7 regulation of metabolism

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7 regulation of metabolism

  1. 1. Mechanism of hormone and neurotransmitter actionTypes of membrane and and intracelular receptors Department of Biochemistry FM MU 2011 (E.T.)
  2. 2. Control of metabolism – Regulation of metabolic events within particular compartment (cellular organelle) that depends only on interactions between molecules in the compartment; – regulations that occur within complete cells without any regard to extracellular signals, in which proteosynthesis and transport across membranes that separate individual compartments have the important roles have; – regulations that are consequences of communication between cells in particular tissues, organs, or the whole organism, depending on extracellular signals – neurotransmitters, hormones, cytokines, and other signal molecules.Numerous metabolic pathways are controlled usually in only one orfew check-points (rate-limiting steps) by more than one differentmechanisms. These formal levels of metabolism control mostly overlap. 2
  3. 3. Mechanism of hormone and neurotransmitter action Signal molecule types in neurohumoral regulations: Signal molecule OrigineHORMONES secreted by endocrine glands, by dispersed glandular cells (eicosanoids by many other cellular types)NEUROHORMONES secreted by neurons into the blood circulationNEUROTRANSMITERS secreted by neurons at nerve endingsCYTOKINES, GROWTH secreted by various types of cellsFACTORS, IKOSANOIDS 3
  4. 4. Action of signal moleculesAction Character of actionendocrine Signal is carried by the blood, may act in the whole body. Typically hormonesparacrine Signals act within short distances of the site of their productionautocrine Signal act on the cells that produce them 4
  5. 5. Signal transductionHow cells receive, process, and respond to information from the environment? Reaction of signal molecule with receptor Membrane receptors Intracelular receptorsProteins and small polar Nonpolar signal moleculessignal molecules (amino (steroids, iodothyronines,acids, peptides, biogenic retinoates)amines, eicosanoids) 5
  6. 6. Membrane and intracellular receptors Nonpolar signal molecule Polar signal molecule bound to the plasma transport protein Transport of signal molecule Membrane receptor Signal transduction Intracellular receptorAmplification Interaction of the complex hormone-receptor with the HRE of nuclear DNA Biological response (prompt effect) Biological response (the effect is slow, either early or late) 6
  7. 7. Main types of membrane receptorsReceptors – ion-channels (ROC, ligand gated ionophores) serve exclusively as receptors for neurotransmitters (see lecture 7).Receptors activating G-proteins (heterotrimeric G-proteins), the result of specific ligand binding is mostly - stimulation or inhibition of adenylate cyclase, - stimulation of phospholipase C, - stimulation of phosphodiesterase.Receptors exhibiting intrinsic catalytic activity - guanylate cyclase activity – receptors for natriuretic peptides, - tyrosine kinase activity - insulin receptor, receptors for insulin-li growth factors (IGF1,2), - dimerizing receptor for epidermal growth factor (EGF).Receptors cooperating with non-receptor tyrosine kinases(e.g., Janus kinase JAK) – receptors for somatotropin (growthhormone), prolactin, erythropoietin, interferons, interleukins and othercytokines. 7
  8. 8. Family of heterotrimeric G-protein-coupled receptors Common structural features : Binding site for the agonist (also accessory bindingAll of them are seven -helical segments sites for antagonists)that span the membrane and are connectedby intra- and extracellular hydrophilic and H2Nmore divergent loops. Intracellular domains - binding site for the specific -COOH G-protein type. 8
  9. 9. Heterotrimeric G-proteinsProteins binding GDP or GTPmostly freely membrane-bound (they can move along the innersurface of the plasma membrane).Subunits ,  a . Subunits G and G are hydrophobic and non specific G subunit is the largest, hydrophilic, it binds GTP or GDP, and It is specific for particularMore than 20 different  subunits have mechanism of second messenger production.been identified. 9
  10. 10. The cycle of G-proteins activation Complex receptor-specific ligand GTP Complex receptor-ligand- -trimer G-GDP,G,G GDP Trimer G-GDP,G,G Dimer G,GDimer G,G Inactive Activated subunit G-GDP subunit G-GTP Interaction with the target protein Pi PRODUCTION OF THE SECOND MESSENGER 10
  11. 11. Selected types of G protein -subunits G subunit type Examples of Effect of activated G activating receptors on the target protein Gs (stimulatory) glucagon stimulation of parathyrin adenylate cyclase -adrenergicGi (inhibitory) somatostatin inhibition of 2-adrenergic adenylate cyclaseGq (activating the PI vazopressin V1 stimulation ofcascade) endotelin ETA,B phospholipase C acetylcholine M1 1-adrenergicGt (inhibitory) rhodopsine stimulation of(for transducin) cGMP phosphodiesterase 11
  12. 12. Hormone receptors that activate Gs or Gi proteins stimulates or inhibit adenylate cyclase Adenylate cyclase, a membrane-bound enzyme, catalyzes the reaction ATP  cAMP + PPi ; the second messenger is cyclic AMP. ligand ligand receptor AMP-cyclase receptor GS Gi   ATP cAMP *phosphodiesterase AMP H2O proteinkinase A inactive (R2C2) phosphorylations active proteinkinase A 2 C + 2 R(cAMP)2 12*Inhibition by caffein, theofyllin
  13. 13. They target cAMP action in the cellsactivity tophysiological AKAPsubstrates AKAP Protein cAMP ATP C R R C ADP C R R Protein-P C Protein Proteinkinase Proteinkinase ATP A (inactive) A (active) ADP Protein-P Phosphorylation of proteins. In cytoplasma - mostly metabolic enzymes (rapid response) In the nucleus – phosphorylation of gene specific transcription factor CREB (cAMP response element-binding protein) (slower response) 13
  14. 14. Localization of cAMP action in specific site of the cellProteins AKAPs (A kinase anchoring proteins)Proteins binding proteinkinase A, they target its activity to physiologicalsubstrates (they serve as a scaffold which localize PAK near tosubstrate).Similar proteins affects also the specific action of phosphatases,phosphodiesterases. 14
  15. 15. http://images.google.cz/imgres?imgurl=http://www.nature.com/nrm/journal/v5/n12/images/nrm1527-f2.jpg&imgrefurl=http://www.nature.com/nrm/journal/v5/n12/fig_tab/nrm1527_F2.html&usg=__Hng6YsRDaJmEJsJrqp2gtuYBJsc=&h=444&w=600&sz=67&hl=cs&start=2&um=1&itbs=1&tbnid=3SbJpzufhbKrrM:&tbnh=100&tbnw=135&prev=/images%3Fq%3Dakap%26um%3D1%26hl%3Dcs%26lr%3D%26sa%3DN%26tbs%3 15Disch:1
  16. 16. Receptors that activate Gq protein stimulatephospholipase C and start the phosphatidylinositol cascade Phospholipase C diacylglycerol Both reaction products are the second inositol 1,4,5- messengers: trisphosphate Inositol 1,4,5-trisphosphate opens the Ca2+ channel in ER membrane, 16 diacylglycerol activates proteinkinase C.
  17. 17. Phosphatidylinositol cascade specific ligand activation of proteinkinase C receptor phospholipase C PIP2 DG Gq  IP3 active proteinkinase C increase of [Ca2+] in cytoplasm phosphorylationsEndoplasmic reticulum Ca2+ IP3 receptors in the membranes of ER act as ligand gated channels for Ca2+ ions 17
  18. 18. Control of metabolism by changes of cytoplasmic concentration of Ca2+•Basal concentration of Ca2+ in cytoplasma  1.10-7 mol/l• Increase to concentration to  1.10-6 rapidly and maximallyagtivates the various Ca2+-regulated cell function• Increse of Ca2+ can be triggered by influx of Ca2+ across the plasmatic membrane (see e.g.smooth muscle contraction) or by release from intracelular stores (ER, mitochondrias) e.g. IP3 gated Ca 2+ channel in ER, or ryanodine receptors in ER/SR of cardiac or skeletal muscle 18
  19. 19. Regulatory protein calmodulineIncreased cytosolic Ca2+ activates various calcium-bindingregultory proteins (family of small, Ca 2+ dependent protein).The most important is calmoduline. It is ubiquitouslyexpressed protein in nearly all cells. Upon binding of Ca2+ (4 binding sites) calmoduline undergoes conformational changes that facilitates its interaction with downstream signaling proteins, e.g. kinases, phosphatases ect. Some Ca-calmodulin-dependent kinases are very specific, the other have a broad substrate specifity. 19
  20. 20. Receptors with guanylate cyclase activityAfter binding of ligand they convert GTP to cGMPcGMP is the second messenger OIt activates proteinkinase G N HNTwo types of receptors: H2N N N•membrane-associated• soluble (cytoplasmic) O O O P O OH OH 20
  21. 21. Membrane receptors with guanylate cyclase activity ANP Receptors for ANP (atrial natriuretic factor) Mainly smooth muscle of vessels and in kidneys ANP is produced GTP cGMP + PPi by cardac atrialphosphodiesterase Protein kinase G tissue in response H2O inactive to increase of GMP blood volume or pressure active protein kinase G (PKG) Phosphorylation of proteins 21
  22. 22. Soluble receptors with guanylate cyclase activity NO NH2 NH2 Receptor je dimeric complex and hem binds hem Binding NO to the hem increases catalytic acitivity guanylate cyclase NO is generated by the action of GTP cGMP nitroxid synthase (NOS)phosphodiesterase NO readily permeates membranes, it can be produced by one type of the cell Activation of and rapidly diffuse into neighboring cell protein kinase G types GMP 22
  23. 23. Proteinkinase GcGMP sensitive proteinkinase GWidely expressed in many cellsIt phosphorylates various proteins (enzymes, transportionproteins ect.) Effect of PKG in smooth muscle Phosphorylation of proteins: • inactivation of proteins attenuating Ca2+ release from ER   Ca2+ • activation of MLC phosphatase  repression of actin-myosin interaction • decrease of K+-channnels activity  decrease of hyperpolarization  increased influx of Ca2+ into the cell 23 Relaxation of smooth muscle
  24. 24. NO/cGMP signaling in smooth muscle of blood vesselscGMP is the key second messenger for induction of vascularsmooth muscle vessels relaxation  vasodilatation and increased bloof flow NO is produced in endothelial cells by the action of nitroxid synthase from arginin (activation e.g. by acetylcholine) and diffuses into adjacent smooth muscle cells NO-synthase L-Arg ·NO + L-citrullin 24
  25. 25. Nitroglycerin and other drugs of organic nitrate type are donors of exogenous NO R-O-NO2 nitrit ·NOGlycerol trinitrateIsosorbide nitrate Activation of soluble guanylate cyclase Therapy of angina pectoris Vasodilatatory effect releases coronary spasmus and normalizes blood perfusion. 25
  26. 26. Inhibition of phosphodiesterase potentiates the effect of NO Several types of phosphodiesterase phosphodiesterase cGMP GMP are known, depending of the type of cell. H2OThe drug sildenafil (Viagra) acts as a selective inhibitor of phosphodiesterase 5(PDE5), that is higly expressed in vascular smooth muscle.Viagra is 80-4000 fold less potent as an inhibitor of other PDE isoforms(including PDE3 that is expessed in heart).During sexual stimulation NO in corpora cavernosa is releasedThe level of cGMP is increased sildenafil prevents decomposition of cGMP 26blood vessel in erectile tissue are dilated and blood flow is increased
  27. 27. Receptors with tyrosin kinase activityCommon features• when the signal molecule binds to the receptor, it triggers conformationalchange of the receptor• this leads to activation of tyrosin kinase activity of the receptor• the first protein substrate is the receptor itself (autophosphorylation of tyrosinemolecules in the receptor), eventually other proteins are phosphorylated• phosphorylated tyrosines and other substrates then acts as a recognition oranchoring site for other proteins, adaptor molecules• adaptor proteins bind to phosphotyrosine residues by SH2 domaines (Srchomology 2 domain) and are also phosphorylated.• adaptor proteins reacts with other molecules and signal is transmitted throughcascade of other reactions mainly phosphorylation/dephosphorylation, exchangeof quanine nucleotides, changes of conformation etc. 27
  28. 28. Insuline receptor Dimeric structure Binding site for insulin on - subunits   -S-S- -S-S- Tyrosin kinase activity on -subunits -S-S-  Binding of insuline to the receptor  tyrosin kinase activityautophosphorylation of -subunits and phosphorylation of IRS 1-4 proteins (insulin receptor substrates 1-4) Insulin   -S-S- -S-S- -S-S-   P- -P P- -P activation and docking of IRS1-4 -P PI-3-kinase on membrane IRS1-4 activation of phosphoprotein phosphatase-1 activation of small G-protein Ras 28
  29. 29. Some signaling pathways of insuline 29http://www.abcam.com/index.html?pageconfig=resource&rid=10602&pid=7
  30. 30. Insuline receptor substrates 1-4 are adaptor proteins.If phosphorylated by the insuline-receptor complex, they bind to other proteins that are activated in this way. Examples of insulin receptor signaling pathways Glykogen synthesis Translocation of glucoseých transportersPhosphorylation of IRS activates Insulin receptor phosphorylates CbIregulatory subunit PIP2-3 kinase Complex CbI-CAP translocates to theCatalytic subunit of PIP2-3 kinase lipid raft in the membranephosphorylates PIP2 to PIP3 CbI reacts with adaptor adaptor proteinPIP3 activates proteinkinase B (AKT), Crkactivation is enabled by PDK(phosphoinositide dependent kinase) Crk is associated with C3Gactivated AKT difuses to cytoplasma C3G activates TC10 (G-protein)and phophorylates (inactivates) TC10 activates translocation ofglykogen synthase kinase transporters into the plasmaticSynthesis of glycogen is activovated membrane(active form is dephosphorylated formof glycogen synthase) 30
  31. 31. Dimerizing receptor for EGF (epidermal growth factor) containing an intrinsic tyrosine kinase activity Dimerization of the receptor after binding of ligand R R RR P- -P Ras–GTP P- -P SoS Raf phosphorylation phosphorylation cascade MAP Dimerization activates tyrosin kinase activity in cytoplazmatic domain. Autophosphorylation of the receptor Adaptor proteins Grb2 (SH-2 domains) bind to phosphorylated sites. G-protein Ras is activated by the action of SOS protein activation of MAP-kinase cascade (Ras/MAP-cascade) 31
  32. 32. RasMonomeric G-protein that binds GTP and at the same time has GTPaseactivity (structural analog of  subunit intrimeric G-protein).It is activated by binding GTP instead GDP SoSActivation of Ras is key step in transmision of signal.Inactive Ras-GDP is converted to Ras –GTP, that activates thenext molecule of the pathway.Inactivation of Ras: hydrolysis of GTP iniciated by otherregulating proteins 32
  33. 33. Superfamily of Ras proteins•5 families: Ras, Rho, Arf, Rab, Ran•They are anchored to lipid membrane by lipid anchors(myristoyl, farnesyl)•Monomeric G-proteins that play important role inregulation of growing, morphogenesis, cell motility,cytokinesis etc.•Mutations in Ras genes induce patologic proliferation andantiapoptosis. About 30 % ofall human tumors involve cellsexpressing mutated Ras oncogenes*.*(Ras genes are named protooncogenes) 33
  34. 34. MAP-kinase signaling pathway(MAP kinase =Mitogen activated protein kinase) Map-kinase cascade It regulates mainly cell growing and Ras–GTP diferentiation. MAP-kinase-kinase-kinase P MAPKKK, Raf ATP ADP phosphorylation of MAP-kinase-kinase P MEK cytosolic or ATP membrane proteins ADP ADP ATP MAP-kinase P ATP phosphorylation of ERK ADP regulatory proteins in nucleus, stimulation of 34 proliferation
  35. 35. Mitogens – grow factors stimulating proliferationExamples of mitogens:Abbr. name FunctionPDGF Platelets derived growth Mitogen for cell of connective factor tissue and non differentiated neurogliaEGF Epidermal growth factor Mitogen for many cells of ektodermal and mesodermal origineFGF-2 Fibroblast growth factor 2 Mitogen for many cells like fibroblasts, endotelhelial cells, myoblasts;IL-2 Interleukine 2 Mitogen for T-lymphocytes 35
  36. 36. Receptors activating non-receptor tyrosine kinasesJAK-STAT receptors (Janus Kinase – Signal Transducer and Activator of Transcription) ligand Receptors of cytokines dimerization – e.g.interferons, interleukines STAT –P STAT STAT –P STAT –P STAT•Receptor does not have kinase activity, but is associated with tyrosinkinase JAK.•After binding of ligand receptors dimerize (homodimers or heterodimers)•Activated JAKs phosphorylates tyrosine residue of the receptor.•The STAT proteins (signal transducers and activators of transcription) associatewith the receptor and are phosphorylated by JAK.•STAT phosphates dimerize, translocate to the nucleus, bind to specific DNA 36elements and regulate transcription.
  37. 37. Intracellular receptors of steroid hormones(and calcitriols), iodothyronines, and retinoatesReceptors are present in cytoplazma or nucleusHormon-receptor complexes binds to the specific sites inDNA and activate the transcription of specific genesComplex hormon-receptor binds to DNA at HRE (hormonresponse element)Superfamily of steroidal and thyroidal receptors – a family ofstructuraly related proteins 37
  38. 38. Example of cortisolcortisol is transported attached on CBG (corticosteroid-binding protein) in ECFhydrophobic molecule penetrates membrane cortisol CBG active complex receptor-ligand (monomer), hsp 90 and other proteins are released GR inactive receptor for glucocorticoids (GR) forms in the cytoplasm active complexes form dimers and complex with hsp 90 dimer are translocated into the nucleus 38 and other proteins through nuclear pores
  39. 39. Glucocorticoid receptor (GR) – function GRE DNA GR DNA binding domain cortisol binding domain (hydrophobic pocket)Active complex cortisol-receptor binds onto DNA at the specific sequenceGRE (glucocorticoid response element, quite generally HRE – hormoneresponse element) 39
  40. 40. Initiation of transcription by cortisolActive complex cortisol-receptor binds onto DNA at the specific sequenceGRE (glucocorticoid response element, one of the HRE – hormone responseelements).The coactivator and specific hormone response element-binding proteins(GREB-proteins) are also attached. This complex acquires the ability to act asenhancer that supports initiation of transcription on the promoter by means ofmediator proteins. cortisol-GR dimer complex GRE enhancer GREB protein coactivator mediator proteins > 1 000 bp CTD Pol II basal TF IID transcription apparatus promoter GR dimer – intracellular glucocorticoid receptor (dimer) GRE – glucocorticoid response element GREB protein – GRE binding protein (a specific transcription factor) 40

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