Structural Network Analysis (1) Jörg Stelling [email protected] CSB Deterministic, SS 2015, 2 Systems Biology & Uncertainty Factors that limit the power of mechanistic (ODE-based) model development for biological systems: Factor 1: Unknown parameter values and model structures for (nearly all) biological systems of interest. .... Answer by structural network analysis methods: Focus on network structure to derive conditions for possible qualitative behavior (parameter-free). Involves radical simplifications of models and scope. 2 Relations Between Spaces Parameter space Flux space State space 3 Structural Analysis: Approach Idea: With well-characterized reaction stoichiometries and reversibilities constrain the possible network behavior (→ constraint-based approaches). Based on first principles: Conservation of mass (and energy and possibly other constraints). Application primarily to metabolic networks. Mathematics: Linear algebra, convex analysis. 4 Metabolic Networks: Definitions R2 Aext R1 B R4 A Bext R3 System definition: Internal versus external metabolites. Reaction stoichiometry: Ratios of products / educts. Reaction directionality: Reversibility / irreversibility. Metabolic fluxes: Rates of metabolic reactions. 5 Metabolic Networks: Definitions R2 Aext R1 B R4 A Bext R3 Number of (internal) metabolites {A,B}: n = 2. Number of metabolic reactions {R1-R4}: q = 4. Sets of reversible and irreversible reactions: rev = {R3}, irrev = {R1,R2,R4}, rev ∩ irrev = 0. 6 Metabolic Networks: Representation R1 R2 R2 Aext B R4 R1 A R3 Bext [ N= R3 R4 1 −1 −1 0 0 1 1 −1 ] Network representation: Stoichiometric matrix N (n x q). Rows → Internal metabolites i ; Columns → Reactions j. Elements nij : Stoichiometric coefficients (>0 for products). A B 7 Metabolic Networks: Representation Metabolic network: 6 (internal) metabolites, 10 reactions. Reaction reversibilities indicated by arrows. Representation through stoichiometric matrix N. 8 Metabolic Networks: Flux Distributions R2 Aext R1 B R4 A Bext R3 [] [] 1 r= 1 0 1 1 r ' = −1 0 1 Flux distribution: Specification of all fluxes in the network → Vector r of q reaction rates. Feasibility criterion: ri ≥ 0 for all irreversible reactions. 9 Metabolic Networks: Mass Balances R2 Aext R1 A B R4 R3 Bext dc A = r 1−r 2 −r 3 dt dc B = r 2 r 3−r 4 dt Mass balances for all internal metabolites; external metabolites assumed to be sources or sinks. Metabolite concentrations: dci/dt = fluxesi,in – fluxesi,out . 10 Metabolic Networks: Balancing Equation d c t = N⋅r t dt Time-dependent concentration changes Stoichiometric matrix: invariant Flux distribution: time-variant r(t) = f(c(t),p,u(t)) Stoichiometric matrix N = well-known systems invariant. Uncertainties in kinetic description of reaction rates. Structural network analysis: Focus on the invariant N. 11 Conservation Relations: Principle A B R1 C D dc A dt dc B dt dc C dt dc D dt = −r 1 = −r1 = r 1 = r1 Conservation relations: Weighted sums of metabolite concentrations that are always constant in the network. Example: [A]-[B]=const.; [A]+[C]=const.; [B]+[D]=const. 12 Conservation Relations: Analysis A B R1 C D [] [ ] −1 N = −1 1 1 1 y= −1 0 0 1 0 1 0 0 1 0 1 m-rank(N) = 4 -1 = 3 CRs y correspond to linearly dependent rows in N. CRs lie in the (left) null-space (or: kernel) of matrix N: T N ⋅y = 0 T y ⋅N = 0 T Maximal number of linearly independent CRs given by the dimension of the null space of N: n-rank(N) . 13 Conservation Relations: Applications A B R1 C D [] [ ] −1 N = −1 1 1 1 y= −1 0 0 1 0 1 0 0 1 0 1 m-rank(N) = 4 -1 = 3 Interpretation: [A]-[B]=const.; [A]+[C]=const.; [B]+[D]=const. Conserved moieties: Positive sum of metabolite concentrations. CRs shrink the possible dynamic behavior: Model reduction. 14 Balanced Networks: Quasi Steady State Metabolic networks: Fast reactions (msec - seconds timescale) and high turnover of reactands. Quasi steady state → Metabolite balancing equation: d c t = N⋅r t dt c t , r t 0 = N⋅r const. Homogeneous systems of linear equations: Consumption of a metabolite equals production. 15 Balanced Networks: Null Space 0 = N⋅r Trivial solution r = 0 → Thermodynamic equilibrium. Metabolic networks: q >> n → Degrees of freedom of the network → Infinite number of compliant vectors r. Linear algebra: All possible solutions lie in the (vector) null space (or: kernel) of N with dimension q-rank(N). 16 Balanced Networks: Null Space r1 Null space 0 = N⋅r r2 17 Balanced Networks: Kernel Matrix R2 R1 A Aext B R4 Bext R3 N= [ 1 −1 −1 0 0 1 1 −1 ] rev = {R3} irrev = {R1 , R2 , R4} [ ] 1 0 K= 1 1 0 −1 1 0 q-rank(N) = 4 -2 = 2 Task: Find q-rank(N) linearly independent solutions → Arrange in a kernel matrix K. Reconstruction of all r by linear combination b of the columns of the kernel matrix: r = Kb. 18 Kernel Matrix: Enzyme Subsets R2 R1 A Aext B R4 Bext R3 [ 1 −1 −1 0 N= 0 1 1 −1 ] rev = {R3} irrev = {R1 , R2 , R4} [ ] 1 K= 0 1 1 0 −1 1 0 R1 R2 R3 R4 Enzyme subset: Set of reactions that (in steady state) always operate together in a fixed ratio. Detection: Rows in K differ only by scalar factor. Strong coupling → Indication of common regulation. 19 Kernel Matrix: Limitations R2 R1 A Aext B R4 Bext R3 [ 1 −1 −1 0 N= 0 1 1 −1 ] rev = {R3} irrev = {R1 , R2 , R4} [ ] 1 K= 0 1 1 0 −1 1 0 b= 1 −1 r T = 1 1 0 1 Caveat #1: Kernel matrix is not a unique representation. Caveat #2: Reaction reversibilities are not considered. Caveat #3: Network degrees of freedom ≤ dimension(K). 20 Metabolic Networks: (Manual) Reconstruction 21 J. Monk et al., Nat. Biotechnol. 32: 447 (2014). Metabolic Networks: Stoichiometric Models Nearly genome-scale level models of metabolic networks. Reconstructions for several organisms including human. 22 Constraint-Based Network Analysis Methods Today N.E. Lewis et al., Nat. Rev. Microbiol. 10: 291 (2012). 23 Flux Balance Analysis: Principles Idea: Incorporate further constraints to limit network behaviour with respect to feasible steady state flux distributions → More predictive models. Examples for constraints: Quasi steady state assumption. Reaction reversibilities / capacities. Optimal feasible steady state. 24 Flux Balance Analysis: Constraints Quasi steady state assumption: 0 = N⋅r Reaction reversibilities / capacities: i r i i Optimal feasible steady state: wT⋅r → max ! Maximal growth rate. Maximal energy (ATP) production. Maximal yield of a desired product. ... 25 Flux Balance Analysis: Constraints r1 Null space 0 = N⋅r Feasible space Reaction reversibilities/ capacities i r i i r2 26 Flux Balance Analysis: Optimization General optimization problem statement for FBA: T Z obj = w ⋅r → max ! s.t. N⋅r = 0 i r i i Linear objective function and system of linear equality/inequality constraints → Linear program → Solution is computationally cheap. 27 Flux Balance Analysis: Optimization Simplex algorithm: Solutions lie on vertices → Start on vertex → Evaluate gradients + move along edges → Continue search or stop at optimal solution. 28 Flux Balance Analysis: Optimization r1 Null space 0 = N⋅r Max r1 Max (r1 + r2) Feasible space Reaction reversibilities/ capacities Max r2 i r i i r2 29 M.A. Oberhardt et al., Molec. Syst. Biol. 5: 320 (2009). Flux Balance Analysis: Applications 30 Oxygen uptake rate J. Edwards et al., Nat. Biotech. 19: 125 (2001). Application #1: Prediction of Phenotypes Stoichiometric model for bacterium E. coli (n=436, q=720). Acetate uptake rate FBA: Maximization of growth rate. Prediction of growth yields and uptake / excretion rates. 31 growth mutant / wild type Application #2: Prediction of Mutant Behavior J. Edwards & B.O. Palsson, PNAS 97: 5528 (2000). Stoichiometric model for bacterium E. coli (n=436, q=720). FBA: Maximization of growth rate after gene deletions. 86% prediction accuracy (viability versus inviability). 32 M.W. Covert et al., Nature 429: 92 (2004). Application #2: Prediction of Mutant Behavior Integration of stoichiometric model with (discrete) representation of regulatory network: Improved predictions. Identification of unknown knowledge gaps by evaluation of the model against high-throughput experimental data. 33 Application #2: Prediction of Mutant Behavior J. Edwards & B.O. Palsson, PNAS 97: 5528 (2000). Prediction of flux distributions: Wild type (black fluxes) versus mutant strain (red). Here: Gene knockout (zwf) in the pentose phosphate pathway (red arrow). 34 Application #3: Mycoplasma pneumoniae Model development: Databases of metabolic reactions (KEGG). Genome analysis. Flux balance analysis. Growth experiments (variable E. Yus et al., Science 326: 1263-68 (2009). conditions). 35 Application #3: Mycoplasma pneumoniae E. Yus et al., Science 326: 1263-68 (2009). 36 E. Yus et al., Science 326: 1263-68 (2009). Application #3: Mycoplasma pneumoniae M. pneumoniae L. lactis E. coli B. subtilis Model analysis in combination with experimental data: Detailed results: Slow growth (8h doubling time) not caused by energy but protein synthesis limitations. Global results: Genome reduction facilitated by multifunctional metabolic enzymes. 37 J. Reed et al., PNAS 103: 17480 (2006). Application #4: Systems Identification Theory-experiment iteration: Identify missing network parts. 38 Caveat #1: Objective Function Strong dependence of results on objective function. Use of 'natural' objective functions such as growth: Not applicable to all organisms (e.g. cells in multicellular organisms → cancer ...). Not applicable under all conditions (e.g. after perturbation of an organism). Alternative / conflicting approaches to optimization. 39 Caveat #2: Alternate Optima Linear programming problem implies that finding a solution can be guaranteed, but: Unique value of the objective function ('growth'). Existence of infinitely many solutions with optimal value of objective function possible. Without incorporating further constraints: Poor performance in predicting flux distributions. 40 Caveat #2: Alternate Optima r1 Null space 0 = N⋅r Max r1 Feasible space Reaction reversibilities/ capacities i r i i r2 41 Flux Variability Analysis (FVA) Identify minimal and maximal fluxes for (optimal) objectiv function value using linear programming: r j →max ! / min ! ∀ j ∈{1…q } s.t. N⋅r = 0 i r i i T w ⋅r = Z obj Returns bounds on feasible fluxes; a solution is unique when bounds for a given flux are identical. 42 Flux Variability Analysis (FVA) r1 Null space 0 = N⋅r r1min=r1max Max r1 Feasible space r2min r2max Reaction reversibilities/ capacities i r i i r2 43 Flux Variability Analysis (FVA) R. Mahadevan & C.H. Schilling, Metabolic Eng. 5: 264 (2003). Example network with multiple inputs: Flux variability depends on network structure and environment. 44 Caveat #2: Alternate Optima E. coli metabolic model: m=625, q=931. Mixed integer linear program (MILP): LP: J. Reed & B.O. Palsson, Genome Res. 14: 1797 (2004). MI: Solution matrix NZJ, Decision variable yi 45 Structural Network Analysis Network structure: Stoichiometric (& other) constraints on behavior → Predictive models rooted in first principles and comprehensive (and available) biological knowledge. Basic definitions: Stoichiometric matrix, mass balances, quasi steady-state assumption. Analysis relying on linear algebra: Null space, kernel matrix, ... → Feasible flux distributions, enzyme subsets, ... Flux balance analysis (FBA) → Optimality assumption → Linear program → Predictions of phenotype, mutants, ... Possible alternative optima → flux variability analysis (FVA). 46 Further Reading J.D. Orth, I. Thiele & B.O. Palsson. What is flux balance analysis? Nature Biotechnology 28: 245-248 (2010). N.E. Lewis, H. Nagarajan & B.O. Palsson. Constraining the metabolic genotype-phenotype relationship using a phylogeny of in silico methods. Nature Reviews Microbiology 10: 291-305 (2012). M. Terzer, N.D. Maynard, M.W. Covert & J. Stelling. Genome-scale metabolic networks. Wiley Interdisciplinary Reviews Systems Biology (2009). (http://www3.interscience.wiley.com/journal/122456827/abstract) 47
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