The Tissue Engineer s Toolkit

Transcription

1 The Tissue Engineer s Toolkit Stimuli Detection and Response Ken Webb, Ph. D. Assistant Professor Dept. of Bioengineering Clemson University

2 Environmental Stimulus-Cellular Response

3 Environmental Stimuli Soluble signaling molecules Hormones Growth factors Adhesion molecules Cell-cell Cell-matrix Material Structure / Mechanical stimulation

4 Six Steps in Cell Signaling (Soluble Factors) Signaling cell 1. Synthesis 2. Release 3. Transport Target cell 4. Binding 5. Signaling 6. Desensitization Enzyme Activity Gene Expression

5 Specificity of Signaling

6 Endocrine Signaling Hormones

7 Paracrine Signaling Neurotransmitters Growth factors

8 Autocrine Signaling Growth factors

9 Receptors Intracellular Ion-channel Surface G protein-linked Receptor with guanylyl cyclases Receptor with tyrosine kinases Enzyme-linked Receptor with serine/threonine kinases Receptor with tyrosine phosphatases Receptor w/o enzyme activity

10 Surface versus Intracellular Receptors

11 Intracellular Receptors Lipophilic-diffuse across plasma membrane and bind cytosolic receptors Cortisol Progesterone Testosterone Thyroxine Retinoic acid

12 Molecules with Intracellular Receptors Low molecular weight, hydrophobic

13 Gene Regulation by Intracellular Receptors

14 Early / Delayed Signal Responses Immediate / early genes Transcription factors

15 Gated Ion Channels

16 (Surface)Trans-membrane Receptors Extracellularligand binding domain Plasma membrane outside inside Ligand Transmembrane domain Intracellular domain Intrinsic enzymatic activity / Binding sites for adapters / enzymes

17 Receptor Characteristics Specificity in general, receptors recognize a single ligand and exhibit minimal non-specific binding High affinity receptors can effectively bind ligands present at very low concentrations (nm pm) Saturability Receptor number is limited and therefore only a finite amount of ligand can be bound. Reversibility ligand-receptor binding is non-covalent and reversible as ligand concentration decreases Coupling receptor intracellular domains either contain intrinsic enzymatic activity or binding sites for enzymes such that extracellular binding results triggers intracellular biochemical reactions

18 Signal Transduction Binding Change in conformation Change in enzymatic activity Amplification Integration Response Down-regulation Key Concept: Multi-step pathway creates multiple opportunities for regulation, integration, inhibition

19 Conserved Mechanisms in Signal Transduction Second messengers GTPase switch proteins Protein kinases / phosphatases Adapter / scaffold proteins

20 Second Messengers camp-cyclic AMP cgmp-cyclic GMP DAG-1,2-diacylglycerol IP 3 -inositol 1,4,5- triphosphate Ca ++ Primarily activated through G-protein receptors in response to hormones However, the activity of these signaling molecules can influence signal transduction by other mechanisms

21 GTPase Switch Proteins GTP-Guanosine triphosphate GDP-Guanosine diphosphate

22 Regulation of GTPase Proteins GAP-GTPase activating proteinsincrease rate of GTP hydrolysis and therefore inactivate Ras GEF-guanine nucleotide exchange factors-promote GDP release and binding of GTP, leading to activation of Ras GDI=guanine nucleotide dissociation inhibitor decreases GTPase activation by blocking interaction with GEF

23 Protein Kinases Contain a highly conserved kinase (enzyme) domain Kinase-transfers a phosphate group from nucleotide triphosphate (usually ATP) to specific amino acid residue containing free hydroxyl group Exhibit high substrate specificity Two types: Tyrosine Kinase Serine/Threonine Kinase

24 Tyrosine Kinases Tyrosine kinases include many growth factor receptors :Platelet-derived growth factor receptor(pdgfr) Epidermal growth factor receptor(egfr) Insulin receptor Insulin-like growth factor 1 receptor(igf1r) Stem cell factor (SCF) receptor

25 Serine / Threonine Kinases

26 Protein Kinases / Phosphatases

27 Phosphorylation Most common mechanism for regulation of protein function Phosphorylation alters the structural conformation of a protein, causing it to become activated, deactivated, or modifying its function Reversible reaction suitable for regulatory control Abnormalities common to many diseases Therapeutic target

28 Regulatory Effects of Phosphorylation Holmberg et al. (2002) Trends Biochem Sci 27:619-27

29 Multiple Phosphorylation Sites Allow fine-tuning of kinase / transcription factor activation and bioactivity Holmberg et al. (2002) Trends Biochem Sci 27:619-27

30 Kinase Expression is Tissue Specific Expression of src-family kinases Src Ubiquitous Fyn Ubiquitous Yes Ubiquitous Lyn Hck Fgr Blk Lck Brain, B-cells, myeloid cells Myeloid cells Myeloid cells, B-cells B-cells T-cells, NK cells, brain

31 Adapter Proteins

32 Tyrosine Kinase Domains SH=src homology-regions that mediate protein-protein binding Facilitate kinase binding and accelerate signaling reactions

33 Signaling Scaffolds

34 Signaling Scaffolds

35 Signal Amplification

36 Second Messengers versus Enzymes Second messengers are very low molecular weight, diffuse rapidly and can act at multiple sites within the cell Kinases act more locally, commonly physically immobilized to plasma membrane, signaling scaffolds, or the cytoskeleton

37 GTPases versus Kinases GTPase activity is regulated by GTP / GDP binding, hydrolysis of GTP leads to inactivation Kinases catalyze ATP hydrolysis and the addition of released phosphate groups to other proteins / enzymes

38 G Protein-coupled Receptors

39 Tyrosine Kinase-linked Receptors

40 G-Protein Coupled Receptors (GPCR)

41 GPCR Ligands Hormones: Glucagon, Leuteinizing hormone, and Thyroid stimulating hormone Neurotransmitters: Epinephrine and norepinephrine Local mediators: Bradykinin, and Vasopressin

42 G Protein Signal Transduction Receptor binding G Protein activation Enzyme activation Second Messenger generation Target enzymes Transcription factors

43 Receptor Activation of Adenylyl Cyclase Activated receptor functions as a guanine nucleotide exchange factor (GEF) activating associated G protein

44 Adenylyl Cylase

45 Generation of camp

46 Activation of Protein Kinase A by camp *PKA : camp-dependent protein kinases, Serine/Threonine kinase *Activated PKA: phospholylates specific substrate protein molecules => Cellular response: rapid response: glycogen metabolism (~ seconds) slow response : gene transcription (~hours)

47 Regulation of blood glucose level

48 PKA Regulates Glycogen Metabolism PKA increases glucose level in blood by i) breaking down glycogen to glucose by activating phosphorlyase ii) Inhibiting glycogen synthesis by inactivating glycogen synthetase

49 PKA Regulates Gene Expression CRE:Cyclic AMP Response Element CREB: CRE-binding protein CBP:CREB-binding protein transcriptional coactivator

50 Amplification of GPCR Signaling

51 Enzymatic Regulation of Inositol Lipids

52 Activation of Inositol Lipid Signaling PI = phosphatidyl inositol

53 Ca-calmodulin dependent kinases Myosin light chain kinase

54 Receptor Tyrosine Kinases

55 RTK Activation Receptor Dimerization

56 RTK Activation PDGF: a dimer => directly cross-link two receptors together FGF: monomer => binds in clusters in proteoglycans => cross-link two receptors and phosphorylate each other Ephrins: a membrane bound protein in a signaling cell => Ephrins cluster in the plasma membrane, so cross-link their receptor

57 RTK to Ras Signaling Ras contains a covalently attached lipid group that serves to anchor the protein to the cytoplasmic face of the plasma membrane Two cytosolic proteins: GRB2 :functions as an adaptor protein Sos: functions as guanine nucleotide excahnge protein (GEF) Convert inactive GDP- bound Ras to active GTP-bound Ras

58 RTK Activation of Ras SOS

59 Activation of MAPK cascade by RTK pathway Raf: MAP-kinase-kinase -kinase MEK: MAP-kinasekinase MAPK-MAP kinase Mitogen activated protein kinase aka ERK-extracellularregulated kinase

60 MAPK Cascade EGFR FGFR PDGFR Trk A/B MAPK=mitogen-activated protein kinase

61 MAPK Stimulates Cell Division Phases of Eukaryotic Cell Cycle

62 Checkpoints in the Cell Cycle

63 Cyclin-cdk Complexes Regulate Cell Cycle Cdk 4 and cdk6 are both MAPK/ERK targets cdk-cyclin-dependent kinase

64 Activation of Cyclin / cdk Complex

65 Mitogen Stimulated Cell Cycle Progression

66 Cyclin-cdk Activity is Regulated by Inhibitors

67 Many RTKs Promote Cell Survival RTK Signaling Blocks Apoptosis Apoptosis-programmed cell death Cytoskeletal collapse Proteolysis of cytoplasmic proteins Disassembly of nuclear envelope DNA fragmentation

68 Apoptosis by caspases Initiator Capases Effector Caspases * Caspases: A family of proteases responsible for apoptosis Synthesized as inactive precusors, procaspases Procaspases are activated by other active caspases and cellular stresses

69 Two Mechanisms of Apoptosis Extrinsic Pathway-Procaspases activtion triggered from outside the cells Intrinsic Pathway- Procaspases activation triggered from within the cells

70 Apoptosis-Extrinsic Pathway

71 Apoptosis-Intrinsic Pathway

72 Phosphatidyl inositol-3-kinase (PI-3-K) Catalyzes phosphate addition from ATP to PIP 2, generating PIP 3 Contains 2 SH2 domains for binding and activation through RTKs Critical role in promotion of cell survival through activation of protein kinase B (PKB) aka Akt

73 Phosphatidyl Inositol-3-Kinase

74 PI -3-Kinase Signaling Prevents Apoptosis :Bcl2 PDK1: Phosphatidyl inositol -dependent protein kinase PKB: Protein kinase B (Akt) BAD(apoptosis promoter ): hold death- inhibitory protein such as bcl2

75 BAD and Bcl2 BAD-pro-apoptotic, if free and activated, binds to mitochondrial membrane and increases permeability-cytochrome c release, intrinsic pathway of apoptosis Bcl2-anti-apoptotic, reduces mitochondrial membrane permeability

76 Signaling Down-regulation Pathway regulation converging inhibitory pathways sequestration of intermediaries Receptor modulation internalization degradation inactivation Inactivation-hydrolysis of cyclic nucleotides / phosphatases Intrinsic short half-life of second messengers

77 Inhibition of GPCR Signaling

78 camp Regulation Activated by Ca-calmodulin

79 Inhibition of GPCR Signaling Activity

80 Down-regulation of Inositol Lipid Signaling 1. IP 3 rapidly dephosphorylated by phosphatases. 2. DAG rapidly hydrolyzed. 3. Ca 2+ rapidly pumped out. 4. Ser/Thr phosphatases dephosphorylate PKC and CaM kinase targets.

81 Negative Control of Signaling

82 Negative Control of Signaling

83 Mechanisms Regulating Signal Specificity Cell-type specific receptor expression levels Variation in cell-type specific kinase / phosphatase expression Variation in adapter proteins Cross-talk with other signaling mechanisms-adhesion / force