Metabolic pathway analysis

Transcription

1 Bioinformatics Metabolic pathway analysis Jacques van Helden

2 Graph-based analysis of biochemical networks Examples of metabolic pathways Jacques van Helden

3 Methionine Biosynthesis in S.cerevisiae Aspartate biosynthesis ATP ADP L-Aspartate Aspartate kinase HOM3 L-aspartyl-4-P NADPH NADP+; Pi L-aspartic semialdehyde Aspartate semialdehyde deshydrogenase HOM2 Threonine biosynthesis NADPH NADP+ AcetlyCoA CoA L-Homoserine O-acetyl-homoserine Homoserine deshydrogenase Homoserine O-acetyltransferase HOM6 MET2 Met31p met32p MET31 MET32 Sulfur assimilation Sulfide O-acetylhomoserine (thiol)-lyase MET17 Cysteine biosynthesis Homocysteine Cbf1p/Met4p/Met28p complex MET28 CBF1 MET4 5-methyltetrahydropteroyltri-L-glutamate 5-tetrahydropteroyltri-L-glutamate L-Methionine Methionine synthase (v it B12-independent) MET6 Gcn4p GCN4 S-adenosyl-methionine H 2 0; ATP synthetase I Pi, PPi S-adenosyl-methionine synthetase II S-Adenosyl-L-Methionine SAM1 SAM2 Met30p MET30

4 Methionine Biosynthesis in E.coli Aspartate biosynthesis L-Aspartate ATP ADP aspartate kinase II/ homoserine dehydrogenase II metl L-aspartyl-4-P Lysine biosynthesis Threonine biosynthesis NADPH NADP+; Pi L-aspartic semialdehyde NADPH NADP+ SuccinylSCoA HSCoA L-Homoserine Aspartate semialdehyde deshydrogenase Homoserine O-succinyltransferase asd meta Methionine repressor metj Cysteine biosynthesis Alpha-succinyl-L-Homoserine L-Cysteine Cystathionine-gamma-synthase Succinate Cystathionine metb H2O Pyruv ate; NH Cystathionine-beta-lyase metc 5-MethylTHF THF Homocysteine L-Methionine Cobalamin-independenthomocysteine transmethylase Cobalamin-dependenthomocysteine transmethylase mete meth metr metr ATP; H2O Pi; PPi S-Adenosyl-L-Methionine

5 Alternative methionine pathways L-Aspartate S.cerevisiae L-aspartyl-4-P L-aspartic semialdehyde E.coli L-Homoserine O-acetyl-homoserine Alpha-succinyl-L-Homoserine Cystathionine Homocysteine L-Methionine S-Adenosyl-L-Methionine

6 KEGG "consensus pathway" for Methionine metabolism

7 Lysine biosynthesis in Escherichia coli Aspartate biosynthesis ATP ADP L-Aspartate L-aspartyl-4-P aspartate kinase III metl Methionine biosynthesis Threnonine biosynthesis NADPH; H+ NADP+; Pi pyruvate 2 H2O L-aspartic semialdehyde dihydropicolinic acid aspartate semialdehyde deshydrogenase asd dihydrodipicolinate synthase dapa NADPH or NADH; H+ NADP+ or NAD+ succinyl CoA CoA tetrahydrodipicolinate N-succinyl-epsilon-keto- L-alpha-aminopimelic acid dihydrodipicolinate reductase tetrahydrodipicolinae N-succinyltransferase dapb dapd glutamate alpha-ketoglutarate succinyl diaminopimelate succinyl diaminopimelate aminotransferase dapc H2O succinate LL-diaminopimelic acid meso-diaminopimelic acid N-succinyldiaminopimelate desuccinylase diaminopimelate epimerase dape dapf CO2 diaminopimelate decarboxylase lysa L-lysine lysr protein lysr

8 Lysine biosynthesis in Saccharomyces cerevisiae 2-Oxoglutarate Acetyl-CoA CoA homocitrate synthase LYS20 1,2,4-Tricarboxylate homocitrate dehydratase LYS7 H2O But-1-ene-1,2,4-tricarboxylate homoaconitate hydratase LYS4 Homoisocitrate NAD+ H+; NADH Oxaloglutarate CO Homoisocitrate dehydrogenase 2-Oxoadipate L-Glutamate 2-Oxoglutarate aminoadipate aminotransferase L-2-Aminoadipate H+ ; NADH (or NADPH) NAD+( or NADP+); H2O amlnoadipate semialdehyde dehydrogenase LYS5 LYS2 L-2-Aminoadipate 6-semialdehyde L-Glutamate ; NADPH (or NADH); H+ NADP+ (OR NAD+); H2O N6-(L-1,3-Dicarboxypropyl)-L-lysine saccharopine dehydrogenase (glutamate forming) LYS9 NADP+ (OR NAD+) ; H2O 2-Oxoglutarate ; NADPH (OR NADH) ; H saccharopine dehydrogenase (lysine forming) LYS1 L-lysine

9 Lysine biosynthesis in KEGG (yeast enzymes in green)

10 EcoCyc example - proline utilization

11 EcoCyc example - proline biosynthesis

12 Ecocyc - metabolic overview

13 KEGG example : proline and arginine metabolism (E.coli) where is proline? how is proline synthesized in E.coli? how is proline catabolized in E.coli? is it obvious that reactions and have distinct side reactants?

14 Graph-based analysis of biochemical networks Pathway reconstruction by reaction clustering Jacques van Helden

15 A graph of compounds and reactions Reactions from KEGG Compound nodes 10,166 compounds (only 4302 used by one reaction) Reaction nodes 5,283 reactions Arcs 10,685 substrate reaction (7,297 non-trivial) 10,621 reaction product (6,828 non-trivial)

16 Metabolic Pathways as subgraphs Escherichia coli 4219 Genes (Blattner) 967 enzymes (Swissprot) 159 pathways (EcoCyc)

17 Reconstructing a pathway from a subset of reactions Input: a set of reactions (the seed reactions) Output: a metabolic pathway including the seed reactions, together with their substrates and products optionally, some additional reactions, interaalated to improve the pathway connectivity the pathway can either be connected, or contain several unconnected components

18 Seed nodes Compound Reaction Seed Reaction

19 Linking seed nodes Compound Reaction Seed Reaction Direct link

20 Enhance linking by intercalating reactions Compound Reaction Seed Reaction Direct link Intercalated reaction

21 Subgraph extraction

22 Validation of the method Take a set of experimentally characterized pathways, and for each one Select a subset of enzymes Use the reactions catalysed by these enzymes as seed nodes Extract the subgraph Compare with known pathway

23 Aspartate biosynthesis ATP ADP Lysine biosynthesis in E.coli L-Aspartate aspartate kinase III lysc L-aspartyl-4-P Methionine biosynthesis Threnonine biosynthesis NADPH; H+ NADP+; Pi pyruvate 2 H2O L-aspartic semialdehyde dihydropicolinic acid aspartate semialdehyde deshydrogenase asd dihydrodipicolinate synthase dapa NADPH or NADH; H+ NADP+ or NAD+ succinyl CoA CoA tetrahydrodipicolinate N-succinyl-epsilon-keto- L-alpha-aminopimelic acid dihydrodipicolinate reductase tetrahydrodipicolinae N-succinyltransferase dapb dapd glutamate alpha-ketoglutarate succinyl diaminopimelate succinyl diaminopimelate aminotransferase dapc H2O succinate LL-diaminopimelic acid meso-diaminopimelic acid N-succinyldiaminopimelate desuccinylase diaminopimelate epimerase dape dapf CO2 diaminopimelate decarboxylase lysa L-lysine lysr protein lysr

24 Example: reconstitution of lysine pathway Gap size: 0 Seeds all Ecs from original pathway are provided as seeds Result: Inferring reaction orientation (reverse or forward) Ordering

25 Example: reconstitution of lysine pathway Gap size: 1 5 seed reactions Result Inferring missing steps Inferring reaction orientation Ordering

26 Example: reconstitution of lysine pathway Gap size: 2 4 seed reactions Result E.coli pathway found Alternative pathways also returned

27 Example: reconstitution of lysine pathway Gap size: 3 3 seed reactions Result E.coli pathway is not found, because the program finds shortcuts between the seed reactions

28 Applications of pathway reconstruction We have the complete genome for dozens of bacteria, for which there is almost no experimental characterization of metabolism For these genomes, enzymes have been predicted by sequence similarity In some cases, one expects to find the same pathways as in model organisms, but in other cases, variants or completely distinct pathways For each known pathway from model organisms Select the subset of reactions for which an enzyme exists in the target organism If a reasonable number of reactions are present Using these as seeds, reconstruct a pathway Preferentially (but not exclusively) intercalate reactions for which an enzyme has been detected in the target organism

29 Graph-based analysis of biochemical networks From gene expression data to pathways Jacques van Helden

30 Reaction clustering and gene expression data Many biochemical pathways are co-regulated at the transcriptional level. Starting from the observation that a group of genes is coregulated, try to find if they could be involved in a common pathway.

31 Gene expression data: cell cycle Alpha cdc15 cdc28 Elu MCM CLB2 SIC1 MAT CLN2 Y' MET Spellman et al. (1998). Mol Biol Cell 9(12), Gilbert et al. (2000). Trends Biotech. 18(Dec),

32 Study case : cluster of co-regulated genes ID name decription YKR069W met1 siroheme synthase YFR030W met10 subunit of assimilatory sulfite reductase YGL125W met13 putative methylenetetrahydrofolate reductase (mthfr) YKL001C met14 adenylylsulfate kinase YPR167C MET16 3'phosphoadenylylsulfate reductase YLR303W MET17 O-Acetylhomoserine-O-Acetylserine Sulfhydralase YJR010W met3 ATP sulfurylase YER091C YIR017C met6 MET28 vitamin B12-(cobalamin)-independent isozyme of methionine synthase (also called N5-methyltetrahydrofolate homocysteine methyltransferase or 5-methyltetrahydropteroyl triglutamate homocysteine Transcriptional methyltransferase) activator of sulfur amino acid metabolism YGR055W MUP1 high affinity methionine permease YJR137C ECM17 ExtraCellular Mutant YER042W YIL074C YLL061W YLL062C YLR302C YNL276C YPL250C YPL274W

33 KEGG - gene search in pathway maps

34 KEGG - reaction coloring in pathway maps

35 KEGG - reaction coloring in pathway maps

36 KEGG - reaction coloring in pathway maps

37 Building pathways from gene clusters Gene chip 1 Experiment chip 2 chip 3... gene gene NA NA NA gene gene gene gene gene gene 1 gene 2 gene 3 gene 4 gene 5 gene 6 gene 7 expr expr expr expr expr expr expr protein 1 protein 2 protein 3 protein 4 protein 5 protein 6 protein 7 cat 1 cat 2 cat 3 cat 4 cat 5 cat 6 react 1 react 2 react 3 react 4 gene expression profiles gene 8 gene 9 expr expr protein 8 protein 9 Classification cluster of co-regulated genes Pathway reconstruction Putative pathway

38 Pathway found in Spellman s MET cluster ATP PPi Sulfate Sulfate adenylyl transferase MET3 Adenylyl sulfate (APS) ATP ADP '-phosphoadenylylsulfate (PAPS) Adenylyl sulfate kinase MET14 NADPH NADP+; AMP; 3'-phosphate (PAP); H '-phosphoadenylylsulfate reductase MET16 3 NADPH; 5H+ 3 NADP+; 3 H2O sulfite sulfide Putative Sulfite reductase Sulfite reductase (NADPH) MET5 MET10 O-acetyl-homoserine O-acetylhomoserine (thiol)-lyase MET17 Homocysteine 5-methyltetrahydropteroyltri-L-glutamate 5-tetrahydropteroyltri-L-glutamate Methionine synthase (vit B12-independent) MET6 L-Methionine

39 Analysis of Gene Expression Data Gene cluster 20 genes Identify genes coding for enzymes 7 enzymes Identify subset of catalyzed reactions 8 reactions Interconnect these reactions to find all possible pathways Automatic Graph Layout Compare with Classical Pathways Pathway Diagram Known Pathways 2 matching pathways

40 Comparison with Sulfur assimilation Sulfate (extracellular) Sulfate transport Sulfate (intracellular) Sulfate transporter Sulfate transporter SUL1 SUL2 ATP PPi Sulfate adenylyl transferase MET3 Adenylyl sulfate (APS) Met31p Met32p MET31 MET32 ATP ADP Adenylyl sulfate kinase MET14 3'-phosphoadenylylsulfate (PAPS) NADPH NADP+; AMP; H+; 3'-phosphate (PAP) 3 NADPH; 5H+ 3 NADP+; 3 H2O 3'-phosphoadenylylsulfate reductase MET16 MET28 sulfite sulfide Putativ e Sulfite reductase Sulfite reductase (NADPH) MET5 MET10 Cbf1p/Met4p/Met28p complex Gcn4p CBF1 MET4 GCN4 Methionine biosynthesis Met31p MET30

41 Comparison with methionine biosynthesis Aspartate biosynthesis ATP ADP L-Aspartate Aspartate kinase HOM3 L-aspartyl-4-P NADPH NADP+; Pi L-aspartic semialdehyde Aspartate semialdehyde deshydrogenase HOM2 Threonine biosynthesis NADPH NADP+ AcetlyCoA CoA L-Homoserine O-acetyl-homoserine Homoserine deshydrogenase Homoserine O-acetyltransferase HOM6 MET2 Met31p met32p MET31 MET32 Sulfur assimilation Sulfide O-acetylhomoserine (thiol)-lyase MET17 Cysteine biosynthesis Homocysteine Cbf1p/Met4p/Met28p complex MET28 CBF1 MET4 5-methyltetrahydropteroyltri-L-glutamate 5-tetrahydropteroyltri-L-glutamate L-Methionine Methionine synthase (v it B12-independent) MET6 Gcn4p GCN4 S-adenosyl-methionine H 2 0; ATP synthetase I Pi, PPi S-adenosyl-methionine synthetase II S-Adenosyl-L-Methionine SAM1 SAM2 Met30p MET30

42 Summary Starting from an unordered cluster of genes, one gets an ordered set of reactions, connected to form a pathway Should permit discovery of novel pathways, that are not stored in any pathway database yet Interpretation of intercalated reactions enzyme is not regulated DNA chip defect for that gene gene was not on the DNA chip enzyme remains to be identified in that organism

43 Analysis of data from Gasch et al. Gasch et al (2000). Molecular Biology of the Cell, 11: yeast genes Various conditions of stress (heat shock, osmotic shock, peroxide, amino acid starvation, nitrogen depletion Steady-state growth on alternative carbon sources Overexpression studies

44 Selected experiments MSN2 MSN2.overexpression..repeat. number of genes MSN4 overexpression number of genes YAP1 overexpression number of genes ethanol log(expression ratio) number of genes galactose number of genes glucose glucose.car.1 number of genes mannose..car.1 number of genes raffinose.car log(expression ratio) log(expression ratio) log(expression ratio) number of genes sucrose.car.1 number of genes ethanol YP.ethanol.vs.reference.pool.car.2 vs reference number of genes fructose YP.fructose.vs.reference.pool.car.2 vs reference number of genes galactose YP.galactose.vs.reference.pool.car.2 vs reference log(expression ratio) log(expression ratio) log(expression ratio) log(expression ratio) number of genes glucose YP.glucose.vs.reference.pool.car.2 vs reference number of genes mannose YP.mannose.vs.reference.pool.car.2 vs reference 0 number of genes raffinose YP.raffinose.vs.reference.pool.car.2 vs reference number of genes sucrose YP.sucrose.vs.reference.pool.car.2 vs reference log(expression ratio) log(expression ratio) log(expression ratio) log(expression ratio)

45 Repressed by mannose (at least 3-fold) Galactose utilization (redundancy in the database?) inferred Citrate cycle with shunt gluconeogenesis Remark: arrows should be displayed as bi-directional

46 Repressed by mannose (at least 2-fold) (redundancy in the database?) Citrate cycle with shunt gluconeogenesis Galactose utilization gluconeogenesis Remark: arrows should be displayed as bi-directional

47 Induced by galactose (at least 2-fold) Galactose utilization Remark: arrows should be displayed as bi-directional

48 Repressed by glucose (at least 2-fold) (redundancy in the database?) gluconeogenesis Galactose utilization gluconeogenesis