北大生化chapter12课件

Chapter 12 Biosignaling,the concept of biosignaling features of biosignaling the four major pathways regulation of pathways,Lectured by Dr. Qin Yongmei (秦咏梅): June 16, 2007,Why do we need biosignaling pathways ?,functional integration of distant organs, tissues and cells requires communication; Signaling is perhaps a primal requirement to respond to our environment; The foundation of any complex response pathway lies with cellular biochemicals.,Cellular response,Growth & Differentiation Growth factors DNA replication,Death apotosis,Degradation oxidation of fatty acids,Release neurotransmitters hormones,Storage glucose glycogen,Requirement of Biosignaling,requires a receptor to detect signals; the receptor must link to or generate an intracellular response; Such linking molecules are known as “second messengers”; This transduction system must meet four specific criteria.,Criterion 1: specificity,High specificity only the target cell is influenced; Receptor binding site ligand (signal molecule) complementary and non-covalent interaction follows the law of mass action,Criterion 2: amplification,often short-lived & low concentration,A single receptor binding event may elicit responses in multiple enzyme,Criterion 3: Desensitization,feedback control,the aim of biosignaling is to produce a rapid and major cellular response to a transient signal.,Criterion 4: Integration,cells frequently receive multiple signals; there are many reciprocal pathways within cells.,Six general types of signal transducers,Gated ion channel,Receptor enzyme,Serpentine receptor (via G-protein),Steroid receptor,Receptor with no intrinsic enzyme activity,Adhesion receptor,Type I: gated-ion channels,a simple signal transduction system mediating neural transmission; Acetylcholine (Ach) diffuses across synaptic cleft of neuromuscular junction to acetylcholine receptor two Ach binding sites exhibit positive co-operativity Entry of Na+, Ca2+ (propagate the initial event): depolarization that affects other channels contraction in muscle Desensitization and channel closure: If the Ach is not rapidly metabolized by acetylcholinesterase, the receptor undergoes a closed conformation with tightly bound to Ach. The slow release of Ach from its binding site eventually allows the receptor to return to its resting state.,Role of voltage-gated and ligand-gated ion channels in neural transmission,axon of presynaptic neuron,Cell body of postsynaptic neuron,Three stages of acetylcholine receptor,Type 2: receptor-enzyme units,(a) tyrosine kinases insulin receptor: prototype for this signaling pathway. the receptor complex: extracellular ligand binding site cytosolic catalytic domain; The enzyme is a tyrosine kinase, which phosphorylate tyrosine residues in specific target proteins.,Physiological role of insulin a. High blood glucose concentration causes increased secretion of insulin and decreased secretion of glucagon; b. Stimulate the glucose transporter, thus the uptake of glucose by muscle and adipose tissue cells; Regulate both fuel metabolism and gene expression: kinase cascade (protein phosphorylation) growth response that include increases in synthesis of DNA and mRNA promoting anabolic processes/inhibiting catabolic ones predominantly in muscle, fat and liver tissue; glycogen (in liver and muscle) triacylglycerols (in adipose tissue),Tyr kinase domain of insulin-receptor,Inactive (unphosphorylated),Active (triply phosphorylated),IRS: insulin receptor substrate protein kinase : Raf-1, MEK, MAPK (phosphorylate Ser or Tyr residue) MAPK (ERK, extracellular regulated kinase): mitogen - activated protein kinase; MEK (MAPKK): mitogen- activated, ERK-activating kinase,Multivalent scaffold proteins and membrane rafts,Many signalling proteins are multivalent and they can interact with several different proteins simultaneously to form multiprotein signalling complexes.,Red: SH2 domain or PTB domain Orange: SH3 domain,Grb2-Sos-Ras-MAPK pathway,PI-3K-PKB pathway,Blue: PH domain (plextrin homology),Some binding modules of signaling proteins,Adaptor,Scaffold,Kinase,Phosphatase,Ras signaling,Transcription,Signal regulation,Phospholipid second- messenger signaling,Protein modules, such as SH2 domain and PTB (phosphotyrosine- binding domain), bind phosphorylated Tyr, Ser or Thr residues in partner proteins.,Activation of glycogen synthase by insulin,Autoinhibited,Active,Autoinhibited,Active,Src Tyr kinase,GSK3: glycogen synthase kinase 3,Dephosphorylation,Membrane rafts and caveolae may segregate signaling proteins,caveolin,GPI linked enzymes,palmitolated,Src-like kinase & trans-membrane receptors,Caveolae associated signalling receptors,Diabetes is a defect in insulin production or action a. Diabetes mellitus is caused by a deficiency in the secretion or action of insulin b.Two major clinical classes of the disease: Type I: insulin-dependent diabetes mellitus (IDDM) Type II: non-insulin-dependent diabetes mellitus (NIDDM) c. Glucosuria: excretion of large amounts of glucose in the urine d. Excessive but incomplete oxidation of FAs in the liver (ketone bodies production acidosis and ketoacidosis) e. glucose-tolerance test,(b) Receptors with no intrinsic enzyme activity (or Type 3 ),JAK: Janus kinase STAT: Signal transducers or activators of transcription,(c) guanylyl cyclases produces cGMP (guanosine 35-cyclic monophosphate ) from GTP, which serve as a second messenger; most cGMP effects are mediated via a cGMP-dependent protein kinase; cGMP levels are restored to normal by a phosphodiesterase that produces GMP.,atrial natriuretic factor,Type 4: receptor/G-protein systems,a complex system: A receptor linked to trimeric GTP binding protein; Binding of the ligand produces a conformational change that causes the G-protein to leave the receptor and “dock” with a membrane bound enzyme. Activity of the enzyme initiates a cascade of events Example: adenylate cyclase phopholipase C,The discovery of G protein,Martin Rodbell found that a transducer provided the link between the hormone receptor (the discriminator) and the amplifier. Alfred G. Gilman used genetic and biochemical techniques to identify and purify the G protein. They used lymphoma cells that normally can be activated by a receptor to form cAMP. A mutated lymphoma cell was found to contain a normal receptor and a normal cyclic AMP-generating enzyme but was yet unable to respond because it lacked the transducer. This was a good system to assay purified G proteins.,normal cell,mutated cell,A G protein could be isolated from normal brain tissue and inserted in the mutated cell, thereby restoring its function.,in resting state,R+L activate G protein,with GTP, G protein is active and separate.,GTP hydrolyzed to GDP, the subunits recombine.,G protein,“G” refers to the fact that protein binds Guanine nucleotides (GDP, GTP) ; G proteins are integral membrane protein, hetertrimers (); G proteins have similar and subunits, but differ in the type of -subunits; When G-protein is activated, the subunit dissociates to interact with an enzymes that generate second messengers (e.g. cAMP).,Various types of G-protein families -subunits,G protein Signal Effected enzyme Effect,Gs epinephrine adenylyl cyclase stimulatory glucagon Gi catecholamines adenylyl cyclase inhibitory Gq acetylcholine phospholipase C stimulatory catecholamines Gt photons cGMP stimulatory phosphodiesterase,An extracellular signal such as epinephrine or PGE1 can have quite different effects on different tissues or cell types. Depend on: The type of receptor; The type of G protein with which the receptor is coupled; The set of PKA target enzymes in the cell.,The Nobel Prize in Physiology and Medicine 1994,Alfred G. Gilman,Martin Rodbell,USA,USA,1941-,1925-1998,“for their discovery of G-proteins and the role of these proteins in signal transduction in cells“,Transduction of the Epinephrine Signal,synthesized in adrenal medulla; belongs to catecholamines; target cells include liver, skeletal muscle, heart muscle and adipose; released in response to acute stress “fight or fight response”,When all subunits are associated together, the G protein is in an inactivate state and in this state the subunit preferentially binds GDP; Once epinephrine or other cAMP-linked hormones bind to the receptor, the hormone-receptor complex interacts with the G protein to bring about its activation; Following interaction of the hormone-receptor complex with the G protein, GTP displaces GDP; Binding of GTP produces a conformational change in the G protein that causes the subunit to dissociate. The subunit then interacts with adenylyl cylcase; The subunit is inactivated when the bound GTP is hydrolyzed to become GDP, which is catalyzed by a GTPase activity that is part of the subunit.,Self-Inactivation of Gs,Cyclic AMP (cAMP),cAMP is synthesized from ATP via adenylate cyclase, and degraded by cyclic AMP phosphodiesterase (inhibited by caffeine); Once cAMP is synthesized, it must ultimately activate or inhibit enzymes involved in fuel metabolism, which is accomplished by the phosphorylation of these regulated enzymes in a reaction catalyzed by PKA.,cAMP acts as a second messenger,Activation of cAMP-dependent protein kinase, PKA,Protein kinase A (PKA) is comprised of R2C2; In the absence of cAMP, R subunits are bound to C subunits and keep the inactivate state; Once cAMP is produced, it binds to R subunits. Binding of cAMP comes the dissociation of active C subunits from the complex; Activation of PKA provides signal amplification by phosphorylation of a variety of proteins; Specific targets of PKA are involved in fat mobilization and carbohydrate metabolism.,A catalytic subunit of PKA,ATP,potent inhibitor peptide (PKI): Arg-Arg-Gln-Ala-Ile (consensus sequence recognized by PKA except Ser is replaced by Ala ),Epinephrine Cascade,Desensitization of the -adrenergic receptor in the continued presence of epinephrine,-adrenergic protein kinase (ARK) -arrestin (arr),prevents interaction between receptor and G protein,b-Arrestin can also initiate a second signaling cascade by acting as scaffold proteins,Phosphoinositide Cascade,IP3,Two second messengers: IP3 and diacylglycerol,IP3 opens channels to release calcium ions from intracellular stores IP3 is able to increase Ca2+ by associating with the IP3 channel or IP3 receptor; At least three molecules of IP3 must bind to sites on the cytosolic side of the membrane protein to open the channel and release Ca2+ (allosteric interaction). Increase Ca2+ activates PKC IP3 is a short-lived messenger (less than a few seconds) 2. Diacylglycerol activates protein kinase C (PKC) PKC phosphorylates Ser or Thr residues of specific target proteins, changing their catalytic activities; Isozymes of PKC: target protein specific and tissue-specific.,Calcium is a second messenger in many signal transductions,Triggering of oscillations in intracellular Ca2+ by extracellular signals,Free Ca2+ can be monitored by using chelators (Fura),norepinephrine,Calcium activates the regulatory protein Calmodulin, which stimulates many enzymes and transporters,Calmodulin (CaM),Ca2+,Calmodulin: - integral subunit of Ca2+/calmodulin-dependent protein kinase (CaM kinase) - regulatory subunit of phosphorylase b kinase of muscle,Regulation of transcription by steroid hormones,Type 5: steroid receptor,Steroid, retinoic acid and thyroid hormones use a signalling process (by-passes the plasma membrane); the steroid hormones reach cells bound to plasma proteins such as albumin; this dependence on albumin for transport is due to their hydrophobicity; the hormones enter cells by passive diffusion along a concentration gradient; the hormones bind to soluble receptors (i.e. not membrane bound) with high affinity/specificity;,the binding energy induces conformational change that result in homo- or heterdimerisation with other receptors; the oligomerized and liganded receptors bind to regulatory regions of DNA known as hormone response elements (HREs); receptor interaction at HREs causes altered rates of gene transcription and subsequently protein levels and cellular effects.,Tamoxifen antagonist of estrogen, to treat hormone-dependent breast cancer,competes with estrogen for binding to the estrogen receptor; tamoxifen-receptor complex has no effect on gene expression.,Regulation of Pathways,External signals,Second messengers,Modulator proteins,Function,Target proteins,Hormones cAMP Transmitters cGMP Drugs Ca2+ Light DAG Odorants IP3 Mitogenic hormones Growth Tyrosine factors kinases,protein kinase And phosphatase,structural proteins And enzymes,metabolic or physiological responses,Plasma membrane,Phosphorylation as a Regulatory Mechanism,Signal transduction,Altered kinase activity,cGMP cAMP Insulin,Ca2+ kinase,There are 120 kinases in yeast i.e. 2% of their entire genome; Several types of kinases are localized to discrete subcellular regions that are in close proximity to the specific target protein;,Many proteins act as targeting or anchoring mediators e.g. AKAP (A-kinase anchoring protein), which associates tightly with the regulatory subunits of PKA; Cells contain many different isoforms of PKC, which are localized by anchoring proteins: e.g. RACKs or receptors for activated C kinase PICKs or proteins that interact with C kinase Phosphatases are also often localized closely to ensure that integrated control is possible.,The Nobel Prize in Physiology and Medicine 1992,Eldwin G. Krebs,Edmond H. Fischer,USA,USA,1920- (born in Shanghai, China),1918-,“for their discovery of concerning reversible protein phosphorylation as a biological regulatory mechanism“,Signaling in Microorganisms and Plants,two-component regulatory system in bacteria,In order to respond to environmental changes, bacteria need to detect signals from environment and transmit them to specific regulator,Signaling systems in plant,Plant signals:,Ethylene (乙烯),Ethylene promotes fiber cell elongation,(Shi et al., Plant Cell 18, 651-664),Ethylene signaling pathway,His kinase,Defects in signaling pathways can lead to cancer and other diseases,Oncogenesis: The process whereby normal cells become cancer cells. Tumor: a population of cells that result from the localized, unregulated growth and division of a single cell. Metastasis: the migration of cells from a tumor and their invasion of other tissues. Only when tumors metastasize are they considered malignant or cancerous.,Oncogenes: mutant forms of the genes for proteins invovled in signal transduction pathway,M