Systems Biology Study Group Chapter 3 Walker Research Group Spring 2007

Overview Review Biochemical Network reconstruction Metabolic Networks –Basic Features Hierarchy Reconstruction Methods Genome-scale Metabolic Reconstructions Multiple Genome-scale Networks Summary

Review Systems Biology: Process of genome scale network reconstruction, followed by synthesis of in silico models describing their functionalities –Enumeration of biological components –Identification of links connecting processes –Modeling –Hypothesis generation and testing

Review Roots of Systems Biology –Biology Molecular biology High throughput sequencing Genome scale analysis –Systems Analog simulations Large scale simulations of metabolic networks Genome scale models and analysis Systems Biology constrained by: –Chemical transformation properties Stoichiometry Relative and absolute rates –Functional states Physiochemical nature Orientation

Biochemical Network Reconstruction Network reconstruction: Process of identifying all reactions that comprise a network Networks are not separate and independent of each other

Metabolic Networks Metabolism – Biochemical modification of chemical compounds within living cells. Metabolic networks are the collection of pathway through which this is accomplished Source: Feigenson, G BIOBM 331

Basic Features Intermediary metabolism: chemical “engine” –Converts raw materials into energy, building blocks for biological structures –Obeys laws of physics and chemistry –Elaborate regulatory structure Source: Feigenson, G BIOBM 331

Hierarchy Simplify conceptualization of network functions Level 1 – Cellular Inputs and Outputs –Coarse description of overall activity First published experiments in human metabolism Italian physician Santorio Santorio in 1614 Used a steelyard balance to weigh himself after eating, sleeping, working etc. Found that most of food intake was lost through “insensible perspiration” Source: Metabolism. Wikipedia, public domain art. 18 July 2005

Hierarchy Level 2 – Sectors –Metabolism has two basic sectors Catabolism – break down substrates into metabolites that cell can use Anabolism – synthesize amino acids, fatty acids, nucleic acids and other cellular building blocks Exchange of chemical groups and redox potentials takes place using carrier molecules, linking the two sectors

Hierarchy Level 2 – Sectors Catabolism Anabolism Growth ATP ADP NADPH NADP+ Sugar- phosphate s PEP Pyruvate AcCoa Α-KG SuccCoA OA Substrates Amino acids Nucleotides Fatty acids Specialty products CO 2, H 2 O O2O2 Energy from catabolism Proteins RNA/DNA Membranes etc Adapted from: Palsson, B Systems Biology

Hierarchy Level 3 – Pathways –Series of chemical reactions occurring within a cell, usually catalyzed by an enzyme –Pathways in catabolism Substrate picked up by cell Hydrolyzed if necessary Activated by cofactor Degraded to yield energy –At this level metabolism relies on basic chemical principles such as stoichiometry and kinetics GLUCOSE GLYCOLYSIS ACETATE CITRIC ACID CYCLE Source: Feigenson, G BIOBM 331

Hierarchy Level 4 – Individual Reactions –High-throughput data makes this level possible –Can reconstruct genome-scale stoichiometric matrices of organisms May be on the order of hundreds of metabolites, thousands of chemical reactions It is at this level that the text is focused Source: Feigenson, G BIOBM 331

Reconstruction Methods Define reaction list – assemble information on all biochemical reactions in network Sources: –Biochemistry –Genomics –Physiology –In silico modeling

Reconstruction Methods Genome annotation –Open reading frames (ORF’s) Identified and assigned functions via experimentation or comparison to known sequences –In silico modeling Can achieve 40 – 70% functional assignment Purely hypothetical

Reconstruction Methods Sequence Data –List of sources –Sequence homology may be evidence of a reaction in an organism Biochemical data –Enzyme isolation and function demonstration –Gives stoichiometry and reversibility of reaction

Reconstruction Methods Enzyme Commission Numbers –Used to systematically and unambiguously characterize reactions –E.C → Glucokinase Protein Database – Crystal structure of E. coli glucokinase in complex with glucose Source: Protein database February 2007http://

Reconstruction Methods Gene – Protein – Reaction Associations –Not all genes have one to one relationship with corresponding enzymes or metabolic reactions May require multiple genes for enzyme to catalyze reaction –Fumerate reductase requires 4 subunits, frdA, frdB, frdC frdD Genes may also encode promiscuous enzymes which catalyze several different reactions –Transketolase I in pentose phosphate pathway

Reconstruction Methods Organism specific sources –E. coli encyclopedia (EcoCyc) database –Yeast Comprehensive Yeast Genome Database Yeast Protein Database Saccharomyces Genome Database Additional issues include: –Demands on the network and composition –Physiological data and ability to reproduce experimental conditions

Genome-scale Metabolic Reconstructions Ongoing process since 1930s –Since glycolytic pathway determined First genome sequenced in 1995 –H. influenzae First reconstruction of genome-scale metabolic network in 1999

Genome-scale Metabolic Reconstructions Table: Genome-scale reconstructions of metabolic networks in microbial cells Number of OrganismGenesMetabolitesReactions H. influenzae E. coli H. pylori S. cerevisiae G. sulfurreducens S. aureus M. succinciproducens Adapted from: Palsson, B Systems Biology

Genome-scale Metabolic Reconstructions Table: Evolution of E. coli metabolic reconstructions Number of metabolitesNumber of reactionsYear , Adapted from: Palsson, B Systems Biology

Multiple Genome-scale Networks Metabolic networks are not isolated –Interact with cellular processes Transcriptional regulation, cell motility –Signaling networks in multicellular organisms –Cell fate processes Mitosis, apoptosis To fully describe a cell, all networks must be reconstructed

Multiple Genome-scale Networks Multiple Network Reconstruction –Common components Same molecules participate in more than one network ATP –Metabolic – energy metabolism –Regulatory – global regulator of DNA coiling –Signaling – phosphate for signaling reactions –Content in Context Integrating all “omics” data –Genomic, transcriptomic, proteomic, metabolomic –Biochemically and genetically accurate framework –Allows for predictions of function in environment

Multiple Genome-scale Networks Regulation of metabolic networks –Modulating enzyme reaction rates, gene expression Activity, concentration or both –Negative: repression or inhibition –Positive: induction or activation –Gene expression is coarse metabolic control –Enzyme activity is fine tuning

Multiple Genome-scale Networks Regulating Enzyme Activity –Allosteric mechanism Enzymes have binding site for substrate and for regulatory molecules –Can activate or inhibit enzyme activity Conformational changes in enzyme molecule Example: Hexokinase –Catalyzes phosphorylation of glucose –Inhibited by ATP, product of glycolysis –Stimulated by ADP, product of ATP stored energy consumption

Summary Complex networks carry out complicated biological functions, like metabolism All networks based on biochemical reactions, described by stoichiometric matrix Hierarchy can be used to conceptualize networks at varying resolutions Metabolism is the best characterized network in terms of biochemistry, kinetics and thermodynamics Network reconstruction requires detailed examination of all components and links the network, many resources can provide this information Metabolic networks do not act independently of other networks, integration of all networks is necessary to describe cellular functions

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