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Computational Investigation of Intracellular Networks

C. E. N. T. E. R. F. O. R. I. N. T. E. G. R. A. T. I. V. E. B. I. O. I. N. F. O. R. M. A. T. I. C. S. V. U. Computational Investigation of Intracellular Networks. Centre for Medical Systems Biology. Systems Approach (components and interactions)

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Computational Investigation of Intracellular Networks

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  1. C E N T E R F O R I N T E G R A T I V E B I O I N F O R M A T I C S V U Computational Investigation of Intracellular Networks Centre for Medical Systems Biology

  2. Systems Approach (components and interactions) Bioinformatic and Statistical Analysis (top-down) Metabolomics and Fluxomics (metabolic flux measurements) Mechanistic Modeling : modular and multiscale (bottom-up) Summary Agenda

  3. Systems Approach (components and interactions) Bioinformatic and Statistical Analysis (top-down) Metabolomics and Fluxomics (metabolic flux measurements) Mechanistic Modeling : modular and multiscale (bottom-up) Summary Agenda

  4. Phenotype 1 Gene 4 Phenotype 1a Gene 3 Phenotype 2 Gene 3 Gene 2 Gene 1 Gene 1

  5. Interfaces of Components May Be Damaged during Isolation Intact System Interfaces Damaged during Isolation Isolation of Components

  6. Study System at Multiple Scales Cell Types FormOrgans or Ecosystems Pathways Form Cells Molecules Form Pathways

  7. Do Not Forget the RNA Level RNA transcription Widespread RNA transcription found in ENCODE project : Nature, June 2007 "Instead of running errands, RNA appears to be running the whole show," said Isidore Rigoutsos, a lead scientist at IBM's Thomas J. Watson Research Center

  8. Systems Approach (components and interactions) Bioinformatic and Statistical Analysis (top-down) Metabolomics and Fluxomics (metabolic flux measurements) Mechanistic Modeling : modular and multiscale (bottom-up) Summary Agenda

  9. Results Rat Boneload vs control C. Reijnders, N. Bravenboer, J van Beek, P. Lips et al.

  10. Bone : Differentially expressed genes 6 hours after mechanical loading Dual Channel 3 significantly down-regulated genes: p21 (c‑Ki‑ras), amino acid transporter system A (ATA2), and phosphate regulating neutral endopeptidase on the X chromosome (PHEX) Single Channel

  11. Changes after loading a bone Local change in loaded bone MEPE-ASARM Enhances mineralization PHEX proteases (cathepsin B) Change in all bones Inhibits mineralization ASARM + MEPE Role creatine kinase and troponin-C yet unknown

  12. Signaling Pathway Map: Epidermal Growth Factor (Kitano) Signaling Pathway Map: Concept Map: MAPK (Van Kampen)

  13. Systems Approach (components and interactions) Bioinformatic and Statistical Analysis (top-down) Metabolomics and Fluxomics (metabolic flux measurements) Mechanistic Modeling : modular and multiscale (bottom-up) Summary Agenda

  14. The Airport Example What do we need to know to determine throughput? Neither the number of airplanes on the ground ... … nor the map of the runways ... is very helpful

  15. The Airport Example What do we need to know to determine throughput? ...but the “radar flux” is informative

  16. Carbon Transition Networks (CTNs) Thomas Binsl

  17. Simulation of glutamate NMR multiplets due to carbon-13 isotope enrichment during infusion of labeled acetate NMR Spectrum 4-carbon glutamate 4-carbon singlet 4-carbon doublet

  18. Anaplerosis 16 % of TCA cycle flux (n=19) Transamination 17.4 µmol/g/min (n=9) In Vivo Metabolic Rates Estimated from 13C NMR Spectrum 58 % acetyl CoA from infused acetate (n=36) Transport time 29.8 sec (n=7) TCA cycle flux = 7.7 µmol/g/min (n = 60) glutamate content 24.6 µmol/g TCA cycle

  19. Systems Approach (components and interactions) Bioinformatic and Statistical Analysis (top-down) Metabolomics and Fluxomics (metabolic flux measurements) Mechanistic Modeling : modular and multiscale (bottom-up) Summary Agenda

  20. Switching from Google Earth, To “Google Human” To “Google Heart” Computational Model of Essential Parts of Molecular System

  21. The K.I.S.S. Principle ("Keep It Simple, Stupid") or ("Keep It Short and Simple") Boehringer Pathways • Big Model Many Trees, Forest Visible? • Small is Beautiful Well Maintained Garden

  22. Model of High Energy Phosphate Group Handling in Energy Metabolism Mitochondrial Matrix • ADP and inorganic phosphate (Pi) enter intermembrane space and stimulate oxidative phosphorylation (OxPhos). • Model Elements : • two creatine kinase (CK) isoforms • diffusion & membrane permeation • Communicating Modules: • ATP consumption (ATPase) as forcing function • ATP production by • mitochondria Pi ATP ADP OxPhos MIM Pi ADP ATP Intermembrane Space Mi-CK PCr Cr MOM MM-CK PCr Cr Cytosol ADP ATP Pi ATPase

  23. Model of High Energy Phosphate Group Handling in Energy Metabolism Mitochondrial Matrix • ADP and inorganic phosphate (Pi) enter intermembrane space and stimulate oxidative phosphorylation (OxPhos). • Model Elements : • two creatine kinase (CK) isoforms • diffusion & membrane permeation • Communicating Modules: • ATP consumption (ATPase) as forcing function • ATP production by • mitochondria Pi ATP ADP OxPhos MIM Pi ADP ATP Intermembrane Space Mi-CK PCr Cr MOM MM-CK PCr Cr Cytosol ADP ATP Pi ATPase Border of the module

  24. Model of High Energy Phosphate Group Handling in Energy Metabolism Mitochondrial Matrix • ADP and inorganic phosphate (Pi) enter intermembrane space and stimulate oxidative phosphorylation (OxPhos). • Model Elements : • two creatine kinase (CK) isoforms • diffusion & membrane permeation • Communicating Modules: • ATP consumption (ATPase) as forcing function • ATP production by • mitochondria Pi ATP ADP OxPhos MIM Pi ADP ATP Intermembrane Space Mi-CK PCr Cr MOM ? ? MM-CK PCr Cr Cytosol ADP ATP Pi ATPase Border of the module

  25. Ingredients for this Simulation Dynamics of response O2 uptake at whole heart level Transport time blood vessels and diffusion Decrease total O2 amount in whole heart Increase O2 uptake heart NMR determined diffusion coefficients Mitochondrial Membrane Permeability Steady state responses isolated mitochondria Enzyme kinetics of two enzymes = Outer Membrane Permeability for ADP = 21 m/s

  26. Our model analysis implies that in rabbit heart at 487 μM/sec ATP hydrolysis, the diffusion flux carried by PCr is 154 μM/sec. This suggests that the PCr shuttle is of minor importance. But what is the function of the two creatine kinase isoforms?

  27. Effect of creatine kinase expression level on metabolic oscillations and on average concentrations

  28. Pictorial Summary Using stable isotopes to trace networks Modular Modeling, Using Information from Multiple Scales, makes Systems Accessible to Deep and Wide Analysis

  29. Networks of Correlation Clish, Van der Greef, Naylor OMICS Vol. 8, 2004

  30. Acknowledgments Thomas Binsl Olav Kongas Hans van Beek Kate Mullen Jaap Heringa Glenn Harrison Lori Gustafson Ivo van Stokkum Koert Zuurbier Johan Groeneveld Bas de Groot Marcel Eijgelshoven David Alders Joli Bussemaker Jan Bart Hak Frits Prinzen Mark Noble

  31. Threshold False Discovery Rate(Benjamini-Hochberg Linear Stepup) P value Linear Stepup Threshold FDR = 20% Rank Order

  32. FluxSimulator (Simulation Results) 1 Metabolite B Time [min] 0 Isotopomer Fraction 1 Metabolite D 0 0 100 200 Time (sec)

  33. COMPARING THE NEW NMR METHOD WITH OXYGEN CONSUMPTION MEASURED BY ‘GOLD STANDARD’ (BLOOD GAS) Oxygen Consumption by NMR Method (μmol / g dry /min) n = 42 pigs Oxygen Consumption by Blood Gas (μmol / g dry /min)

  34. Adenine Nucleotide – Creatine - Phosphate Module ATP ATP PCr ADP ADP creatine kinase Mitochondria Myofibril • Module : set of molecular processes performing a cellular function • Functions of the adenine nucleotide-creatine-phosphate module: • transfer high-energy phosphate bonds from mitochondria to cytosolic ATPases • dynamic adaptation of ATP synthesis to time-varying hydrolysis • emergency buffer system

  35. Predicted oscillation of ADP in cytosol for low permeability of mitochondrial outer membrane Membrane Permeability 0.16 m/s (Vendelin, Saks et al. 2000) ADP levels much too high Response much too slow

  36. Isolating the Module under Study Experimentally Response measured of venous oxygen in mouse heart Activation time of oxidative phosporylation 3.7 s … and Measuring the Response of the System as a Whole

  37. The response is simplified by removing pulsatility from the ATP hydrolysis forcing function Membrane Permeability 1 =  m/s (for sake argument) 2 = 85 m/s (Beard 2006) 3 = 21 m/s (optimised tmito = 3.7 s) 4 = 3.5 m/s (Saks 2003) 5 = 0.16 m/s (Saks 2000) 1 2 3 4 5

  38. experiment: normal CK experiment: both CK isoforms inhibited Partitioning the Contributions of Mitochondrial and Muscle CK Isoforms Mi-CK changes MM-CK changes Both CKs change

  39. Time varying load on the mitochondria at two levels: First level: ATP hydrolysis in beating heart muscle pulsates during each heartbeat ATP Hydrolysis (μM/s) ATP Hydrolysis (μM/s) Time ( s ) ATP Synthesis (μM/s) Time ( s )

  40. Time varying load on the mitochondria at two levels: Second level: Heart rate is variable At t=0 paced heart rate is stepped from 135 to 220 beats/min

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