Unfinished business from April 6! Metabolomics, spring 06 Hans Bohnert ERML 196...
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Unfinished business from Unfinished business from April 6April 6!!Metabolomics, spring 06
Hans BohnertERML 196
265-5475333-5574
http://www.life.uiuc.edu/bohnert/
Metabolite profiling = a static picture, a snapshot!
Does it matter?*
*Fernie AR et al. (2005) Flux an important, but neglected, component of functional genomics. Curr. Opin. Plant Biology 8, 174.
*Fell DA (2005) Enzymes, metabolites and fluxes. J. Exptl Botany 56, 267.
Crossed out belowwhat has beencovered already!
… one leftover case study and new material.
class April 11
Metabolite profiling = a static picture, a snapshot! Does it matter?
Static (steady-state) “knowledge units” - genome sequence, microarray profile, proteome composition
How to understand cellular dynamics?
Flux – where to measure, how and what is the most important “link”?
Metabolites – intermediates in pathways to end-products (starch, cellulose, proteins, fats, lipids, second. products)
Enzyme activity changes: steady-state of intermediates or flux?What is affected?yeast metabolomics (mutants) metabolites do change.
Plants – metabolites +/- constant, flux altered photosynthesis – Calvin cycle – [NAD(P)H] – [ATP] – sucrose to starch [ADP-glucose pyrophosporylase]
Steady state alone can be misleadingpool size constant but coordinated increase in flux (activities altered)
Monitoring fluxRate of depletion of an initial substrate
Rate of accumulation of an end product
Isotope labeling of (a) metabolite(s) (complete or in certain atoms)
radioactive or stable isotopes (2H, 3H, 13C, 14C, 15N, 18O, 32P, 35S)
Can we infer flux from changes in intermediates? think allosteric effects of metabolites measuring regulated steps in a pathway is intermediates [conc](consider the Mark Stitt lecture)
Pathways branch (label lost)Different pathway(s) provide(s) intermediate (label diluted by unknown)Tracer addition may change the equilibrium of the systemPlants: where, and how, to introduce the tracerPool size – dilution of labelIs end-product transported – loss of labelDo we know the pathway, or assume we know, and are we right
Need certainty about pathway structures – (MapMan, TAIR, KEGG) – do we?Need certainty about pathway structures – (MapMan, TAIR, KEGG) – do we?
More pitfalls and traps!
Measuring (labeled) substrate consumption – insensitive, inaccurate
Measuring end-product – stable, transported or metabolized (e.g., disappear in cell wall; does CO2 production and glycolysis)
Branched pathways – do we know
Linear relationship between product level and time (growth!)
Experimental material – entire plant, organ (or part of organ), tissue slice, cells, organelles
How “big” is the flux, the pathway – can we actually measure it? NMR (stable isot.), GC-MS, LC-MS - sensitivity and accuracy
Positional information of tracer substrate modification may be important
Long-term feeding expt, or pulse labeling, or pulse/chase expts
Schwender et al. (2004) Rubisco without the Calvin cycle improves the carbon efficiency of developing green seeds. Nature 432, 779. (on web as: Shachar-Hill-Nature-2004)
Figure 1a
what a waste!
C3
C2
Gly
coly
sis
Fixation
non-oxidative PPP
C1 lost
Some green seedsSome green seeds(mostly oil seeds) (mostly oil seeds)
have Rubisco – why?have Rubisco – why?
Figure 1b
Expanded part 1a
fluxesVgapdh - TCA
Vpdh - PDH
Vrub - refix
Vx - OPPP
+ other
Label from Rubisco - always C1 in PGA
Figure 1 Metabolic transformation of sugars into fatty acids.
a, Conversion of hexose phosphate to pentose phosphate through the non-oxidative steps of the pentose phosphate pathway and the subsequent formation of PGA by Rubisco bypasses the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase while recycling half of the CO2 released by PDH. PGA is then further processed to pyruvate, acetyl-CoA and fatty acids.
b, Part of a expanded to indicate carbon skeletons and to define relationships between
V PDH (flux through PDH complex); V X (additional CO2 production by the OPPP, the TCA, and so on); V Rub (refixation by Rubisco). Metabolites: Ac-CoA, acetyl coenzyme-A; DHAP, dihydroxyacetone-3-phosphate; E4P, erythrose-4-phosphate; Fru-6P, fructose-6-phosphate; GAP, glyceraldehydes-3-phosphate; Glc-6P, glucose-6-phosphate; PGA, 3-phosphoglyceric acid; Pyr, pyruvate; R-5P, ribose-5-phosphate; Ru-1,5-P2, ribulose-1,5-bisphosphate; Ru-5P, ribulose-5-phosphate; S-7P, sedoheptulose-7-phosphate; Xu-5P, xylulose-5-phosphate. Enzymes: Aldo, fructose bisphosphate aldolase; Eno, 2-phosphoglycerate enolase; Xepi, xylulose-5-phosphate epimerase; FAS, fatty-acid synthase, PGM, phosphoglyceromutase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, phosphoglucose isomerase; Riso, ribose-5-phosphate isomerase; PDH, pyruvate dehydrogenase; PFK, phosphofructokinase; PK, pyruvate kinase, PGK, phosphoglycerate kinase; PRK, phosphoribulokinase; TA, transaldolase; TK, transketolase; TPI, triose phosphate isomerase.
Results of NMR and GC-MS analyses
Remember – MS can “see” isotopomers! i.e., can observe which carbon is 13C.
Conclusions Rubisco operates as part of a previously un-described metabolic routebetween carbohydrate and oil (Fig. 1a). Three stages:(1) conversion of hexose phosphates to ribulose-1,5-bisphosphate by the non-oxidative reactions of the OPPP together with
phosphoribulokinase.
(2) conversion of ribulose-1,5-bisphosphate and CO2 (most produced by PDH3) to PGA by Rubisco
(3) metabolism of PGA to pyruvate and then to fatty acids (Fig. 1a).
The net carbon stoichiometry of this conversion: 5 hexose phosphate > 6 pentose phosphate > 12 acetyl-CoA + 6 CO2
The conversion of the same amount of hexose phosphates by glycolysis:5 hexose phosphate >10 Acetyl-CoA + 10 CO2
PGA
PEP
Phe
Val
Ac-CoA
Pyruvate[1-13C]Alanine
Ru1,5-P2Triose-P
1 1
1
11 1
PDH
PK
PGM, ENO
Val
1Val(1-5) Val(2-5)
Phe
1PHE(1-2)
PHE(2-9)PHE(1-9)
B D
CO2
PGA
PEP
Phe
Val
Ac-CoA
PyruvateAlanine
External 13CO2
Ru1,5-P2Triose-P
1 1
1
11 1
PDH
PK
PGM, ENO
A
CO2
PGA
PEP
Phe
Val
Ac-CoA
Pyruvate[U-13C3]Alanine
Ru1,5-P2Triose-P
1 1
1
11 1
PDH
PK
PGM, ENO
OAA
C
CO2
Fate of labeled COFate of labeled CO22 in fatty acid biosynthesis in fatty acid biosynthesis
Scenarios and calculations
externalfeed
feed by13C1-ala
feed byU-13C1-ala
calculateratios
from MS
A summary
3 pathways
glycolysis (not TCA) OPPP Calvin cycle
loss 1C
fatty acids
C5needs NAPH
C2
Rubisco
needs light+
chloroplasts
Only flux through Rubisco leads to increased efficiencyby providing 40-50% ofthe PGA for FA-biosynthesis
15% of NADPH/ATPused in FAbiosynthesis
How about non-green seeds? (sunflower)
TPs
• To arrive at the total carbon balance, all reactions leading to amino acidshad to be quantified (count isotopomers).
• The contribution of carbon from OAA (which would be unlabeled in short-time experiments) had to be tested.
• The lack of re-utilization of pyruvate back to PEP had to be ruled out
• 5 Glucose molecules (30 C) are transformed to 24 C-atoms in acetyl-CoA with 6 CO2 being released. Therefore 80 % of the carbon provided as carbohydrate is incorporated into fatty acid by this novel route, compared to 66.7% by the conventional glycolytic route. Bypass of GAPDH and PGA kinase requires that ATP and reductant must be provided by light. Light doesindeed affect the ratio of carbon to oil.
• Non-green plastids showed a ratio of carbon to oil of 1.2-16 (instead of ~3),i.e., they operated through glycolysis and TCA cycle to generateseed-deposited oil.
Summarizing statements
HP PP
TP
PGA
Pyruvate
CO2
Hexose
Ac-CoA
PPP
HP PP
TP
PGA
Pyruvate
CO2
CO2
Hexose
Ac-CoA
PPP
A. Glycolysis C. Non-oxidativebypass
HP PP
TP
PGA
Pyruvate
CO2
Hexose
Ac-CoA
PPP
C18:0 C18:0 C18:0
HP PP
TP
PGA
Pyruvate
CO2
Ac-CoA
PPP
D. Autotrophy
C18:0
B. Oxidativebypass
CO2 CO2 CO2 CO2
3791Number of modes of this type
-52
-71
∞
-7
-8
80 %
+2
-8
66.7 %66.7 %
Carbon in C18:0 / Carbon uptake
(glucose)
+2NADPH balance
+1ATP balance
3791Number of modes of this type
-52
-71
∞
-7
-8
80 %
+2
-8
66.7 %66.7 %
Carbon in C18:0 / Carbon uptake
(glucose)
+2NADPH balance
+1ATP balance
HP PP
TP
PGA
Pyruvate
CO2
CO2
Hexose
Ac-CoA
PPP
1
3
4
2
5
6
7
C18:08
CO2
Flux mode analysis of seed metabolism.
C-use efficiency and 4 characteristic fluxes shown relative to one mol C18:0
Roessner-Tunali et al. (2004) Kinetics of labeling of organic and amino acids in potato tubers by gas chromatography-mass spectrometry following incubation in (13)C labeled isotopes. Plant J. 39, 668.
Where does the label go?
• primary metabolism
• potato tubers
• wild type and transgenics
• EI GC-MS (not CI)
• U-13C/14C glucose feeding
• pathway verification
As much a science project as a test of the sensitivity of GC-MS
• What tissue to use
• Type of MS to use
• Type of ionization to use
• Do we need fractionation?
• Can global analysis provide data that are equivalent to single metabolite analysis in accuracy?
• Can global analysis provide an accurate picture to evaluate the exchange of carbon between pools?
• Can we use GC-MS for phenotyping/fingerprinting and/orGMO-typing?
Objectives (Roessner-Tunali)
Possible reaction ratesto measure
What is U-13C or U-14C glucose?
Three lines:
Wt
INV-2-30 ??
SP-29 ??
Sucrose Phosphorylase
Sucrose phosphorylase is the enzyme responsible for the conversion of sucrose to fructose and glucose-1-phosphate. This reaction is reversible. The enzyme is reported to have broad specificity, and so it may be possible for many other substrates to replace fructose as the glucosyl acceptor. Sucrose phosphorylase has the potential, therefore, to covert sucrose to a number of industrially useful glucoslyated derivatives, with the
commercially important sugar fructose as the by-product
2nd take home, and final essay, for the remaining and dedicated undergraduate students.
(1) The paper uses an INV line (INV-2-30). Please collect information on thefunction of this enzyme, its position and function in metabolism, and its effect(s) on carbon distribution in plants.
(2) What is the reaction catalyzed by INV, which comes in several forms and may be found in different compartments. Please explain.
(3) Describe reactions and enzymes that counteract the presence of INV.
(4) You should consult one reference each that report on the over- and under-expression of INV in transgenic plants (mainly tobacco and potato) and discuss the results. Please identify the references.
Please return your essay, preferable typed and short and concise, by the first week of May (i.e., before study day).
Amounts over time (up to 12h)
(P < 0.05)
bold - transgenic difference to wild type
tuber slices5h incubationwash – GC-MS
Hex-P determined:
P-ester pool/ hex-P
Mean +/- SE (n=3)
Sucrose reductionenzymes
-may increase amino acids
increase sucrose re-synthesis
reduce starch amounts(not synthesis)
reduce hex-P pool
reduce cell wall material(not synthesis)
nmol x g FW-1
important – watch differences in rates of synthesis (Δf = >100)
predominant fluxes
isotopomer counting
[CH3-O-NH3]+ Cl-
absolute amounts bystandard dilutions
• results comparable to conventional methods measuring individual metabolites; faster & at least as accurate• starch turnover in tubes• sucrose low in transgenics leads to increased carbon partitioning• identify uni-directional flux in patterns
• distribution of a single isotopically labeled precursor
Problems?• constant rate assumed• assume one cell type• no further use of products• accuracy of side reactions• maybe stable isotopes
A different experiment
Arabidopsis ecotypes in high CO2 in FACE rings
Attempts at correlating
gene expression and
metabolite concentrations
Raffinose
SucroseStarch
Galactose
Glucose Fructose Melibiose
3-Phosphoglycerate
Pyruvate
Acetyl-CoA
Citrate
Isocitrate
alpha-Ketoglutarate
Succinate
Fumarate
Malate
Oxaloacetate
Chorismate
Serine
Cysteine
Tyrosine
Phenylalanine
Prephenate
PEP
Tryptophan
Glycine
Leucine
Valine
Aspartate
Asparagine
Aspartate-4-semialdehyde
Lysine
Homoserine-4-phosphate
Threonine
Isoleucine
Methionine
Glutamate
Glutamine
Proline
2.3.3.1
4.2.1.3
1.1.1.42
6.2.1.41.3.5.1
4.2.1.2
1.1.99.16
5.4.2.1
4.2.1.11
2.7.1.40
1.1.1.952.6.1.523.1.3.32.1.2.1
2.3.1.30
4.2.99.8
3.2.1.26Neutral
Invertase
Invertase, cell wall
Invertase, vacuole
5.3.1.9
2.7.1.1
1.2.1.12
2.7.2.3
Maltose
Oxaloacetate
4.1.1.49
4.1.1.31
4. 1.3.8
2.2.1.61.1.1.862.6.1.42
4.1.3.124.2.1.331.1.1.852.6.1.42
2.6.1.1
6.3.5.4
2.7.2.4
1.2.1.11
4.2.1.52
1.3.1.26
2.6.1.17
3.5.1.18
5.1.1.7
1.1.1.3
2.7.1.39
4.2.3.1
2.2.1.6
1.1.1.86
2.6.1.42
4.2.99
4.4.1.8
2.1.1.142.1.1.10
1.4.7.1
6.3.1.2
4.1.3.27
4.2.1.10
2.7.1.71
2.5.1.194.2.3.5
2.4.2.18
5.3.1.24
4.1.1.48
5.4.99.5
1.3.1.12
4.2.1.51
2.6.1.5
4.2.3.4
3.2.1.1
3.2.1.22.4.1.25MEX1
DEP2
Galactinol2.4.1.123
2.4.1.82
AT5G65750
At4g02610At4g27070
At5g14800At5g62530
Figure 7.
Co
l 21
Transcripts
Metabolites
-0.6 0 0.6
(log2 - fold change)C
ol
27
Cvi
21
Cvi
27
isoforms
Adding an introduction to the next topic – single cell analysis, using NMR a a major tool.
Hoping to get here!
Metabolomics-edited transcriptomics analysis ofMetabolomics-edited transcriptomics analysis ofSe anticancer action in human lung cancer cellsSe anticancer action in human lung cancer cells
Fan, Bandura, Higashi & Lane (2005) Metabolomics 1, 325-339
(META)
Transcriptomic analysis is an essential tool for systems biology but it has been stymied by a lack of global understanding of genomic functions, resulting in the inability to link functionally disparate gene expression events. Using the anticancer agent selenite and human lung cancer A549 cells as a model system, we demonstrate that these difficulties can be overcome by a progressive approach which harnesses the emerging power of metabolomics for transcriptomic analysis. We have named the approach Metabolomics-edited transcriptomicanalysis (META). The main analytical engine was 13C isotopomer profiling using a combination of multi-nuclear 2-D NMR and GC-MS techniques. Using 13C-glucose as a tracer, multiple disruptions to the central metabolic network in A549 cells induced by selenite were defined. META was then achieved by coupling the metabolic dysfunctionsto altered gene expression profiles to: (1) provide new insights into the regulatory network underlying the metabolic dysfunctions; (2) enable the assembly of disparate gene expression events into functional pathways that was not feasible by transcriptomic analysis alone. This was illustrated in particular by the connection of mitochondrial dysfunctions to perturbed lipid metabolism via the AMP-AMPK pathway. Thus, META generated both extensive and highly specific working hypotheses for further validation, thereby accelerating the resolution of complex biological problems such as the anticancer mechanism of selenite.
Key words (3-6) two-dimensional NMR; GC-tandem MS; 13C isotopomer profiling; selenite; lung adenocarcinoma A549 cells.
Abbreviations 1H–13C HMBC: 1H–13C heteronuclear multiple bond correlation spectroscopy; 1H–13C HSQC: 1H–13C heteronuclear single quantum coherence spectroscopy; 2-D 1H TOCSY: two dimensional 1H total correlation spectroscopy; [U)13C]-glucose: uniformly 13C-labeled glucose; MSn: mass spectrometry to the nth dimension; MTBSTFA: N-methyl-N-[tert-butyldimethylsilyl]trifluoroacetamide; P-choline or PC: phosphorylcholine; PDA: photodiode array; TCA: trichloroacetic acid.
Knowledge: Se is an essential atom, high amounts affect (cancer) growth, Se inproteins is related to ROS homeostasis (somehow)
Experiment: The addition of Se to lung cells affects growth – what is the basis?Use genomics platforms (transcript analysis), GC-MS & esp. NMR
Hypothesis: gene expression is altered, and metabolite analysis can be correlated with transcript changes – can it, is the question!
Approaches Microscopy, NMR, GC-MS, transcripts
Se interferes with the cytoskeleton and mitochondrial activity
Selenite effects proliferating cells;
Selenite-rich diets may have anti-cancerapplications.
Se leads to degradation of DNA
TUNEL assay?