Signal networks and pathways

1. Signal networks and pathways Chitta Baral Arizona State University

2. Goal: Decoding the cell and … • Ultimate goal • Decoding the cell (understand what is happening inside the cell) • Control cell behavior • It involves • Accurate description of cellular biochemistry that allows • Explanation of particular cell behaviors and phenotypes (such as proliferation, cell migration, cell death, etc.) • Prediction of future behavior • Planning interactions with the cell to control the cell behavior

3. Biochemistry, network of interactions • Relation between various molecules is often expressed using network of interactions (called Biochemical networks) • Three kinds of biochemical networks • Metabolic networks • Represent the chemical transformation between metabolites • Example: Glycolysis (breakdown of glucose) • Signal networks (also called protein networks) • Representing protein-protein interactions • Gene networks • Representing relationship between genes that encode the impact of the expression level of one gene on another • Pathways: Specific series of interaction in a network

4. G proteins • 1994 Nobel prize. Discovery of G-protein coupled receptors and their role in signal transduction. Gilman, Alfred G. and Rodbell, Martin. http://www.nobel.se/medicine/laureates/1994/index.html • 43,409 articles in Medline that mention G proteins. • 1. Basic facts about G proteins • Each G-protein consists of 3 sub-units: Ga, Gb, and Gg. • These sub-units can be together or separated. • Each G-protein has a binding site that can be occupied by either GDP or GTP. • A G-protein is said to be inactive when it is bound to GDP. • A G-protein is said to be active when it is bound to GTP.

5. cyclicAMP (cAMP) pathway • 2. Up regulation: When a ligand binds to a receptor in a cell membrane the effect is that • A. the receptor becomes activated • B. the nucleotide binding site on the G-protein is altered, • C. GTP replaces GDP, • D. GDP is released, • E. and Ga-GTP gets disassociated from GbGg. • F. Ga being active triggers the binding of Ga-GTP to a membrane bound adenylate cyclase molecule, • activating it for production of cyclicAMP (cAMP).

6. G protein activation/inactivation cycle

7. cAMP

8. cyclicAMP (cAMP) pathway • 3. Down regulation – when the ligand dissociates from the receptor the effects are • A. GTP is hydrolyzed by a GTPase activity on Ga • B. Ga-GTP becomes Ga-GDP, • C. and disassociates from adenylate cyclase molecule, making the later inactive. • D. Ga then reassociates with GbGg • E. CyclicAMP (in the cytoplasm) is then inactivated by the enzyme phosphodiesterase, which hydrolyzes it to AMP.

9. Interference with cAMP • A. Cholera toxin inhibits the GTPase activity of the G-proteins of Gs subfamily, thus impacting 3A. • B. The ras gene produces a G-protein that lacks GTPase activity, thus impacting 3A. • C. Pertussis toxin inactivates the process that downregulates adenylate cyclase activity with respect to G-proteins of the Gi subfamily, with a few exceptions such as Gz, thus impacting 3C. • D. G-proteins of the Gq subfamily are not modifiable by pertussis toxin or cholera toxin, thus nullifying A and C above.

10. A specific example of CAMP mediated regulation – Glycogen degradation • In liver or muscle cells in presence of the ligand epinephrine hormone (also called adernalin) increase in the cAMP concentration (in the cytoplasm) • activates protein kinase, • active protein kinases then converts inactive phosphorylase kinase to active form by ATP dependent phosphorylation, • active phosphorylase kinase then phosphorylates (i.e., converts) less active phosphorylase-b to more active phosphorylase-a, and • phosphorylase-a then catalyzes the phosphorolytic cleavage of glycogen into molecules of glucose-1-phosphate. (i.e., glycogen breakdown happens.)

11. References (for stuff so far) • The world of cell 2nd ed. Becker, Deamer. Chapter 21. (Latest one is 5th ed, by Becker, Kleinsmith and Hardin; has a nice Cd-Rom with the book and explains signal transduction very well) • Biochemistry of signal transduction and regulation. G. Krauss. Sec 5.5.1 • http://www.mun.ca/biology/desmid/brian/BIOL2060_W2003/CellBiol10/CB10.html • Look for signal transduction, pathways, signal pathways, G protein, biochemical networks etc.

12. Reasoning about cAMP and Glycogen degradation • Observation: cAMP concentration way above normal • Possible explanation: presence of Cholera toxin or Pertussis toxin • Planning: How to overcome it using drugs. • Predicting the impact of not having enough adernalin. • Observation: Lack of glycogen breakdown. • Explained by low cAMP concentration • Explained by G proteins not getting activated in adequate concentration

13. Computer Science (AI) challenges • Represent signal networks • Such that we can reason with it • We can elaborate on it (add more details) without making wholesale changes • Reason with them • Explain observations. • Predict effect of particular actions. • Plan to make the cell behave a particular way.

14. Analogy with current AI research • Goal: To represent effect of actions on the world, executability condition of actions, relation between objects in the world, etc. • An example: • Description D. • S1 Load causes loaded. • S2 Shoot causes ~ alive if loaded. • S3 Intially alive. • S4 Initially ~loaded. • Planning: D |= ~alive after X. X=Load;Shoot. • Explanation: {S1, S2, S3, ~alive after shoot} |= Initially loaded • Prediction: • D |= ~alive after Load? D |= loaded after Load?

15. Home work 3 (Due Feb 17th) – 100 pts • Describe a particular signal pathway. • Draw the figure. • Write in English similar to the slides • Write in English-like syntax. (such as in the previous slide and make up specific terms like `causes’ that you may need.) • Source (journal: Cellular signaling; signal transduction chapter in books on cell biology; www.afcs.org. etc.) 50% bonus if you use a journal instead of a book.

16. Glossary • Activation • A process of (i) initiating a chemical or biochemical reaction (ii) converting an inactive component to a functionally active form. • Adenylate Cylase • The enzyme that catalyzes the synthesis of cyclic AMP (cAMP) from ATP, • ATP <--> cAMP + PP • Allosteric • Pertaining to the topologically distinct sites on a protein or an enzyme molecule. • AMP • An Adenosine 3'-monophosphate or Adenosine 5'-monophosphate nucleotide with the phosphate group linked to the carbon 3 (or 5 resp.) of the ribose. • Arrestin • Family of inhibitory proteins that bind to tyrosine-phosphorylated receptors, thereby blocking their interaction with G-proteins and effectively terminating the signaling.

17. Glossary (cont) • Channel Protein • Proteins that form water-filled pores or channels across the membrane and are responsible for transporting solutes across the membrane. • conformational change • change in the form differing in secondary or tertiary structure. • cyclic AMP (cAMP) • An abbreviation for adenosine 3',5'-monophosphate or adenosine 2',3' -monophosphate. • An important intracellular regulator or second messenger for a number of cellular processes in animals, bacteria, fungi and plants. • Effector molecule • Small, biologically active molecule that acts as a regulator to control the activity of a protein or an enzyme by binding to a specific region on the protein or enzyme. • Enzyme • Bioactive protein that catalyzes the biochemical reactions in the living cell. • GAPs (growth-associate proteins) • Promote the hydrolysis of bound GTP, thereby switching the G-protein to the inactive form. • GDP (Guanosine 5'-Diphosphate) • GEFs (guanine nucleotide exchange factors) • Family of proteins that facilitate the exchange of bound GDP or GTP on small G-proteins such as ras and rho and thus activate them. (act in the opposite way to GAPs.)

18. Glossary (cont.) • G-Protein • A GTP binding membrane protein that is capable of hydrolyzing GTP, activating membrane bound CAMP, and mediating a variety of signal transducing systems. • GRK: G-Protein receptor kinase • GTP: Guanosine 5'-Triphosphate • GTPase (Guanosine triphosphatase) • Enzyme that catalyzes the reaction GTP +H20 <---> Guanosine + Triphosphate • Guanine: A constituent base in nucleic acids. • Guanosine: A nucleoside and constitutent of nucleotides • Hydrolysis: Splitting of 1 molecule to 2 by incorporation of 1 water molecule. • Induction • An increase in the rate of enzyme synthesis due to the presence of substrate or inducer. • Inhibition • (i) reduction or prevention in the rate of enzymatic activity (ii) repression of physical or chemical activity. • Kinase • The enzyme that catlyzes the transfer of a phosphate group from one compound to another. • Nucleoside • A component of a nucleotide that consists of a nitrogenous base (purine or pyrimidine) linked to a pentose sugar (ribose or deoxyribose)

19. Glossary (cont.) • Nucleotide: The basic building blocks of nucleic acids it consists of a nucleoside and a phosphate. • phosphatase (two kinds: acidic and alkaline) • acidic phosphatase is an enzyme that catalyzes the hydrolysis of a number of phosphomonoesters at acid pH but not phosphodiesters. • Phosphoric monoester +H2O <--> Alcohol + Phosphoric acid. • alkaline phosphatase is an enzyme that catalyzes the hydrolysis of phosphomonoester at alkaline pH. • phosphodiesterase • The enzyme that catlyzes the hydrolysis of phosphodiester bond in the polynucleotides or cyclic nucleotides. • PKA: Protein kinase A • Protein • A polymer of L-amino acids that folds into a conformation specified by the linear sequence of amino acids nd functions as an enzyme, a hormone, an antibody or a structural component of the cell. • Regulatory enzyme • The enzyme that possesses a regulatory site for binding effector molecules in addition to the catalytic binding site. • RGS (Regulators of G-Protein signalling) • RGS is a protein that can increase the GTPase activity by more than one order of magnitude. • Transport protein: A protein that mediates the entry of specific substances into a cell.