (2) Cell’s response to environment; (3) Intracellular homeostatsis – internal communication. What is signal transduction? 3

4 Generic Signal Transduction Signal Sensor Transduction cascade Metabolism Migration Survival …

External message to the cell  Peptides / Proteins - Growth Factors  Amino acid derivatives - epinephrine, histamine  Other small biomolecules - ATP  Steroids, prostaglandins  Gases - Nitric Oxide (NO)  Photons  Damaged DNA  Odorants, tastants Signal = LIGAND: (a molecule that binds to a specific site on another molecule, usually a protein, ie. receptor) What is the signal? 5

Sensors, what the signal/ligand binds to initiate ST Hydrophillic Ligand Cell-Surface Receptor Plasma membrane Hydrophobic Ligand Carrier Protein Intracellular Receptor Nucleus Alberts et al. Adapted from Molecular Biology of the Cell, 4th edition, What is the receptor?

1)Ligand-gated ion channel Cell surface receptor types 7 (1) ACh binding; (2) Gate open; (3) Ion pass & membrane depolarization; (4) ACh reduces in concentration; (5) Gate close and desensitization.

Cell surface receptor types 8 (1) Ligand-binding; (2) Receptor conformation change; (3) G protein binding and activation (GTP to GDP); (4) Activated G-protein binds to effectors and trigger further cellular response. 2)G-protein coupled receptor (GPCR) Effectors Cellular responses

Cell surface receptor types 9 (1) Signal binds to activated receptor dimer; (2) Adaptors with SH2 domain bind specifically to each phosphorylated tyrosine (PT) on receptor dimer; (3) Unique cellular response for each Adaptor-PT. 3)Receptor tyrosine kinase

Ligands binding enzyme linked receptors; signaling diverse cellular responses including:  Proliferation  Differentiation  Growth  Survival  Angiogenesis Capable of sending signal to multiple cell types or be specific Growth factor & receptor 10

Growth factor FactorPrincipal SourcePrimary ActivityComments PDGF Platelets, endothelial cells, placenta Promotes proliferation of connective tissue, glial and smooth muscle cells Two different protein chains form 3 distinct dimer: AA, AB and BB EGF Submaxillary gland, Brunners gland Promotes proliferation of mesenchymal, glial and epithelial cells Not clear TGF-  Common in transformed cells May be important for normal wound healingRelated to EGF FGF Wide range of cells; protein is associated with the ECM Promotes proliferation of many cells; some stem cells inhibition; early embryos development At least 19 family members, 4 distinct receptors NGFNot clearPromotes neural cell growth and survival Related proto-oncogenes; trkA (trackA), trkB, trkC ErythropoietinKidneyPromotes proliferation, differentiation of erythrocytesNot clear TGF-  Activated TH 1 cells (T-helper) and natural killer (NK) cells Anti-inflammatory (suppresses cytokine production and class II MHC expression), promotes wound healing, inhibits macrophage and lymphocyte proliferation At least 100 different family members IGF-IPrimarily liverPromotes proliferation of many cell types Related to IGF-II and proinsulin, also called Somatomedin C IGF-IIVariety of cells Promotes proliferation of many cell types primarily of fetal origin Related to IGF-I and proinsulin 11

12 Growth factor receptors

Most growth factors bind to RTK 13

14 RTK signal transduction Dimerization & activation of EGFR SH2 domains

15 Signaling pathways activated by RTKs MAPK Apoptosis & metabolic processes Multiple cytoplasmic targets

16 Signal activation by RTKs via membrane translocation Activation of PKB (Akt): (1) PIP3 serves as a binding site for PH domains of PDK1 and PKB leading to activation of PDK1 and PKB activities; (2) Full activation of PKB requires phosphorylation by PDK1. (3) Activated PKB prevents apoptosis and regulates various metabolic processes.

17 Signal activation by RTKs via conformation changes Binding of the SH2 domains of p85, the regulatory subunit of PI-3 kinase to pTyr sites on activated receptors releases an autoinhibitory constraint that stimulates the catalytic domain (p110). PI-3 kinase catalyzes the phosphorylation of the 39 positions of the inositol ring of PtdIns(4)P and PtdIns(4,5)P2 to generate PtdIns(3,4)P2 and PtdIns(3,4,5)P3, respectively.

18 Signal activation by RTKs via tyrosine phosphorylation Binding of the SH2 domains of PLC gamma to pTyr sites in activated receptors facilitates tyrosine phosphorylation of PLC gamma as well as membrane translocation. Tyrosine phosphorylation is essential for PLC gamma activation leading to hydrolysis of PtdIns(4,5)P2 and generation of the two second messengers Ins(1,4,5)P3 and diacyglycosol.

19 Mechanisms for amplifying RTK activation (1) FGF receptor substrate-2 (FRS2α) forms a complex with activated FGF or NGF receptors via its phosphotyrosine-binding domain (PTB). (2) FGFR phosphorylates FRS2α on multiple tyrosines, and the resulting pTyr recruit Grb2 and Shp2, which bring Gab1 into the complex. (3) Gab1 is tyrosine phosphorylated and recruits PI-3K. (4) PI-3K initiates a positive feedback loop by generating PIP3, then recruiting more Gab1 and leading to further PI-3K activation. FRS2α Gab1

20 Mechanisms for attenuating & terminating RTK activation (1) EGFR’s activity is attenuated by PKC at the membrane region and by PTP. (2) Mechanism for signal termination is via receptor endocytosis and degradation. (3) The RING finger domain of Cbl functions as a ubiquitin- ligase leading to receptor ubiquitination and degradation by proteosome.

21 Protein domains controlling RTK signaling (1) SH2 and PTB bind to tyrosine phosphorylated sites in activated RTKs (2) PH binds to phosphoinositides (PIPs) leading to membrane association (3) SH3 and WW bind to proline-rich sequences in target proteins (4) PDZ binds to hydrophobic residues at the C-termini of target proteins (5) FYVE specifically binds to Pdtlns(3)P leading to membrane association Activated RTKs Grb2– SH3 – SH2 – SH3 – c-Src– SH3 – SH2 – Kinase – PI3-K– SH3 ––––– SH2 –– SH2 – PLC-γ–– PH –––– PLC –––– p – SH2 –– SH2 –– SH3 – H –– PLC –– SHP-2– SH2 –– SH2 –––– PTPase ––––

22 Multiple domains of PLCγ cooperate to integrate multiple signals (1) SH2 forms complex of RTKs. (2) C2 and PH are in charge of PLCγ’s membrane association. (3) RTK-mediated tyrosine phosphorylation of PLCγ leads to intramolecular binding of the C-terminal SH2 domain to phosphotyrosine 783, stimulating enzymatic activity of PLCγ and then leading to hydrolysis of PIP2, and consequently leads to the formation of IP3 and DAG.

23 Activate MAPK pathway by RTKs Apoptosis Growth, differentiation, inflammation Shape, migration Tumor genesis

24 Constructing a pathway model Dynamic nature of biological networks More than a topological linkage of molecular networks. Pathway models can be based on network characteristics including those of invariant features.

25 Constructing a pathway model Dynamic nature of biological networks Abstraction & Resolution  How much do we get into details?  What building blocks do we use to describe the network?

26 Constructing a pathway model Step I – Definitions A simple imaginary metabolic network represented as a directed graph Vertex (A, B, C): concentration of protein/substrate. Edge (b1, b2, b3): - flux (conversion mediated by proteins of one substrate into the other) Internal flux edge External flux edge How do we define a biologically significant system boundary?

27 Constructing a pathway model Step II – Interaction kinetics A simple imaginary metabolic network represented as a directed graph Enzyme + Substrate Kinase-ATP complex + inactive-enzyme ==> Kinase + ADP + active enzyme K ATPADP P

28 Reversibility & equilibrium of chemical reactions  Chemical reactions are reversible  Under certain conditions (concentration, temperature) both reactants and products exist together in equilibrium state H 2  2H

29 Reaction rates Net reaction rate = forward rate (consumption) – reverse rate (production)  In equilibrium: Net reaction rate = 0  When reactants “just” brought together: Far from equilibrium, focus only on forward rate  But, same arguments apply to the reverse rate

30 The differential rate law How does the rate of the reaction depend on the concentration? e.g. 3A + 2B  C + D rate = k [A] m [B] n (Specific reaction) rate constant Order of reaction with respect to A Order of reaction with respect to B m+n: Overall order of the reaction Stoichiometric coefficient

31 Rate constants and reaction orders  Each reaction is characterized by its own rate constant, depending on the nature of the reactants and the temperature  In general, the order with respect to each reagent must be found experimentally (not necessarily equal to stoichiometric coefficient)

32 Elementary processes and rate laws  Reaction mechanism: The collection of elementary processes by which an overall reaction occurs  The order of an elementary process is predictable Unimolecular A*  B K + [A] First order Bimolecular A + B  C + D K + [A] [B] Second order Trimolecular A + B + C  D + E K + [A] [B] [C] Third order

33 Elementary processes and rate laws  Reaction mechanism: The collection of elementary processes by which an overall reaction occurs  The order of an elementary process is predictable Unimolecular A*  B K + [A] – K – [B] First order Bimolecular A + B  C + D K + [A] [B] – K – [C] [D] Second order Trimolecular A + B + C  D + E K + [A] [B] [C] – K – [D] [E] Third order

34 Constructing a pathway model Step III – Dynamic mass balance Stoichiometry Matrix Flux vector Concentration vector

A ‘simple’ ODE model of yeast glycolysis 35

Example and its time-dependent behavior Two genes and their products 36 Positive feedback loop g1g2 P1P2 k2k2 k1k1

Example and its time-dependent behavior Two proteins react and have negative feedback on g1 transcription 37 g1g2 P1P2 k1k1 k2k2 P1P2 k3k3

Example and its time-dependent behavior The reaction product has also a positive feedback on g2 transcription 38 g1g2 P1P2 k1k1 k2k2 P1P2 k3k3 k4k4

Growth signal autonomy, insensitivity to anti- growth signals, resistance to apoptosis, uncouple cell’s growth program from signals in the environment. Growth factors in normal cells serve as environmental signals. Growth factors regulate growth, proliferation, and survival. These are all deregulated in cancer. Hanahan and Weinberg, Hallmarks of Cancer, Cell (100) 57, Growth factor ST and cancer 39

EGFR, kinase activity stimulated by EGF-1 and TGF-a involved in cell growth and differentiation, was linked via sequence homology to a known avian erythroblastosis virus onocgene, v- erbB. Since then, many oncogenes have been shown to encode for GFRs. GF receptors with oncogenic potential 40 EGFR familyInsulin Receptor family erbB1 (c-erbB)IGF-1 (c-ros) erbB2 (neu) Neurotrophins FGF FamilyNGFR (trk) FGFR-1(fig)BDNFR (trk-B) FGFR-2(K-sam)NT3 R (trk-C) PDGFR Family CSF-1R (c-fms) SLF R (c-kit)

PDGF, originally shown to regulate proliferation, was also shown to have homology to v-sis, the simian sarcoma virus. Other viral oncogenes encoded protein products that were growth factors that often over-expressed in cancer such as TGF- a. Autocrine signalling leads to deregulated growth. GF receptors with oncogenic potential 41 PDGF familyNeurotrophins A chainNGF B chain (c-sis)BDNF FGF FamilyNT3 acidic FGFCytokines (Hematopoietic) basic FGFIL-2 EGF Family IL-3 EGFM-CSF TGF-aGM-CSF

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Function of (mutated) Ras in cancer Cancer 46

47 Ras recruits Raf to the membrane

ST intermediates can be targets for anti-cancer drugs 48

Projects Q&A! 1.Biological pathway simulation 2. Computer-aided anti-cancer drug design 3. Disease-causing mutation on drug target 49 Any questions? Thank you!